Vitamin K and Teeth

Published Research – Vitamin K and Teeth

• Teeth and bone are dynamic tissues, constantly maintaining and renewing themselves with the help of vitamin K. 
• Osteocalcin and matrix Gla protein are critical to bone mineralization, formation and turnover.  They make the jaw healthy!
• Vitamin K dependent proteins have been found throughout bones and teeth.  These proteins require vitamin K to activate them.  
• Vitamin K has been proposed as a treatment for BRONJ, and as an alternate to bisphosphonates.
• Osteocalcin significantly accelerates tooth movement during orthodontic therapy.
• Some of the highest concentrations of vitamin MK4 are found in saliva.  
• Vitamin K has antimicrobial effects and some believe vitamin K reduces plaque buildup through its presence in saliva, Fetuin A and matrix Gla protein.

On this page, we will discuss mineralized tissues, with a focus on teeth and bones, and the role of vitamin K.  We will begin by reviewing the components of each, and the importance of the vitamin K dependent proteins, osteocalcin and matrix Gla protein in teeth and bones.  We will review the abundance of research on vitamin K and bone health, and the emerging study of vitamin K and teeth health. Bone and teeth are dynamic tissues, and remodeling and remineralization are an ongoing process.  There is emerging research on how vitamin K is an important, effective, and safe contributor to those dynamic processes.  

Mineralized Tissues
To begin, teeth and bone are mineralized tissues, which are biological tissues of the body have incorporated minerals into a collagen matrix, hence the term biomineralization. Biomineralization is a dynamic, complex, lifelong process.  

Collagen is an important structural protein found in many tissues.  More than twenty human collagens have been reported.  Of these, type I collagen is the most abundant protein in the human body and it is essential to the proper development and function of mineralized tissues.  Type I collagen forms into a matrix that serves as the structural scaffold for mineralization. Calcium and phosphorus are deposited into this matrix and they complex together as crystalline salts which act collectively to harden and stabilize the collagen matrices within which they reside.  Together this constitutes crystals of hydroxyapatite, which is the mineral found in vertebrate bones, mammalian teeth, and fish scales (Feng et al, 2009; Prasad et al, 2010; Gorski, 2011).  The result is a hardened, mineralized tissue which is rigid and strong (Lin et al, 1993; McKee et al, 2005; McKee et al, 2012).

Mineralized tissues combine stiffness, low weight, strength, flexibility, and toughness.  Mineralized tissue is able to withstand substantial loads and impacts, and is able to respond dynamically – through the actions of cells – to a variety of mechanical challenges.  It gives bones and teeth their greater loadbearing capacity (Barthelat, 2007; Qin et al, 2012).      

The process of biomineralization forms unique hybrid biological tissues, for example, enamel, dentin, cementum, and bone.  Biomineralization is precisely regulated by proteins.  

These remarkable tissue construction events are orchestrated by the tissue-forming “blast” cells – the osteoblasts, the odontoblasts (a misnomer, in fact should be dentinoblasts) and the cementoblasts – with each being associated with a thin layer of unmineralized matrix which subsequently mineralizes at the “mineralization front” to form the completed tissue. (Mckee, et al, 2013). 
 

Teeth
First, let’s review the components of teeth, or dentes, which is the Latin term.  A tooth consists of a crown and a root. The crown of a tooth is what is evident when you look in your mouth.  The root is anchored in the bone of the jaw, surrounded by the gums. Another name for the gums is gingiva.  The part of the jawbone where the teeth are anchored is referred to as the aveolar bone. Inside the tooth are chambers in the crown and a canal in the root, which together forms the pulp cavity that houses dental pulp.  Dental pulp furnishes the blood and nerve supply of the tooth. The term periodontium refers to all the specialized tissues that both surround and support the teeth, maintaining them in the jawbone. 

The tooth and its periodontium represent a remarkable tooth-suspension and masticatory apparatus, functionalized by four key mineralized tissues – enamel, dentin, cementum and bone  - along with a periodontal ligament, the loss of any one of which will render the entire apparatus nonfunctional (Abou, et al, 2016).

-Enamel
The crown, or the portion of the tooth that is visible in the mouth, is composed of dentin and is covered by a layer of enamel, the hardest tissue of the body.  Enamel is a unique, highly mineralized material that is designed to resist the forces of mastication, chewing, biting, and crunching. It also helps to resist mechanical abrasion and grinding, and chemical (dietary and bacterial) attack during the entire life of an individual (Nancy & Smith, 2000; Simmer et al, 2010; Bar-on & Wagner, 2012).  

Enamel is produced by ameloblast cells which deposit enamel on the tooth, while it develops within the jawbone.  Ameloblasts cells secrete the proteins which will later mineralize to form enamel. Ameloblast cells are present only during tooth development.  As the enamel matures, the enamel proteins are removed, and the enamel turns into a highly mineralized tissue.  Half of the ameloblasts die during enamel formation, and the rest die after the process ends, at the point of tooth eruption.  

Tooth enamel can be weakened and damaged by bacterial acids, which can result in dental cavities.  Because ameloblasts, which make enamel, are not present on the surfaces of erupted teeth, it is not possible to create or produce secondary or regenerative enamel (Smith, 1998; Lacruz et al, 2017).  Biological repair is not possible, however weakened enamel can be restored to some degree by improving its mineral content.   

-Dentin
Beneath the enamel and around the pulp lies the dentin, a hard, dense, bone-like matrix which makes up the bulk of each tooth.  Dentin is both harder and denser than actual bone. Dentin surrounds the pulp cavity that holds the nerves and blood vessels necessary for tooth function.  The rigid dentin provides powerful protection for dental pulp and support for enamel, while also allowing flex when forces from chewing are placed upon the enamel, preventing its fracture (Ten Cate, 1994). 

Dentin consists of a complex mixture of proteins and other molecules attached to mineralized tissue, which are organized as microscopic channels, called dentinal tubules.  Dentinal tubules radiate outward through the dentine from the pulp to the outer layer of the enamel border. There are about three miles of tubules in each tooth (Bronckers et al, 1985; Linde & Goldberg, 1993; Butler & Ritchie, 1995; Ten Cates, 2017). Although your teeth look solid, they are actually porous, but the dentinal tubules are not visible to the eye. 

The tooth is fed by elements delivered by fluid flow through the dentinal tubules.  This fluid flow is typically from the inside out, however it can be halted or reversed, when impacted by a systemic stressor such as excessive sugar intake (Leonara et al, 1993; Zhang et al, 2005). 

Dentin is produced by odontoblast cells.  Odontoblasts are specialized cells which produce and deposit the organic matrix that will serve as the structural skeleton in dentin.  Odontoblasts also synthesize dentine matrix proteins, collagens and non-collagen proteins like osteonectin, and osteocalcin into mineralizing dentin.  The majority of dentin is composed of proteins common to both dentin and bone (Dahl & Mjor, 1973; Bronckers et al, 1985; Dimuzio et al, 1983; Finkelman & Butler, 1985; Gehron-Robey, 1989; Butler & Ritchie, 1995; D’Souza et al 1997; Papagerakis et al, 2002). 

Unlike enamel, dentin continues to form throughout life as it is a living tissue responsible for the constant growth and repair of the tooth.  The dentin has live cells that monitor what is happening in the mouth. These live cells transmit pain, especially when you eat or drink something that's hot or cold because the dentin stimulates your tooth pulp or nerve.  Because it is softer than enamel, dentin decays more rapidly and is subject to severe cavities. 

The potential of pulp tissue to self-regenerate lost dentin is well known. When pulp tissue is exposed due to the loss of the overlaying dentin, direct pulp capping therapy can allow the pulp to form new dentin, known as a dentin bridge.  Dentin mineral is remodeled to a much lesser extent.         

- Cementum
The third mineralized tissue is cementum.  Cementum is a bone-like tissue that forms a thin surface layer over the roots of teeth.  Cementum performs essential functions: first to provide the attachment of the tooth to the alveolar bone, or jaw bone, by the periodontal ligament fibers (also called Sharpey’s fibers); and second to prevent the root from being resorbed during remodeling of the periodontium.  The aveolar bone is the thickened ridge of the jaw bone that contains the tooth sockets and it comes in direct contact with the root of the tooth. The aveolar process refers to the bony portion of the jawbone, which houses the developing tooth buds and later the roots of the teeth.  The periodontal ligament is specialized connective tissue that joins the cementum to the alveolar bone. (Schroeder, 1991; Freeman, 1994; Bosshard & Selvig, 1997; Hashimoto et al, 2001; Nanci & Bosshardt, 2006; Ripamonti, 2007). The periodontal ligament completes the suspension apparatus for the tooth, which allows for the flexible distribution of forces from chewing.  Thirdly, cementum helps support the tooth, along with the alveolar bone and the periodontal ligament. The amalgam of cementum and tooth crown dentin, flex readily when forces are placed upon the enamel (Herring, 2012; Ho et al, 2000; McCulloch et al, 2000; Naveh et al, 2012).  

Similar to the function of osteoblast cells in bone tissue, cementoblasts form the organic matrix with collagen fibers, and noncollagenous proteins such as osteocalcin, bone sialoprotein, osteopontin, glycoproteins and proteoglycans (Bosshardt & Selvig 1997; D’Errico et al, 2000; Bosshardt et al, 2006; Choi et al, 2016).  The hydroxyapatite becomes crystallized and the cementum mineralization proceeds.   

During the later steps, many of the cementoblasts become entrapped by the cementum they produce, becoming cementocytes.

The cementum is a dynamic and responsive tissue and has adaptive and reparative functions.  Cementum has a very slow regenerative capacity and is not resorbed under normal conditions.  Unlike ameloblasts and odontoblasts, which leave no cellular bodies in their secreted products, many cementoblasts and osteoblasts become entrapped by the formed cementum and bone they produce, becoming cementocytes and osteocytes, respectively.  Unlike bone tissue, this tissue does not undergo remodeling but grows thicker with constant cell renewal (Bosshardt & Selvig 1997).  

Stimulating or augmenting cementum formation is a goal of restorative dentistry

Bone
The fourth mineralized tissue is bone.  Bones perform multiple functions, such as structural support for the body, allowing movement, and protecting our vital organs.  Bone tissue also serves as an important vital ion reservoir.  Bone stores abundant quantities of key nutrients, lipids, and mineral ions such as calcium and phosphate which can be mobilized upon demand and released for systemic use by cells throughout the body (Wysolmerski, 2010).

Teeth are anchored in the jawbone, which is also referred to as the maxilla (upper jawbone) and the mandible (lower jawbone).  The jawbone acts to maintain and stabilize teeth during adulthood (McKee et al, 2002). The jawbone has a high turnover (modeling and remodeling) rate, presumably resulting from the substantially large and frequent mastication forces placed upon it (Bertoldo et al, 2007; Tricker et al, 2002).  

Osteogenesis is the formation of bone tissue.  Bone tissue begins with osteoblast cells, which secrete mainly type 1 collagen, and important noncollagen proteins, including osteocalcin, osteopontin, and bone sialoprotein, among others.  Osteoblasts are also involved in the mineralization process (Palmer et al, 2008).

Collagen provides a soft, unmineralized matrix, as a template for mineralization.  The mineral portion of bone is calcium and phosphorus which together constitute crystals of hydroxyapatite, which give it strength and density (Feng, 2009; Prasad et al, 2010; Gorski, 2011).  Almost 70% of bone is made up of hydroxyapatite, and it gives bones and teeth their greater load bearing capacity (Boskey, 2006).  

Before the extracellular matrix is calcified, it is called osteoid (meaning bone-like) tissue. When the concentrations of calcium and phosphate ions rise high enough, they are deposited into the extracellular matrix, and the bone calcifies, converting the osteoid into mineralized bone.  Calcification occurs only in presence of collagen. Impaired calcification (i.e. in diseases such as rickets) leads to higher levels of osteoid tissue than normal.  

In the process of mineralization, some osteoblasts are trapped within the mineralizing matrix and become a different type of cell, called osteocytes.  Osteocytes are the most abundant and long-lived cells within bone. Osteocytes regulate both remodeling and mineralization processes (Shimada et al 2001; Balcerzak et al 2003).  Osteocytes communicate with each other via interconnecting long channels that can send messages throughout the tissue to other cells that maintain bone. The primary function of these cells appears to be signaling for bone resorption or formation in response to mechanical stress.  Thus, both osteoblasts and osteocytes remain viable and active during the mineralization process (Lanyon, 1993; Bonewald, 2006; Feng et al, 2009; Gorski, 2011). 

Ultimately, bone is composed of bone forming cells, osteblasts and osteocytes and bone resorbing cells, osteoclasts, a matrix of collagen and noncollagenous proteins and inorganic mineral salts.  Bone resorption is essential for the growth, healing, and remodeling of adult bone and to regulate the calcium available to the body. 

Vitamin K Dependent Proteins
Now for the good stuff!
Vitamin K dependent proteins (VKDPs) are a group of proteins that require vitamin K to carboxylate or biologically activate them.  Vitamin K dependent proteins have in common the presence of Glutamic acid residues.  These Glu residues require vitamin K to be present and available in order to become carboxylated or biologically activated.  Carboxylation means that the Glu residues are converted into Gla residues and are then referred to as Gla proteins. Gla-proteins have a high affinity for mineral ions such as calcium and for hydroxyapatite crystals.  Carboxylation is critically important, as it now allows the proteins to bind calcium (Suttie, 1985; Nimptsch et al 2009).  

Vitamin K dependent proteins play an important role in regulating mineralization in bone.  Without vitamin K, the proteins are not carboxylated or biologically activated and do not attract or bind calcium.  

Osteocalcin and matrix Gla protein (MGP) are the two major Gla-containing proteins associated with calcified hard tissue like teeth and bones.  Both have a high affinity for calcium and hydroxyapatite through interaction with the Gla residue, and they are important in the regulation of mineral accumulation in calcified tissues.  Matrix Gla protein and osteocalcin are two vitamin K dependent proteins that also play important roles in tooth development and maintenance (Hauschka et al, 1975; Hauschka & Reddi, 1980; Price et al, 1981; Price et al, 1983; Otawara & Price, 1986; Hauschka et al, 1989; Price, 1989).  Another important is periostin (Balli et al, 2015; Cobo et al, 2015).  And in both teeth and in bones, vitamin K dependent proteins regulate the mineralization process, particularly matrix Gla protein (Zhu et al, 2007; Li et al, 2012).  

Below is a brief review of some of the key research done on osteocalcin and matrix Gla protein as it relates to mineralized tissues.

OSTEOCALCIN
Osteocalcin (OC) is one of the most abundant, vitamin K-dependent proteins found in people   It is also referred to as bone Gla protein or BGP.  The distribution of osteocalcin in the mammalian body is virtually limited to mineralized tissues such as bone, dentin and cementum (Cole & Hanley, 1991).  Recent research has demonstrated that osteocalcin is not only involved in bone remodeling but plays a critical function in multiple physiological processes, including as a hormone and important in regulating glucose, cognitive functioning and male fertility (Price et al, 1976; Schwetz et al, 2012; Lombardi et al, 2015).  

Osteocalcin and Bone
Osteocalcin is crucial for bone mineralization.  When osteocalcin is secreted into the hydroxyapatite crystal grid, and with the availability of vitamin K, osteocalcin becomes carboxylated and activated.  Carboxylated osteocalcin has a high affinity for calcium ions and binds calcium, incorporating it into the bone mineral matrix.  This precedes the mineralization of bone. This way, osteocalcin seems to be able to exert its regulatory effects on the organization of the bone (Koshihara et al 1997; Allison, et al 2000; Young, 2003; Gorski, 2011; Shiraki, et al 2015; Zoch et al 2016; Akbari & Rasouli-Ghahroudi, 2018). 

Osteocalcin and Bone Formation
Most osteocalcin is incorporated into the organic matrix that will later ossify into bone, however, a small fraction is secreted into the circulation. For this reason, osteocalcin was initially studied as a bone formation marker, and, indeed, osteocalcin concentrations correlate with direct measurements of bone formation (Delmas et al 1986).  The first piece of evidence that OC is a determinant of bone formation was obtained in mice lacking osteocalcin genes whom developed a higher bone mass (Ducy et al, 1996), suggesting that OC directly regulates osteoblastic bone formation.  

Osteocalcin levels in the blood (serum) are an established biomarker of bone turnover and bone formation (Delmas 1993; Chapurlat & Confavreux, 2016) as most osteocalcin is bound to bone minerals and only small amounts are detected in the circulation.   Serum or blood, levels of osteocalcin reflect intact or total osteocalcin (tOC), which refers to the sum of both cOC (carboxylated osteocalcin) and ucOC (uncarboxylated osteocalcin). The percentage circulating osteocalcin that is not γ-carboxylated (percent ucOC) is used as a biomarker of vitamin K status (O’Connor & Durack, 2017) and considered the inactive form in terms of bone metabolism in humans).  Currently, serum OC levels are used to evaluate bone metabolism, as a bone formation marker, and are often considered one of the most sensitive markers (Delmas et al, 1990; Delmas 1993; Cantatore et al, 2004; Reinehr & Roth, 2010; Neve et al, 2012). 

Subsequent studies suggested that OC accelerates and increases bone formation. It was demonstrated that OC improved the initial adherence of osteoblast‐like cells on biocement (Knepper‐Nicolai et al, 2002).  Similarly, the addition of OC enhanced the appearance of active osteoblasts and bone healing around hydroxyapatite/collagen composites in an animal model (Rammelt et al, 2005).  

Osteocalcin and Bone Turnover
In addition to the circulating osteocalcin derived from bone formation in the circulation, there is evidence that intact as well as osteocalcin fragments are liberated from the bone matrix during osteoclastic bone resorption (Salo et al, 1997; Ivaska et al, 2004; Ferron et al, 2010).  Hence, in a clinical setting, osteocalcin has also been used as a surrogate marker to evaluate bone turnover in terms of bone loss and to describe the effects of antiosteoporotic drugs (Vergnaud et al 1997; Szulc & Delmas 2008; Chapurlat et al, 2000; Gorski, 2011).  

Osteocalcin and Bone Mineralization
Osteocalcin also seems to be involved in the process of mineralization as well as bone matrix production.  It is believed to act in the bone matrix to stimulate bone mineral maturation and to regulate mineralization  (Hauschka, et al 1989), and in calcium homeostasis (Boskey et al 1998; Neve, et al 2013).

Rodents who have been bred to not have functioning osteocalcin undergo increased bone mineralization, followed by an increase in trabecular thickness, density and bone volume. During skeletal development, bone mass increases due to the dominant function of osteoblasts which secrete OC, amongst other proteins, enabling bone to grow (Ducy, et al 1996). 

Osteocalcin and Vitamin K Status
It has also been speculated that serum ucOC levels are a marker of both bone turnover as well as vitamin K status in bone.  Low dietary intake of vitamin K is related to elevated ucOC levels in the blood (Aonuma et al, 2009). The failure of carboxylation results in accumulation of uncarboxylated OC, with less affinity to calcium ions and consequently reduced bone quality (Hauschka et al, 1989).  Serum ucOC levels increased with age and after menopause in elderly women and predicted the risk of hip fracture in the same patients).  Recent data demonstrated that higher levels of ucOC, was associated with increased susceptibility to vertebral fracture and a greater risk of low BMD (Plantalech et al, 1991; Szulc et al, 1996; Vergnaud et al, 1997; Knapen et al, 1998; Booth et al, 2004; Tsugawa et al, 2008; Emaus et al, 2011), suggesting their bones are not as strong when the osteocalcin was not activated and functional.  In Japan, the levels of ucOC has been used clinically to decide whether supplementing with vitamin K is indicated for the treatment of osteoporosis. 

A recent work on bones from single and double genetic knockout mice shows that osteocalcin helps direct mineral growth and thus determines the quality of bone.  Especially, it is proposed that osteocalcin helps regulate bone mineral crystal size, shape, and organization (Simon et al, 2018).

Osteocalcin and Teeth
Specifically in teeth, osteocalcin is produced by odontoblasts and cementoblasts.  Odontoblasts are responsible for the formation of dentin and pre-dentin, and cementoblasts are responsible for the formation of cementum, which covers the tooth and root.  Osteocalcin has been found to determine the expression pattern of DSPP, which stands for dentin sialophosphoprotein. DSPP is necessary for proper dentin formation and the regulation of mineral deposits in teeth.  The proteins it produces become the major noncollagenous components of teeth and are distributed in the collagen matrix. (Papagerakis et al, 2002).  

Osteocalcin has also been found within the enamel matrix, specifically when the maturation stage was reached (Bronckers et al 1985; Price, 1985; Hauschka & Wians, 1989; Hauschka et al 1989; Bronckers et al, 1994; Bronckers et al, 1998; Papagerakis et al, 2002).  

In the dental root, osteocalcin expression is localized in cells lining cementum. And cells at the inter‐radicular area also express osteocalcin (Kagayama et al, 1997).  

Dental pulp has an important ability to form mineralized hard tissue in response to a variety of external stimuli.  Recently, several studies revealed that the source of cells for hard tissue formation is the dental pulp itself.  Dental pulp tissue contains stem and progenitor cells that differentiate into odontoblasts as well as osteoblasts, and which participate in dentin and pulp regeneration.  When injured, pulp cells migrate, proliferate, and differentiate into odontoblast-like cells to lay down new reparative dentin and osteocalcin is one of the reparative molecules commonly expressed in response to injury of the dental pulp (Shigehara et al, 2006; Abd-Elmeguid et al, 2013).  

A goal of orthodontics is to improve the rate, quality and stability of tooth movement.  The presence of osteocalcin has been found to accelerate tooth movement. When purified osteocalcin was injected into rat molars, it enhanced the rate of orthodontic tooth movement (Kobayashi et al, 1998).  It enhanced osteoclastogenesis, or bone resorption, on the pressured side. Later research, with slightly different methodology (coil spring vs elastic band) for more precision, showed that injections of osteocalcin into rats elicited a significantly greater rate of tooth movement than the controls and increased the total amount of tooth movement.  The accelerated orthodontic tooth movement lasted at least ten days. Later examination revealed that this acceleration of tooth movement was caused by enhanced recruitment of osteoclasts, augmented by osteocalcin on the pressure side. The conclusion drawn was that OC may be an effective agent for facilitating orthodontic tooth movement (Hashimoto et al, 2001).

MATRIX GLA PROTEIN
Matrix Gla protein (MGP) plays an important role in the development of tooth and bone.  Matrix Gla protein, is an essential inhibitor of mineralization and regulates the potentially fatal accumulation of calcium (Romberg et al, 1986; Price, 1989; Luo et al, 1997; Pudota et al, 2000;  Bandyopadhyay et al, 2002; Roy & Nishimoto, 2002;  Scheller et al, 2009; Sasaki et al, 2010; Jussila & Thesleff, 2012; Theuwissen, et al, 2012; Ma et al, 2015; Wang et al,  2017).

MGP and Calcification
According to the findings in both animal and human studies, MGP facilitates normal bone metabolism, while inhibiting the calcification of the soft tissues it is expressed from.  Early research in genetically modified mice bred to lack MGP, demonstrated that they developed spontaneous and extensive calcification of arteries and cartilage, premature calcification in bone, calcification of normally noncalcifying cartilage, such as the trachea, increased calcification of growth plate cartilage, short stature, osteopenia, and fractures.  As a result of excessive abnormal calcification of their arteries, leading to blood vessel rupture and death, the MGP-deficient mice died around two months of age. This was ground breaking research as it illustrated that the absence of MGP unleashed a rapid calcification process, while confirming that in the presence of MGP the calcification process was regulated (Luo et al, 1995, 1997; Price et al, 1998; Howe & Webster, 2000). 

Research using the drug warfarin, provided further insight into the regulatory role of matrix Gla protein.  Warfarin is a vitamin K antagonist, meaning it interferes in the function of vitamin K. For example, treatment of pregnant rats with warfarin, reduced Gla residue synthesis in the fetus and newborn and caused severe skeletal malformations (Suttie, 1985).  These defects include disorganization and excessive mineralization of growth plate and nasal septum cartilages, early closure of the growth plate, growth retardation, and abnormalities in, ossification centers (Price et al, 1982; Price, 1989; Feteih et al, 1990).  A similar prenatal exposure to warfarin is associated with embryopathy and skeletal defects in humans (Hall et al, 1980).  

The Keutel syndrome, an autosomal recessive disorder that is associated with abnormal cartilage calcification, short terminal finger bones, pulmonary stenosis and underdeveloped midface, was found to be based on genetic mutations which resulted in a nonfunctional MGP protein.  This was further evidence on the importance of MGP in regulating calcification (Yasui et al, 1999; Munroe et al, 1999).

MGP and Calcification Regulation
It appears that MGP regulates calcification with a specific timing and spatial pattern of expression.

The developmental expression of MGP in cartilage and bone formation was studied and they found that MGP was expressed at its highest level during cartilage formation and calcification, as the developing cartilage was replaced by bone to form the skeleton.  The results demonstrated that MGP is not expressed randomly by various cells in non-calcified tissues; on the contrary it has an organized spatial pattern of expression (Barone et al, 1991).

In the fish, Sparus aurata, the timing of MGP gene expression was related to the development of skeletal structures.  The expression of MGP mRNA was first detected at 2 days posthatching and thereafter continuously detected until 130 days post hatch.  Testing indicated that until 45 days post hatch, the MGP gene was highly expressed in a number of different tissues including skull, jaw, neural and spinal arches, heart and cartilage issues. At 85 days post hatch, a stage when most skeletal structures are mineralized, MGP gene expression and protein accumulation were restricted to the remaining cartilage structures, whereas osteocalcin gene expression and protein accumulation were localized in most mineralized structures. MGP gene expression was also detected in heart and kidney (Pinto et al, 2003). 

MGP expression was studied in developing chickens from 1 to 14 days old.  They found that MGP was expressed in different locations, on a different schedule.  The pattern suggested that during normal bone development, MGP was expressed in areas that are about to undergo ossification immediately prior to its initiation. Once ossification takes place (3 and 4 weeks), MGP expression diminishes from the center and goes to margins of ossification.  MGP’s role was to inhibit calcification in non-calcified tissues (cartilage). Once the tissue had undergone calcification, MGP was no longer needed. (Dan et al, 2009).

A study of MGP during the development of the avian growth plate was done using Tibial dyschondroplasia (TD), which is a skeletal abnormality of the avian species.  The findings showed MGP is widely expressed in the pre-ossified future bone, but after hatching, its expression gradually diminishes. They concluded that MGP expression was timely and spatially regulated.  They suggest that MGP, directly or through interaction with BMP2, plays a role as ossification regulator that acts prior to ossification. (Dan et al, 2012).

MGP and Bone Morphogenetic Protein-2
As the research advanced, studies showed that MGP inhibition of calcification was done via two mechanisms.  

One, MGP binds and inhibits the growth of calcium crystals.  Once activated by vitamin K, MGP is attracted to hydroxyapatite crystals, forming a coat on the surface of the crystals. This results in the inhibition of crystal growth by preventing the aggregation of the crystals (Hauschka et al, 1989; Spronk et al, 2001; Price et al, 2006; Price et al, 1998).  

Two, Matrix Gla protein (MGP) has been recognized as a regulator for bone morphogenetic protein-2 (BMP-2) (Zebboudi et al, 2002; Li et al, 2012).  

What is BMP
BMP stands for Bone Morphogenetic Structure.  Morpho means form. Genetic refers to the development of form and structure, in this case of bone.  BMP-2 belongs to a growth factor superfamily, which is a potent inducer of bones, cartilage, and connective tissues formation.   BMP-2 also promotes the process of calcification by inducing apoptosis or cell death (Wozney & Rosen 1998; Proudfoot et al 2000; Boström et al, 2001).  

BMP-2 coordinates early tooth mineralization.  BMP-2 promotes and regulates odontoblast differentiation and dental pulp cell differentiation.  When BMP-2 is absent, odontoblasts don’t differentiate, they don’t produce appropriately and the quality of dentin is altered, with patchy unmineralized areas and malformed dentinal tubules.  The result is a thinner disorganized dentin and pulp obliteration. (Saito et al, 2004; Iohara et al, 2004; Yang et al, 2012; Malik et al, 2018).  And odontoblasts are capable of producing and secreting BMP-2 (Bègue-Kirn et al, 1994).

Also, BMP-2 can induce dental pulp cell differentiation.  Dental pulp is important as it has the ability to regenerate and form reparative dentin, making it possible to retain the vitality of dental pulp.  Without that pulp cell differentiation, there are reduced blood vessels and capillaries (Gronthos et al, 2000; Smith & Lesot, 2001; Sloan & Smith, 2007; Peng et al, 2009; Yang et al, 2012; Yildurim et al, 2012; Washio et al, 2012; Qin et al, 2012).   

The effect of MGP is dose-dependent and appears to involve direct binding between the proteins as well as a matrix association.   MGP interacts, binds and inhibits BMP-2 to generate an inactive complex. When BMP-2 was inhibited, then human bone formation was inhibited in a dose dependent manner.  And high levels of MGP results in a strong enhancement of BMP-2 activity (Wallin et al, 2000, Bostrom et al, 2001; Zebboud et al, 2002; Sweatt et al, 2003; Zebboudj et al, 2003).  

Other research suggested that regulation of the physical proximity between MGP and BMP2 expression zones might play a role in calcification inhibition or initiation.  Therefore, when MGP is inactive or absent from tissues, the action of BMP becomes pronounced, causing extensive calcification (Ran et al, 2012).  

MGP and BMP-2 together may regulate the differentiation of human periodontal ligament cells. 

The periodontal ligament (PDL), a thin non-mineralized connective tissue, is located between two hard tissues: the alveolar bone (jawbone) and the roots of our teeth.  The PDL anchors the tooth to the inner wall of the alveolar socket and plays a crucial role in providing support, protection, and sensory input to the masticatory system (Beertsen et al, 2000).   In a study looking at the expression pattern of MGP, and BMP-2 on the periodontal ligament, they found that MGP and BMP-2 were co-localized, with similar genetic expression patterns, suggesting the possibility of direct binding of MGP to BMP-2, and concluding that MGP might regulate the differentiation of human PDL Cells. (Selvig et al, 2002; Khanna-Jain et al, 2010).  

MGP and Mineralization Regulation
MGP has also been identified as a negative regulator of mineralization, meaning that the expression of MGP in nearby cells may prevent and stop mineralization (Yagami et al, 1999).  Using transgenic mice studies, MGP was demonstrated to repress alveolar bone and tooth matrix mineralization (Kaipatur et al, 2008).

Summary
We have discussed the two Vitamin K dependent proteins that are primarily involved with calcification and mineralization in the body, osteocalcin and matrix Gla protein (Wen, Chen et al, 2018).  They are key to the regulation of mineralization in both the teeth and bones. Osteocalcin is involved in bone formation and bone turnover. In teeth, osteocalcin helps manage proper dentin formation and the regulation of mineral deposits in teeth.  And it is found in all elements of teeth, enamel, cementum, dentin and pulp. And it hastens orthodontic treatments. Matrix Gla protein facilitates normal bone metabolism and ensures that calcification is regulated in a developmentally timely and spatial manner.  MGP also regulates BMP-2, which is a potent inducer of bones, cartilage, and connective tissues formation.   BMP-2 coordinates early tooth mineralization and can induce dental pulp differentiation.  

Without these activated proteins, the body is not able to regulate the process of calcium uptake and bone mineralization.

*** Keep in mind, the body makes sure that the clotting factors are given vitamin K first.  Only when you have sufficient vitamin K,
beyond the clotting factors, will it be distributed around the body to the other tissues that need it. ***

Vitamin K – necessary to activate these proteins
Vitamin K is an essential fat-soluble vitamin that is naturally present in some foods and as a dietary supplement.  Naturally, it includes vitamin K1, also known as phylloquinone, and vitamin K2, a series of menaquinones. Phylloquinone is present primarily in vegetables and some fruits.  Menaquinones can be found in animal-based, and fermented foods. Almost all menaquinones, in particular the long-chain menaquinones, are also produced by bacteria in the human gut (Suttie, 2010).  A form of vitamin K2, MK-4, is unique in that it is produced by the body from phylloquinone via a conversion process that does not involve bacterial action or the gut. In fact, tissues that accumulate high amounts of MK4 have a remarkable capacity to convert up to 90% of the available K1 into MK4 (Davidson et al, 1998; Ronden et al, 1998) (Shearer & Newman, 2008; Suttie, 2014).  To date, at least 20 vitamin K dependent proteins have been identified, including the seven involved in blood coagulation (Azuma & Inoue, 2019).

Vitamin K functions as a cofactor for the conversion of glutamic acid (Glu) residues to gamma-carboxyglutamic acid (Gla) in vitamin K-dependent proteins (VKDP).  These VKDPs require carboxylation to become biologically active, and they have been identified as having an active role in mineralization.   One outcome of this carboxylation is that the proteins can now bind calcium, which in turn, promotes the correct deposition of calcium.  (Poser et al, 1980; Price & Williamson, 1985; Coutu et al, 2008; Akbari et al, 2018).   Although vitamin K-dependent gamma-carboxylation occurs only on specific glutamic acid residues in a small number of proteins, it is critical to the calcium-binding function of those proteins.

Bioavailability
Both vitamins K1 and K2 can activate VKDPs.  However, long-chain menaquinones such as MK7 and Mk9, have a higher bioavailability and longer half-life, in comparison to K1.  Thus, MK4 and MK7 are available longer in circulation, to be absorbed by extrahepatic tissue. Of all the menaquinones, MK7 is absorbed most efficiently and exhibits greatest bioavailability.  In a comparative study, both vitamin K1 and MK7 were readily absorbed within 2 hours after ingestion. However, the serum concentrations of K2 (MK7) were 10-fold higher than K1 (Schurgers & Vermeer 2001).  MK7 serum levels remain increased for several days (Schurgers & Vermeer, 2000; Schurgers & Vermeer, 2002; Schurgers et al, 2007; Sato et al, 2012).

Not all menaquinones are equally well absorbed.  For example, MK4 does not reflect an increased serum concentration after administration, while MK9 has a very long half-life, but due to the lipophilicity is poorly absorbed (lipophilic means combines with fats) (Schurgers & Vermeer, 2002).

K1 shows large inter-individual variation in fasting plasma concentrations.  Vitamin K1 also showed inferior absorption in comparison to MK4, as well as longer chain menaquinone food sources (MK8 and MK9) (Schurgers & Vermeer, 2001). 

Although vitamin K is fat soluble, only a little is stored in the body and without a regular dietary intake its reserve would be rapidly depleted.  For this reason, the human body recycles Vitamin K, compensating its limited storage capacity (Stafford, 2005). However, the typical diet contains insufficient amounts of vitamin K.

While there is not yet research linking vitamin K with teeth health, there is an abundance of research showing that vitamin K dependent proteins are part of all the mineralized components of teeth.  This means that the availability of vitamin K during tooth development is key to their functionality. Also, vitamin K dependent proteins are critical for bone health, including the jaw. And there is speculation that vitamin K dependent proteins play important roles in the health of the oral environment.

However, there is a wealth of evidence on vitamin K and bone health. 

Vitamin K2 and Bone 
There is wealth of evidence supporting the beneficial effects of vitamin K2 on bone metabolisms, formation, and mineralization. 

Lab Studies - In Vitro (in a culture dish – not in a living organism)
In vitro studies using tests from various species demonstrate that vitamin K2 inhibits osteoclastogenesis, or resorption of bone on a dose dependent manner (Akiyama et al, 1994). 

Other research has shown that vitamin K2, especially MK4, protects osteoblasts from apoptosis or cell death, in a dose-dependent manner.  It also stimulates the differentiation of the osteoblast, upregulates the expression of the bone marker genes, and inhibits osteoclastogenesis, or the creation of osteoclasts which resorb bone.  (Kameda et al, 1996; Urayama, et al, 2000; Koshihara et al, 2003; Kim et al, 2013; Ichikawa et al, 2006; Wu et al, 2015).  It inhibits bone resorption by targeting osteoclasts to undergo apoptosis, which leads to cell death.  Overall, it has an anabolic effect on bone.

On the other hand, vitamin K2 (either MK4 or Mk7 in this context) exert beneficial or anabolic effects towards osteoblasts, as reviewed in a couple of recent articles (Schwalfenberg, 2017; Myneni & Mezey, 2017).

In lab studies, vitamin K2 has been shown to be the most important inducer of bone mineralization in human osteoblasts.  In 2001, Yamaguchi et al. unraveled the stimulatory effect of MK‐7 on osteoblastic bone formation in vitro, and they also discovered the suppressive effect of MK‐7 on osteoclast‐like cell formation and osteoclastic bone resorption in rat bone tissues.  The presence of MK-7 resulted in a significant decrease in the number of osteoclasts. Finally, in 2011, it was shown that MK‐7 reinforces the synthesis of various bone‐specific proteins (Yamaguchi & Sugimoto, 2001; Yamaguchi & Ma, 2001; Yamaguchi & Weilzmann, 2011).

MK4 has also been shown to promote the maturation of osteoblast cells.  Using osteocalcin as an indicator of osteoblast function, MK4 increases the level of osteocalcin, improved bone formation markers, and reduced bone resorption (Akedo et al, 1992; Kim, 2013; Poon et al, 2015).  Similar to MK-4, MK-7 has been found to increase bone calcium content in vitro (Ehara et al, 1996).

-Bone Remodeling
In addition to enhancing bone formation, vitamin K2 regulates bone remodeling, an important process necessary to maintain adult bone (Myneni & Mezey, 2017).

In vitro animal studies demonstrated that vitamin K2 promotes the transition of osteoblasts to osteocytes. They observed that incubation of human osteoblasts with vitamin K2 in a collagen gel medium increased the number of mature osteocyte-like cells.  This is important as it suggests that sufficient vitamin K contributes to optimal osteocyte density in newly formed bone. Appropriate osteocyte density is essential for bone health as osteocytes appear to initiate bone repair in response to microcracks, as well as new bone formation in response to increased loading of bone (Koshihara & Hoshi, 1997; Koshihara et al, 2003; Igarashi et al, 2007; Atkins et al, 2009). 

Lab Studies - Rodent models - In Vivo (taking place in living organisms)
Evidence from animal studies supports the role of vitamin K2 in bone health. In an effort to study osteoporosis, surgical and chemical procedures have been done with rodents to induce osteopenia and osteoporotic-like conditions.  These procedures included ovariectomy, orchidectomy, glucocorticoids, removal of the sciatic nerve and calcium or magnesium deficient diets. (Akiyama et al, 1993; Kobayashi et al, 2002; Iwamoto et al, 2003b; Asawa et al, 2004; Iwamoto et al, 2010; Iwasaki-Ishizuka et al, 2005).  As an example, because ovariectomy in rats results in estrogen deficiency, this leads to increased osteoclast formation, increased bone resorption and bone turnover, and a significant decrease of bone mass and bone strength, and can be used as a model for estrogen-deficient, postmenopausal bone loss.  Or an orchidectomy which results in a testosterone deficiency, which in turn reduces periosteal bone formation, while also increasing cancellous bone turnover.

The results of these animal studies show MK4 at 50 mg/kg per day (or 3400 mg per 150 pound weight) inhibited the expected decreases in bone mineral density of the femur, and improved other bone parameters caused by ovariectomy in only a two-week period.  It was also shown that vitamin K2 prevented bone loss, decreases in bone mineral density (BMD), loss of bone mass, and reductions in bone mineral content of lumbar vertebrae and stimulated bone formation (Hara et al 1993; Akiyama et al, 1999; Yamaguchi et al, 1999; Tsukamoto et al, 2000; Yamaguchi et al, 2000; Iwasaki et al, 2002; Shiraishi et al, 2002; Asawa et al, 2004; Iwamoto et al, 2010; Nagura et al, 2015; Wu et al, 2015).  The prevention of bone loss due to the inhibition of bone resorption found with in vitro studies has been verified by in vivo studies with ovariectomized rats fed vitamin K2 (50 mg/kg) for two weeks (Akiyama et al, 1993).  

The sciatic-neurectomized (surgical removal of nerve) rat is used as a model of immobilization osteopenia in humans and is used to study prevention of osteopenia from disuse.  In neurectomized rats, oral administration of vitamin K2 (10 or 30 mg/kg) for seven to 42 days increased bone mass and maintained bone microstructure. In the K2 30-mg/kg group, bone thickness increased and mineralization was enhanced.  Low-dose vitamin K2 increased mineral apposition rate and bone formation rate, without increasing bone resorption (Akiyama et al, 1993; Shiraishi et al, 2002; Iwasaki et al, 2002). It also showed improved osteocyte density and better cortical structures (Iwamoto et al, 2008; Iwamoto et al, 2010). 

In orchidectomized rats, whose testicles have been removed, vitamin K2 (30 mg/kg) administered twice weekly for 10 weeks, resulted in suppressed bone resorption and bone turnover.  Vitamin K2 in sciaticneurectomized and orchidectomized rats suppressed bone resorption and stimulated bone formation. The evidence indicates K2 has potential to enhance bone formation and inhibit bone resorption in orchidectomized or neurectomized rats (Iwamoto, et al, 2003; Yeah, et al, 2003).  

Vitamin K2 treatment has been reported to have an inhibitory effect on osteoclastic bone resorption in murine osteogenic culture (Hara, et al 1993; Akiyama et al, 1994; Hara et al, 1995). 

Research has established that MK-7 directly stimulates calcification in the bones of normal rats in vitro (Ehara et al, 1996; Wu et al, 2015).  More recent research showed that MK-7 had promotes metabolic activity on bone components and that it may be able to stimulate bone formation and calcification. It increased the DNA content, calcium content, and alkaline phosphatase activity in vitro in bones of young rats.  They concluded that MK7 can directly stimulate the function of osteoblasts to differentiate. Bone mineralization was stimulated by increasing γ-carboxylated osteocalcin in osteoblasts, a marker of bone formation (Katsuyama et al, 2005; Katsuyama et al, 2007).  MK7 was also found to facilitate bone mineralization, including cortical bone structure (Yamaguchi et al, 1999.  They concluded that MK7 can be used as a therapeutic agent for bone disease.  (Zhu et al, 2017).

Studies have indicated that VK2 has a more pronounced osteoprotective effect than VK1 (Igarashi et al, 2007; Kim et al, 2013).  As illustrated by the results of the studies in vitro, vitamin K (K2 in particular) improves the function of osteoblasts by inducing their proliferation, decreasing their apoptosis, and increasing the expression of osteogenic genes. It also has positive effects on the bone turnover and accordingly regulates bone metabolism

Clinical Studies - Human Models
There is a growing body of research showing the beneficial effects of vitamin K on bone health in people, with attempts to refine the specific improvements.

-Increased Bone Mineral Density (BMD)
From middle-age onward, BMD decreases and bone quality deteriorates with advancing age, resulting in loss of bone strength.  Especially in women, BMD decreases sharply in the perimenopausal period and for several years thereafter.  A randomized two-year study investigated the therapeutic effect of MK4 and vitamin D2 on vertebral bone mineral density in 172 postmenopausal women with osteoporosis.  Four groups received the following; either (a) MK4, 45 mg/day, (b) standard vitamin D3 supplement, (c) combined MK4 and vitamin D3 therapy, and (d) control group receiving dietary therapy alone. In the combined MK4 and D3 group, collagen measures were unchanged for the first twelve months, then they significantly increased at 24 months with improvements in BMD.  They concluded that combined therapy with MK4 and vitamin D, given for 24 months, significantly increased bone mineral density. However, at 18- and 24-month evaluations, BMD was significantly higher in the K2 group compared to the control group (Ushiroyama et al, 2002).

In 2007, Kapen from the Netherlands presented the result of a 3-year randomized clinical intervention study of 325 healthy postmenopausal non-osteoporotic women receiving MK4, 45mg/day, or placebo.  Computations showed that MK4 significantly improved the hip bone strength, bone mineral content and femoral neck width. In the placebo group, bone strength decreased significantly. They concluded that the high dose of vitamin K2 prevented the postmenopausal bone loss (Knapen et al, 2007).  

Studies show that increased consumption of MK7, in the form of natto, leads to an increase in activated osteocalcin.  This was linked to increased bone matrix formation and bone mineral density, and a lower risk of hip fracture (Kaneki et al 2001).  The results were confirmed in a 3-year study with 944 women aged 20 to 79 years, which showed that intake of MK-7-rich natto was associated with the preservation of bone mineral density (Ikeda et al 2006).  Moreover, both MK-4 and MK-7 supplementation resulted in an increase of carboxylated osteocalcin and a decrease of uncarboxylated osteocalcin, and improved BMD (Schurgers, et al, 2007; Shiraki, Itabashi, 2009; Binkley et al, 2009; Koitaya et al, 2009; Iwamoto, 2014; Koitaya et al, 2014; Ishida et al, 2018)

Reduced Hip Fracture Incidence
Population studies have shown that low levels of vitamin K are significantly correlated with hip fracture incidence.  In an early study, three types of vitamin K levels in the blood (K1, MK7, and MK8) were measured in patients with hip fractures, compared to healthy controls.  All patients with hip fractures had significantly low levels of all three types of vitamin K. They concluded that patients with hip fractures have vitamin K deficiency (Hodges et al, 1993).  

The correlation between low intake of vitamin K and increased fracture rate was also revealed by prospective analysis within the Nurses’ Health Study cohort of 1984.  The diet was assessed in 72,327 women, aged 38-63 years, with a food frequency baseline-questionnaire. During the subsequent 10 years of follow-up, 270 hip fractures were reported.  Women in the quintiles of 2-5 of vitamin K intake had a significantly lower age-adjusted risk of hip fracture (Feskanich et al, 1999).  

Japanese population studies show that the lower the vitamin K levels, the greater the risk of fractures.  Studies with MK-7, which is high in natto, showed that in Japanese postmenopausal women, low dietary intake of the vitamin was associated with an increased risk of hip fracture (Kaneki et al, 2001).  Furthermore, the fracture-preventing effect of vitamin K was observed in several clinical studies in Japan, which was confirmed by meta-analysis (Yaegashi et al, 2008). A systematic review has shown that 45 mg of MK4 decreased fractures in vertebra by 60%, hip fractures by 77%, and nonvertebral fractures by 81% in Japanese patients (Cockayne et al, 2006).

In the Netherlands and Finland, data clearly demonstrated the association between measures of vitamin K deficiency (uncarboxylated osteocalcin) and fracture risk.  Vitamin K was found to be a predictor of bone health (Knapen et al, 1998; Luukinen et al, 2000). The same outcomes were shown in a French study, yielding a positive association between ucOC and fracture risk (Szulc et al, 1993).  

 The most clinically relevant end-point is reduction of fractures in prospective, randomized clinical trials. In this regard only MK4 has demonstrated the ability to decrease fractures in clinical trials (Shiraki, et al 2000; Sato, et al, 2005; Cockayne, Adamson et al 2006).  In addition to decreasing fractures by up to 87%, clinical trials in Japan and Indonesia show that MK4 attenuates medication- and disease-induced bone loss as determined by bone density scans (Somekwawa et al, 1999; Sugiyama et al, 1999; Yonemura et al, 2004; Purwosunu et al, 2006; Ushiroyama et al ,2003; Shiraki et al, 2000; Cockayne et al, 2006;  Ichikawa et al, 2007; Iketani et al, 2003; Iwamoto et al, 1999; Iwamoto et al, 2000; Nishiguchi et al, 2000; Sato et al, 2002; Shimoi et al, 2002).  

A 2006 meta-analysis published in the Archives of Internal Medicine by Cockayne et al at the University of York in England evaluated clinical trials on MK4 and fracture risk.  They identified 13 randomized, controlled trials of the effect of MK4 on osteoporosis. Of those, seven had fracture risk as an end point and thus were included in their meta-analysis. They concluded that 45 mg of MK4 decreases vertebral fracture by 60%, hip fracture by 73%, and all nonvertebral fractures by 81% (Cockayne et al, 2006).

In an observational study, vitamin K levels were assessed in 387 women on hemodialysis.  Important proportions of patients had deficiency of MK7 (35.4%), vitamin K1 (23.5%), and MK4 (14.5%), vertebral fractures (55%), and vascular calcification (aortic calcification 80%, iliac calcification 56%). Vitamin K compound deficiency was found to be a predictor of both vertebral fractures and vascular calcification.  This is the first study to relate vitamin K1 and K2 deficiency directly both to vertebral fractures and vascular calcification. Vitamin K1 deficiency was the strongest predictor of vertebral fractures compared to vitamin K2 deficiency (Fusaro et al., 2012).

Several clinical trials have tested the effect of vitamin K2 supplements on bone health. The majority were done using MK-4 as it has been reported to have protective effect on bone loss and fracture risk in both healthy and osteoporotic postmenopausal women. A dose of 45 mg day was used in these studies, which is about 150–180 times greater than the recommended daily dietary intake of vitamin K (250– 300 lg) (Orimo et al, 2012).  Forty-five milligram was the minimum effective dose for improving bone mass parameters in postmenopausal women with osteoporosis. This dosage was determined as an optimal dose in a dose finding study of MK-4 in Japan, where the patients were administered daily doses of 15, 45, 90, and 135 mg.  No toxic effects of MK-4 (45 mg day) have been reported (Shiraki et al, 2000; Iwamoto et al, 2001; Ushiroyama et al, 2002; Ishida et al, 2004; Inoue, Fujita et al, 2009; Iwamoto, 2014; Jiang et al, 2014)

MK4 has also been shown to stop and reverse bone loss, and reduce fracture risk, from medical conditions and medications. In clinical trials MK4 (45 mg daily) prevented bone loss and/or fractures caused by corticosteroids (eg, prednisone, dexamethasone, prednisolone), anorexia nervosa, cirrhosis of the liver, osteoporosis, menopause (estrogen deficiency), disuse from stroke, immobilization (eg, extended illness, hospitalization), Parkinson disease, phenytoin therapy, testosterone deficiency (eg, aging, prostate cancer treatment), primary biliary cirrhosis, leuprolide treatment (for prostate cancer), and other diseases and medications (Sato et al, 1998; Iwamoto et al, 1999; Somekawa et al, 1999; Iwamoto et al, 2000; Nishiguchi et al, 2001; Sato et al, 2002; Shiomi et al, 2002;  Sugiyama et al, 1999; Yonemura et al, 2004; Yonemura et al, 2000; Ushiroyama et al, 2002; Iketani et al, 2003; Purwosuni et al, 2006).  

-Increased Collagen Production
The degradation of collagen is an important element in the development of osteoporosis.  MK7 and MK4 have been shown to increase collagen production. As collagen forms the matrix on which calcium and other minerals are accumulated, then increased deposition of collagen makes the bone more flexible.  This is very important for the attainment of “higher” or better bone quality (Koshihara et al, 1997).  

Other research shows that MK4 may also decrease the time it takes to heal a fracture, by stimulating bone collagen production. In a letter to Nature in 1960, Bouckaert and Said reported that complete transverse fractures experimentally induced into rabbit and rat femurs completely healed in the presence of vitamin K (Bouckaert & Said, 1960). 

While no clinical trials to date have been conducted to confirm this finding, the aforementioned basic science provides the underlying biochemical understanding for their observation. Promoting collagen production using MK4 may decrease the predisposition to microfractures and promote the healing of microfractures when they do occur, thereby playing a fundamental role in preventing and treating bone weakening.

There is a paradigm shift underway regarding the definition of bone quality.  Historically it was equated with bone mineral density. However, an argument is being made that bone quality needs to reflect collagen and osteocytes.  Neustadt and Pieczenik have presented the Neustadt-Pieczenik Collagen Deficit and Restoration hypothesis, where the fundamental premise is that bone health and recovery can be accomplished by stimulating bone collage production.  All three forms (phylloquinone, MK4, and MK7) may be useful in this regard. (Neustadt & Pieczenik, 2011; Kuroshima et al, 2017).

-Osteoporosis Treatment
The incidence of osteoporosis is high in postmenopausal women.  A number of human trials have found vitamin K2 effective in the treatment of osteoporosis (Orimo et al, 1992; Orimo et al, 1998; Iwamoto et al, 2000; Shiraki et al, 2000; Asakura et al, 2001; Ushiroyama, 2002).

In 1998, the effects of MK4 on bone and calcium metabolism in osteoporosis patients was evaluated in a 24 week, double-blind, placebo-controlled study, where 80 osteoporotic patients were included.  Treatment was MK4, 90 mg or placebo. In the MK4 group, bone density increased at least 7%, while it decreased by at least 7% in the placebo group. The results suggest that MK4, at a dosage of 90 mg/day is effective in maintaining cortical bone density and is a safe treatment for osteoporosis (Orimo et al, 1998).

A longitudinal study of 17 postmenopausal women given vitamin K2 (45 mg/day) for one year showed a slight increase in spinal BMD, compared to the control group of 19 postmenopausal women who experienced a decrease (-2.87 ± 0.51%) in BMD (Iwamoto et al, 1999).

In a randomized, open-label study, 241 osteoporotic women were given either 45 mg/day vitamin K2 and 150 mg elemental calcium (treatment group; n=120) or 150 mg elemental calcium (control group; n=121). After two years, vitamin K2 was shown to maintain lumbar BMD.  Patients receiving just calcium lost 2.5% of their lumbar bone density. Furthermore, patients receiving K2 also experienced significantly lower fracture incidence (10% versus 30%, in the treatment and control groups, respectively) (Shiraki, Shiraki et al, 2000).  

Ninety-two postmenopausal women, ages 55-81 years, were randomly assigned to one of four groups: vitamin K2 (45 mg/day), vitamin D3 (0.75 mcg/day), a combination of vitamins K and D3 (same dosage as above), or calcium lactate (2 g/day). The vitamin-K and -D groups both experienced significant increases in BMD compared to the calcium group over a two-year period, while combined treatment was synergistic, significantly increasing lumbar BMD by 1.35 percent. These findings indicate that combined administration of vitamin D3 and MK‐4, compared with calcium administration alone, appears to be instrumental in increasing the BMD‐values of the lumbar spine in postmenopausal women with osteoporosis (Iwamoto et al, 2000; Ushiroyama et al, 2002; Iwamoto et al, 2003)

Vitamin K2 has been used in the treatment of osteoporosis in Asian countries for a number of years (Koitaya et al, 2009; Koitaya, et al, 2014).  In Japan, a high dose of MK4, 45 mg/day (15 mg x 3/day) has been used as a therapeutic treatment for osteoporosis. In 1995, the Japanese Society of Osteoporosis included MK4, together with vitamin D, as the first line of treatment for osteoporosis, and it has also been approved by the Ministry of Health for the prevention and treatment of osteoporosis (Japanese 2011 Guidelines, 2012; Orimo et al, 2012; Okano, 2016).  

A traditional Japanese food, “natto” (fermented soybeans) contains high concentrations of MK&, a form of vitamin K2 (menaquinone), synthesized by microorganisms.  Epidemiological study conducted in Japan have shown a negative correlation of Natto intake and incidence of hip fracture which drew attention towards a possible link between vitamin K and osteoporosis (Kaneki et al, 2001).  

MK-7 has a longer circulating half-life and greater potency compared to MK-4. In a randomized double-blind placebo-controlled trial of 334 healthy postmenopausal Norwegian women given 360 μg/day of vitamin K2 (a low dose) in the form of Natto capsules (rich in MK-7) for one year, showed no effect on bone loss (Emaus et al, 2010).   In contrast, another placebo-controlled trial in 244 healthy Dutch postmenopausal women supplemented with 180 μg/day of MK-7 for three years showed a small but significant reduction in the age-related declined of bone mineral density and bone strength in femoral neck and lumbar spine BMD and BMC (bone mineral content) loss compared to placebo.  Vitamin K2 (MK-7) also prevented the loss in vertebral height in the lower thoracic spine.   These results confirm the hypothesis that long-term supplementation with MK-7 beneficially affects bone health (Knapen et al, 2013). 

Recent meta-analysis of randomized controlled trials encompassing 6759 participants concluded that vitamin K2 does play a role in the maintenance and improvement of vertebral BMD, and in the prevention of fractures in postmenopausal women with osteoporosis. However, vitamin K2 did not show any effect in postmenopausal women without osteoporosis (Huang et al, 2015). 

All of this research on vitamin K improving bone health would apply to the jawbone.

REMODELING AND REMINERALIZATION
Throughout the life, teeth and bone are at risk of injury, degradation, and demineralization.  Teeth and bone are both dynamic tissues, responding to their internal biochemical environment as well as external forces, and undergo constant renewal.   Bone is always being regenerated and remodeled.

BONE
The skeleton renews itself in two ways: bone modeling and bone remodeling. Bone modeling occurs during growth and development in childhood. It involves formation of bone by osteoblasts, or resorption of bone by osteoclasts.  The primary function of bone modeling is to increase bone mass and to maintain or reshape the bone, or change the position of the cortex relative to its central axis (called bone drift) in response to various physiological stimuli (Clarke, 2008; Allen & Burr, 2014; Myneni & Mezey, 2017).  

In the process of bone formation, the skeleton requires essential nutrients such as amino acids, fatty acids, carbohydrates, minerals, vitamins, and water (Weaver & Gallant, 2014).  Inadequate skeletal nutrition in infants and young children results in growth retardation and bony deformities. In adolescents and young adults, it results in the failure of individuals to attain their genetically prescribed, maximum peak bone mineral density, and in middle‐aged and older adults, the consequence is rapid bone loss that can cause or aggravate osteoporosis (Holick & Nieves, 2015). 

In contrast, bone remodeling occurs after the skeleton has reached maturity during adulthood.  It consists of a series of highly regulated steps; directed by osteoblasts and osteoclasts, occurring sequentially on the bone surface.  Bone-resorbing cells, osteoclasts, enter the blood vessels of bone, and remove bone mineral and bone matrix, old or micro damaged bone.  Each osteoclast acidifies the bone surface, dissolving mineralized matrix, and creating a void.  This void is subsequently filled with strong, new bone matrix deposited by osteoblasts.  Upon resorption, bone-matrix embedded osteocalcin is released contributing to its circulating levels (Gilbert, 2000; Sims & Martin, 2014; Udagawa et al, 1992; Allen & Burr, 2014; Paldanius, 2017; Wasilewski et al, 2019).

Bone remodeling is necessary to maintain structural integrity, bone volume, regulate calcium and phosphate homeostasis, and repair micro-fractures, preventing accumulation of old bone.  Normal bone remodeling balances bone formation and bone resorption to ensure that there is no net change in bone mass or quality after each bone remodeling event (Clarke, 2008; Feng & McDonald, 2011). The process of bone remodeling occurs throughout life and can occur upon or within any of the bone surfaces.  The entire adult skeleton is replaced every 10 years in humans and in doing so, the skeleton ultimately repairs and replaces itself throughout the lifespan.  Cortical bone makes up 75% of all bone, but trabecular or cancellous bone is ten times more metabolically active than the cortical bone.  The jaw bone has an inner region of cancellous bone (Sims & Martin, 2014).     

The orthodontic treatment of braces for the teeth are a prime example of bone formation and remodeling.  In orthodontic treatment, teeth are rearranged in response to applied mechanical forces that cause remodeling of the periodontal tissues (Waldo & Rothblatt, 1954; Storey, 1973; King & Theims, 1979).  When orthodontic force is applied to a tooth, the jawbone on the pressure side undergoes successive cycles of bone resorption and formation, while bone on the tension side predominantly undergoes continuous bone formation.  During application of orthodontic forces on the teeth, osteoclasts are increased on the compressed side and osteoblasts on the traction side. The increased number of cells stimulates bone remodeling around root and leads to tooth movement (Goulart et al, 2006; Yamaguchi, et al, 2007; Yoshida et al, 2009; Marquezan et al, 2010).

Bone and tooth health is a goal.  The medical and dental fields are always looking at how to maintain, if not restore health to their respective mineralized tissues.  Pharmaceutical interventions have been attempted to help with bone health, with toxic outcomes. We will present some of the causes of bone and teeth disease, and treatments, including the role of vitamin K.  

Bone restoration
Some of the factors that affect the process of bone formation and remodeling are physical activity, nutrition, local and systemic factors, disease, age, and medications   Bone diseases such as rickets and osteoporosis cause a significant reduction in bone mineralization and bone mineral density, which lead to increased fracture risk and skeletal deformity, respectively ( Meunier & Boivin, 1997; Simonet et al, 1997).  

Age affects bone health.  Bone loss is most typical in women after reaching the age of 50 years following menopause.  The post-menopausal period is accompanied by substantial reductions in estrogen levels, resulting in an imbalance in bone turnover markers, and leading to bone resorption.  This makes postmenopausal women susceptible to osteoporosis and fractures (Davis et al, 2015).

Osteoporosis, whether caused by biological changes from aging, or from disease, or from medications, affects some 200 million people worldwide.  More than 25 million Americans are at risk of osteoporosis and its consequences. In the USA alone, there are more than 2 million fractures caused by osteoporosis per year, costing $19 billion (McGowen et al, 2004). 

-Bone and Steroids
Some medications cause bone loss.  Corticosteroids and glucocorticosteroids (GC) are common prescription medications used to fight inflammation.  However, they are also associated with the side effect of bone loss, an increased risk of fracture. and osteonecrosis of the head of the femur bone.   While they may reduce inflammation, GCs also modify osteoblasts, osteocytes and osteoclasts, and interfere with bone remodeling (Jowsey & Riggs, 1970; Meunier et al, 1984; Van Staa et al, 2002; Kerachian, et al, 2009; Kim et al, 2011; Weinstein, 2012; Koromila et al, 2014)  

Medications for the prevention and treatment of these complications have been investigated for many years, and supplementing with vitamin K seems promising (Van Staa et al, 2002). 

An early study with rodents found that treating with both the steroid prednisolone (7 mg/kg/day) for nine weeks along with vitamin MK4 (17 mg/kg/day) significantly inhibited the decrease in length, weight, and bone density of femurs and tibiae.  Follow up research showed that a larger dose of 21 mg of vitamin Mk4 a day reduced the bone loss caused by prednisolone (Hara, Akiyama et al 1993). Later work showed that vitamin Mk4 inhibited the loss of bone mineral density loss and also improved the rate of bone formation (Hara, Kobayashi et al, 2002).  Studies have shown that vitamin K2 shows the same improvements as glucocorticosteroids (GCs) (Tanana et al, 2006; Iwamoto et al, 2008).  

In human clinical trials MK4 (45 mg daily) prevented bone loss and/or fractures caused by corticosteroids (eg, prednisone, dexamethasone, prednisolone), osteoporosis, menopause (estrogen deficiency), Parkinson disease, testosterone deficiency (eg, aging, prostate cancer treatment), and other diseases and medications (Ushiroyama et al, 2002; Iketani et al, 2003; Iwamato et al, 1999; Iwamoto et al, 2000; Nishiguchi et al, 2001; Sato et al, 1998; Sato, Honda, et al, 1998; Shiomi et al, 2002; Somekawa et al, 1998; Sugiyama et al, 1999; Yonemura et al, 2000; Yonemura et al, 2004).

Vitamin K2 was found to ameliorate the adverse effects of steroid treatment on osteocyte density (Iwamoto et al, 2008; Iwamoto et al, 2010).   Several studies have indicated that GCs inhibit osteoblast proliferation (bone building cells) in vivo, while VK2 has also been reported to promote osteoblast proliferation (Yamaguchi et al, 2001; Sanderson et al, 2015; Shi et al, 2015).  A recent study demonstrated that VK2 promoted osteoblast proliferation and osteogenic differentiation, and inhibited cell death and enhanced cell survival, supporting the view that VK2 is a promising option for the prevention and treatment of GC-induced osteoporosis and osteonecrosis (Zhang et al, 2016).

-Bone and Bisphosphonates
Osteoporosis is caused by an imbalance between new bone formation by osteoblast cells and bone resorption by osteoclast cells.  If there is an imbalance, the result is a highly porous structure that is fragile (Zhao et al, 2018).  There has been much research into treatments and remedies for the bone loss that comes with age, particularly for women.  All treatments aim at restoration of the original innate balance by enhancing bone formation or/and inhibiting bone absorption. Hence, osteoblasts and osteoclasts are a popular target for such a purpose.

For a while, calcium supplements were recommended, but research has illustrated the serious side effects of that regimen, including an 30% increased risk of myocardial infarction, stroke, and sudden death, a 92% increased risk of gastrointestinal symptoms, and a 17% increased risk of kidney stones, a 50% increased risk of hip fracture and an increased risk of hypercalcemia (calcium overload)  (Jackson et al, 2006; Bischoff-Ferrari et al, 2007; Bolland et al, 2008; Bolland et al, 2010; Lewis et al, 2012; Gallagher et al, 2014; Yang et al, 2019)

A recently popular treatment for osteoporosis has been bisphosphonates, medications which impair osteoclast function.  Bisphosphonates (BP) have become the standard of care for millions of men and women with osteoporosis. Bisphosphonates impair osteoclast function which is the resorption of old damaged bone so that new bone can be created.   The rationale for using bisphosphonates was that these medications increase bone mineral density, and decrease fracture risk by inhibiting resorption and preventing bone turnover (Sato et al, 1991; Greenspan et al, 2000).   

Oral bisphosphonates prescribed include etidronate (Didronel®) risedronate (Actonel), tiludronate (Skelid®), alendronate (Fosamax), Zoledronic acid (ZA) and ibandronate (Boniva®). In 2006 alendronate was the most widely prescribed oral BP, accounting for 37.7% of the osteoporosis drug market and generating $2 billion in sales in the United States alone. (Hughes et al, 1995; Neustadt & Pieczenik, 2011).   T

However, less than a decade after the first pivotal clinical trial with alendronate in 1995, reports regarding serious complications, began to appear in the literature.  In the short term, slowing bone resorption increases bone density. But in the long run, it appears that bisphosphonates suppress the bone turnover rate, impair new bone formation and reduce the bone's ability to repair microscopic cracks from normal wear and tear, with serious, unpredicted side effects (Russell et al, 2007).

Research showed that ZA directly blocked bone metabolism when applied locally at grafted bones, hence decreasing new bone formation (Olejnik et al, 2016; Belfrage et al, 2012).  Research showed that when ZA was added to an osteoblast precursor cells, it induced cell death (Patntirapong et al, 2012).  When ZA was added to cultures of human epithelial cells and gingival cells, at levels equivalent to human osteoporosis treatment, the effect was toxic.  Even at low concentration, ZA had a negative impact on osteoblast proliferation (Basso et al, 2013; Yang et al, 2013). 

Research with bisphosphonates found that zolendronic acid and pamidronate (two widely used bisphosphonates) are directly toxic to osteoblasts, fibroblasts and significantly negatively affect collagen production – a key element in bone formation.  The research indicated that type 1 collagen was reduced in all three cell lines treated with zoledronate and pamidronate. If you remember from earlier discussion, collagen is a central ingredient of the matrix which becomes mineralized into bone.  Since collagen is not detected by x-rays it is not part of a bone density scan report. Thus, in only reporting the density of bones the scans are evaluating bone quantity and not bone quality. Any discussion of bone quality as a benefit from bisphosphonates would be incomplete without considering the pivotal role of bone collagen (Simon et al, 2010).  In a rat bone defect model, ZA given at doses equivalent to human osteoporosis treatments showed no effect on properties of newly formed bone BUT high dose ZA disrupted collagen and apatite crystal organization, thereby hindered bone healing (Olejnik et al, 2016).

The prolonged use of BP therapy causes the accumulation of microdamage in bone, reduced heterogeneity of the organic matrix and mineral properties, and a deterioration of bone quality, which can lead to atypical femoral fractures (ATFs).   Other studies showed that the risk of atypical fracture increased along with the yearly ZA administration (Mashiba et al, 2000; Saito et al, 2008; Donnelly et al, 2012; Ataoglu et al, 2016.

The most alarming side effect to physicians and patients alike, is osteonecrosis of the jaw (ONJ), first reported by dentists and oral surgeons in 2003, and atypical femoral fractures (AFFs), first reported in 2007. Many subsequent publications have appeared on both conditions, including 3 major reports from American Society for Bone and Mineral Research (ASBMR) Task Forces (Khosla, et al, 2007; Shane, Burr et al 2014; Shane et al, 2010). 

Osteonecrosis of the jaw, is where bone tissue fails to heal after minor trauma such as tooth extraction. BRONJ (bisphosphonate related osteonecrosis of the jaw), has been defined as “exposed bone in the either or both jaw bones that persists for at least 8 weeks, in the absence of previous radiation and of metastases in the jaws.” (Khosla et al, 2007; Rizzoli et al, 2008). This can lead to bone infection and fracture, and may require long-term antibiotics and surgery to remove the dead bone.  

The incidence of osteonecrosis of the jaw is a serious clinical concern after invasive dental treatment procedures (Marx, 2003; Ficarra et al, 2005).  In 2007 the risk of BRONJ in cancer patients treated with high doses of intravenously applied BPs was estimated in the range of 1% to 10% (Khosla et al, 2007). The relative risk of BRONJ from oral bisphosphonates has historically been calculated at 0.05 to one case each 100,000 persons-years of exposure for oral BPs.  However, a retrospective article published in 2009 concluded that the actual risk of BRONJ from oral bisphosphonates is 4%. This was the first large institutional study in the United States evaluating the epidemiology of BRONJ (Sedghizadeh et al, 2009).

 Alarmingly, a more recent article published in 2011 concluded that the risk for BRONJ from oral bisphosphonates was actually 8%, double what had been previously reported. The increased incidence of BRONJ was based on a retrospective analysis of data collected from 11 different clinical centers across Europe.  Importantly, the majority of patients (57%) who experienced BRONJ had no risk factors as defined by the American Association of Oral and Maxillofacial Surgery (Otto, et al, 2011). 

The jaw bones have a high rate of turnover, given their activity level and the forces impacting them from chewing.  While it has not been directly studied, it is likely that the jawbone death seen in BRONJ is only the first evidence of the bone damage from bisphosphonates, and that if tested, bone damage in other parts of the skeleton would emerge.

An important new theory is that the BRONJ reflects a “collagen deficit”, and that bisphosphonates lead to decreased collagen production, which in turn creates a microenvironment more susceptible to microfractures. This in turn provides a pocket for bacteria to seed and reproduce.  There is compelling evidence that jaw microcracks are a fundamental requirement for the etiology of BRONJ (Hoefert et al, 2010; Allen & Burr, 2008; Mashiba et al, 2005)

There have been proposals to move the occurrence of BRONJ away from a risk management and quality assurance model, to a preventive model.  Now, there are calls for research to explore whether vitamin K can function as a treatment for patients with BRONJ or who have suffered other damage from bisphosphonates (Neustadt & Piezenik, 2011).

Vitamin K is renowned for being a vital nutrient, and crucial for bone health, as the reviewed research has indicated.  Vitamin K2 has shown effectiveness as a therapeutic agent for bone ailments and diseases, like bone loss or brittleness (osteomalacia and osteoporosis).  Research has already established that vitamin K stimulates collagen production. (Bouckaert & Said, 1960; Ichikawa et al, 2007; Amizuka et al, 2009).  Promoting collagen production using MK4 may decrease the predisposition to microfractures and promote the healing of microfractures when they do occur, thereby playing a fundamental role in preventing and treating BRONJ (Hoefert et al, 2010).

And since microfractures are a component of BRONJ pathogenesis and only MK4 has been shown to reduce fractures, it thus represents the leading therapeutic candidate for preventing and treating osteoporosis and BRONJ (Neustadt & Pieczenik, 2011; Kuroshima et al, 2017).

In addition, Vitamin K acts on bone using different pathways than bisphosphonates, which inhibit osteoclast functioning and bone resorption.  The mechanism of osteoclastic inhibition by vitamin K2 is not due to cell toxicity and did not affect cell proliferation. The osteoclastic inhibitory action by K2 is unique to its structure. Vitamin K1 does not affect bone resorption and osteoclast-like cell formation.29 In vitro studies with mouse cell cultures found a dose-dependent inhibition by K2 of prostaglandin E2 (PGE2) synthesis, which in turn was shown to inhibit bone resorption.  So vitamin K improves bone density and strength without the destructive effects if bisphosphonates (Hara et al, 1995; Akiyama et al, 1993).

In addition, K2’s safety profile makes it an attractive alternative to bisphosphonates, which have been linked to a litany of adverse effects, ranging from serious heart rhythm abnormalities to stomach ulcers and oesophageal damage. Vitamin K has no upper limits and has no side effects. 

While no clinical trials to date have been conducted to confirm this finding, studies from the medical literature support the safety and efficacy of MK4 as a potential therapeutic agent in preventing and treating osteoporosis and BRONJ.  While the approach outlined herein requires additional study, the basic science and biochemical foundation provides the conceptual framework the conceptual framework to begin to address this osteonecrosis of the jaw in a proactive, rather than reactive, way.

Given the specific effects of vitamin K2 on osteoclasts and in bone remodeling, vitamin K2 appears to be a safe alternative to use instead of bisphosphonates, Vitamin K2 randomized controlled trials need to be done to see whether K2 can prevent periodontal bone loss.

Summary
The skeleton is a dynamic tissue.  Throughout life, the skeleton renews itself in two ways: bone modeling and bone remodeling.  Some of the factors that affect the process of bone formation and remodeling are physical activity, nutrition, local and systemic factors, disease, age, and medications.  Osteoporosis is one outcome when bone development becomes imbalanced. Osteoporosis, whether caused by biological changes from aging, or from disease, or from medications, affects some 200 million people worldwide.   

Some medications cause bone loss.  Corticosteroids and glucocorticosteroids (GC) are common prescription medications used to fight inflammation.  However, they are also associated with the side effect of bone loss, an increased risk of fracture. and osteonecrosis of the head of the femur bone
For a while, calcium supplements were recommended, but research has illustrated the serious side effects of that regimen and they are no longer recommended for osteoporosis.  A recently popular treatment for osteoporosis has been bisphosphonates, medications which impair osteoclast function – which is the resorption of old damaged bone so that new bone can be created.   

Bisphosphonates, which impair osteoclast function and bone resorption, have been widely prescribed.  However, the prolonged use of BP therapy causes the accumulation of microdamage in bone and a reduced bone quality which can lead to atypical femur fractures, and necrosis (Saito et al, 2008; Otto, et al, 2011; Donnelly et al, 2012), increased advanced glycation end products (Mashiba et al, 2000; Saito et al, 2008), and a deterioration of bone quality, which can lead to atypical femoral fractures (AFFs).   Alarmingly, a more recent article published in 2011 concluded that the risk for BRONJ from oral bisphosphonates was actually 8%, double what had been previously reported (Otto et al, 2011).  Now, there are calls for research to explore vitamin K can function as a treatment for patients with BRONJ or who have suffered other damage from bisphosphonates (Neustadt & Piezenik, 2011). 

Bone and tooth health is a goal.  The medical and dental fields are always looking at how to maintain, if not restore health to their respective mineralized tissues.  Pharmaceutical interventions have been attempted to help with bone health, with toxic outcomes. Now the dental field is exploring other interventions, including vitamin K in the diet and the role that vitamin K plays in dental health.

TEETH
Similar to bone, teeth are mineral composites comprised of the hydroxyapatite in the enamel, collagen in the dentine, and living tissues.  However, it is the anatomical arrangement and location of teeth that sets them apart from bones (Vanderby & Provenzano, 2003; Burr, 2004; Ji et al, 2013; Shepherd et al, 2012).
 

Throughout the life, teeth are at risk of demineralization, due to their anatomical arrangement and location.  Teeth are exposed to food, drink, and the microbiota of the mouth; therefore, they have developed a high resistance to localized demineralization that is unmatched by bone.  This resistance is chiefly due to the enamel layer that covers the crown of the teeth (Meredith et al, 1996; Barbour et al, 2006; Ren, 2011; Scaramucci et al, 2015). 

When resistance is lost, tooth decay begins and cavities develop.  Loss of functionality within teeth and periodontal tissues can be inherited or caused by degenerative or infectious disease, by trauma, by dietary deficiencies, or as a consequence of surgical, radiological or chemical/drug treatments. 

Tooth decay is the destruction of your tooth enamel, the hard, outer layer of your teeth. are permanently damaged areas in the hard surface of your teeth that develop into tiny openings or holes, called cavities.

Teeth restoration

-Teeth and Saliva
Saliva is the most important natural factor to protect teeth, and prevent tooth decay.  Saliva is regarded by some as a magic biofluid (Ilea et al 2019). Saliva is not just water.  Saliva contains active ingredients like minerals and enzymes such as calcium phosphate and fluoride, and proteins like matrix Gla protein, and fetuin A (references).  Saliva is the medium for nourishment from the outside of the tooth and the transport medium by which preventive agents are distributed around the mouth. When saliva is reduced either by disease or medications, and ‘dry mouth’ ensues, the presence of cavities increases (Kumar et al, 2012).

Saliva has a buffering capability.  It bathes the teeth, and in the process restores minerals to the tooth surface, and prevents the continuous corrosion of the enamel surface.  Saliva is a constant source for calcium and phosphate that helps in maintaining supersaturation with respect to tooth mineralization. If saliva were just water it would leach minerals from the tooth surface and the enamel would start to erode.  

Also, when saliva bathes the tooth, it neutralizes the acids released by decay-causing bacteria.  Foods such as refined carbohydrates like sugar feed the bacteria, which produces acid as a by-product (references).  

Saliva also delivers building blocks for remineralization “on request”.  As an example, saliva constantly delivers fluoride to the tooth surface; salivary fluoride is a key player in preventing tooth demineralization and enhancing mineralization (Dowd, 1999). 

In the open areas of the oral cavity, saliva can keep the tooth surface clean.  In the crevices it becomes more difficult and dental plaque can form. Dental plaque is a biofilm of bacteria that grows on surfaces within the mouth, as a defense against acidic challenges.  It begins as a complex of calcium phosphate and a glycoprotein called precipitin. It is a sticky, colorless deposit at first, but it can build up and form a tartar, becoming a hard, yellow scale around teeth.  (It is also referred to as a pellicle). Plaque is always forming, starting immediately after tooth cleaning or toothbrushing.  However, depending on diet, plaque can become acidic, causing demineralization.  

However, plaque also has a protective function.  It can maintain calcium ions near the enamel surface and act as a reservoir.  Thus, plaque can simultaneously regulate the uptake and release of calcium and phosphate between the tooth surface and saliva. In this way, the pellicle acts as a semipermeable membrane and maintains the integrity and mineral homeostasis of the enamel surface (Hara & Zero, 2014; Ionta et al, 2014; Hannig et al, 2014).  

There are three major salivary glands, the parotid, the sublingual and the submandibular.  The submandibular is located beneath the lower jaws and produces 70% of saliva. The salivary glands are innervated by the parasympathetic and sympathetic branches of the autonomic nervous system.

-/-Teeth and Saliva and Vitamin K
It has been observed that K2 has antimicrobial effects and reduces the number of cavity-causing bacteria, which helps to prevent tooth decay. Dr. Price, a dentist, discovered a fat soluble nutrient that he labelled ‘activator x’.  He practiced in Ohio, and after concluding that folks had too many cavities, he set out around the world, searching to understand what made people healthy. The world over, Dr. Price, found groups of people, isolated from the modern world who were healthy.  He found that when they moved into modern culture, they quickly developed cavities. He found that their diets contained at least four times more minerals and water-soluble vitamins than the standard American diet of the 1930s, and ten times more fat-soluble vitamins.  He concluded that processed food lacked the nutrition that nourished the body.

Price’s research showed that patients with active tooth decay had a count of 323,000 bacteria per milliliter in their saliva.  After eating Activator X, vitamin K2 rich butter oil, the count dropped to 15,000 per ml, a 95% decline. In some cases, the bacteria disappeared completely. 

The addition of dietary K2 changes the quality of saliva in another way that fights tooth decay.  The saliva of patients with cavities, tends to rob the teeth of minerals. When saliva from patients with tooth decay was mixed with powdered bone or tooth chips, minerals moved from the tooth or bone into the saliva.  Then the experiment was repeated with saliva from patients treated with vitamin K2. Then the minerals moved from the saliva into the bone tissue.

And in patients with cavities, the minerals moved from teeth into the saliva, while after taking vitamin K2, the minerals moved from saliva into bone.  Saliva has more K2 than any other organ other than pancreas (Price, 1939; Rheume-Bleue, 2012).

Vitamin K has been tested as possible anti-cavity agent by virtue of its enzyme inhibiting activity in the carbohydrate degradation cycle.  Vitamin K was found to prevent acid formation in incubated mixtures of glucose and saliva during in vitro studies (Fancher et al, 1944). In 1945, Burrill and group had subjects chew a commercial gum product or gum containing vitamin K every day for ten minutes after each meal.  These groups were compared to a control group. At the end of eighteen months, those who had chewed gum with vitamin K had 69% fewer cavities than the control group, and 54% lower than those who had chewed commercial gum (Fosdick, 1942; Burrill et al, 1945; Sivapathasundharam et al, 2006).

It is believed that vitamin K2 exerts an impact on salivary signaling and composition.  When calcium is needed it's transported via the saliva and blood and then deposited into the teeth, bones, nerves and muscles. Vitamin K is essential to activate the proteins that bind and pick up the calcium so it can be delivered throughout the body to your teeth and bones.  If you don’t have enough Vitamin K2, the calcium remains in your blood and saliva instead of going to its preferred destination of the bones and teeth to harden them

Fetuin A
There are anecdotal reports of folks taking vitamin K and having less dental plaque.  It appears that the presence of vitamin K activates matrix Gla protein (MGP) in the saliva.  Activated MGP is known both to bind calcium and also to complex with the protein fetuin-A, also found in saliva (Price et al, 2003).  

Fetuin A is a protein synthesized in the liver and secreted into the blood.  Fetuin A acts as a mineral carrier and a potent inhibitor of mineralization (Jahnen-Dechent, et al, 2011).  Fetuin-A has a high affinity for apatite mineral and is an inhibitor of new apatite formation from supersaturated mineral solutions.  (The mineralized surfaces of our teeth, the enamel and dentin, consist of very hard crystal called hydroxyapatite, which is made from calcium, phosphate and hydroxyl ions (Schinke et al, 1996; Jahnen-Dechent et al, 1997; Reynolds et al, 2004; Heiss et al, 2007; Heiss et al, 2008).  

Fetuin A can rapidly form a fetuin-A calcium phosphate complex, which includes carboxylated matrix Gla protein.  Both fetuin and matrix Gla protein function as potent inhibitors of calcification in vitro.  This complex inhibits the growth, aggregation, and precipitation of the plaque, then clearing it from blood, and preventing it from being deposited.  Fetuin A does not complex with uncarboxylated matrix Gla protein, highlighting the necessity of vitamin K to be present (Price et al, 2003; Price et al, 2004; Reynolds et al, 2005; DoÄŸan et al, 2016).

 

*** So if you have sufficient amounts of vitamin K, it will activate the proteins that bind calcium, 
which are found in saliva, and which seems to effect plaque build-up. ***

-Teeth and Dentinal Fluid Flow

+ + Tooth Decay and the Acid Theory
The traditional explanation of tooth decay is the Acid Theory, which is that oral bacteria collect on the tooth’s surface as plaque.  Refined carbohydrates like sugar feed these bacteria which produce acid. These bacteria ferment sugars as their normal way of feeding and form acid as a by-product.  Acid produced by bacteria dissolves or demineralizes the hard tissues of the teeth, causing tooth decay (Ruby et al, 2010).  

Thus, tooth decay is the rotting of teeth due to bacterial infection.  If the infection in enamel goes untreated, the disease can spread internal to the tooth, involving dentin and pulp tissue (Lussi et al, 2008, Youravong et al, 2008; Scaramucci, Carvalho et al, 2015; Kwang & Abott, 2014; Ren, 2011).  

The Acid Theory confines tooth decay to the immediate oral environment, as an isolated process revolving around the bacteria-acid axis as the primary cause and effect.

+ + Tooth Decay and the System Theory
A tooth is a living structure. It needs nutrients supplied on a daily basis just like any other tissue in the body in order to maintain good, decay-free health.  This is accomplished through the mechanism of dentinal fluid flow. The dentin of a tooth is comprised of tiny tubules that run from the inside of the tooth, out through the hard dentin and ending beneath the enamel.  The tubules are too small to see, and if your teeth are healthy, they are also covered by enamel.

In a healthy tooth, the blood supply comes in, brings nutrients and delivers them to the pulp, which then flows the nutrients from the inside to the outside of the tooth through the dentinal tubules.  This fluid flow functions to prevent the influx of bacteria and acid into the tooth. And it allows new dentin to be created and maintained. Nerves also pass through these tubules allowing dentin to transmit pain, unlike enamel (Jacobson, 2002).

In addition, the odontoblasts contain an additional mechanism that allows them act like a pump, so dentinal fluid flow is centrifugal in motion.  Because the pressure inside the tooth is greater than in the mouth, the tooth has a built-in defense system against bacteria.  Since dentin has no direct blood source, the outward flow draws nutrients from blood vessels in the pulp, into the dentin, keeping it healthy.  And the outward flow repels acids and microbes, keeping them from penetrating the tooth.

An original study in 1970 by Dr. Ralph Steinman and endocrinologist Dr. Leonara found that injections of dye into the stomach, flowed through the dentinal tubules into the mouth within one hour (Steinman, 1977).  But when fed a decay-producing diet, the fluid flow reversed.  Fluids flowed from the surface of the tooth, through the enamel (bringing bacteria and debris along with it), through the dentine, and into the pulp chamber. These rats experienced lots of decay. When dentinal fluid flows inward, toward the pulp, it draws acids and pathogens into the tooth. 

This flow could be turned one way or the other just by altering the diet. Steinman found that he could alter the diet and alter the amount of decay in a perfect parallel.  He could feed the diet as food they ate, or feed the rats through a stomach tube so that food never touched their teeth. Results were the same either way. What they ingested controlled the amount of tooth decay generated. 

Steinman and Leonora were the first to isolate, purify and crystallize a hormone called "parotid hormone" which is manufactured in the parotid gland, one of the salivary glands. The fluid flow through the tooth is stimulated by an endocrine hormone secreted by the parotid gland, hence the term ‘parotid hormone’.  The parotid hormone can inhibit or enhance nutrient uptake by the tooth (Steinman & Leonara, 1971; Zhang et al, 2005; Southward, 2015).    

When there is a proper died, the hormone was produced in adequate amounts and the fluid flow brings nutrients into all parts of the tooth.  When the diet is high in sugars and refined carbohydrates, the fluid flow is reversed, dragging sludge from the saliva into the tooth where a chemical breakdown took place.  

The endocrine system is a collection of glands that produce hormones.  Hormones can be thought of as chemical messages that communicate with the body and bring about changes.  When stressed, hormones can be released into the body. The hypothalamus/parotid axis is the endocrine axis most relevant to dental health (Leonara et al, 1993).  The hypothalamus plays a significant role in the endocrine system and it helps control the level of sugar in the blood (Chan & Sherwin, 2012; Pozo & Claret, 2018).  

The endocrine axis related to dentinal fluid flow is called the HPEA axis (Hypothalamus-Parotid gland Endocrine Axis).

The System Theory of dental caries proposes a much more inclusive view of tooth decay, beyond the oral cavity.  The System Theory acknowledges the effect of refined carbohydrates and sugar on the oral cavity, but also recognizes the influence of the hypothalamus/pituitary/adrenal axis and the endocrine system. And it posits vitamin K as a key modulator of the endocrine system when there is a dietary sugar spike.  The Systemic Theory of dental caries recognizes that the process is multi-factorial and involves the entire body, rather than just the oral cavity (Southward, 2011).

As an example, sugar has a significant impact on the body when it is absorbed, beyond the acid it creates.  Under the System Theory, the impact of sugar and diet starts in the hypothalamus part of the brain. Blood sugar spikes need to be managed, and when they register in the hypothalamus, the hypothalamus initiates changes in the dentinal fluid flow.  This response is chiefly endocrine and affects the entire body. Disruption of the metabolic cycle causes a disruption in the tooth metabolic cycle.

High sugar intake also creates an increase in reactive oxygen species and oxidative stress in the hypothalamus. When this signaling mechanism halts or reverses the dentinal fluid flow, it renders the tooth vulnerable to oral bacteria, which attach to the tooth surface.  The acid attack stimulates an inflammatory response. Vitamin K2 (K2) has been shown to have an antioxidant potential in the brain and may prove to be a potent way to preserve the endocrine controlled, centrifugal dentinal fluid flow (Li et al, 2003; Oldenburg et al, 2008).   Not by coincidence, some of the highest concentrations of vitamin K2 are found in the pancreas and the salivary glands.  It can be construed that there exists a close relationship between both of these glands through the hypothalamus.  

Nutrition plays a significant role in both the systemic and traditional theories of tooth decay (Southward, 2015).  Nutrients play an important regulatory role in preserving health of the human body and of all metabolically active tissues. Micronutrients, vitamins and antioxidants play an essential role for constant regenerative processes, for coping with oxidative stress, and also for adequate immune responses. Undernutrition or malnutrition concerning certain food components can lead to defects of the dental hard tissues, the oral mucosa and the periodontium.  Research shows that vitamin K2 and vitamin D together result in a far greater reduction of tooth decay than does either vitamin alone. Sound nutritional habits and a sufficient supply of essential vitamins and minerals are of considerable importance for oral health (Vanishree et al, 2015).  

In the System Theory, vitamin K2 has been proposed as an important nutrient supplement to the diet.  Vitamin K has already been established as an important component of the insulin cycle, and it is believed to modulate the disruptions on teeth caused by sugar.  Vitamin K2 (K2) has been shown to have an antioxidant potential in the brain and may prove to be a potent way to preserve the endocrine controlled centrifugal dentinal fluid flow (Vanishree et al, 2015).  Much research, however, is needed to determine why this vitamin can enhance defenses locally through changing saliva composition and systemically through its influence on the hypothalamus and the endocrine aspect of the parotid gland.

In the future, dental disease will be construed and recognized as an inflammation related degenerative lifestyle disease in line with cardiovascular incidents, as well as with bone brittleness (osteoporosis) and diabetes mellitus. 

Summary of Teeth
Throughout the life, teeth are at risk of demineralization, due to their anatomical arrangement and location. Teeth are exposed to food, drink, and the microbiota of the mouth; therefore, they have developed a high resistance to localized demineralization that is unmatched by bone.  This resistance is chiefly due to the enamel layer that covers the crown of the teeth.  Saliva plays a pivotal role in the health of the mouth and in the prevention of tooth decay, and is regarded by some as a magic biofluid (Ilea et al, 2019).  Saliva has a buffering capability.  It bathes the teeth, and in the process restores minerals to the tooth surface, and prevents the continuous corrosion of the enamel surface.  Also, when saliva bathes the tooth, it neutralizes the acids released by decay-causing bacteria.  

There are numerous anecdotal reports of folks taking vitamin K and having less dental plaque.  It appears that the presence of vitamin K activates matrix Gla protein (MGP) in the saliva which complexes with Fetuin A.  This complex appears to remove and reduce calcium and plaque building up from the tooth.

The traditional model of tooth decay is the Acid Theory, which is that oral bacteria collect on the tooth’s surface as plaque and dissolves or demineralizes the hard tissues of the teeth, causing tooth decay (Ruby et al, 2010).  The new System Theory of dental caries, acknowledges the effect of refined carbohydrates and sugar on the oral cavity, but also recognizes the influence of the hypothalamus/pituitary/adrenal axis and the endocrine system and the impact on saliva flow. The systemic concept of dental caries recognizes that the process is multi-factorial and involves the entire body, rather than just the oral cavity (references).  In this model, vitamin K2 has been proposed as an important supplement to the diet. 

GENETICS
The dream and drive for the future of tooth health is genetics, using the technology of genetics to recreate and restore the damage from caries.  

Within our bodies, we house trillions of cells, all busily going about doing their jobs while we enjoy our days. Each of those cells has a nucleus that contains our DNA -- genetic material passed on to us from our parents. DNA is composed of different sequences of our genes. These sequences hold directions for making the proteins that will carry out a cell's particular function. This is how one cell might end up being important to your kidneys, while another cell makes bone.

All cells contain exactly the same genetic material and yet, some specialize as skin cells, muscle cells and so on. Cell specialization occurs because cells are able to express, or turn on, only a fraction of their genes while the others are turned off.  Each cell expresses, or turns on, only a fraction of its genes. The process of turning genes on and off is known as gene regulation.  

Gene regulation is an important part of normal development.  Genes are turned on and off in a coordinated fashion during development in order to make cells look and function as specialized cells, such as brain or nerve cells.  Genes are also turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example.  When a gene is turned off, it no longer provides the directions for making proteins. This means that the proteins needed to fulfill a particular job -- say, tolerate lactase -- aren't produced.  Gene regulation also allows cells to react quickly to changes in their environments.  

Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription.  Transcription when the information in a gene’s DNA is transferred to messenger Ribo Nucleac Acid (mRNA)). Messenger RNA conveys the genetic information and the cellular machinery uses the mRNA instructions to create proteins with specific jobs.

Transcription factors are proteins that regulate the transcription of genes, that is their copying into RNA , on the way to making a protein.  Transcription factors identify and help activate, or turn specific genes “on” or “off” by binding to to regulatory regions of DNA and literally switch the gene on or off by inhibiting or attracting the molecular machinery that allows for gene expression.  If a gene is not transcribed in a cell, it can’t be used to make a protein in that cell. By controlling the level of transcription, this process can determine the amount of protein product that is made by a gene at any given time.

Vitamin K has been identified as a transcription factor, with cell signaling capability.  Vitamin K2 activates the steroid and xenobiotic receptor (SXR) and operates as a transcriptional regulator of the number of osteoblastic biomarker genes and extracellular matrix related genes. The SXR, also known as the pregnane X receptor (PXR), modulates gene transcription and its protective role in bone metabolism has been shown in rodent studies (Hara et al, 1993; Akiyama et al, 1994; Hara et al, 1995; Kliewer et al, 2002; Tabb et al, 2003; Ichikawa et al, 2006; Katsuyama et al, 2007; Igarashi et al, 2007; Shearer & Newman, 2008; Azuma et al, 2010).  

So while the essential role of vitamin K through the carboxylation pathways is well established, vitamin K2 performs some of its osteoprotective functions by upregulating bone marker genes.

As an example, a study investigated the effects of vitamin K1, MK4, and K3 on human osteoblast cells.  They found that vitamin K1least potently induced mineralization and had negligible effects on gene expression.  Vitamins K2 and K3 had more potent effects on mineralization: at low doses they were only marginally inhibited by warfarin, and at high doses they were unaffected by warfarin. They identified at least two mechanisms through which vitamin K acts on osteoblasts and on osteocyte-like cells: mineralization, which appears to be mediated in part through a γ-carboxylation-dependent event, and effects on gene expression.  However, vitamin K had a distinct effect at the level of expression of certain genes, with vitamins K2 and K3 profoundly downregulating RANKL mRNA. These results suggest that vitamin K influences the behavior of mature osteocytes. This is consistent with a significant body of literature that has identified vitamin K as an activator of the SXR-mediated transcription pathway (Atkins, Welldon et al, 2009). 

The effects of vitamin K are not limited to these pathways. Ichikawa et al. (2007) observed that MK-4, through a pathway independent of SXR and carboxylation, resulted in activation of two genes, namely, growth differentiation factor 15 and Stanniocalcin. Induction of these genes is exclusive to MK-4, and vitamin K1 and MK-7 do not have such effects (Ichikawa et al, 2007).

The effect of vitamin K on gene regulation in teeth is being explored.  A recent study on tooth epigenetics specifically looked at the impact of K2 on dental health.  This study identified at least 18 dental genes that are affected directly or indirectly by vitamin K2 (Le Bechek et al, 2012).  

Recent advances in tissue engineering have drawn scientists to test the possibility of regeneration a whole tooth or part of the tooth structure (Mantesso & Sharpe, 2009; Chen & Jin, 2010; Huang et al, 2009).

In a study, Mk7 was shown to effect gene expression during tooth development.  Three levels of 3 levels of mk7 was injected into mice (0.2, 2, and 10mg/kg body weight).  The analysis indicated that all dosages resulted in significantly altered expression.  281 genes were differentially expressed, associated with decreased cell death or aptosis. And osteoclast and osteoblast signaling was altered.  Interestingly also some genes related to enamel/dentine biosynthesis exhibited differential expression such as Amelx and Ambn. (Amelx in instrumental in the building and maintenance of tooth enamel and its resilience to enmal erosion.)  They concluded that MK7 increased the transcription of genes involved in development of bone and of enamel/dentin and that vitamin K2 (Mk7) upregulates Amelx and DKK1 in tooth germs. DKK1 is instrumental in building and maintenance of tooth enamel and contributes to resistance to erosion.  In further detail, the data showed that a plethora of genes were upregulated, and enhanced several times over by vitamin K2 treatment. As there was a broad spectrum of beneficial effects from vitamin K2, they concluded that K2 was a true regulator of genes involved in the assembly of collagen related to the development of bone and enamel and dentin (Tai et al, 2014).  

The prospective of tooth tissue engineering is very attractive, but the field is in its infancy and we are far from performing routine clinical procedures. Predictable periodontal tissue regeneration needs to ideally recapitulate embryonic development, following similar morphogenetic gene expression patterns. Currently being explored are techniques ranging from stem cell therapy, the use of growth factors, pulp implants, implant of 3D cell printed in hydrogels, injectable scaffolds, bioactive materials, the use of co-enzymes, and root canal revascularization.

Despite the large amount of interest in this field, no clinical trials have been performed for dentine repair and very limited clinical applications are available in periodontal disease treatment.

Although we know that the regulation of genes is critical for life, this complex process is not yet fully understood (Mooney et al, 1996; Bohl et al, 1998; Buurma et al, 1999; Gronthos et al, 2000; Selvig et al, 2002; Young et al, 2002; Young et al, 2005; Yen et al, 2008).

References.

Fosdick LS, Fancer OE, Calandra JC.  The effect of synthetic vitamin K on the rate of acid formation in the mouth.  Science. 1942 Jul 10;96(2480):45. 

Fosdick LS, Fancher OE, Calandra JC.  The effect of synthetic vitamin K and related compounds on the rate of acid formation in saliva. J Dent Res.  1944;23(1):31-37.

Burrill DY, Caladra JC, Tilden EB, Fosdick LS.  J Dental Res. The effect of 2-Methyl-1,4-Napthoquinone on the incidence of dental caries.  1945;24(6):273-92.  

Vitamin K and tooth decay.  Nutr Revs. 1946 Jul;4(7): 213-216.

Waldo CM, Rothblatt JM.  Histologic response to tooth movement in the laboratory rat; procedure and preliminary observations.  J Dent Res. 1954 Aug; 33(4):481-6.

Bouckaert JH, Said AH.  Fracture healing by vitamin K. Nature. 1960;185:849.

Jowsey J, Riggs BL.  Bone formation in hypercortisonism.  Acta Endocrinol (Copenh). 1970 Jan;63(1):21-8. 

Steinman RR, Leonara J.  Relationship of fluid transport through the dentin to the incidence of dental caries.  J Dent Res. 1971;50(6):Part 2:1536-1543.  

Dahl E, Mjör IA.  The structure and distribution of nerves in the pulp-dentin organ.  Acta Odontol Scand. 1973 Dec;31(6):349-56.


Storey E.  Tissue response to the movement of bones.  Am J Orthod. 1973 Sep;64(3):229-47.  

Hauschka PV, Lian JB, Gallop PM.  Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue.  Proc Natl Acad Sci USA. 1975 Oct;72(10):3925-9. 
In view of the recently discovered requirement of vitamin K for generation of calcium binding sites (gamma-carboxyglutamate) by gamma-carboxylation of specific glutamic acid residues in prothrombin, our findings may implicate vitamin K metabolism in normal bone development. 

Steinman RR, Leonara J.  Effect of selected dietary additives on the incidence of dental caries in the rat.  J Dent Res. 1975;54(3):570-7. 

Price PA, Otsuka AA, Poser JW, Kristaponis J, Raman N.  Characterization of a gamma-carboxyglutamic acid-containing protein from bone.  Proc Natl Acad Sci USA.  1976 May;73(5);1447-51.
A gamma-carboxyglutamic acid-containing protein has been purified from the calcified tissue sof several vertebrates.  The presence of three-gamma-carboxyglutamic acid residues in the bovine protein was established.  

Steinman RR.  The control of dental caries by manipulating dentinal fluid movement.  J. Missouri Dent Assn. 1977;57(7):15-18.

Glimcher MJ.  Phosphopeptides of enamel matrix.  J Dent Res. 1979 Mar;58(spec Issue B):790-809.

King GJ, Theims S.  Chemical mediation of bone resorption induced by tooth movement in the rat.  Arch Oral Biol. 1979;24:811-15.

 

1980

Hall JG, Pauli RM, Wilson KM.  Maternal and fetal sequelae of anticoagulation during pregnancy.  Am J Med. 1980 Jan;68(1):122-40.
Review of published cases of pregnancies in which coumarin derivatives (warfarin) or heparin were administered demonstrates that use of either class of anticoagulant carries substantial risks. Of 418 reported pregnancies in which coumarin derivatives were used, one-sixth resulted in abnormal liveborn infants, one-sixth in abortion or stillbirth and, at most, two-thirds in apparently normal infants. In addition to the expected hemorrhagic complications, fetal effects of coumarin derivative administration include a specific embryopathy and central nervous system abnormalities. The use of heparin during gestation does not result in a significantly better outcome of pregnancy. Heparin does not appear to be a clearly superior alternative to coumarin derivatives.

Hauschka PV, Reddi AH.  Correlation of the appearance of gamma-carboxyglutamic acid with the onset of mineralization in developing endochondral bone. Biochem Biophy Res Commun.  1980 Feb 12;92(3):1037-41.

Poser JW, Esch FS, Ling NC, Price PA.  Isolation and sequence of the vitamin K-dependent protein from human bone.  Undercarboxylaton of the first glutamic acid residue. J Biol Chem. 1980;255:8685-91. 

Price PA, Lothringer JW, Baukol SA, Reddi AH.  Developmental appearance of the vitamin K-dependent protein of bone during calcification. Analysis of mineralizing tissues in human, calf, and rat.  J Biol Chem. 1981 Apr 25;256(8):3781-4.
Several mineralizing tissues have been analyzed for the vitamin K-dependent protein of bone (BGP)  (aka osteocalcin) in order to establish the temporal relationship between initial mineral deposition and the appearance of BGP.  In fetal human bone, the level of BGP rises from 5% of the adult level at 10 weeks gestational age to the adult level at 15 weeks. Thus, adult levels of BGP are reached in human bone shortly after the initial appearance of mineral and long before birth. The relative absence of BGP in initially deposited bone mineral and its subsequent appearance several days later may be causally related to the maturation of bone mineral to hydroxyapatite, a structure which binds BGP. The implications of the timing of BGP appearance in mineralizing tissues to its possible function in bone are discussed.

Price P.A.  Williamson. M.K. Haba. T.Dell. R.B. Lee. W.S.  Excessive mineralization with growth plate closure in rats on chronic warfarin treatment. Proc. Natl. Acad. Sci. USA.  1982;79(2):7734-38.
Rats maintained for 8 months on a level of warfarin sufficient to decrease the vitamin K-dependent protein of bone (bone Gla protein or osteocalcin) to 2% of normal have an excessive mineralization disorder characterized by complete fusion of the proximal tibial growth plate and cessation of longitudinal growth. The general features of this abnormality resemble the fetal warfarin syndrome in humans, a disorder also characterized by excessive mineralization of the growth plate. These excessive mineralization disorders may be caused by the decreased levels of bone Gla protein, a protein that potently inhibits mineralization in vitro.

DiMuzio MT, Bhown M, Butler WT.  The biosynthesis of dentine gamma-carboxyglutamic acid-containing proteins by rat incisor odontoblasts in organ culture.  Biochem J. 1983 Nov 15;216(2):249-57.
Rat odontoblasts were shown to synthesize and secrete gamma-carboxyglutamic acid (Gla)-containing proteins into dentine after organ culture in the presence of radio labelled amino acid precursors. The present results are significant in identifying dentine gla-containing protein as endogenous to mineralizing dentine and may relate to the commonality between calcifying connective tissues in general.


Price PA, Urist MR, Otawara Y.  Matrix Gla protein, a new gamma-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone.  Biochem Biophys Res Commun. 1983 Dec 28;117(3):765-71.
A new protein has been isolated from demineralized bovine bone matrix. This protein has five to six residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid (Gla), and we have accordingly designated it matrix Gla protein. 

Meunier PJ, Dempster DW, Edouard C, Chapuy MC, Arlot M, Charhon S.  Bone histomorphometry in corticosteroid-induced osteoporosis and Cushing’s syndrome.  Adv Exp Med Biol. 1984;171:191-200. 

Bronckers ALJJ, Gay S, Dimuzio MT, Butler WT. Immunolocalization of gamma-carboxyglutamic acid-containing proteins in developing rat bones.  Collagen Rel Res 1985;5:273-281.


Finkelman RD, Butler WT.  Appearance of dentin gamma-carboxyglutamic acid-containing proteins in developing rat molars in vitro.  J Den Res. 1985 Jul;64(7):1008-15.

Freeman.  1985. Periodontium.  In: Ten Cate AR, editor.  Oral Histology, Development, Structure and Function.  St. Louis, MO: CV Mosby Co. p. 234-263.

Price PA. Vitamin K-dependent formation of bone Gla protein (osteocalcin) and its function.  Vitam. Horm. 1985; 42:65-108.
This chapter discusses the vitamin K–dependent formation of bone γ-carboxyglutamic acid (Gla) protein (BGP, osteocalcin) and its function.  BGP is among the most abundant noncollagenous bone proteins and appears to be a universal constituent of the skeleton and tooth dentin of all vertebrates. 

Price PA, Williamson MK.  Primary structure of bovine matrix Gla protein, a new vitamin-K dependent bone protein.  J Biol Chem. 1985 Dec 5;260(28):14971-5.
The complete amino acid sequence of bovine bone matrix Gla protein (MGP) was determined.  This 79-residue, vitamin K-dependent protein contains a single disulfide bond and 4.8 gamma-carboxyglutamate (Gla) residues, one each at positions 37, 41, 48, and 52, and 0.8 Gla and 0.2 Glu at position 2. MGP is the first vitamin K-dependent protein to be discovered which has several non-gamma-carboxylated residues to the NH2-terminal side of its Gla residues. The presence of NH2-terminal Glu residues suggests that the gamma-carboxylase may have additional, as yet unrecognized, specificity requirements which determine the susceptibility of Glu residues for gamma-carboxylation.


Suttie JW.  Vitamin K-dependent carboxylase.  Ann Rev Biochem. 1985 Jul;54:459-477. 


Delmas PD, Demiaux B, Malaval L, Chapuy MC, Meunier PJ.  Osteocalcin (or bone gla-protein), a new biological marker for studying bone pathology.  Presse Med. 1986 Apr 5; 15(14):643-6.
Osteocalcin, also called bone gla-protein, is a bone matrix protein synthesized specifically by osteoblasts. It circulates in blood. We measured osteocalcin serum levels in 169 adult controls and 161 patients with different disseminated or localized bone diseases. The normal concentration of 6.2 +/- 0.2 ng/ml increases significantly with age. Serum osteocalcin levels are considerably increased in renal osteodystrophy, and to a lesser degree in primary hyperparathyroidism, and Paget's disease, all diseases characterized by increased bone turnover. High levels are also encountered in osteomalacia. Conversely, serum osteocalcin levels are significantly decreased in patients under long-term corticosteroid therapy; they remain normal in patients with bone myeloma and bone metastases under treatment. The circulating osteocalcin therefore is the first specific and sensitive marker for bone turnover. 

Otawara Y, Price PA.  Developmental appearance of matrix GLA protein during calcification in the rat.  J Biol Chem.  1986 Aug 15;261(23):10828-32. 
A marked dissociation has been observed between the timed accumulation in calcified tissues of two related vitamin K-dependent proteins, bone Gla protein (BGP) and the recently discovered matrix Gla protein (MGP).  In long bone diaphyses, total levels of MGP were essentially equivalen tin newborn, juvenile and adult rats.  In agreement with previous studies, BGP levels were only 5% of adult levels in newborn rat bones and increased to 90% of adult levels by 19 days of age.  These differences in the timed accumulation of MGP and BGP in calcifying tissues indicate that MGP could function earlier in bone formation than does BGP.. 


Romberg RW, Werness PG, Riggs BL, Mann KG. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins. Biochemistry. 1986;25:1176–1180. 

Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al.  Novel regulators of bone formation: molecular clones and activities. Science.  1988 Dec;242(4885):1528-34.
Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), direct cartilage and bone formation.  Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.

Gehron-Robey P.  The biochemistry of bone.  Endrocrinol Metabol Clin N Am.  1989;18:858-902.

Hauschka PV, Lian JB, Cole DE, Gundberg CM. Osteocalcin and matrix Gla protein: vitamin K‐dependent proteins in bone. Physiol Rev 1989: 69: 990– 1047.

Hauschka PV, Wians FH Jr.  Osteocalcin-hydroxyapatite interaction in the extracellular organic matrix of bone.  Anat Rec. 1989 Jun;224(2):180-8.
Osteocalcin, a major noncollagenous matrix protein of bone, dentin, and cementum, is found in tight association with the calcium phosphate mineral phase of these tissues. This article reviews the structural data for osteocalcin relevant to mineral adsorption. The equilibrium-binding properties for Ca2+ ions and hydroxyapatite are considered, along with the apparent physicochemical effects of osteocalcin on bone mineral dynamics. Several of osteocalcin's possible biological activities (involvement in mineralization, chemoattraction, and leukocyte elastase inhibition) are discussed in relation to the mineral-adsorption characteristics of this protein.

Price P.A.Gla-containing proteins of bone.  Conn. Tissue Res. 1989;21;51-60.
Bone has high levels of two proteins which contain the vitamin K dependent, calcium binding amino acid, γ-carboxyglutamic acid (Gla), bone Gla protein and matrix Gla protein. BGP (osteocalcin) is synthesized only by calcified tissues while MGP is synthesized by calcified tissues, cartilage, and all soft tissues tested. The synthesis of both proteins in osteoblastic cells is stimulated by 1,25(OH)2D3. Treatment of rats with the vitamin K antagonist Warfarin causes secretion of a non-γ-carboxylated BGP which cannot bind to hydroxyapatite. Warfarin treatment reduces bone levels of BGP to 2% of normal, but does not appear to affect the structure of bone. The pattern of cartilage calcification is similar to that seen in the fetal Warfarin syndrome in humans, and may be due to abnormal synthesis of MGP. 

1990

Delmas PD, Price PA, Mann KG.  Validation of the bone Gla protein (osteocalcin) assay.  J Bone Miner Res.  1990 Jan;5(1):3-4.

Feteih R., Tassinari M.S., Lian J.B. Effect of sodium warfarin on vitamin K-dependent proteins and skeletal development in the rat fetus. J. Bone Min. Res. 1990;5:885–894.
Warfarin was administered daily to Sprague-Dawley rats from gestational day 8 to day 22 to examine the effects of this compound on the fetal skeleton and on the vitamin K-dependent bone and cartilage proteins.  There was a 43% mortality rate among the dams. In the surviving litters, fetal bone osteocalcin and gamma-carboxyglutamic acid were significantly reduced (50 and 57%, respectively, on gestational day 22) when compared to age- and/or weight-matched control pups. These results suggest that the growth plate abnormalities seen with prenatal warfarin exposure relate to the inhibition of the vitamin K-dependent proteins of the skeletal system.

Barone L.M., Owen T.A., Tassinari M.S., Bortell R., Stein G.S., Lian J.B. Developmental expression and hormonal regulation of the rat matrix GLA protein (MGP) gene in chondrogenesis and osteogenesis. J. Cell. Biochem. 1991;46:351–365. 
Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintenance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintenance of a collagenous matrix.

Cole DEC, Hanley DA.  Osteocalcin. In: Hall BK (ed) Bone, Vol.3; Bone matrix and bone specific products.  CRC Press, Boca Raton, 1991; 239-294.

Plantalech L, Guillaumont M, Vergnaud P, Leclercq M, Delmas PD.  Impairment of gamma carboxylation of circulating osteocalcin (bone gla protein) in elderly women.  J Bone Miner Res. 1991;6:1211-1216. 

Sato M, Grasser W, Endo N, Akins R, Simmons h, Thompson DD, et al.  Bisphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.  J Clin Invest. 1991 Dec;88(6):2095-105. 
Studies of the mode of action of the bisphosphonate alendronate showed that 1 day after the injection of 0.4 mg/kg [3H] alendronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts.

Schroeder HE. 1991. Oral Structural Biology.  New York, NY: Thieme Medical Publications. Cementogenesis and radicular cementum; p. 144-171.  

Akedo Y, Hosoi T, Inoue S, Ikegami A, Mizuno Y, Kaneki M, et al. Vitamin k2 modulates proliferation and function of osteoblastic cells in vitro. 1992;187:814–20. 
A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.

Orimo H, de Souza AC, Ouchi, Nakamura T, Shiraki M.  Skeletal tissue and nutrition in the aging process: an overview.  Nutr. Rev. 1992 Dec;50(12):382-4.

Udagawa N, Takito J, Suda T. Mechanism of acid production and secretion by osteoclasts. Nihon Rinsho. 1992;50:2133–8. 

Akiyama Y, Hara K, Ohkawa I, Tajima T.  Effects of menatetrenone on bone loss induced by ovariectomy in rats.  Jpn J Pharmacol. 1993;62:145-153 .
The effects of menatetrenone, MK4, on bone loss induced by ovariectomy in rats was studied in 3 experiments.  MK4 was given as a dietary supplement.  In experiment 1, at 2 weeks postovariectomy, menatetrenone (10mg/kg/day given for 2 weeks) inhibited the decrease in bone density of the femoral metaphysis induced by the ovariectomy.  In experiment 2, MK4 (3 or 30mg/km/day given for 6 months) inhibited the decrease in bone strength of the femur and the decrease in calcium and hydroxproline content of the femoral diaphysis at 6 months post-ovariectomy.  In experiment 3, MK4 treatment, at 30 or 100 mg/kg/day for 6 months, protected against the decrease in bone strength and calcium and hydroxyproline content in the bone loss model induced by ovariectomy and calcium-deficient diet.  These findings suggest that MK4 protects against the bone loss induced by ovariectomy.

Delmas PD.  Biochemical markers of bone turnover.  Bone Miner Res. 1993 Dec;8 Suppl 2:S549-55.
The noninvasive assessment of bone turnover has received increasing attention over the past few years because of the need for sensitive markers in the clinical investigation of osteoporosis. For assessment of the level of bone turnover in women with vertebral osteoporosis, serum osteocalcin and urinary pyridinoline appear to be the most sensitive markers so far. 

Hara K, Akiyama Y, Ohkawa I, Tajima T.  Effects of menatetrenone on prednisolone-induced bone loss in rats.  Bone. 1993 Nov Dec;14(6):813-8. 
This study was carried out to evaluate the effect of MK4 on prednisolone-induced bone loss.  Three experiments were performed in rats which received menatetrenone as a dietary supplement. In experiment 1, a soluble form of prednisolone, dissolved in drinking water, was administered to rats at 7 mg/kg/day for 9 weeks. The length, dry weight, and bone density of femurs and tibiae, as well as urinary gamma-carboxyglutamic acid (Gla) content, were significantly lower in the prednisolone-control group than in the intact group. Menatetrenone (17 mg/kg/day) significantly inhibited the decrease in these bone parameters, especially in tibiae. In experiments 2 and 3, prednisolone (10 mg/kg), dissolved in cottonseed oil, was given to rats intramuscularly three times a week for 4 and 10 weeks, respectively. In experiment 2, bone length, bone strength and calcium content in the femur were reduced by 4-week prednisolone treatment. These reductions were significantly improved by menatetrenone (21 mg/kg/day). In experiment 3, 10-week prednisolone treatment reduced bone length and the calcium and hydroxyproline content of the femur. Menatetrenone (0.4, 10, and 50 mg/kg/day) significantly inhibited the reduction of calcium content in the femur. These results suggest that menatetrenone may inhibit the bone loss induced by corticosteroid treatment.

Hodges SJ, Akesson K, Vergnaud P, Obrant K, Delmas PD.  Circulating levels of vitamin K1 and K2 decreased in elderly women with hip fracture.  J Bone Miner Res. 1993 (Oct);8(10):1241-5.
This study measured the serum levels of phylloquinone (vitamin K1) and of the menaquinones, MK-7 and MK-8, in a group of 51 women with a mean age of 81 years who were studied within a few hours after a hip fracture. A group of 38 healthy age-matched women randomly chosen from the same population served as controls. Patients with hip fracture had a marked reduction in serum vitamin K1, MK-7, and MK-8, and a large number had undetectable levels, especially of MK-8. These data suggest that patients with hip fracture have vitamin K deficiency, an abnormality that could affect bone metabolism through an impairment of the gamma carboxylation of the gla-containing proteins of bone.

Lanyon LE. Osteocytes, strain detection, bone modeling and remodeling.  Calcif. Tissue Int. 1993;53(Suppl 1):S102. 

Leonara J, Tieche JM, Steinman RR.  Further evidence for a hypothalamus-parotid gland endocrine axis in the rat.  Arch Oral Biol. 1993;38(10):911-916.

Lin CP, Douglas WH, Erlandsen SL.  Scanning electron microscopy of type collagen at the dentin-enamel junction.  J Histochem Cytochem. 1993 Mar;41(3):381-8.
This study was undertaken to identify the presence, type, and distribution of collagen at the dentin-enamel junction as an initial step in understanding its structural-functional role in dental occlusion and they concluded that the dentin-enamel junction can be regarded as a fibril-reinforced bond which is mineralized to a moderate degree.

Linde A, Goldberg M.  Dentinogenesis. Crit Rev Oral Biol Med.  1993;4(5):679-728.  
The formation of dentin, dentinogenesis, comprises a sophisticated interplay between several factors in the tissue, cellular as well as extracellular. Dentin may be regarded as a calcified connective tissue. In this respect, as well as in its mode of formation, it is closely related to bone. After describing dentin structure and composition, this review discusses items such as the morphology of dentinogenesis.

Orimo H, Shiraki M, Inoue S.  Estrogen and bone. Osteoporos Int.  1993;3 Suppl 1:153-6.

Szulc P, Chapuy MC, Meunier PJ, Delmas PD.  Serum undercarboxylated osteocalcin is a marker of the risk of hip fracture in elderly women.   J Clin Invest. 1993;91(4):1769-1774.

Akiyama Y, Hara K, Tajima T, Murota SI, Morita I.  Effect of vitamin K2 (menatetrenone) on osteoclast-like cell formation in mouse bone marrow cultures.  European Journal of Pharmacology. 1994;263(1-2):181-85. 

Bègue-Kirn C, Smith AJ, Loriot M et al . Comparative analysis of TGF beta s, BMPs, IGF1, msxs, fibronectin, osteonectin and bone sialoprotein gene expression during normal and in vitro-induced odontoblast differentiation. Int J Dev Biol. 1994;38(3):405–420.

Bronckers ALJJ, Farach-Carson MC, Van Waveren E, Butler WT.  Immunolocalization of osteopontin, osteocalcin and dentin sialoprotein during dental root formation and early cementogenesis in the rat.  J Bone Miner Res. 1994;9:833-841.

Ten Cate AR. 1994. Oral Histology. Development, Structure, and Function. 4th ed. St. Louis, MO:  Mosby.

Butler WT, Ritchie H.  The nature and functional significance of dentin extracellular matrix proteins.  Int J Dev Biol. 1995;39:169-179.

Hara K, Akiyama Y, Nakamura T, Murota S, Morita I. The inhibitory effect of vitamin K2 (menatetrenone) on bone resorption may be related to its side chain. Bone. 1995;16(2):179-84.

Hughes DE, Wright KR, Uy HL, Sasaki A, Yoneda T, Roodman GD, et al. Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Miner Res. 1995;10(10):1478–87. 

Luo G, D’Souza R, Hogue D, Karsenty G.  The matrix Gla protein gene is a marker of the chondrogenesis cell lineage during mouse development.  J Bone Miner Res. 1995 Feb;10(2):345-324.
Matrix Gla protein (MGP) is a skeletal member of the family of mineral-binding Gla proteins.  In a study of mice, MGP gene expression is detectable as early as day 10.5 of embryonic development, before any skeletal structures are identifiable. As development proceeds, MGP gene is predominantly expressed in cells of the chondrocytic lineage in areas that will undergo endochondral ossification as well as in areas that will remain cartilaginous, such as the trachea and bronchi. In growth plate cartilage, MGP mRNA is present in resting, proliferative, and late hypertrophic chondrocytes. Finally, the MGP gene is expressed at a lower level in kidney medulla and uterus smooth muscle but not in brain, spleen, or heart during development. This study demonstrates that during development MGP gene expression occurs early and is predominant in lung and limb buds, and in cells of the chondrocytic lineage. 

Shearer MJ.  Vitamin K. Lancet.  1995;345(8944(:229-234.

Vermeer C, Jie K-S, Knapen M. Role of vitamin K in bone metabolism. Annual review of nutrition. 1995;15(1):1-21.

Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, et al.  Increased bone formation in osteocalcin-deficient mice. Nature. 1996 Aug 1;382(6590):448-52.
To investigate the role of osteocalcin, the most abundant osteoblast-specific non-collagenous protein, we have generated osteocalcin-deficient mice. These mice develop a phenotype marked by higher bone mass and bones of improved functional quality. Before and after studies showed that the absence of osteocalcin leads to an increase in bone formation without impairing bone resorption. To our knowledge, this study provides the first evidence that osteocalcin is a determinant of bone formation.

Ehara Y, Takahashi H, Hanahisa Y, Yamaguchi M. Effect of vitamin K2 (menaquinone-7) on bone metabolism in the femoral-metaphyseal tissues of normal and skeletal-unloaded rats: enhancement with zinc. Res Exp Med (Berl) 1996; 196: 171-178.

Kameda T, Miyazawa K, Mori Y, et al.  Vitamin K2 inhibits osteoclastic bone resorption by inducing osteoclast apoptosis.  Biochemical and Biophysical Research Communications. 1996;220(3):515-91.

Meredith N, Sherriff M, Setchell DJ, Swanson SA. Measurement of the microhardness and young’s modulus of human enamel and dentine using an indentation technique. Arch Oral Biol. 1996;41(6):539–545. 

Mooney DJ, Powell C, Piana J, Rutherford B. Engineering dental pulp-like tissue in vitro. Biotechnol Prog. 1996;12:865–8. 

Szulc P, Chapuy MC, Meunier PJ, Delmas PD.  Serum undercarboxylated osteocalcin is a marker of the risk of hip fracture:  a three year follow-up study. Bone. 1996;18:487-488.
We have previously shown that elderly women with an increased serum undercarboxylated osteocalcin (ucOC) level have an increased risk of sustaining a hip fracture as compared to those with normal serum ucOC. We reassessed our findings on a larger number of hip fractures that occurred over 3 years in 183 institutionalized women (aged 70–97 years) belonging to a large prospective clinical trial. UcOC was still predictive of the hip fracture when age and parathyroid hormone concentration were included into the model (odds ratio = 2.6, 95 % C.I. = 1.05–6.4). These data confirm that ucOC is a marker of the increased risk of hip fracture in elderly institutionalized women. Serum ucOC may reflect some nutritional deficiency associated with increased bone fragility. 

Beertsen W, McCulloch CAG, Sodek J.  The periodontal ligament: A unique, multifunctional connective tissue.  Periodontology 2000: 1997;13:20-40.
The periodontal ligament is the soft connective tissue interposed between the roots of our teeth and the inner wall of the alveolar socket. Its fibers form a meshwork that stretches out between the cementum and the bone and is firmly anchored by Sharpey’s fibers (Fig. 1). The periodontal ligament links the teeth to the alveolar bone proper, providing support, protection and provision of sensory input to the masticatory system. Our objective is to illustrate how the unique properties of the periodontal ligament endow this tissue with functional attributes that are not replicated by other tissues. 

Bosshard DD, Selvig KA.  Dental cementuma; the dynamic tissue covering the root.  Periodontol 2000. 1997 Feb;13:41-75.  

D'Souza RN, Cavender A, Sunavala G, Alvarez J, Ohshima T, Kulknari AB, et al.  Gene expression patterns of murine dentix matrix protein 1 (Dmp 1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo.  J Bone Miner Res.  1997;12:2040-2049.

Kagayama M, Li HC. Zhu J, Sasano Y, Hatakeyama Y, Mizoguchi I.  Expression of osteocalcin in cementoblasts forming acellular cementum.  J Periodontal Res. 1997 Apr;32(3):273-8.
To determine the phenotypic expression of cementoblasts responsible for acellular cementum, a study was performed of rat osteocalcin (OC). Maxillary first molars of Wistar male rats aged 2 and 3 wk were used for observations. Osteocalcin staining was detected in cells lining root surface in both 2- and 3-wk-old rats. Almost all cells lining cellular cementum were positive for OC. The present results suggest that the OC expression of cementoblasts forming acellular cementum is similar to that of cells forming cellular cementum as well as osteoblasts and odontoblasts, and has a role for calcification of acellular cementum.

Koshihara Y, Hoshi K.  Vitamin K2 enhances osteocalcin accumulation in the extracellular matrix of human osteoblasts in vitro.  J Bone Miner Res. 1997 Mar;12(3):431-8.
The role of vitamin K in osteocalcin accumulation in the extracellular matrix of normal human osteoblasts in culture was investigated by using a human intact osteocalcin-specific assay system. The results proved that vitamin K2 increased Gla-containing osteocalcin, which accumulated osteocalcin in the extracellular matrix, and facilitated mineralization in vitro. Vitamin K2 also enhanced the 1,25(OH)2D3-induced osteocalcin mRNA level, but vitamin K2 alone did not show osteocalcin mRNA expression. We thus demonstrated that vitamin K2 enhanced not only the accumulation of Gla osteocalcin, but also the osteocalcin production induced by 1,25(OH)2D3 in human osteoblasts in culture.

Koshihara Y, Hosti K.  Vitamin K2 enhances osteocalcin accumulation in the extracellular matrix of human osteoblasts in vitro.  J Bone Miner Res. 1997 Mar;12(3):431-8.
The role of vitamin K in osteocalcin accumulation in the extracellular matrix of normal human osteoblasts in culture was investigated.  Human osteoblasts produced osteocalcin by treatment with 10(-9) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) for 20 days in culture. With the addition of vitamin K2 (1.5-5.0 microM), osteocalcin accumulation in the extracellular matrix of the osteoblasts was increased.  These results proved that vitamin K2 increased Gla-containing osteocalcin, which accumulated osteocalcin in the extracellular matrix, and facilitated mineralization in vitro. We thus demonstrated that vitamin K2 enhanced not only the accumulation of Gla osteocalcin, but also the osteocalcin production induced by 1,25(OH)2D3 in human osteoblasts in culture.

Luo G, Ducy P, McKee MD, Pinero GJ, Lover E, Behringer RR, et al.  Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein.  Nature. 1997 Mar 6;386(6620):78-81.
Calcification of the extracellular matrix (ECM) can be physiological or pathological. Physiological calcification occurs in bone when the soft ECM is converted into a rigid material capable of sustaining mechanical force; pathological calcification can occur in arteries and cartilage and other soft tissues. No molecular determinant regulating ECM calcification has yet been identified. A candidate molecule is matrix GLA protein (Mgp), a mineral-binding ECM protein synthesized by vascular smooth-muscle cells and chondrocytes.  Mice that lack Mgp develop to term but die within two months as a result of arterial calcification which leads to blood-vessel rupture. Chondrocytes that elaborate a typical cartilage matrix can be seen in the affected arteries. Mgp-deficient mice additionally exhibit inappropriate calcification of various cartilages, including the growth plate, which eventually leads to short stature, osteopenia and fractures. These results indicate that ECM calcification must be actively inhibited in soft tissues. To our knowledge, Mgp is the first inhibitor of calcification of arteries and cartilage to be characterized in vivo.

Meunier P, Boivin G. Bone mineral density reflects bone mass but also the degree of mineralization of bone: therapeutic implications. Bone. 1997;21(5):373–377.

Salo J, Lehenkari P, Mulari M, Metsikkö K, Väänänen HK.  Removal of osteoclast bone resorption products by transcytosis.  Science. 1997;276;270-73. 

Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, et al.  Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.  Cell. 1997 Apr 18;89(2):309-319.
In vivo, hepatic expression of OPG in transgenic mice results in a profound yet nonlethal osteopetrosis. These same effects are observed upon administration of recombinant OPG into normal mice. Furthermore, OPG blocks ovariectomy-associated bone loss in rats. These data show that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis associated with increased osteoclast activity.

Vergnaud P., Garnero P., Meunier P.J., Breart G., Kamihagi K., Delmas P.D.  Undercarboxylated osteocalcin measured with a specific immunoassay predicts hip fracture in elderly women: the EPIDOS Study. J Clin Endocrinol Metab. 1997;82:719–724. 

Vervoort LM, Ronden JE, Thijssen HH.  The potent antioxidant activity of the vitamin K cycle in microsomal lipid peroxidation.  Biochem Pharmacol. 1997:54(8):871-876.

Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P, Karsenty G.  Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin.  Bone. 1998 Sep;23(3):187-96.
To test the hypothesis that osteocalcin is involved in regulating mineral properties, a more sensitive assay of mineralization, Fourier transform infrared microspectroscopy (FT-IRM) was used to study thin sections of femora of 4-week-, 6-month- (intact and ovariectomized), and 9-month-old wild-type and osteocalcin-knockout mice. No differences were detected in the mineral properties of the 4-week-old knockout and wild-type mice indicating that the mineralization process was not altered at this time point, however differences emerged from six month-old animals.  These spatially resolved data provide evidence that osteocalcin is required to stimulate bone mineral maturation.

Bohl KS, Shon J, Rutherford B, Mooney DJ. Role of synthetic extracellular matrix in development of engineered dental pulp. Journal of Biomaterials Science Polymer Edition. 1998;9:749–64. 

Bronckers AL, Price PA, Schrijvers A, Bevoets TJ, Karsenty G.  Studies of osteocalcin function in dentin formation in rodent teeth.  Eur J Oral Sci. 1998 Jun;106(3):795-807.
Osteocalcin (OC) is a major non-collagenous protein synthesized by osteoblasts, odontoblasts and cementoblasts. We examined the function of OC in dentinogenesis by exposing rat and hamster tooth organ cultures to 1,25(OH)2vit D3 or to bovine OC added to the culture medium. We furthermore examined dentinogenesis in tooth explants cultured in the presence of warfarin (an inhibitor of gamma-carboxylation of OC). Finally, we analyzed dentin from osteocalcin null mutant mice. Exposure to 1,25(OH)2vit D3 increased OC synthesis by odontoblasts in vitro at the transcriptional and protein levels. High levels of bovine OC temporarily suppressed the initial formation of dentin and enamel. This effect was not seen when tooth explants were exposed to thermally decarboxylated OC.  Exposure of tooth explants to warfarin had no significant effect on dentinogenesis. Dentin obtained from two-month-old OC null mutants looked structurally normal and did not show marked differences in dentin matrix thickness and mineral content compared to wild type. We concluded that, although OC at supraphysiological levels has the potential to affect dentin mineralization probably through its Gla-residues.

Davidson RT, Foley AL, Engelke JA, Suttie JW.  Conversion of dietary phylloquinone to tissue menaquinone-4 in rats is not dependent on gut bacteria.  J Nutr. 1998 Feb;128(2):2203.
The ability of male rats to accumulate menaquinone-4 (MK-4) in tissues when fed a vitamin K-deficient diet supplemented with intraperitoneal phylloquinone (K) as the sole source of vitamin K for 14 d was assessed.  Supplementation with the equivalent of 1500 microg vitamin K/kg diet increased tissue MK-4 concentrations above those of controls fed a vitamin K-deficient diet. MK-4 concentrations were approximately 5 ng/g (11 pmol/g) in liver, 14 ng/g in heart, 17 ng/g in kidney, 50 ng/g in brain and 250 ng/g in mandibular salivary glands of gnotobiotic rats. Cultures of a kidney-derived cell line (293) converted K to the expoxide of MK-4 in a manner that was dependent on both time of incubation and concentration of vitamin K in the media. A liver-derived cell line (H-35) was less active in carrying out this conversion. These data offer conclusive proof that the tissue-specific formation of MK-4 from K is a metabolic transformation that does not require bacterial transformation to menadione as an intermediate in the process.

Knapen MH, Neuwenhuijzen Kruseman AC, Wouters RSME, Vermeer C.  Correlation of serum osteocalcin fractions with bone mineral density in women during the first 10 years after menopause. Calcif Tissue Int, 1998. 63(5): 375–379.

Kobayashi Y, Takagi H, Sakai H, Hashimoto F, Mataki S, Kobayashi et al.  Effects of local administration of osteocalcin on experimental tooth movement.  Angle Orthod. 1998;68:259-66.

Munroe P.B., Plgunturk R.O., Fryns J.-P., Maldergem L.V., Ziereisen F., Yuksel B., Gardiner R.M., Chung E.  Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nat. Genet. 1999;21:142–144.

Orimo H, Sugioka Y, Fukunaga M, Muto Y, et al.  Diagnostic criteria of primary osteoporosis. J of Bone and Min Metab.  1998 Aug;16(3):139-150.

Orimo H, Shiraki M, Tomita A, Morii H, Fujita T, Ohata M.  Effects of menatetrenone on the bone and calcium metabolism in osteoporosis:  A double-blind placebo-controlled study. J Bone Miner Metab. 1998 June;16(s):106-112.
To evaluate efficacy of MK4 on cortical bone mineral density and its safety, a 24-week double-blind placebo-controlled study was conducted by enrolling 80 osteoporotic patients. Patients were given either 90 mg/day of vitamin K2 or a placebo. Bone density was assessed. In the vitamin K2 group, bone density increased by 2.20% ± 2.48% from the baseline; in the placebo group, it decreased by −7.31% ± 3.65% (P = .037, K2vs placebo). Our findings suggest that vitamin K2 at a dosage of 90 mg/day is effective in maintaining peripheral cortical bone density and is safe in treatment for osteoporosis.

Price PA, Faus SA, Williamson MK.  Warfarin causes rapid calcificiaton of the elastic lemallae in rat arteries and heart valves.  Arterioscler Thromb Vasc Biol. 1998 Sep;18(9):1400-7.
High doses of warfarin cause focal calcification of the elastic lamellae in the media of major arteries and in aortic heart valves in the rat. Aortic calcification was first seen after 2 weeks of warfarin treatment and progressively increased in density at 3, 4, and 5 weeks of treatment. By 5 weeks, the highly focal calcification of major arteries could be seen on radiographs and by visual inspection of the artery. The calcification of arteries induced by warfarin is similar to that seen in the matrix Gla protein (MGP)-deficient mouse, which suggests that warfarin induces artery calcification by inhibiting gamma-carboxylation of MGP and thereby inactivating the putative calcification-inhibitory activity of the protein. Warfarin treatment markedly increased the levels of MGP mRNA and protein in calcifying arteries and decreased the level of MGP in serum.


Ronden JE, Drittij-Reijnders MJ, Vermeer C, Thijssen HH.  Intestinal flora is not an intermediate in the phylloquinone-menaquinone-4 conversion in the rat.  Biochim Biophys Acta. 1998 Jan 8;1379(1):69-75.  
To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinone-4 (MK-4) we investigated the tissue distribution of vitamin K in germ-free rats. The rats were made vitamin K deficient by feeding a vitamin K-free diet for 13 days. In a subsequent period of 6 days, phylloquinone and menadione were supplied via the drinking water in concentrations of 10 and 50 micromol l(-1). Menadione supplementation led to high levels of tissue MK-4, particularly in extrahepatic tissues like pancreas, aorta, fat and brain. Liver and serum were low in MK-4. Phylloquinone supplementation resulted in higher phylloquinone levels in all tissues when compared with vitamin K-deficient values. The main target organs were liver, heart and fat. Remarkably, tissue MK-4 levels were also higher after the phylloquinone supplementation. The MK-4 tissue distribution pattern after phylloquinone intake was comparable with that found after menadione intake. Our results demonstrate that the conversion of phylloquinone into MK-4 in extrahepatic tissues may occur in the absence of an intestinal bacterial population and is tissue specific.

Sato Y, Honda Y, Kuno H, Oizumi K. Menatetrenone ameliorates osteopenia in disuse-affected limbs of vitamin D- and K-deficient stroke patients. Bone. 1998;23(3):291-296.

Smith CE, Cellular and chemical events during enamel maturation.  Crit Rev Oral Biol Med. 1998;9(2):128-61.  
This review focuses on the process of enamel maturation, a series of events associated with slow, progressive growth in the width and thickness of apatitic crystals. This developmental step causes gradual physical hardening and transformation of soft, newly formed enamel into one of the most durable mineralized tissues produced biologically. 

Akiyama Y, Hara K, Kobayashi M, Tomiuga T, Nakamura T.  Inhibitory effects of vitamin K2 (menatetrenone) on bone resorption in ovariectomized rats: a histomorphometric and dual energy X-ray absorptiometric study.  Jpn J Pharmacol. 1999 May;80(1):67-74.
To clarify how vitamin K2 prevents bone loss in vivo, it was given to ovariectomized 20-week-old rats for 2 weeks. Bone mineral density (BMD) in the whole femur and in 7 specific portions (F1 to F7 from the proximal to the distal end) was determined by dual-energy X-ray absorptiometry, and histomorphometry was also performed in proximal tibial metaphysis. Ovariectomy (OVX) resulted in significant decreases in the BMD in the whole femur and the F1, F2, F6 and F7 portions. The data indicate that the bone loss within 2 weeks is due to the enhancement of bone resorption. Vitamin K2 at 50 mg/kg inhibited the decrease in the BMD of the whole femur together with the F6 and F7 portions. These results suggest that vitamin K2 prevents bone loss through the inhibition of bone resorption and osteoclast formation in vivo.

Buurma B, Gu K, Rutherford RB.  Transplantation of human pulpal and gingival fibroblasts attached to synthetic scaffolds.  Eur J Oral Sci. 1999 Aug;107(4):282-9.
These data revealed that three adult human dental pulp and gingival cell populations, each from individual donors, attached to PGA scaffolds and cultured for 24 h in vitro, survive implantation, and express genes indicative of a capacity to produce extracellular matrix. The implanted cells may also express genes associated with responsiveness to BMP-mediated tissue inductive signals.

Dowd FJ.  Saliva and dental caries.  Dent Clin North Am. 1999;43(4):579-597.

Feskanich D, Weber P, Willett WC, Rockett H, Booth SL, Colditz GA. Vitamin K intake and hip fractures in women: a prospective study. Am J Clin Nutr. 1999. 69(1): 74–79.

Iwamoto I, Kosha S, Noguchi S, et al. A longitudinal study of the effect of vitamin K2 on bone mineral density in postmenopausal women a comparative study with vitamin D3 and estrogen-progestin therapy. Maturitas. 1999;31(2):161-164.

Somekwawa Y, Chigughi M, Harada M, Ishibashi T.  Use of vitamin K2 (menatetrenone) and 1,25-dihydroxyvitamin D3 in the prevention of bone loss induced by leuprolide.  Endocrinol Metab. 1999 Aug;84(8):2700-4.
The purpose of this study is to evaluate the efficacy of vitamin K2 and vitamin D3 in preventing bone loss induced by estrogen deficiency during therapy with the GnRH agonist (GnRH-a) leuprolide. One hundred ten women (mean age, 46.2+/-0.5 yr), receiving leuprolide therapy for estrogen-dependent diseases (such as endometriosis and uterine leiomyomas), were randomly allocated into four groups (group A, leuprolide only; group B, leuprolide with vitamin K2; group C, leuprolide with 1,25-(OH)2D3; and group D, leuprolide with vitamin K2 and 1,25-(OH)2D3). Bone mineral density was reduced in all four groups. Bone formation markers were significantly increased in all four groups, and the percent changes of bone formation markers were highest in group B. Bone resorption markers also increased significantly in all four groups after treatment of 6 months. Vitamin K2, especially when combined with 1,25-(OH)2D3, can partially prevent bone loss caused by estrogen deficiency. 

Sugiyama T, Tanaka H, Kawai S. Clinical vignette. Vitamin K plus vitamin D treatment of bone problems in a child with skeletal unloading. J Bone Miner Res. 1999;14(8):1466-1467.

Yagami K, Suh JY, Enomoto-Iwamoto M, Koyama E, Abrams WR, Shapiro IM, et al.  Matrix Gla protein is a developmental regulator of chrondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb.  J Cell Biol. 1999 Nov 29;147(5):1097-108.  
Matrix GLA protein (MGP), a vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Yamaguchi M, Taguchi H, Gao YH, Igarashi A, Tsukamoto Y.  Effect of vitamin K2 (menaquinone-7) in fermented soybean (natto) on bone loss in ovariectomized rats.  J Bone Miner Metab. 1999;17(1):23-9.
The effect of dietary vitamin K2, MK7, on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12mg/100g diet) or menaquinone-7 (MK-7; 18.1mg/100g diet) for 24 days. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4.

Yasui T, Fujita K, Sasaki S, Sato M, Sugimoto M, et al.  Expression of bone matrix proteins in urolithiasis model rats.  Urological Research. 1999 Aug;27(4):255-61.
Urinary calcium stones are a pathological substance, and they show similarities to physiological mineralization and other pathological mineralizations. The expression of messenger (m) RNAs of osteopontin (OPN), matrix Gla protein (MGP), osteonectin (ON) and osteocalcin (OC) in bones and teeth has been described. We previously identified OPN as an important stone matrix protein. In addition, the spontaneous calcification of arteries and cartilage in mice lacking MGP was recently reported, a finding which indicates that MGP has a function as an inhibitor of mineralization. Here, we examined the mRNA expressions of OPN, MGP, ON, and OC in the kidneys of stone-forming model rats administered an oxalate precursor, ethylene glycol (EG) for up to 28 days. We suggest that OPN acts on calcification and MGP acts on suppression.

 

2000

Allison Jane Lee, Stephen Hodges and Richard Eastell.  Measurement of osteocalcin. Ann Clin Biochem. 2000;37:432-446.  
Bone turnover may be assessed by the measurement of enzymes or matrix proteins produced by osteoblasts (which form bone) or osteoclasts (which resorb bone). The introduction of reliable, specific tests for the biochemical markers of bone metabolism would aid in the clinical management of metabolic bone diseases, including osteoporosis. Osteocalcin, also known as bone Gla protein, is a marker of bone formation. It is a vitamin K and vitamin O-dependent protein produced by osteoblasts and is the most abundant and most widely studied of the non-collagenous proteins in bone.

Chapurlat R.D., Garnero P., Breart G., Meunier P.J., Delmas P.D.  Serum type I collagen breakdown product (serum CTX) predicts hip fracture risk in elderly women: the EPIDOS Study. Bone.  2000;27:283–286. 

D’Errico JA, Berry JE, Ouyang H, Strayhorn CL, Windle JJ, Somerman MJ.  Employing a transgenic animal model to obtain cementoblasts in vitro.  J. Periodontol. 2000 Jan;71(1):63-72.  
Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. The mechanisms controlling development and regeneration of this tissue are not well understood. This study looked at mice cells that express osteocalcin as a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Gilbert SF. 2000. Osteogenesis: The Development of Bones. In: Developmental Biology. 6th ed. Sunderland, MA:  Sinauer Associates. Available online at: http://www.ncbi.nlm.nih.gov/books/NBK10056/.

Greenspan SL, Harris ST, Bone H, et al.  Bisphosphonates: safety and efficacy in the treatment and prevention of osteoporosis.  Am Fam Physician. 2000;61:2731-36.

Gronthos S, Mankani M, Brahim J et al . Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA. 2000;97(25):13625–630.
In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

Ho AM, Johnson MD, Kingsley DM.  Role of the mouse ank gene in control of tissue calcification and arthritis. Science. 2000;289:265–270.

Howe AM, Webster WS.  Warfarin exposure and calcification of the arterial system in the rat.  Int J Exp Pathol. 2000 Feb;81(1):51-6. 
There is evidence from knock-out mice that the extrahepatic vitamin K-dependent protein, matrix gla protein, is necessary to prevent arterial calcification. The aim of this study was to determine if a warfarin treatment regimen in rats, designed to cause extra-hepatic vitamin K deficiency, would also cause arterial calcification. Sprague-Dawley rats were treated from birth for 5-12 weeks with daily doses of warfarin and concurrent vitamin K1. This treatment causes an extrahepatic vitamin K deficiency without affecting the vitamin K-dependent blood clotting factors. At the end of treatment the rats were killed and the vascular system was examined for evidence of calcification. All treated animals showed extensive arterial calcification. The cerebral arteries and the veins and capillaries did not appear to be affected. It is likely that humans on long-term warfarin treatment have extrahepatic vitamin K deficiency and hence they are potentially at increased risk of developing arterial calcification.

Iwamoto J, Takeda T, Ichimura S.  Effect of combined administration of vitamin D3 and vitamin K2 on bone mineral density of the lumbar spine in postmenopausal women with osteoporosis.  J Orthop Sci. 2000;5(6):546-1. 
The effect of the combined administration of vitamin D3 and vitamin K2 on bone mineral density (BMD) of the lumbar spine was examined in postmenopausal women with osteoporosis. Ninety-two osteoporotic women who were more than 5 years after menopause, aged 55-81 years. These findings indicate that combined administration of vitamin D3 and vitamin K2, compared with calcium administration, appears to be useful in increasing the BMD of the lumbar spine in postmenopausal women with osteoporosis.

Luukinen H, Kakonen SM, Pettersson K, Koski K, Laippala P, Lovgren T, Kivela SL, Vaananen HK. Strong prediction of fractures among older adults by the ratio of carboxylated to total serum osteocalcin. J Bone Miner Res, 2000. 15(12): 2473–2478.

Mashiba T., Hirano T., Turner C., Forwood M., Johnston C., Burr D. Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces some biomechanical properties in dog rib. J Bone Miner Res. 2000;15:613–620.

McCulloch CA, Lekic P, McKee MD. Role of physical forces in regulating the form and function of the periodontal ligament. Periodontol 2000. 2000;24:56–72. 
Vitamin K-dependent matrix Gla protein (MGP) has been suggested to play a role in the inhibition of soft tissue calcification. Our data demonstrate the close association between MGP and calcification. It is suggested that undercarboxylated MGP is biologically inactive and that poor vascular vitamin K status may form a risk factor for vascular calcification. 

Nanci,A. and Smith,C.E. (2000): Matrix-mediated mineralization in enamel and the collagen-based hard tissues. In: Proc. 6th Intl. Con. on Chem. Biol. Min. Tis. (C. Robinson, A. Boskey, N. Goldberg Eds.), Am. Acad. Orthoped. Sug., pp. 217-224. 

Nishiguchi S, Shiomi S, Tamori A, Habu D, Takeda T, Tanaka T, et al.  Effect of ethanal on bone mineral density of rats evaluated by dual-photon X-ray absorptiometry.  J Bone Miner Res. 2000;18(6):317-20. 

Proudfoot D, Skepper JN, Hegyi L, Bennett MR, Shanahan CM, Weissberg PL.  Apoptosis regulates human vascular calcification in vitro. Evidence for initiation of vascular calcification by apoptotic bodies.  Circ Res. 2000;87:1055-62.

Pudota BN, Miyagi M, Hallgren KW, West KA, Crabb JW, Misono KS, et al.  Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation.  Proc Natl Acad Sci USA. 2000 Nov 21;97(24):13033-8.
The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. 

Schurgers LJ, Vermeer C. Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations. Haemostasis. 2000;30:298–307. 

Shiraki M, Shiraki Y, Aoki C, Miura M. Vitamin K2 (menatetrenone) effectively prevents fractures and sustains lumbar bone mineral density in osteoporosis.  J Bone Miner Res. 2000;15(3):515-522. 
Their findings suggest that vitamin K2 treatment effectively prevents the occurrence of new fractures, although the vitamin K2-treated group failed to increase in LBMD. Furthermore, vitamin K2 treatment enhances gamma-carboxylation of the OC molecule.

Tsukamoto Y, Ichise H, Kakuda H, Yamaguchi M.  Intake of fermented soybean (natto) increases circulating vitamin K2 (menaquinone-7) and gamma-carboxylated osteocalcin concentration in normal individuals.  J Bone Miner Metab. 2000;18(4):216-22.
Changes in circulating vitamin K2 (menaquinone-7, MK-7) and gamma-carboxylated osteocalcin concentrations in normal individuals with the intake of fermented soybeans (natto) were investigated. Serum MK-7 and gamma-carboxylated osteocalcin concentrations in men with the occasional or frequent dietary intake of natto were significantly higher than those without any intake. The present study suggests that intake of fermented soybean (natto) increases serum levels of MK-7 and gamma-carboxylated osteocalcin in normal individuals.

Urayama S, Kawakami A, Nakashima T, Tsuboi M, Yamasaki S, Hida A, et al. Effect of vitamin K 2 on osteoblast apoptosis: Vitamin K 2 inhibits apoptotic cell death of human osteoblasts induced by Fas, proteasome inhibitor, etoposide, and staurosporine. Journal of Laboratory and Clinical Medicine. 2000;136(3):181-93.
Vitamin K2 is used for the treatment of osteoporosis, but the precise mode of action is still not clear. We investigated the effects of vitamin K2 on apoptosis of human osteoblasts. Cells were cultured with or without various concentrations of vitamin K2 and tumor necrosis factor-α (TNF-α). Our results suggest that vitamin K2 inhibits apoptotic cell death of osteoblasts and maintains the number of osteoblasts. These actions may explain the therapeutic efficacy of vitamin K2 in osteoporosis.

Wallin R, Cain D, Hutson SM, Sane DC, Loeser R.  Modulation of the binding of matrix Gla protein (MGP) to bone morphogenetic protein-2 (BMP-2).  Thromb Haemost. 2000 Dec;84(6):1039-44. 
Matrix Gla protein (MGP) is an inhibitor of calcification of the arterial wall but the mechanism of inhibition has not been resolved. Since chondrogenesis has been identified in calcified arteries from MPG null mice, we hypothesized that locally produced MGP might inhibit calcification by neutralizing the known effect of bone morphogenetic proteins (BMPs) as promotors of chondrogenesis and bone formation. The data propose that MGP matures earlier in the secretory pathway than other vitamin K-dependent proteins. 

Yamaguchi M, Kakuda H, Gao YH, Tsukamoto Y.  Prolonged intake of fermented soybean (natto) diet containing vitamin K2 (menaquinone-7) prevents bone loss in ovariectomized rats.  J Bone Miner Metab. 2000;18(2):71-6.
The effect of the prolonged intake of dietary vitamin K2 (menaquinone-7, MK-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing the fermented soybean (natto) without or with supplemental MK-7, for 150 days. Feeding produced a significant elevation of MK-7 concentration in the serum of OVX rats. In this case, the femoral MK-4 content was significantly increased, but MK-7 was not detected in the femoral tissues, indicating degradation of MK-7. Serum gamma-carboxylated osteocalcin concentration was significantly decreased by OVX. This decrease was significantly prevented by the feeding of the natto diets with supplemental MK-7 (18.8 micrograms/100 g diets). This study demonstrates that the prolonged intake of natto dietary including MK-7 has a preventive effect on bone loss induced by OVX. Dietary MK-7 may be useful in the prevention of osteoporosis.

Yonemura K, Kimura M, Miyaji T, Hishida A. Short-term effect of vitamin K administration on prednisolone-induced loss of bone mineral density in patients with chronic glomerulonephritis. Calcif Tissue Int. 2000;66(2):123-128.

Asakura H, Myou S, Ontachi Y, Mizutani T, Kato M, Saito M, et al.  Vitamin K administration to elderly patients with osteoporosis induces no hemostatic activation even in those with suspected vitamin K deficiency.  Osteoporos Int. 2001 Dec;12(12):996-1000. 
The administration of menaquinone-4 (MK-4), significantly reduces bone loss in postmenopausal osteoporotic women. However, concerns have been raised about whether vitamin K administration alters the hemostatic balance by inducing a thrombotic tendency. We investigated were whether the administration of vitamin K in the form of MK-4 induced a thrombotic tendency in 29 elderly patients with osteoporosis. Patients were administered 45 mg/day (three times a day, 30 min after each meal) of MK-4 for 12 weeks. Blood samples were obtained from the patients at 0, 4 and 12 weeks after the start of MK-4 administration. A number of hemostatic parameters remained stable under the markedly increased plasma levels of MK-4. These results indicate that MK-4 can be administered safely, with regard to maintaining the hemostatic balance, to osteoporotic patients receiving no anticoagulant therapy.

Boström K, Tsao D, Shen S, Wang Y, Demer LL.  Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells.  J Biol Chem. 2001 Apr 27;276(17):14044-52.  
Matrix GLA protein (MGP) is ubiquitously expressed with high accumulation in bone and cartilage, where it was found to associate with bone morphogenetic proteins (BMP) during protein purification. To test whether MGP affects BMP-induced differentiation, three sets of experiments were performed.  The results suggest that MGP modulates BMP activity.


Hashimoto F, Kobayashi Y, Mataki S, Kobayashi K, Kato Y, Sakai H.  Administration of osteocalcin accelerates orthodontic tooth movement induced by a closed coil spring in rats.  Eur J Orthod. 2001 Oct;23(5):535-45.
The effect of local administration of osteocalcin (OC) on experimental tooth movement was examined in the rat. The maxillary first molar was first moved mesially with an initial tipping force of 30 g with a closed-coil spring anchored to the incisor for 10 days (n = 48). Three experimental groups (n = 8) were injected daily with purified rat OC at doses of 0.1, 1, and 10 micrograms, respectively. Tooth movement was evaluated daily.  Acceleration of tooth movement by OC was significant in the early experimental period. A significantly larger number of osteoclasts accumulated on the mesial alveolar bone surface in the 1-microgram OC-injected group on day 3 than that observed in control group. These results suggest that administration of OC accelerates orthodontic tooth movement due to enhancement of osteoclastogenesis on the pressure side, primarily in the early experimental period.

Iwamoto J., Takeda T., Ichimura S. Effect of menatetrenone on bone mineral density and incidence of vertebral fractures in postmenopausal women with osteoporosis: A comparison with the effect of etidronate. J. Orthop. Sci. 2001;6:487–492. 

Kaneki M, Hodges SJ, Hosoi T, et al. Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk. Nutrition. 2001;17(4):315–321. 
A statistically significant inverse correlation was found between incidence of hip fractures in women and natto consumption in each prefecture throughout Japan. These findings indicate that the large geographic difference in MK-7 levels may be ascribed, at least in part, to natto intake and suggest the possibility that higher MK-7 level resulting from natto consumption may contribute to the relatively lower fracture risk in Japanese women.

Kim BY, Yoon, HY, Yun SL, Woo ER, Song NK, Kim Hg et al.  In vitro and in vivo inhibition of glucocorticoid-induced osteoporosis by the hexane extract of Poncirus trifoliata.  Phytother Res. 2001 Jul;25(7):1000-10.


Nishiguchi S, Shimoi S, Kurooka H, et al.  Randomized pilot trial of vitamin K2 for bone loss in patients with primary biliary cirrhosis. J Hepatol. 2001;35(4):543-545.

Shimada T, Mizutani S, Muto T, Yoneya T, Hino R, Takeda S, et al.  Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia.  Proc Natl Acad Sci. USA. 2001;98:6500-6505.

Smith AJ, Lesot H.  Induction and regulation of crown dentinogenesis:  embryonic events as a template for dental tissue repair?  Crit Rev Oral Biol Med. 2001;12(5);425-37.

Schurgers LJ, Vermeer C. Determination of Phylloquinone and Menaquinones in Food. Pathophysiol. Haemost. Thromb. 2001;30:298–307. 

Spronk HMH, Soute, BAM, Schurgers LJ, Cleutjens JPM, Thijssen HWH, De Mey JGR, Vermeer C.  Matrix Gla protein accumulates at the border of regions of calcification and normal tissue in the media of the arterial vessel wall.  Biochem and Biophys Res Comms. 2001;298:485-490.  

Yamaguchi M, Sugimoto E, Hachiya S. Stimulatory effect of menaquinone‐7 (vitamin K2) on osteoblastic bone formation in vitro. Mol Cell Biochem, 2001. 223: 131–137. 

Yamaguchi M and Ma ZJ. Inhibitory effect of menaquinone‐7 (vitamin K2) on osteoclast‐like cell formation and osteoclastic bone resorption in rat bone tissues in vitro. Mol Cell Biochem, 2001. 228: 39–47.

Bandyopadhyay, P.K., J.E. Garrett, R.P. Shetty, T. Keate, C.S. Walker, and B.M. Olivera. Gamma-Glutamyl carboxylation: an extracellular posttranslational modification that antedates the divergence of mollusks, arthropods, and chordates. Proc. Natl. Acad. Sci. USA. 2002;99:1264–1269. 

Iwasaki Y, Yamato H, Muraya H, Takahashi T, Ezawa I, Kurokawa K, et al.  Menetetrenone prevents osteoblast dysfunction in unilateral sciata neuroectomized rats.  Jpn J Pharmacol. 2002;90:88-93.
These data suggested that MK-4 reduced the loss of trabecular bone, prevented osteoblast dysfunction to a certain extent, and contributed to preservation of the trabecular microstructure in this rat model of osteopenia induced by sciatic neurectomy.

Kliewer SA, Goodwin B, Willson TA.  The nuclear pregnane X receptor: a key regulator of xenobiotic metabolism.  Endocrine Reviews. 2002 Oct 1;23(5):687-702.

Kobayashi M, Hara K, Akiyama Y.  Effects of vitamin K2 (menatetrenone) on calcium balance in ovariectomized rats.  Jpn J Pharmacol. 2002 Jan;88(1):55-61.
MK4 has been used for the treatment of osteoporosis in Japan. We investigated the effects of ovariectomy (OVX) and vitamin K2 on the calcium (Ca) balance in 20-week-old female Fischer rats. MK4 (31 mg/kg per day) was given to animals as a dietary supplement. At weeks 4 and 8 after OVX, a Ca balance study was performed for 5 days. The Ca balance improved significantly in the vitamin K2 groups as compared with the sham- and OVX-control groups. The administration of vitamin K2 significantly inhibited an OVX-induced decrease in cortical area and cortical thickness in the femur. These findings suggest that the poor Ca balance observed in ovariectomized rats may be improved by vitamin K2; vitamin K2 may be involved in preventing bone loss in vivo.

Jacobson A.  Oral development and histology.  3rd edition.  New York: Thieme Medical Publishers, Inc.  2002.

Knepper‐Nicolai B, Reinstorf A, Hofinger I, Flade K, Wenz R, Pompe W. 2002. Influence of osteocalcin and collagen I on the mechanical and biological properties of Biocement D. Biomol Eng.  2002;19:227– 231.

McKee IW, Williamson PC, Lam EW, Heo G, Glover KE, Major PW.  The accuracy of 4 panoramic units in the projection of mesiodistal tooth angulations.  Am J Orthod Dentofactial Ortho. 2002 Feb;121(2):166-75.

Papagerakis P, Berdal A, Mesbah M, Peuchmaur M, et al.  Investigation of osteocalcin, osteonectin and dentin sialophosphoprotein in developing human teeth.  Bone. 2002;30(2):377-385.

Papagerakis P, Berdal A, Mesbah M, Peuchmaur M, Malaval L, Nydegger J, Simmer J, et al.  Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth.  Bone. 2002 Feb;30(2):377-385.  
Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum.  The aim of this study was to identify the expression pattern of the two major proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. 

Roy ME, Nishimoto SK.  Matrix Gla protein binding to hydroxyapatite is dependent on the ionic environment: calcium enhances binding affinity but phosphate and magnesium decrease affinity.  Bone. 2002 Aug; 31(2):296-302.
Matrix Gla protein (MGP) is an inhibitor of mineralization found in bone, cartilage, developing tissues, smooth muscle, and atherosclerotic plaques. MGP interaction with hydroxyapatite (HA) has been inferred by its function, but has never been measured directly. In this study, the influence of MGP antibody (x-MGP) binding, plasmin digestion, and various ions, including calcium and phosphate, on (125)I-labeled MGP-HA binding was examined. MGP binding to HA is sensitive to protein binding, limited proteolysis, and the surrounding ionic environment.

Sato Y, Honda Y, Kaji M, et al. Amelioration of osteoporosis by menatetrenone in elderly female Parkinson's disease patients with vitamin D deficiency. Bone. 2002;31(1):114-118.

Schurgers LJ, Vermeer C. Differential lipoprotein transport pathways of K-vitamins in healthy subjects. Biochim Biophys Acta - Gen Subj. 2002;1570:27–32. 

Selvig KA, Sorensen RG, Wozney JM, Wikesjo UME.  Bone repair following recombinant human bone – Morphogenetic Protein-2 stimulated periodontal regeneration.  J Period. 2002 Sep;73(9):1020-9.Recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable sponge (ACS) carrier is currently being evaluated as candidate therapy for periodontal regeneration. The objective of this study was to characterize, in some detail, tissue reactions following surgical implantation of rhBMP-2/ACS into periodontal defects.  This study highlighted the challenges in regenerating bone tissue, ranging from spaces devoid of collagen, or ankylosis. Lamellated trabecular bone was the predominant regenerated tissue. A typical cementum-periodontal ligament-alveolar bone relationship was a rare observation.

Shiomi S, Nishiguchi S, Kubo S, et al. Vitamin K2 (menatetrenone) for bone loss in patients with cirrhosis of the liver. Am J Gastroenterol. 2002;97(4):978-981.

Shiraishi A, Higashi S, Masaki T, Saito M, Ito M, Ikedo S, et al.  A comparison of alfacalcidol and menatetrenone for the treatment of bone loss in an ovariectomized rat model of osteoporosis.  Calcif Tissue Int. 2002 Jul;71(1):69-79.  
This study was carried out to evaluate the effect of menatetrenone, a vitamin K2 with 4 isoprene units, on prednisolone-induced bone loss. Three experiments were performed in rats which received menatetrenone as a dietary supplement. In experiment 1, a soluble form of prednisolone, dissolved in drinking water, was administered to rats at 7 mg/kg/day for 9 weeks. Menatetrenone (0.4, 10, and 50 mg/kg/day) significantly inhibited the reduction of calcium content in the femur. These results suggest that menatetrenone may inhibit the bone loss induced by corticosteroid treatment.

Tricker ND, Dixon RB, Garetto LP. Cortical bone turnover and mineral apposition in dentate bone mandible. In: Garetto LP, Turner CH, Duncan RL, Burr DB, editors. Cortical bone turnover and mineral apposition in dentate bone mandible. Indiana University School of Dentistry; 2002. pp. 226–227. 

Ushiroyama T, Ikeda A, Ueki M. Effect of continuous combined therapy with vitamin K(2) and vitamin D(3) on bone mineral density and coagulofibrinolysis function in postmenopausal women. Maturitas. 2002;41(3):211-221.

van Staa TP, Leufkens HG, Cooper C. The epidemiology of corticosteroid-induced osteoporosis: a meta-analysis. Osteoporos Int. 2002;13:777–787. 

Yonemura K, Kimura M, Miyaji T, Hishida A. Short-term effect of vitamin K administration on prednisolone-induced loss of bone mineral density in patients with chronic glomerulonephritis. Calcif Tissue Int. 2000;66(2):123-128.

Young CS, Terada S, Vacanti JP, Honda M, Bartlett JD, Yelick PC. Tissue engineering of complex tooth structures on biodegradable polymer scaffolds. J Dent Res. 2002;81:695–700. 

Zebboudj AF, Imura M, Bostrom K.  Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2.  J Biol Chem. 2002 Feb 8;277(6):4388-94.
Matrix GLA protein (MGP) has been identified as a calcification inhibitor in cartilage and vasculature. Part of this effect may be attributed to its influence on osteoinductive activity of bone morphogenetic protein-2 (BMP-2). Inhibitory levels of MGP yielded increased matrix binding of BMP-2, suggesting that MGP inhibits BMP-2 in part via matrix association. These results suggest that MGP is a BMP-2 regulatory protein.

Vanderby R, Provenzano PP. Collagen in connective tissue: from tendon to bone. J Biomech. 2003;36(10):1523–1527.

Yonemura K, Fukasawa H, Fujigaki Y, Hishida A. Protective effect of vitamins K2 and D3 on prednisolone-induced loss of bone mineral density in the lumbar spine. Am J Kidney Dis. 2004;43(1):53-60.

Cockayne S, Adamson J, Lanham-New S, et al. Vitamin K and the prevention of fractures: systematic review and meta-analysis of randomized controlled trials. Arch Intern Med. 2006;166(12):1256-1261.)
This systematic review suggests that supplementation with vitamin K1 and MK4 reduces bone loss. In the case of the latter, there is a strong effect on incident fractures among Japanese patients.

Balcerzak M, Hamade E, Zhang L, Pikula S, Azzar G, Radisson J, Bandorowicz-Pikula J, Buchet R. The role of annexins and alkaline phosphatase in mineralization process. Acta Biochim. Pol. 2003;50:1019.  

Iwamoto J, Takeda T, Ichmura S. Treatment with vitamin D3 and/or vitamin K2 for postmenopausal osteoporosis. Keio J Med, 2003. 52: 147–150. 

Iketani T, Kiriike N, Murray, et al. Effect of menatetrenone (vitamin K2) treatment on bone loss in patients with anorexia nervosa. Psychiatry Res. 2003;117(3):259-269.

Iwamoto, M., H. Kandori, and N. Kamo. Photochemical properties of pharaonis phoborhodopsin (sensory rhodopsin II). Recent Res. Dev. Chem. 2003B;1:15–30. 

Iwamoto J, Yeh JK, Takeda T.  Effect of vitamin K2 on cortical and cancellous bones in orchidectomized and/or sciatic neuroectomized rats.  J Bone and Miner Res. 2003;18(4):776-783.
We examined the effect of vitamin K2 on cortical and cancellous bones in orchidectomized and/or sciatic neurectomized rats. Ninety male Sprague-Dawley rats, 3 months of age.  MK4 was administered orally twice a week at a dose of 30 mg/kg each. After 10 weeks of feeding, the tibial shaft and proximal tibia were processed for cortical and cancellous bone histomorphometric analyses, respectively. Vitamin K2 administration in NX and ONX rats suppressed bone resorption and stimulated bone formation (mineralization), with retardation of a reduction of trabecular thickness without any significant effect on cancellous bone mass, and suppressed endocortical bone resorption, retarding a reduction in maturation-related cortical bone gain. The present study provides evidence indicating that vitamin K2 has the potential to suppress bone resorption or bone turnover.

Iwamoto J, Yeh JK, Takeda T, Ichimura S, Sato Y.  Comparative effects of vitamin K and vitamin D supplementation on prevention of osteopenia in calcium-deficient young rats.  Bone. 2003 Oct;33(4):557-66.
The aim of this study was to clarify the difference in the effects of vitamin K and vitamin D supplementation on the development of osteopenia in young rats under mild calcium deficiency. Sixty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into six groups with 10 rats in each group.  This study shows the differential effects of vitamin K and vitamin D supplementation on the development of osteopenia in young rats under mild calcium deficiency. Vitamin K supplementation stimulates renal calcium reabsorption, increases maturation-related cancellous bone gain, and retards the reduction in maturation-related cortical bone gain.

Koshihara Y, Hoshi K, Okawara R, Ishibashi H.  Vitamin K stimulates osteoblastogenesis and inhibits osteoclastogenesis in human bone marrow cell culture.  J of Endo. 2003;176:399-48.
Accumulating evidence indicates that menaquinone-4 (MK-4), inhibits osteoclastogenesis in murine bone marrow culture, but the reason for this inhibition is not yet clear, especially in human bone marrow culture. To clarify the inhibitory mechanism, we investigated the differentiation of colony forming-unit fibroblasts (CFU-Fs) and osteoclasts in human bone marrow culture.  They concluded that vitamin K might stimulate osteoblastogenesis in bone marrow cells, regulating osteoclastogenesis through the expression of RANKL/ODF more than through that of OPG/OCIF.

Li J, Lin JC, Wang H, Peterson JW, Furie BC, Furie B, et al.  Novel role of vitamin k in preventing oxidative injury to developing oligodendrocytes and neurons.  J Neurosci. 2003 Jul 2;23(13):5816-26.
Oxidative stress is believed to be the cause of cell death in multiple disorders of the brain, including perinatal hypoxia/ischemia. Although vitamin K is not a classical antioxidant, we report here the novel finding that vitamin K1 and K2 (menaquinone-4) potently inhibit glutathione depletion-mediated oxidative cell death in primary cultures of oligodendrocyte precursors and immature fetal cortical neurons with EC50 values of 30 nm and 2 nm, respectively. The mechanism by which vitamin K blocks oxidative injury is independent of its only known biological function as a cofactor for gamma-glutamylcarboxylase, an enzyme responsible for posttranslational modification of specific proteins. Furthermore, the vitamin K antagonists warfarin and dicoumarol and the direct carboxylase inhibitor 2-chloro-vitamin K1 have no effect on the protective function of vitamin K against oxidative injury. Vitamin K does not prevent the depletion of intracellular glutathione caused by cystine deprivation but completely blocks free radical accumulation and cell death. The protective and potent efficacy of this naturally occurring vitamin, with no established clinical side effects, suggests a potential therapeutic application in preventing oxidative damage to undifferentiated oligodendrocytes in perinatal hypoxic/ischemic brain injury.

Marx RE.  Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws:  a growing epidemic. J Oral Maxillofac Surg. 2003;61:1115-17.

Pinto JP, Conceicao N, Gavaia PJ, Cancela ML.  Matrix Gla protein gene expression and protein accumulation colocalize with cartilage distribution during development of the teleost fish Sparus aurata.  Bone. 2003 Mar;32(3):201-10.
Matrix Gla protein (MGP) is a member of the family of extracellular mineral-binding Gla proteins, expressed in several tissues with high accumulation in bone and cartilage.  All available evidence indicates that MGP plays a role as an inhibitor of mineralization. We investigated the sites of gene expression and protein accumulation of MGP throughout development of the bony fish Sparus aurata. The expression of MGP mRNA was first detected at 2 days posthatching (dph) by Northern analysis, RT-PCR amplification, and in situ hybridization, and thereafter continuously detected at various levels of intensity, until 130 dph.  The MGP gene was highly expressed in a number of different tissues including skull, jaw, neural and hemal arches, and heart and the protein accumulated in cartilaginous tissues. At 85 dph, a stage when most skeletal structures are mineralized, MGP gene expression and protein accumulation were restricted to the remaining cartilaginous structures, whereas osteocalcin gene expression and protein accumulation were localized in most mineralized structures. MGP gene expression was also detected in heart and kidney. 

Price PA, Nguyen TMT, Williamson MK.  Biochemical characterization of the fetuin-mineral complex.  J Biol Chem. 2003;278:22153-22160.
The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP).   

Sweatt A, Sane DC, Hutson SM, Wallin R.  Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats.  J Thromb Haemost. 2003 Jan;1(1):178-85. 
The vitamin K-dependent protein, matrix Gla protein (MGP) is a binding protein for bone morphogenetic protein-2 (BMP-2). Recombinant BMP-2 binds to the Gla-containing region of MGP in the presence of Ca2+. Immunohistochemistry showed that calcified lesions in the aortic wall of aging rats contained elevated concentrations of MGP that was poorly gamma-carboxylated and did not bind BMP-2. These results demonstrate that the BMP-2/MGP complex exists in vivo, consistent with a role for MGP as a BMP-2 inhibitor. Age-related arterial calcification may be a consequence of under-gamma-carboxylation of MGP, allowing unopposed BMP-2 activity.

Tabb MM, Sun A, Zhou C, et al. Vitamin K2 Regulation of Bone Homeostasis Is Mediated by the Steroid and Xenobiotic Receptor SXR.  The Journal of Biological Chemistry. 2003;278(45):43919–43927.

Yeah HHS, Rasgon M, Rutkowski et al.  Management of patients with chronic kidney disease at Kaiser Permanente.  Nephrology News & Issues. 2003 Aug;25-28.  

Young MF. Bone matrix proteins: their function, regulation, and relationship to osteoporosis. Osteoporos Int. 2003;14:35–42. 10.1007/s00198-002-1342-7. 

Zebboudj AF,  Shin V, Boström K.  Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells.  J Cell Biochem. 2003 Nov 1;90(4):756-65.
Expression of matrix GLA protein (MGP), an alleged calcification inhibitor, is increased in calcified arteries. We used calcifying vascular cells (CVC) that form calcified nodules in vitro to clarify the importance of MGP in vascular cell calcification and differentiation. Thus, addition of MGP either promoted or inhibited calcification, depending on the relative amounts of BMP-2 and MGP. Together, our results suggest that the effect of MGP on calcification and osteogenic differentiation is determined by availability of BMP-2.

Asawa Y, Amizuka N, Hara K, Kobayashi M, Aita M, Li M, et al.  Histochemical evaluation for the biological effect of menatetrenone on metaphyseal trabeculae of ovariectomized rats.  Bone. 2004 Oct;35(4):870-80.
To evaluate the biological effects of MK-4 on ovariectomy (OVX)-induced bone loss, we have examined histological alterations of femoral metaphyses of sham-operated (sham group), ovariectomized (OVX group), and MK-4 dietary-supplemented OVX (MK-4 group; 50 mg/kg per day) female Fischer rats 1, 2, 5, and 8 weeks after OVX.  MK-4 appeared to lessen the increase in osteoclastic bone resorption induced by OVX, as well as to maintain the accelerated osteoblastic activity. Thus, MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy.

Booth SL, Broe KE, Peterson JW, Cheng DM, Dawson-Hughes B, Gundberg CM, et al.  Associations between vitamin K biochemical measures and bone mineral density in men and women.  J Clin Endocrinol Metab. 2004 Oct;89(10):4904-9.
Few data exist on the association between vitamin K status and bone mineral density (BMD) in men and women of varying ages. We examined cross-sectional associations between biochemical measures of vitamin K status and BMD at the hip and spine in 741 men and 863 women (mean age, 59 yr; range, 32-86 yr) who participated in the Framingham Heart Study (1996-2000). Vitamin K status was assessed by plasma phylloquinone and percentage undercarboxylated osteocalcin (%ucOC). 

Burr DB. Anatomy and physiology of the mineralized tissues: role in the pathogenesis of osteoarthrosis. Osteoarthr Cartil. 2004;12(A):20–30.

Cantatore FP, Corrado A, Grano M, Quarta L, Colucci S, Melillo N.  Osteocalcin synthesis by human osteoblasts from normal and osteoarthritic bone after vitamin D3 stimulation.  Clin Rhematol. 2004 Dec;23(6):490-5.
In this study we correlated osteocalcin production from human osteoblasts isolated from healthy and osteoarthritic subjects to the degree of cartilage damage, before and after stimulation with 1,25(OH)2-vitamin D3, the active metabolite of vitamin D3. Thus, after vitamin D3 stimulation, a significant increase in osteocalcin production by maximally damaged osteoblasts compared to the minimally damaged ones was observed. This study confirms abnormal osteoarthritic osteoblast behaviour and indicates that osteoblasts from different areas of the same affected joint may be metabolically different, supporting the hypothesis that subchondral osteoblasts may play an essential role in the pathogenesis of OA.

Iohara K, Nakashima M, Ito M, Ishikawa M, Nakasima A, Akamine A.  Dentin regeneration by dental pulp stem cell therapy with recombinant human bone morphogenetic protein 2.  J Dent Res. 2004 Aug;83(8):590-5. 
Regenerative medicine is based on stem cells, signals, and scaffolds. Dental pulp tissue has the potential to regenerate dentin in response to noxious stimuli, such as caries. The progenitor/stem cells are responsible for this regeneration. Thus, stem cell therapy has considerable promise in dentin regeneration. Culture of porcine pulp cells, as a three-dimensional pellet, promoted odontoblast differentiation compared with monolayers. The expression of dentin sialophosphoprotein (Dspp) and enamelysin/matrix metalloproteinase 20 (MMP20) mRNA confirmed the differentiation of pulp cells into odontoblasts and was stimulated by the morphogenetic signal, bone morphogenetic protein 2 (BMP2). In conclusion, BMP2 can direct pulp progenitor/stem cell differentiation into odontoblasts and result in dentin formation.

Ishida Y, Kawai S.  Comparative efficacy of hormone replacement therapy; etidronate, calcitonin, alfacalcidol, and vitamin K in postmenopausal women with osteoporosis:  The Yamaguchi Osteoporosis Prevention Study. Am J Med. 2004;177:549-555.

Ivaska KK, Hentunen TA, Vääräniemi J, Ylipahkala H, Pettersson K, Väänänen HK.  Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro.  J Biol Chem. 2004;279:18361-69.
In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation.

Iwamoto J, Takeda T, Sato Y.  Effects of vitamin K2 on osteoporosis.  Curr Pharm Des. 2004;10(21):2557-76.
Findings suggest that vitamin K2 may not only stimulate bone formation but also suppress bone resorption in vivo. Clinically, vitamin K2 sustains the lumbar bone mineral density (BMD) and prevents osteoporotic fractures in patients with age-related osteoporosis, prevents vertebral fractures in patients with glucocorticoid-induced osteoporosis, increases the metacarpal BMD in the paralytic upper extremities of patients with cerebrovascular disease, and sustains the lumbar BMD in patients with liver-dysfunction-induced osteoporosis. Vitamin K deficiency, as indicated by an increased circulating level of undercarboxylated osteocalcin, may contribute to osteoporotic fractures. Even though the effect of vitamin K2 on the BMD is quite modest, this vitamin may have the potential to regulate bone metabolism and play a role in reducing the risk of osteoporotic fractures. No randomized well-controlled prospective studies conducted on a sufficiently large number of patients have been reported yet, therefore, further studies are needed to confirm the efficacy of vitamin K2 in the treatment of osteoporosis.

McGowen JA, Raisz LG, Noonan AS, ALE . Bone Health and Osteoporosis: A Report of the Surgeon General. Office of the Surgeon General (US); Rockville (MD): 2004. The frequency of bone diseases. 

Price PA, Williamson MK, Nguyen TMT, Than TN.  Serum levels of the fetuin-mineral complex correlate with artery calcification in the rat.  J Biol Chem. 2004;279:1594-1600.

Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol. 2004;15:2857– 2867.

Saito A, Suzuki Y, Ogata S, Ohtsuki C, Tanihara M.  Prolonged ectopic calcification induced by BMP-2-derived synthetic peptide.  J Biomed Mater Res A. 2004 Jul 1;70(1):115-21.

Wajih N, Borras, Wei X, Hutson SM, Wallin R.  Processing and transport of matrix Gla protein (MGP and bone morphogenetic protein-2 (BMP-2) in cultured human vascular smooth muscle cells: evidence for an uptake mechanism for serum fetuin.  JB Papers in Press. 2004 Oct 8; 279(41):43052-60.
Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.

Yonemura K, Fukasawa H, Fujigaki Y, Hishida A. Protective effect of vitamins K2 and D3 on prednisolone-induced loss of bone mineral density in the lumbar spine. Am J Kidney Dis. 2004;43(1):53-60.

Ficarra G, Beninati F, Rubino I, Vannucchi A, Longo G, Tofnelli P, et al.  Osteonecrosis of the jaws in periodontal patients with a history of bisphosphonates treatment.  J Clin Periodontol. 2005 Nov;32(11):1123-8.
Osteonecrosis of the jaws is being increasingly reported in patients receiving intra-venous bisphosphonates. A series of nine periodontally involving patients showing osteonecrosis of the jaws that appeared following the intra-venous use of bisphosphonates is reported.  Jaw osteonecrosis appears to be associated with the intra-venous use of bisphosphonates. Dental professionals should be aware of this potentially serious complication in periodontal patients receiving long-term treatment with bisphosphonates.

Iwasaki-Ishizuka Y, Yamato H, Murayama H, Ezawa I, Kurokawa K, Fukagawa M.  Menatetrenone rescues bone loss by improving osteoblast function in rats immobilized by sciatic neurectomy.  Life Sci. 2005 Feb 25:76(15):1721-34.
MK-4 is a vitamin K2 homologue that has been used as a therapeutic agent for osteoporosis in Japan. However, there is no far any reported evidence that MK-4 ameliorates a pre-existing condition of reduced bone mineral density (BMD) in vivo. In this study, we evaluated the effect of MK-4 in a rat model of established bone loss through immobilization caused by sciatic neurectomy. Four weeks treatment of MK-4 increased bone formation and suppressed bone resorption. In addition, increased carboxylated osteocalcin and decreased undercarboxylated osteocalcin in serum were observed in MK-4-administered rats. These results indicated that MK-4 rescued bone volume by improving osteoblast dysfunction and accelerating gamma carboxylation of osteocalcin. MK-4 may be useful for treating disuse osteopenia.

Katsuyama H., Otsuki T., Tomita M., et al. Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells. International Journal of Molecular Medicine. 2005;15(2):231–236. 
Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

Mashiba T, Mori S, Burr DB, et al. The effects of suppressed bone remodeling by bisphosphonates on microdamage accumulation and degree of mineralization in the cortical bone of dog rib. J Bone Miner Metab. 2005;23(suppl):36-42.

McKee MD, Addison WN, Kaartinen, MT.  Hierarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton.  Cells, Tissues, Organs. 2005;181(3-4):176-88.

Plaza SM, Lamson DW.  Vitamin K2 in bone metabolism and osteoporosis.  Alt Med Rev. 2005;10(1): 24-35.
This article covers in vitro, in vivo, and human data on the positive effect of vitamin K2 on osteoporosis. Data is available on vitamin K2 for osteoporosis caused by a number of conditions, including postmenopausal osteoporosis, Parkinson’s disease, biliary cirrhosis, stroke, and drug-induced osteoporosis. The activity of vitamin K2 involves both an increase in the bone-building process and a separate decrease in the bone loss process. Vitamin K2 exerts a more powerful influence on bone than vitamin K1, and should be considered for prevention or treatment in those conditions known to contribute to osteoporosis. 

Rammelt S, Neumann M, Hanisch U, Reinstorf A, Pompe W, Zwipp H, Biewener A. Osteocalcin enhances bone remodeling around hydroxyapatite/collagen composites. J Biomed Mater Res A.  2005;73:284– 294.

Sato Y, Kanoko T, Satoh K, Iwamoto J. Menatetrenone and vitamin D2 with calcium supplements prevent nonvertebral fracture in elderly women with Alzheimer's disease. Bone. 2005;36(1):61-68.

Stafford DW.  The vitamin K cycle.  J Throm & Haemostasis.  2005 Aug;3(8):1873-78.
Post‐translational modification of glutamate to gamma carboxyl glutamate is required for the activity of vitamin K‐dependent proteins. Carboxylation is accomplished by the enzyme gamma glutamyl carboxylase (GGCX) which requires the propeptide‐containing substrate and three co‐substrates: reduced vitamin K, CO2, and O2. Most propeptides bind tightly to GGCX and all of the Glu residues that will be modified are modified during one binding event. Carboxylation requires the abstraction of a proton from the 4‐carbon of glutamate by reduced vitamin K and results in the conversion of vitamin K to vitamin K epoxide. The vitamin K epoxide must be recycled to vitamin K before it can be reused, a reaction catalyzed by the enzyme vitamin K epoxide reductase (VKOR).

Young CS, Abukawa H, Asrican R, Ravens M, Troulis MJ, Kaban LB, Vacanti JP, Yelick PC. Tissue-engineered hybrid tooth and bone. Tissue Eng. 2005;11:1599–610. 

Zhang Q, Szalay AA, Tieche JM, et al.  Cloning and functional study of porcine parotid hormone, a novel proline0rich protein.  J Biol Chem. 2005;280(23):22233-44.

Bonewald LF.  Mechanosensation and Transduction in Osteocytes.  Bonekey Osteovision. 2006 Oct;3(10):7-15.


Bosshardt DD, Sculean A, Donos N, Lang BP. Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins.  Eur J Oral Sci. 2006 May;114 Suppl 1:225-31. discussion 254-6, 381-2.


Nanci A, Bosshardt DD.  Structure of periodontal tissues in health and disease.  Peridontol 2000. 2006;40:11-28.

Barbour ME, Finke M, Parker DM, Hughes JA, Allen GC, Addy M. The relationship between enamel softening and erosion caused by soft drinks at a range of temperatures. J Dent. 2006;34(3):207–213. 

Boskey AL.  Assessment of bone mineral and matrix using backscatter electron imaging and FTIR imaging.  Curr Osteoporos Rep. 2006 Jun;4(2):71-5.
The resistance of bone to fracture is determined by its geometric and material properties. The geometry and density can be determined by radiographic methods, but material properties such as collagen structure, mineral composition, and crystal structure currently require analysis by invasive techniques. Backscatter electron imaging provides quantitative information on the distribution of the mineral within tissue sections, and infrared and other vibrational spectroscopic methods can supplement these data, providing site-specific information on mineral content as well as information on collagen maturity and distributions of crystal size and composition. This information contributes to the knowledge of "bone quality."

Goulart CS, Nouer PR, Mouramartins L, Garbin IU, de Fátima Zanirato Lizarelli R. Photoradiation and orthodontic movement: experimental study with canines. Photomed Laser Surg. 2006 Apr;24(2):192–6. 

Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S, Steroid and xenobiotic receptor SXR mediates vitamin K2- activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells. The Journal of Biological Chemistry.  2006;281(25):16927–16934, 2006.  

Ikeda Y, Iki M, Morita A, et al. Intake of fermented soybeans, natto, is associated with reduced bone loss in postmenopausal women: Japanese Population-Based Osteoporosis (JPOS) Study. J Nutr. 2006;136(5):1323–1328. 
Natto intake may help prevent postmenopausal bone loss through the effects of menaquinone 7 or bioavailable isoflavones, which are more abundant in natto than in other soybean products.

Jackson RD, Lacroix AZ, Gass M, Wallace RB, Robbins J, Lewis CE, et al. Calcium plus vitamin D supplementation and the risk of fractures. New England Journal of Medicine. 2006;354:669–683. 

Price PA, Roublick AM, Wiliamson MK.  Artery calcification in uremic rats is increased by a low protein diet and prevented by treatment with ibandronate.  Kidney Int. 2006 Nov;70(9):1577-83.

Purwosunu Y, Muharram, Rachman IA, et al. Vitamin K2 treatment for postmenopausal osteoporosis in Indonesia. J Obstet Gynaecol Res. 2006;32(2):230-234.

Sivapathasundharam B, Raghu A R. Dental Caries. In: Rajendran R, Sivapathasundharam B (ed.) Shafer’s Textbook of Oral Pathology New Delhi: Elsevier Publishing, 2006; p. 595-601.

Shigehara S, Matsuzaka K, Inoue T.  Morphohistological change and expression of HSP70, osteopontin and osteocalcin mRNAs in rat dental pulp cells with orthodontic tooth movement.  Bull Tokyo Dent Coll. 2006;47:117-24.

Tanana I, Oshima H.  Vitamin K2 as a potential therapeutic agent for glucocorticoid-induced osteoporosis.  Clin Calcium. 2006;16:1851-57.
Most of the guidelines for management of glucocorticoid-induced osteoporosis (GIOP) recommend bisphosphonates as therapeutic agents. However, bisphosphonates have a gastrointestinal side effect, and a potential risk for pregnant women and children. An analog of vitamin K(2) reduced the fracture risk independent from the bone mineral densities in GIOP. Since GIOP induced bone fracture even in the high bone mass, the vitamin K(2) analog should be an effective therapeutic agent for GIOP through increasing bone strength without an increase in bone mineral density.

Tsugawa N, Shiraki M, Suhara Y, Kamao M, Tanaka K, Okano T. Vitamin K status of healthy Japanese women: age-related vitamin K requirement for γ-carboxylation of osteocalcin. The American journal of clinical nutrition. 2006;83(2):380-6.

Barthelat F. et al. On the mechanics of mother-of-pearl: a key feature in the material hierarchical structure. Journal of the Mechanics and Physics of Solids.  2007;55(2):225–444.

Bertoldo F, Santini D, Lo Cascio V. Bisphosphonates and osteomyelitis of the jaw: a pathogenic puzzle. Nat Clin Pract Oncol. 2007;4:711–721. 
The maxillary and mandibular bones undergo high-turnover remodeling to maintain mechanical competence. Common dental or periodontal diseases can increase local bone turnover. Bisphosphonates (BPs) accumulate almost exclusively in skeletal sites that have active bone remodeling. The maxillary and mandibular bones are preferential sites for accumulation of BPs, which become buried under new layers of bone and remain biologically inactive for a long time. Surgical odontostomatological procedures create open bony wounds that heal quickly and without infection, as a result of activation of osteoclasts and subsequently osteoblasts. Once BPs are removed from the bone via activation of osteoclasts after a tooth extraction or a periodontal procedure, they induce osteoclast apoptosis. This inhibition of osteoclast bone resorption impairs bone wound healing because of decreased production of cytokines derived from the bone matrix, and the bone is exposed to the risk of osteomyelitis and necrosis. 

Bischoff-Ferrari HA, Dawson-Hughes B, Baron JA, Burckhardt PLiR, Spiegelman D, Specker B, et al. Calcium intake and hip fracture risk in men and women: a meta-analysis of prospective cohort studies and randomized controlled trials. American Journal of Clinical Nutrition 2007 86 1780–1790.


Heiss A, Jahnen-Dechent W, Endo H, Schwahn D: Structural dynamics of a colloidal protein-mineral complex bestowing on calcium phosphate a high solubility in biological fluids. Biointerphases.  2007;2:16–20.  

Ichikawa T, Horie-Inoue K, Ikeda K, et al. Vitamin K2 induces phosphorylation of protein kinase A and expression of novel target genes in osteoblastic cells. J Mol Endocrinol. 2007;39(4):239-247.

Igarashi M, Yogiashi Y, Mihara M, Takada I, Kitagawa H, Kato S. Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene. Molecular and cellular biology. 2007;27(22):7947-54.

Katsuyama H., Saijoh K., Otsuki T., Tomita M., Fukunaga M., Sunami S. Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells. International Journal of Molecular Medicine. 2007;19(2):279–284. 
Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. 

Khosla S, Burr D, Cauley J, Dempster DW, Ebeling PR, Feisenberg D, et al.  Bisphosphonate-associated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research.  J Bone Miner Res. 2007 Oct;22(10):1479-91.
ONJ has been increasingly suspected to be a potential complication of bisphosphonate therapy in recent years. Thus, the ASBMR leadership appointed a multidisciplinary task force to address key questions related to case definition, epidemiology, risk factors, diagnostic imaging, clinical management, and future areas for research related to the disorder. This report summarizes the findings and recommendations of the task force.  The task force defined ONJ as the presence of exposed bone in the maxillofacial region that did not heal within 8 wk after identification by a health care provider. The risk of ONJ in patients with cancer treated with high doses of intravenous bisphosphonates is clearly higher, in the range of 1-10 per 100 patients (depending on duration of therapy). A research agenda aimed at filling the considerable gaps in knowledge regarding this disorder was also outlined.

Knapen MH, Schurgers LJ, Vermeer C.  Vitamin K2 supplementation improves hip bone geometry and bone strength indices in postmenopausal women.  Osteoporosis International. 2007 July;18(7):963-72.
Vitamin K mediates the synthesis of proteins regulating bone metabolism. We have tested whether high vitamin K2 intake promotes bone mineral density and bone strength. Results showed that K2 improved BMC and femoral neck width, but not DXA-BMD. Hence high vitamin K2 intake may contribute to preventing postmenopausal bone loss.

Komai M, Shirakaw H.  Vitamin K metabolism. Menaquinone-4 (MK-4) formation from ingested VK analogues and its potent relation to bone function.  Clin Calcium. 2007;17(11):1663-1672.

Ripamonti U.  Recapitulating development:  a template for periodontal tissue engineering.  Tissue Eng. 2007 Jan;13(1):51-71. 

Russell R., Xia Z., Dunford J., Oppermann U., Kwaasi A., Hulley P., et al. Bisphosphonates: an update on mechanisms of action and how these relate to clinical efficacy. Ann N Y Acad Sci. 2007;1117:209–257.

Schurgers LJ, Spronk HM, Soute BA, Schiffers PM, DeMey JG, Vermeer C. Regression of warfarin-induced medial elastocalcinosis by high intake of vitamin K in rats. Blood. 2007;109(7):2823–2831. 
Arterial calcification (AC) is generally regarded as an independent risk factor for cardiovascular morbidity and mortality. Matrix Gla protein (MGP) is a potent inhibitor of AC, and its activity depends on vitamin K (VK). In rats, inactivation of MGP by treatment with the vitamin K antagonist warfarin leads to rapid calcification of the arteries. Here, we investigated whether preformed AC can be regressed by a VK-rich diet. Rats received a calcification-inducing diet containing both VK and warfarin (W&K). During a second 6-week period, animals were randomly assigned to receive either warfarin & vitaminK (3.0 mg/g and 1.5 mg/g, subsequently), a diet containing a normal (5 microg/g) or high (100 microg/g) amount of VK (either K1 or K2). Increased aortic calcium concentration was observed in the group that continued to receive W&K and also in the group changed to the normal dose of VK and AC progressed. Both the VK-rich diets decreased the arterial calcium content by some 50%. In addition, arterial distensibility was restored by the VK-rich diet. Using MGP antibodies, local VK deficiency was demonstrated at sites of calcification. This is the first study in rats demonstrating that AC and the resulting decreased arterial distensibility are reversible by high-VK intake.

Schurgers L.J., Teunissen K.J.F., Hamulyák K., Knapen M.H.J., Vik H., Vermeer C. Vitamin K-containing dietary supplements: Comparison of synthetic vitamin K1 and natto-derived menaquinone-7. Blood. 2007;109:3279–3283. 
The purpose of this paper was to compare in healthy volunteers the absorption and efficacy of K(1) and MK-7. Serum vitamin K species were used as a marker for absorption and osteocalcin carboxylation as a marker for activity. Both K(1) and MK-7 were absorbed well, with peak serum concentrations at 4 hours after intake. A major difference between the 2 vitamin K species is the very long half-life time of MK-7, resulting in much more stable serum levels, and accumulation of MK-7 to higher levels (7- to 8-fold) during prolonged intake.

Sloan AJ, Smith AJ . Stem cells and the dental pulp: potential roles in dentine regeneration and repair.  Oral Diseases. 2007;13(2):151-57.
The dentine–pulp complex displays exquisite regenerative potential in response to injury. The postnatal dental pulp contains a variety of potential progenitor/stem cells, which may participate in dental regeneration. A population of multipotent mesenchymal progenitor cells known as dental pulp stem cells with high proliferative potential for self‐renewal has been described and may be important to the regenerative capacity of the tissue. The nature of the progenitor/stem cell populations in the pulp is of importance in understanding their potentialities and development of isolation or recruitment strategies, and allowing exploitation of their use in regeneration and tissue engineering. 

Yamaguchi M, Fujita S, Yoshida T, Oikawa K, Utsunomiya T, Yamamoto, H, Kasai K.  Low-energy laser irradiation stimulates the tooth movement velocity via expression of M-CSF and c-fms.  Orthodontic Waves. 2007 Dec;66(4):139-148.

Yamaguchi M, Kasai K.  The effects of orthodontic mechanics on the dental pulp.  Seminars in Orthodontics. 2007 Dec;13(4):272-80.
Orthodontic forces are known to produce mechanical damage and inflammatory reactions in the periodontium, as well as cell damage, inflammatory changes, and circulatory disturbances in dental pulp. It is known that orthodontic forces are capable of stimulating the whole vascular system in the dental pulp. Results of published histological data demonstrated that the dental pulp is affected by the orthodontic forces in the form of circulatory vascular stasis to necrosis. This article reviews the current knowledge of the biological aspects of dental pulp tissue changes incident to orthodontic tooth movement.

Zhu W, Boachie-Adjei O, Rawlins BA, Frenkel B, Boskey AL, Ivashkiv LB, et al.  A novel regulatory roe for stromal-derived factor-1 signaling in bone morphogenic protein-2 osteogenic differentiation of mesenchymal C2C12 cells.  J Biol Chem. 2007 Jun 29;282(26):18676-85.

Allen MR, Burr DB. Mandible matrix necrosis in beagle dogs after 3 years of daily oral bisphosphonate treatment. J Oral Maxillofac Surg. 2008;66(5):987-994.

Bolland MJ, Barber PA, Doughty RN, Mason B, Horne A, Ames R, et al.  Vascular events in healthy older women receiving calcium supplementation: randomised controlled trial. BMJ 2008 336 262–266. 
1471 postmenopausal women (mean age 74): 732 were randomized to calcium supplementation and 739 to placebo.   In the calcium group, myocardial infarction was more common, followed by stroke or sudden death.  They concluded that calcium supplementation in healthy postmenopausal women is associated with upward trends in cardiovascular event rates. This potentially detrimental effect should be balanced against the likely benefits of calcium on bone.

Clarke B.  Normal bone anatomy and physiology.  Clin J Am Soc Nephrol. 2008;3(Suppl 3):S131-S139. 

Coutu DL, Wu JH, Monette A, Rivard GE, Blostein MD, Galipeau J.  Periostin: a member of a novel family of vitamin K-dependent proteins is expressed by mesenchymal stromal cells.  J Biol Chem. 2008;283:17991-18001.

Heiss A, Eckert T, Aretz A, Richtering W, van Dorp W, Schäfer C, Jahnen-Dechent W: Hierarchical role of fetuin-a and acidic serum proteins in the formation and stabilization of calcium phosphate particles. J Biol Chem. 2008;283:14815–14825. 

Iwamoto J., Matsumoto H., Takeda T., Sato Y., Liu X., Yeh J. K. Effects of vitamin K2 and risedronate on bone formation and resorption, osteocyte lacunar system, and porosity in the cortical bone of glucocorticoid-treated rats. Calcified Tissue International. 2008;83(2):121–128. 

Kaipatur NR, Murshed M, McKee MD.  Matrix Gla protein inhibition of tooth mineralization.  J Dent Res. 2008 Sep;87(9):839-44.
Extracellular matrix (ECM) mineralization is regulated by mineral ion availability, proteins, and other molecular determinants. To investigate protein regulation of mineralization in tooth dentin and cementum, and in alveolar bone, we expressed matrix Gla protein (MGP) ectopically in bones and teeth in mice.  Mineralization was virtually absent in tooth root dentin and cellular cementum, while crown dentin showed "breakthrough" areas of mineralization. Acellular cementum was lacking in Col1a1-Mgp teeth, and unmineralized osteodentin formed within the pulp. These results strengthen the view that bone and tooth mineralization is critically regulated by mineralization inhibitors.

Lussi A, Jaeggi T. Erosion – diagnosis and risk factors. Clin Oral Investig.  2008;12(S1):5–13. 

Oldenburg J, Marinova M, Muller-Reible C. Watzka M.  The vitamin K cycle. Vitam Horm. 2008;78:35-62.

Palmer LC, Newcomb CJ, Kaltz S, Spoerke ED, et al.  Biomimetic systems for hydroxyapatite mineralization inspired by bone and enamel.  Chem Rev. 2008 Nov;108(11):4754-4783.

Price W.  2008. Nutrition and Physical Degeneration, 8th ed. Weston Price DDS. Price-Pottenger Nutrition Foundation Inc. p. 362–369, 398. 


Rizzoli R, Boonen S, Brandi M, Burlet N, Delmas P, Reginster JY.  The role of calcium and vitamin D in the management of osteoporosis.  Bone. 2008 Feb;42(2):246-9.

Saito M.  Assessment of bone quality.  Effects of bisphosphonates, raloxifene, alfacalcidol, and menatetrenone on bone quality:  collagen cross-links, mineralization, and microdamage. Clin Cal. 2008 mar;18(3):364-72.


Shearer MJ, Newman P.  Metabolism and cell biology of vitamin K.  Thromb Haemost. 2008 Oct:100(4):530-47.  
Structural differences in the isoprenoid side chain govern many facets of metabolism of K vitamins including the way they are transported, taken up by target tissues, and subsequently excreted. In the post-prandial state, phylloquinone is transported mainly by triglyceride-rich lipoproteins (TRL) and long-chain MKs mainly by low-density lipoproteins (LDL). TRL-borne phylloquinone uptake by osteoblasts is an apoE-mediated process with the LRP1 receptor playing a predominant role. One K(2) form, MK-4, has a highly specific tissue distribution suggestive of local synthesis from phylloquinone in which menadione is an intermediate. Both phylloquinone and MKs activate the steroid and xenobiotic receptor (SXR) that initiates their catabolism, but MK-4 specifically upregulates two genes suggesting a novel MK-4 signalling pathway. Many studies have shown specific clinical benefits of MK-4 at pharmacological doses for osteoporosis and cancer.   Other putative non-cofactor functions of vitamin K include the suppression of inflammation, prevention of brain oxidative damage and a role in sphingolipid synthesis. Anticoagulant drugs block vitamin K recycling and thereby the availability of reduced vitamin K. Under extreme blockade, vitamin K can bypass the inhibition of Gla synthesis in the liver but not in the bone and the vessel wall. In humans, MK-7 has a greater efficacy than phylloquinone in carboxylating both liver and bone Gla proteins. A daily supplement of phylloquinone has shown potential for improving anticoagulation control.

Szulc P, Delmas PD.  Biochemical markers of bone turnover: potential use in the investigation and management of postmenopausal osteoporosis.  Osteoporosis Int. 2008 Dec;19(12):1683-704.

Tsugawa N, Shiraki M, Suhara Y, Kamao M, Osaki R, Tanaka K, et al.  Low plasma phylloquinone concentration is associated with high incidence of vertebral fracture in Japanese women.  J Bone Min Metab. 2008;26:79-85.
It has been reported that vitamin K supplementation effectively prevents fractures and sustains bone mineral density in osteoporosis. However, there are only limited reported data concerning the association between vitamin K nutritional status and bone mineral density (BMD) or fractures in Japan. The objectives were to evaluate the association between plasma phylloquinone (K1) or menaquinone (MK-4 and MK-7) concentration and BMD or fracture in Japanese women prospectively. These results suggest that subjects with vitamin K1 insufficiency in bone have increased susceptibility for vertebral fracture independently from BMD.

Yaegashi Y, Onodo T, Anno K, Kuribayashi T, Sakata K, Orimo H.  Association of hip fracture incidence and intake of calcium, magnesium, vitamin D, and vitamin K.  European J of Epidemiology. 2008 March;23(3):219-225.
To analyze the association between hip fracture incidence in 12 regional blocks within Japan and dietary intake of four key nutrients: calcium, magnesium, vitamin D, and vitamin K. A review of the data from a 2002 national survey showed significant correlations between the standardized incidence ratio by region and magnesium, vitamin D, and vitamin K in both men and women, and calcium in women. The strongest inverse correlations were found in vitamin K in both men and women (r = −0.844, P = 0.001, and r = −0.834, P = 0.001, respectively). After adjusting for calcium, magnesium, and vitamin D, the partial correlation between the standardized incidence ratio by regional block and vitamin K was strongest in both men and women They concluded that a significant correlation between hip fracture incidence and vitamin K intake. 

Yen A, Sharpe P. Stem cells and tooth tissue engineering. Cell and Tissue Research. 2008;331:359–372. 

Youravong N, Teanpaisan R, Norén JG, et al. Chemical composition of enamel and dentine in primary teeth in children from Thailand exposed to lead. Sci Total Environ. 2008;389(2–3):253–258.

Amizuka N, Li M, Guy Y, Liu Z, Suzuki R, Yamamoto T.  Biological effects of vitamin K2 on bone quality. Clin Calcium.  2009 Dec;19(12):1788-96.
Post-transcriptional maturation with the presence of vitamin K(2) promotes gamma-carboxylation of osteocalcin, enabling further binding to hydroxyapatite, from which one could infer that vitamin K(2) increased the quality of bone matrix. For instance, vitamin K(2) rescued the impaired collagen mineralization caused by Mg insufficiency, by promoting a re-association of the process of collagen mineralization with mineralized nodules. Sodium warfarin, which antagonizes the function of vitamin K(2), reduced the binding of osteocalcin to bone matrices, and consequently resulted in crystalline particles being dispersed throughout the osteoid without forming mineralized nodules. Therefore, gamma-carboxylated Gla proteins mediated by vitamin K(2) appear to play a pivotal role in normal mineralization in bone.

Anouma H, Miyakoshi N, Hongo M, Kasukawa Y, Shimada Y.  Low serum levels of undercarboxylated osteocalcin in postmenopausal osteoporotic women receiving an inhibitor of bone resorption.  Tohoku J Exp Med. 2009;218:201-05.

Atkins GJ, Welldon KJ, Wijenayaka AR, Bonewald LF, Findlay DM.  Vitamin K promotes mineralization, osteoblast-to-osteocyte transition, and on anticatabolic phenotype by γ-carboxylation-dependent and -independent mechanisms.  Am J of Physiology. 2009 Dec;297(6):C1358-67.

Binkley N, Harke J, Krueger D, Engelke J, Vallarta-Ast N, Gemar D, et al. Vitamin K treatment reduces undercarboxylated osteocalcin but does not alter bone turnover, density, or geometry in healthy postmenopausal North American women. J Bone Miner Res. (2009) 24:983–91. 

Dan H, Simsa-Maziel, S, Hisdai, A, Sela-Donenfeld, D, and Monsonego Ornan, E. Expression of matrix metalloproteinases during impairment and recovery of the avian growth plate. J. Anim. Sci. 2009;87, 3544–3555.

Feng X.  Chemical and Biochemical Basis of Cell-Bone Matrix Interaction in Health and Disease.  Curr Chem Biol. 2009 May 1;3(2):189-196.
Bone, a calcified tissue composed of 60% inorganic component (hydroxyapatite), 10% water and 30% organic component (proteins), has three functions: providing mechanical support for locomotion, protecting vital organs, and regulating mineral homeostasis. A lifelong execution of these functions depends on a healthy skeleton, which is maintained by constant bone remodeling in which old bone is removed by the bone-resorbing cell, osteoclasts, and then replaced by new bone formed by the bone-forming cell, osteoblasts. This remodeling process requires a physical interaction of bone with these bone cells. In this review, I will discuss the current understanding of the molecular mechanism underlying the cell-bone interaction.

Feng JQ, Ye L, Schiavi S.  Do osteocytes contribute to phosphate homeostasis?  Curr Opin Nephrol Hypertens. 2009 Jul;18(4:285-91.
Osteocytes, the terminally differentiated cell of the osteoblast lineage, account for over 90% of all bone cells. Due to their relative inaccessibility within mineralized matrix, little is known regarding their specific functions in comparison to the well studied surface bone cells, osteoblasts and osteoclasts. Furthermore, bone is often viewed as a mineral reservoir that passively releases calcium and phosphate in response to hormones secreted from remote organs. Noncollagenous matrix proteins produced in osteocytes, such as dentin matrix protein 1 (DMP1), have also been viewed as inert scaffolds for calcium-phosphate deposition. Recent discoveries of new genetic mutations in human diseases and development of genetically engineered animal models challenge these classic paradigms, suggesting that the osteocyte plays an active role in both mineralization and total systemic phosphate regulation. In this review, we will focus on roles of osteocytes in mineralization.

Huang GTJ, Gronthos S, Shi S. Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources: Their Biology and Role in Regenerative Medicine. Journal of Dental Research. 2009;88:792–806. 

Inoue T, Fujita T, Kishimoto H, Makino T, Nakamura T, Sato T, et al.  Randomized controlled study on the prevention of osteoporotic fractures (OF study):  a phase IV clinical study of 15-mg menatetrenone capsules. J Bone Miner Metab. 2009;27(1):66-75.


Kerachian MA, Séguin C, Harvey EJ.  Glucocorticoids in osteonecrosis of the femoral head:  a new understanding of the mechanisms of action. Steroid Biochem Mol Biol.  2009 Apr;114(3-5):121-8. 

Koitaya N, Ezaki J, Nishimuta M, Yamauchi J, Hashizume E, Morishita K, Miyachi M, Sasaki S, Ishimi Y. Effect of low dose vitamin K2 (MK-4) supplementation on bio-indices in postmenopausal Japanese women. J Nutr Sci Vitaminol (Tokyo). 2009;55:15–21. 
These results suggest that supplementation with 1.5 mg/d MK-4 accelerated the degree of OC gamma-carboxylation. The concentrations of serum lipids and other indices were not different between the groups at either intervention period. Thus, the additional intake of MK-4 might be beneficial in the maintenance of bone health in postmenopausal Japanese women.

Mantesso A, Sharpe P.  Dental stem cells for tooth regeneration and repair.  Expert Opin Biol Ther. 2009 Sep;9(9):1143-54.
Mesenchymal stem cells (MSCs) resident in bone marrow are one of the most studied and clinically important populations of adult stem cells. Cells with, similar properties to these MSCs have been described in several different tooth tissues and the potential ease with which these dental MSCs could be obtained from patients has prompted great interest in these cells as a source of MSCs for cell-based therapeutics. In this review we address the current state of knowledge regarding these cells, their properties, origins, locations, functions and potential uses in tooth tissue engineering and repair. 

Nimptsch K, Rohrmann S, Nieters A, Linseisen J. Serum undercarboxylated osteocalcin as biomarker of vitamin K intake and risk of prostate cancer: a nested case-control study in the Heidelberg cohort of the European prospective investigation into cancer and nutrition. Cancer Epidemiol Biomarkers Prev. 2009;18:49–56.
From cell studies, Vitamin K is known to exert anticancer effects on a variety of cancer cell lines, including prostate cancer cells. Recently, we reported an inverse association between dietary intake of vitamin K2 and risk of prostate cancer. In this nested case-control study including 250 prostate cancer cases and 494 matched controls, we aimed to confirm this cancer-protective effect using serum undercarboxylated osteocalcin (ucOC), a biomarker of vitamin K status inversely associated with vitamin K intake. They found that there were increased risks of advanced-stage and high-grade prostate cancer in folks with higher levels of uncarboxylated osteocalcin, indicating insufficient vitamin K intake.  

Peng L., Ye L., Zhou X.D. Mesenchymal stem cells and tooth engineering. Int. J. Oral. Sci. 2009;1:6–12.

Scheller EL, Krebsbach PH, Kohn DH. Tissue engineering: state of the art in oral rehabilitation. J Oral Rehabil 2009; 36: 368-389.

Sedghizadeh PP, Stanley K, Caligiuri M, Hefkes S, Lowry B, Shuler CF.  Oral bisphosphonate use and the prevalence of osteonecrosis of the jaw.  An institutional inquiry. JADA. 2009 Jan;140:61-66.
Background. Initial reports of osteonecrosis of the jaw (ONJ) secondary to bisphosphonate (BP) therapy indicated that patients receiving BPs orally were at a negligible risk of developing ONJ compared with patients receiving BPs intravenously. The authors conducted a study to address a preliminary finding that ONJ secondary to oral BP therapy with alendronate sodium in a patient population at the University of Southern California was more common than previously suggested. This is the first large institutional study in the United States with respect to the epidemiology of ONJ and oral bisphosphonate use. The findings from this study indicated that even short-term oral use of alendronate led to ONJ in a subset of patients after certain dental procedures were performed. These findings have important therapeutic and preventive implications.

Shiraki M, Itabashi A.  Short-term menatetrenone therapy increases gamma-carboxylation of osteocalcin with a moderate increase of bone turnover in postmenopausal osteoporosis:  a randomized perspective study. J Bone Miner Metab. 2009;27(3):333-40.
The effect of MK4 on bone turnover was investigated in postmenopausal patients with osteoporosis. A 6-month open-label, randomized prospective study was conducted in 109 patients. The control group received calcium aspartate while the MK4 group received 45 mg of menatetrenone daily for 6 months. Significant differences of uncarboxylated and carboxylated osteocalcin between the two groups were observed from 1 month onward. In addition, a higher level of intact osteocalcin was found in the MK4 group compared with the control group after 6 months (P = 0.006). In conclusion, one month of MK4 therapy enhanced the secretion and gamma-carboxylation of osteocalcin, while urinary NTX excretion was increased after 6 months of treatment. 

Yoshida T, Yamaguchi M, Utsunomiya T, Kato M, Arai Y, Kaneda T, et al.  Low-energy laser irradiation accelerates the velocity of tooth movement via stimulation of the alveolar bone remodeling.  Orthod Craniofac Res. 2009 Nov;12(4):289-98.

2010

Azuma K, S. C. Casey SC, Ito M, et al.  Pregnane X receptor knockout mice display osteopenia with reduced bone formation and enhanced bone resorption.  Journal of Endocrinology. 2010;207(3):257–263, 2010.  

Bolland MJ, Avenell A, Baron JA, Grey A, Maclennan GS, Gamble GD, et al.  Effect of calcium supplements on risk of myocardial infarction and cardiovascular events: meta-analysis. BMJ. 2010;341:c3691. 
Calcium supplements are associated with an increased risk of myocardial infarction.  As calcium supplements are widely used these modest increases in risk o cardiovascular disease might translate into a large burden of disease in the population.  A reassessment of the role of calcium supplement sin the management of osteoporosis is warranted.

Chen F-M, Jin Y.  Periodontal tissue engineering and regeneration:  current approaches and expanding opportunities. Tissue Engineering Part B:  Reviews. 2010;16(2):219-55.

Ferron M, Wei J, Yoshizawa T, Del Fattore A, De Pinho RA, Teti A, et al.  Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism.  Cell. 2010 Jul 23;142(2):296-308.
The broad expression of the insulin receptor suggests that the spectrum of insulin function has not been fully described. A cell type expressing this receptor is the osteoblast, a bone-specific cell favoring glucose metabolism through a hormone, osteocalcin, that becomes active once uncarboxylated. We show here that insulin signaling in osteoblasts is necessary for whole-body glucose homeostasis because it increases osteocalcin activity. To achieve this function insulin signaling in osteoblasts takes advantage of the regulation of osteoclastic bone resorption exerted by osteoblasts. Indeed, since bone resorption occurs at a pH acidic enough to decarboxylate proteins, osteoclasts determine the carboxylation status and function of osteocalcin. Accordingly, increasing or decreasing insulin signaling in osteoblasts promotes or hampers glucose metabolism in a bone resorption-dependent manner in mice and humans. Hence, in a feed-forward loop, insulin signals in osteoblasts activate a hormone, osteocalcin, that promotes glucose metabolism.

Hoefert S, Schmitz I, Tannapfel A, Eufinger H. Importance of microcracks in etiology of bisphosphonate-related osteonecrosis of the jaw: a possible pathogenetic model of symptomatic and non-symptomatic osteonecrosis of the jaw based on scanning electron microscopy findings. Clin Oral Investig. 2010;14(3):271-284.

Iwamoto J, Matsumoto H, Takeda T, Sato Y, Uzawa M.  A radiographic study on the associations of age and prevalence of vertebral fractures with abdominal aortic calcification in Japanese postmenopausal women and men.  J Osteoporos. 2010; 2010: 748380.

Iwamoto J, Seki A, Sato Y, Matsumoto H, Tadeda T, Yeh JK.  Vitamin K2 promotes bone healing in a rat femoral osteotomy model with or without glucocorticoid treatment.  Calcif Tissue Int. 2010 Mar;86(3):234-41.
The purpose of the present preclinical study was to determine whether vitamin K(2) would promote bone healing in a rat femoral osteotomy model with or without glucocorticoid (GC) treatment. MK4 at a dose of 30 mg/kg) was administered orally five times a week for eight weeks.  The results suggested that vitamin K(2) downregulated bone turnover and stimulated lamellar bone formation in GC-untreated rats and prevented an increase in bone resorption while maintaining bone formation and prevented a decrease in lamellar bone formation in GC-treated rats. Thus, vitamin K(2) appears to be effective for promoting bone healing in a rat femoral osteotomy model with or without GC treatment.

Khanna-Jain R, Agata H, Vuorinen A, Sándor GK, Suuronen R, Miettinen S.  Addition of BMP-2 or BMP-6 to dexamethasone, ascorbic acid, and β-glycerophosphate may not enhance osteogenic differentiation of human periodontal ligament cells.  Growth Factors. 2010 Dec;28(6):437-46.

Marquezan M, Bolognese AM, Araujo MT. Effects of Two Low-Intensity Laser Therapy Protocols on Experimental Tooth Movement. Photomed Laser Surg. 2010 Dec;28(6):757–62. 

Prasad M, Butler WT, Qin C.  Dentin sialophosphoprotein (DSPP) in biomineralization.  Connect Tissue Res. 2010 Oct;51(5):404-417.
Two of the proteins found in significant quantity in the extracellular matrix (ECM) of dentin are dentin phosphoprotein (DPP) and dentin sialoprotein (DSP). The discoveries in the past 40 years with regard to DPP, DSP and DSPP have greatly enhanced our understanding of biomineralization and set a new stage for future studies. In this review, we summarize the important and new developments made in the past four decades regarding the structure and regulation of the DSPP gene, and the biochemical characteristics of DSPP, DPP.

Reinehr T, Roth CL.  A new link between skeleton, obesity and insulin resistance: relationships between osteocalcin, leptin and insulin resistance in obese children before and after weight loss.  Int J Obes (Long). 2010 May;34(5):852-8. 
The skeleton is regarded recently as an endocrine organ that affects energy metabolism.  We analyzed osteocalcin, adiponectin, leptin and insulin resistance (IR) index homeostasis model assessment (HOMA) in 60 obese and 19 age- and gender-matched normal weight children. Osteocalcin levels were lower in obese children.

Jussila M, Theseff I.  Signaling networks regulating tooth organogenesis and regeneration, and the specification of dental mesenchymal and epithelial cell lineages.  Cold Spring Harb Perspect Biol. 2010 Apr 1;4(4):a008425. 
The dental hard tissues, dentin, enamel, and cementum, are formed by unique cell types whose differentiation is intimately linked with morphogenesis. During evolution the capacity for tooth replacement has been reduced in mammals, whereas teeth have acquired more complex shapes. Mammalian teeth contain stem cells but they may not provide a source for bioengineering of human teeth. Therefore it is likely that nondental cells will have to be reprogrammed for the purpose of clinical tooth regeneration. Obviously this will require understanding of the mechanisms of normal development. The signaling networks mediating the epithelial-mesenchymal interactions during morphogenesis are well characterized but the molecular signatures of the odontogenic tissues remain to be uncovered.

Ruby JD, Cox CF, Momoi Y, et al.  The caries phenomenon: a timeline from witchcraft and superstition to opinions of the 1500s to today’s science.  Int J Dent. 2010;432767. 

Sasaki H, Muramatsu T, Kwon HJ, Yamamoto H, Hashimoto S, Jung HS, et al.  Down-regulated genes in mouse dental papillae and pulp. J Dent Res. 2010 Jul;89(7):679-83.
Important factors involved in odontogenesis in mouse dental papillae disappear between the pre- and post-natal stages of development. Therefore, we hypothesized that certain genes involved in odontogenesis in dental papillae were subject to pre-/post-natal down-regulation. Our goal was to identify, by microarray analysis, which genes were down-regulated. 

Simmer JP, Papagerakis P, Smith CE, Fisher DC, Rountrey AN, Zheng L, Hu JC. Regulation of dental enamel shape and hardness. J Dent Res. 2010;89:1024–1038. 

Suttie JW. Vitamin K. In: Coates PM, Betz JM, Blackman MR, et al., eds. Encyclopedia of Dietary Supplements. 2nd ed. London and New York: Informa Healthcare; 2010:851-60.


Shane E, Burr D, Ebeling PR, Abrahamsen B, Adler RA, Brown TD, et al. American Society for Bone and Mineral Research. Atypical subtrochanteric and diaphyseal femoral fractures: report of a task force of the American Society for Bone and Mineral Research. J Bone Miner Res. 2010;25(11):2267–2297. 

Simon MJ, Niehoff P, Kimmig B, et al. Expression profile and synthesis of different collagen types I, II, III, and V of human gingival fibroblasts, osteoblasts, and SaOS-2 cells after bisphosphonate treatment. Clin Oral Investig. 2010;14(1):51-58.
Bisphosphonates (BP) are used in the treatment of malignant osteolytic processes and postmenopausal osteoporosis. There have been a number of incidents in patients treated with BP. The incidents are described as an osteonecrosis of the jaw (ONJ). The main medications associated with these reports are zoledronic acid (ZOL) and pamidronate (PAM). The clinical presentations describe a deterioration of the jaw bones and surrounding tissues. The purpose of this study was to investigate expression of collagen and they found principally, a decreased production of collagen was measured. With this in vitro study, we demonstrated how negatively influencing a long exposure to ZOL and PAM can be. 

Wysolmerski JJ.  Interactions between breast, bone, and brain regulate mineral and skeletal metabolism during lactation.  Ann N Y Acad Sci. 2010 Mar;1192:161-9.
Mammalian reproduction requires that nursing mothers transfer large amounts of calcium to their offspring through milk. As a result, lactation is associated with dramatic alterations in bone and mineral metabolism, including reversible bone loss. One theme that has emerged from recent studies examining these adaptations is that the lactating breast actively participates in regulating bone and mineral metabolism. This review will detail our current knowledge of interactions between the breast, skeleton, and hypothalamus during lactation and will consider implications that this reproductive physiology has for the pathophysiology of osteoporosis and breast cancer.

Emaus N, Nguyen ND, Almaas B, Bernsten GK, Center JR, Christensen M, et al.  Serum level of under-carboxylated osteocalcin and bone mineral density in early menopausal Norwegian women.  Eur J Nutrl. 2011 Feb;52(1):49-55.  

Feng X, McDonald JM.  Disorders of bone remodeling.  Ann Rev Pathol. 2011;6:121-45. 

Gorski JP.  Biomineralization of bone:  a fresh view of the roles of non-collagenous proteins.  Front Biosci (Landmark Ed). 2011 Jun 1;16:2598-621.  
This review emphasizes the view that secreted non-collagenous proteins in mineralizing bone actively participate in the mineralization process and ultimately control where and how much mineral crystal is deposited, as well as determining the quality and biomechanical properties of the mineralized matrix produced.

Herring S.  2011. Biomechanics of teeth in bone:  Function, movement, and prosthetic rehabilitation.  In: McCauley LK, Somerman MJ. Mineralized Tissues in Oral and Craniofacial Science: Biological Principles and Clinical Correlates.  Hoboken, NJ: Wiley-Blackwell. p. 255-268.

Jahnen-Dechent W, Heiss A, Schafer C, Ketteler M, Towler DA.  Fetuin-A regulation of calcified matrix metabolism. Circ Res.  2011;108(12):1494-1509.
The final step of biomineralization is a chemical precipitation reaction that occurs spontaneously in supersaturated or metastable salt solutions. Genetic programs direct precursor cells into a mineralization-competent state in physiological bone formation (osteogenesis) and in pathological mineralization (ectopic mineralization or calcification). Therefore, all tissues not meant to mineralize must be actively protected against chance precipitation of mineral. Fetuin-A is a liver-derived blood protein that acts as a potent inhibitor of ectopic mineralization and binds small clusters of calcium and phosphate. This interaction results in the formation of prenucleation cluster-laden fetuin-A monomers, calciprotein monomers, and considerably larger aggregates of protein and mineral calciprotein particles. Hence, fetuin-A is a mineral carrier protein and a systemic inhibitor of pathological mineralization complementing local inhibitors that act in a cell-restricted or tissue-restricted fashion. Fetuin-A deficiency is associated with soft tissue calcification in mice and humans.

Neustadt J, Pieczenik S.  Bridging the gap between osteoporosis and osteonecrosis of the jaw: preventing and treating BRONJ with MK4.  Compend Contin Educ Dent. 2011 Oct;32(8):e125-31.
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a significant concern to patients and dentists. Bisphosphonates are used for millions of men and women with osteoporosis, with the rationale being that these medications increase bone mineral density and decrease fracture risk. This article proposes a rational approach for moving BRONJ away from a risk management and quality assurance model that is currently being used by dentists, to a preventive model. The role of collagen as postulated in this article cannot be ignored in the pathophysiology and potential prevention and treatment of osteoporosis and BRONJ. Studies from the medical literature support the safety and efficacy of MK4 as a potential therapeutic agent in preventing and treating osteoporosis and BRONJ. While the approach outlined herein requires additional study, the conceptual framework based on a broad review of the osteonecrosis of the jaw (ONJ) literature provides a means to begin to address this situation in a proactive, rather than reactive, way.

Otto S, Abu-Id MH, Fedele S, et al. Osteoporosis and bisphosphonates-related osteonecrosis of the jaw: not just a sporadic coincidence—a multi-centre study. J Craniomaxillofac Surg. 2011;39(4):272-277.

Ren, YF, Liu X, Fadel N, Malmstrom H, Barnes V, Xu T.  Preventive effects of dentifrice containing 5000ppm fluoride against dental erosion in situ.  J Dent. 2011 Oct;39(10):672-8. 

Southward K.  The systemic theory of dental caries.  Gen Dent. 2011;59(5):367-73.

Yamaguchi M and Weilzmann MN. Vitamin K 2stimulates osteoblastogenesis and suppresses osteoclastogenesis by suppressing NF‐kappa B activation. Int J Mol Med, 2011. 27: 3–14.


Bar-On B, Wagner HD.  Enamel and dentin as multi-scale bio-composites.  J Mech Behav Biomed Mater. 2012 Aug;12:174-83.

Belfrage O, Isaaksson H, Tägil M.  Local treatment of a bone graft by soaking in zoledronic acid inhibits bone resorption and bone formation.  A bone chamber study in rats. BMC Musculoskeletal Disord. 2012 Dec 5;13:240.
In the present study we evaluate different ways of applying bisphosphonates locally to the graft in a bone chamber model, and compare that with systemic treatment.  This study found a strong inhibitory effect on bone resorption by bisphosphonates but also a limited inhibition of the ingrowth of new bone.

Chan O, Sherwin RS.  Hypothalamic Regulation of Glucose-Stimulated Insulin Secretion.  Diabetes. 2012 Mar;61(3):564-65.

Dan H, Simsa-Maziel S, Reich A, Sela-Donenfeld D, Monsonego- Ornan E.  The role of matrix Gla protein in ossification and recovery of the avian growth plate.  Front Endocrinol. 2012;3:79.
Matrix Gla protein (MGP), an inhibitor of mineralization, is expressed by chondrocytes and vascular smooth muscle cells to inhibit calcification of those soft tissues. Tibial dyschondroplasia (TD), a skeletal abnormality can serve as a good model for studying process and genes involved in matrix mineralization and calcification. In this work, we studied the involvement of MGP in the development of TD.  First, we found that during normal bone development, MGP is expressed in specific time and locations, starting from wide-spread expression in the yet un-ossified diaphysis during embryonic development, to specific expression in hypertrophic chondrocytes adjacent to the chondro-osseous junction and the secondary ossification center just prior to calcification. In addition, we show that MGP is not expressed in the impaired TD lesion, however when the lesion begins to heal, it strongly express MGP prior to its calcification. Moreover, we show that when calcification is inhibited, a gap is formed between the expression zones of MGP and BMP2 and that this gap is closed during the healing process. To conclude, we suggest that MGP, directly or through interaction with BMP2, plays a role as ossification regulator that acts prior to ossification, rather then simple inhibitor.

Donnelly E., Meredith D., Nguyen J., Gladnick B., Rebolledo B., Shaffer A., et al. Reduced cortical bone compositional heterogeneity with bisphosphonate treatment in postmenopausal women with intertrochanteric and subtrochanteric fractures. J Bone Miner Res. 2012 27:672–678. 

Fusaro M, Noale M, Viola V, Galli F, Tripepi G, et al.  Vitamin K, vertebral fractures, vascular calcifications, and mortality: Vitamin K Italian (VIKI) dialysis study.  J Bone Min res. 2012;27(11):2231-2416.
Vitamin K (vitamin K1 or phylloquinone and vitamin K2, a series of menaquinones [MKs]) is involved in the production of bone and matrix amino acid γ‐carboxy‐glutamic acid (Gla) proteins, regulating bone and vascular calcification. Low vitamin K concentrations are associated with increased risks of fractures and vascular calcification, and frequent complications in hemodialysis patients. We carried out an observational study to establish the prevalence of vitamin K deficiency and to assess the relationship between vitamin K status, vertebral fractures, vascular calcification, and survival in 387 patients on hemodialysis for ≥1 year. Vitamin K1 deficiency was the strongest predictor of vertebral fractures. MK4 deficiency was a predictor of aortic calcification.  MK7 deficiency was a predictor of iliac calcification. TOur study suggests that the vitamin K system may be important for preserving bone mass and avoiding vascular calcification in hemodialysis patients, pointing out a possible role of vitamin K in bone and vascular health. Based on our results, we suggest that the general population should also be studied for vitamin K deficiency as a possible cause of both vertebral fractures and vascular calcification. 

Hocking LJ, Whitehouse C, Helfrich MH. Autophagy: a new player in skeletal maintenance? J Bone Miner Res. 2012; 27(7):1439-47.

Japanese 2011 guidelines for prevention and treatment of osteoporosis‐ executive summary. Arch Osteporos, 2012. 7: 3–20.

Kumar P, Mastan KMK, Chowdhary R, Shanmugam K.  Oral manifestations in hypertensive patients: A clinical study. J Oral Maxillofac Pathol.  2012 May-Aug;16(2):215-221.

Le Bechek A, Portales-Casmar E et al.  Mir@nt@n: a framework integrating transcription factors, microRNAs and their targets to identify sub-network motifs in a meta-regulatory networkmode.  Bioinformatics. 2012;12:67.  

Lewis JR, Zhu K, Prince RL. Adverse events from calcium supplementation: relationship to errors in myocardial infarction self-reporting in randomized controlled trials of calcium supplementation. Journal of Bone and Mineral Research. 2012;27:719–722. 

Li R, Li X, Shou M, Han N, Zhang Q.  Quantitative determination of matrix gla protein (MGP) and BMP-2 during the osteogenic differentiation of human periodontal ligament cells.  Archives of Oral Biology. 2012 Oct;57 (10):1408-1417.
Matrix Gla protein (MGP) has been recognized as a potent calcification inhibitor and a regulator for bone morphogenetic protein-2 (BMP-2). The periodontal ligament (PDL) is a non-mineralized connective tissue located between two mineralized tissues, the cementum and the alveolar bone. However, the mechanism by which PDL prevents mineralization has yet to be defined. This study aims to examine the expression pattern of MGP and BMP-2 during human periodontal ligament cells (hPDLCs) osteogenic differentiation in vitro, preliminarily exploring their roles in this process. Our results indicate that MGP might regulate hPDLCs osteogenic differentiation which might keep a potential relationship with BMP-2 in this process.

McKee MD, Murshed M, Kaartinen MT. Extracellular matrix and mineralization of craniofacial bone. In: McCauley LK, Somerman MJ, editors. Extracellular matrix and mineralization of craniofacial bone. Ames, IA, USA: Wiley-Blackwell; 2012. pp. 99–109. 

McKee MD, Cole WG.  2012. Bone matrix and mineralization.  In: Francis H. Glorieux, John M Pettifor, Harold Juppner.  Pediatric Bone, Biology & Diseases, 2nd Ed.   Cambridge, MA.  Academic Press. P. 9-37.

Naveh GR, Lev-Tov Chattah N, Zaslansky P, Shahar R, Weiner S. Tooth-PDL-bone complex: Response to compressive loads encountered during mastication - A review. Arch Oral Biol. 2012. 

Neve A, Corrado A, Cantatore FP.  Osteocytes: central conductors of bone biology in normal and pathological conditions.  Acta Physiol 2012;204:317-330.    

Orimo H, Nakamura T, Hosi T, Iki M, Uenishi K Endo N.  Japanese 2011 guidelines for prevention and treatment of osteoporosis – executive summary.  Archives of Osteoporosis. 2012 Dec;7(1-2):3-20. 

Orimo H, Nakamura T, Hosoi T, Iki M, Uenishi K, Endo N, et al.  Japanese 2011 guidelines for prevention and treatment of osteoporosis--executive summary.  Arch Osteoporos. 2012;7:3-20.  
The present guidelines provide information for the managements of primary osteoporosis in postmenopausal women and men over 50 years old, a summary of the evidence for the treatment of secondary osteoporosis, and a summary of the evidence for the prevention of osteoporosis in younger people.  The essential points of the Japanese practice guidelines on osteoporosis were translated into English for the first time. It is hoped that the content of the guidelines becomes known throughout the world.

Patntirapong S, Singhatanadgit W, Chanruangvanit C, Lavanrattanakul K, Satravaha Y. Zoledronic acid suppresses mineralization through direct cytotoxicity and osteoblast differentiation inhibition. J Oral Pathol Med. 2012;41(9):713–20. pmid:22563819.

Qin C, Feng JQ. Dentin. In: McCauley LK, Somerman MJ, editors. Dentin. Ames, IA, USA: Wiley-Blackwell; 2012. pp. 135–141. 

Qin W, Yang F, Deng R et al . Smad 1/5 is involved in bone morphogenetic protein-2-induced odontoblastic differentiation in human dental pulp cells. J Endod 2012;38 (1):66–71.

Ran L et al.  Quantitative determination of matrix Gla protein (MGP) and BMP-2 during the osteogenic differentiation of human periodontal ligament cells.  Archives of Oral Biology. 2012;57(10):1408-1417.
Quantitative determination of matrix Gla protein (MGP) and BMP-2 during the osteogenic differentiation of human periodontal ligament cells, and the results indicate that MGP might regulate hPDLCs osteogenic differentiation which might keep a potential relationship with BMP-2 in this process.

Rheume-Bleue K.  Vitamin K2 and the Calcium Paradox.  John Wiley & Sons, Canada. 2012.

Sato T, Schurgers LJ, Uenishi K.  Comparison of menaquinone-4 and menaquinone-7 bioavailability in healthy women.  Nutr J. 2012 Nov 12;11:93.
To investigate MK-4 and MK-7 bioavailability, nutritional doses were administered to healthy Japanese women.  MK-7 was well absorbed and reached maximal serum level at 6 h after intake and was detected up to 48 h after intake. MK-4 was not detectable in the serum of all subjects at any time point. Consecutive administration of MK-4 (60 μg; 135 nmol) or MK-7 (60 μg; 92 nmol) for 7 days demonstrated that MK-4 supplementation did not increase serum MK-4 levels. However, consecutive administration of MK-7 increased serum MK-7 levels significantly in all subjects.  We conclude that MK-4 present in food does not contribute to the vitamin K status as measured by serum vitamin K levels. MK-7, however significantly increases serum MK-7 levels and therefore may be of particular importance for extrahepatic tissues.

Schwetz V, Pieber T, Obermayer-Pietsch, B.  Mechanisms in endocrinology: the endocrine role of the skeleton: background and clinical evidence.  Eur J Endocrinol, 166 (2012), pp. 959-967.

Shepherd TJ, Dirks W, Manmee C, et al. Reconstructing the life-time lead exposure in children using dentine in deciduous teeth. Sci Total Environ. 2012;425:214–222. 

Theuwissen E, Smit E, Vermeer C. The role of vitamin K in soft-tissue calcification. Adv Nutr. 2012;3(2):166–173. 
Circulating desphospho-uncarboxylated matrix Gla protein was found to be predictive of cardiovascular risk and mortality, whereas circulating total uncarboxylated matrix Gla protein was associated with the extent of prevalent arterial calcification. Vitamin K intervention studies have shown that MGP carboxylation can be increased dose dependently.  This study showed maintenance of vascular elasticity during a 3-y supplementation period, with a parallel 12% loss of elasticity in the placebo group. 

Yang W, Harris MA, Cui Y, Mishina Y, Harris SE, Gluhak-Heinrich J.  Bmp2 is required for odontoblast differentiation and pulp vasculogenesis.  J Dent Res. 2012 Jan;91(1):58-64.
Using the Bmp2 floxed/3.6Col1a1-Cre (Bmp2-cKO(od)) mouse model, we have observed severe defects in odontogenesis and dentin formation with the removal of the Bmp2 gene in early-polarizing odontoblasts. The odontoblasts in the Bmp2-cKO(od) do not mature properly and fail to form proper dentin with normal dentinal tubules and activate terminal differentiation.  There is less dentin, and the dentin is hypomineralized and patchy. The complex roles of Bmp2, postulated to be both direct and indirect, lead to permanent defects in the teeth throughout life, and result in teeth with low quantities of dentin and dentin of poor quality.

Yildirum S.  Isolation methods of dental pulp stem cells.  Dental Pulp Stem Cells. 2012;p.41-51. In Dental Pulp Stem Cells. New York; Springer. Springerbriefs in Stem Cells book series.  
Easy and quick isolation of the most primitive stem cell populations from available tissues is warranted for tissue engineering. The most reliable cell source for dental tissue engineering is that of autologous pulp stem/progenitor cells isolated from deciduous or primary teeth. The ease of isolation and high expansion potential in vitro has made them very attractive as a model system for many researchers.

Washio A, Kitamura C, Morotomi T, Terashita M, Nishihara T.  Possible involvement of smad signaling pathways in induction of odontoblastic properties in KN-3 cells by bone morphogenetic protein-2:  A growth factor to induce dentin regeneration. Int J of Dentistry. 2012;article id 258469.

Weinstein RS.  Glucocorticoid-induced osteoporosis and osteonecrosis.  Endocrinol. Clin North Am. 2012 Sep;41(3):595-611.
Glucocorticoid administration is the most common cause of secondary osteoporosis and the leading cause of nontraumatic osteonecrosis. In patients receiving long-term therapy, glucocorticoids induce fractures in 30% to 50% and osteonecrosis in 9% to 40%. This article reviews glucocorticoid-induced osteoporosis and osteonecrosis, addressing the risk factors, pathogenesis, evaluation, treatment, and uncertainties in the clinical management of these disorders.

Abd-Elmeguid A, Abdelayem M, Kline LW, Mogbel R, Vliagofti H, Yu DC.  Osteocalcin expression in pulp inflammation. J Endod. 2013 Jul;39(7):865-72.  
Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation.  Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment.

Basso FG, Silveira Turrioni AP, Hebling J, de Souza Costa CA. Zoledronic acid inhibits human osteoblast activities. Gerontology. 2013;59(6):534–41. pmid:23867757.
Bisphosphonates are potent inhibitors of bone resorption. These kinds of drugs, which are used for the treatment of osteolytic diseases, have been associated with the occurrence of oral osteonecrosis, especially in patients over 60 years old. Current studies have demonstrated that the cytotoxic effects of bisphosphonates on osteoblasts play an important role in oral osteonecrosis development.  Bisphosphonates are potent inhibitors of bone resorption. These kinds of drugs, which are used for the treatment of osteolytic diseases, have been associated with the occurrence of oral osteonecrosis, especially in patients over 60 years old. Current studies have demonstrated that the cytotoxic effects of bisphosphonates on osteoblasts play an important role in oral osteonecrosis development.

Ji W, Yang F, Ma J, et al. Biomaterials Incorporation of stromal cell-derived factor-1 a in PCL/gelatin electrospun membranes for guided bone regeneration. Biomaterials. 2013;34(3):735–745. 

Kim M, Na W, Sohn C. Vitamin K1 (phylloquinone) and K2 (menaquinone-4) supplementation improves bone formation in a high-fat diet-induced obese mice. J Clin Biochem Nutr. 2013;53:108–113. 
Several reports suggest that obesity is a risk factor for osteoporosis. Vitamin K plays an important role in improving bone metabolism. This study examined the effects of vitamin K1 and vitamin K2 supplementation on the biochemical markers of bone turnover and morphological microstructure of the bones by using an obese mouse model. These findings suggest that vitamin K supplementation reversed the high fat diet induced bone deterioration by modulating osteoblast and osteoclast activities and prevent bone loss in a high-fat diet-induced obese mice.

Knapen MH, Drummen NE, Smit E, Vermeer C, Theuwissen E.  Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women.  Osteoporosis Int. 2013 Sep;24:2499-507.
We have investigated whether low-dose vitamin K2 supplements (menaquinone-7, MK-7) could beneficially affect bone health. Next to an improved vitamin K status, MK-7 supplementation significantly decreased the age-related decline in bone mineral density and bone strength. Our results showed that MK-7 intake significantly improved vitamin K status and decreased the age-related decline in BMC and BMD at the lumbar spine and femoral neck, but not at the total hip. Bone strength was also favorably affected by MK-7. MK-7 significantly decreased the loss in vertebral height of the lower thoracic region at the mid-site of the vertebrae.

McKee MD, Hoac B, Addison WM, Barros NMT, Millan HL, et al.  Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.  Periodontal 2000;2013 Oct;63(1). 
The formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth – where calcium phosphate crystals are deposited and grow within an extracellular matrix – is essential to dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition such that teeth become loose and prone to infection and are lost prematurely. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins (SIBLINGs), and matrix vesicles.

Neve A, Corrado A, Cantatore FP.  Osteocalcin: skeletal and extra-skeletal effects.  J Cell Physiol. 2013 Jun;228(6):1149-53.
Osteocalcin (OC) is a non-collagenous, vitamin K-dependent protein that binds calcium and consequently hydroxyapatite. The dual role of OC in bone can be presumed as follows: firstly, OC acts as a regulator of bone mineralization; secondly, OC regulates osteoblast and osteoclast activity. Recently the metabolic activity of OC, restricted to the un-carboxylated form has been demonstrated in osteoblast-specific knockout mice. This effect is mediated by the regulation of pancreatic β-cell proliferation and insulin secretion and adiponectin production by adipose tissue and leads to the regulation of glucose metabolism and fat mass. This review highlights the recent studies concerning skeletal and extra-skeletal effects of OC.

Ten Cate’s Oral Histology, Nanci, Elsevier, 2013;70-94.

Yang X, Lu Y, Li Z, Wang Y, Zhao F, Han J. Low concentrations of zoledronic acid are better at regulating bone formation and repair. Intractable & rare diseases research. 2013;2(1):18–23. pmid:25343096; PubMed Central PMCID: PMC4204573.

Jiang Y, Zhang ZL, Zhang ZL, Zhu HM, Wu YY, Cheng Q, Wu FL, Xing XP, Liu JL, Yu W, Meng XW. Menatetrenone versus alfacalcidol in the treatment of Chinese postmenopausal women with osteoporosis: a multicenter, randomized, double‐blinded, double dummy, positive drug‐controlled clinical trial. Clin Interv Aging, 2014. 9: 121–127.

Allen MR, Burr DB.  2014. Bone modeling and remodeling.  In: Allen MR, Burr DB. Basic and Applied Bone Biology, Chapter 4.  Elsevier: Oxford, UK. P. 75-90.

Gallagher JC,Smith LM, Yalamanchili V. Incidence of hypercalciuria and hypercalcemia during vitamin D and calcium supplementation in older women. Menopause.  2014;21:1173–1180. 

Gröber U, Reichrath J, Holick MJ, Kisters K.  Vitamin K: an old vitamin in a new perspective, Dermato-Endocrinology. 2014;6:1.
Current research increasingly indicates that vitamin K has a considerable benefit in the prevention and treatment of bone and vascular disease. Vitamin K1 (phylloquinone) is more abundant in foods but less bioactive than the vitamin K2 menaquinones (especially MK-7.  Vitamin K-dependent (VKD) proteins support calcium homeostasis, inhibit vessel wall calcification, support endothelial integrity, facilitate bone mineralization, are involved in tissue renewal and cell growth control, and have numerous other effects. The following review describes the history of vitamin K, the physiological significance of the K vitamers, updates skeletal and cardiovascular benefits and important interactions with drugs.

Hara AT, Zero DT.  The potential of saliva in protecting against dental erosion. Monogr Oral Sci. 2014;25:197-205.
Saliva is the most relevant biological factor for the prevention of dental erosion. It starts acting even before the acid attack, with an increase of the salivary flow rate as a response to the acidic stimuli. This creates a more favorable scenario, improving the buffering system of saliva and effectively diluting and clearing acids that come in contact with dental surfaces during the erosive challenge. Saliva plays a role in the formation of the acquired dental pellicle, a perm-selective membrane that prevents the contact of the acid with the tooth surfaces. Due to its mineral content, saliva can prevent demineralization as well as enhance remineralization. These protective properties may become more evident in hyposalivatory patients. Finally, saliva may also represent the biological expression of an individual's risk for developing erosive lesions; therefore, some of the saliva components as well as of the acquired dental pellicle can serve as potential biomarkers for dental erosion.

Hannig, M. & Hannig, C. The pellicle and erosion. Monographs in Oral Science. 2014;25:206–214.  
All tooth surfaces exposed to the oral environment are naturally coated by the acquired salivary pellicle. The pellicle is composed of adsorbed macromolecular components from saliva, gingival crevicular fluid, blood, bacteria, mucosa and diet. The pellicle (formed in situ/in vivo) functions as a semipermeable network of adsorbed salivary macromolecules and provides partial protection against acidic challenges; however, it cannot completely prevent demineralization of the tooth surface. The physiological pellicle reduces calcium and phosphate release from the enamel, and much less from the dentinal surface. Improvement of the pellicle's protective properties by dietary components (e.g. polyphenolic agents) might be a promising erosion-preventive approach that, however, needs validation by in situ experiments.

Ionta FQ, Mendonca FL, de Oliviera GC, de Alencar CR, Honorio HM, et al. In vitro assessment of artificial saliva formulations on initial enamel erosion remineralization. Journal of Dentistry. 2014;42:175–179.   

Iwamoto J.  Vitamin K2 therapy for postmenopausal osteoporosis.  Nutrients. 2014 May 16;6(5):1971-80.
Vitamin K may play an important role in the prevention of fractures in postmenopausal women with osteoporosis. Menatetrenone is the brand name of a synthetic vitamin K2 that is chemically identical to menaquinone-4. The present review study aimed to clarify the effect of menatetrenone on the skeleton in postmenopausal women with osteoporosis, by reviewing the results of randomized controlled trials (RCTs) in the literature. RCTs that investigated the effect of menatetrenone on bone mineral density (BMD).  Eight studies met the criteria for RCTs. Small RCTs showed that menatetrenone monotherapy decreased serum undercarboxylated osteocalcin (ucOC) concentrations, modestly increased lumbar spine BMD, and reduced the incidence of fractures (mainly vertebral fracture), and that combined alendronate and menatetrenone therapy enhanced the decrease in serum ucOC concentrations and further increased femoral neck BMD. This review of the literature revealed positive evidence for the effects of menatetrenone monotherapy on fracture incidence in postmenopausal women with osteoporosis. 

Koitaya N, Sekiguchi M, Tousen Y, Nishide Y, Morita A, Yamauchi J, Gando Y, Miyachi M, Aoki M, Komatsu M, et al. Low-dose vitamin K2 (MK-4) supplementation for 12 months improves bone metabolism and prevents forearm bone loss in postmenopausal Japanese women. J Bone Miner Metab. 2014;32:142–150. 

Koromila T, Baniwai SK, Song YS, Martin A, Ziong J, Frenkel B.  Glucocorticoids antagonize RUNX2 during osteoblast differentiation in cultures of ST2 pluripotent mesenchymal cells.  J Cell Biochem. 2014 Jan;115(1):27-33.

Kwang S, Abbott P. The presence and distribution of bacteria in dentinal tubules of root filled teeth. Int Endod J. 2014;47(6):600–610. 

Shane E, Burr D, Abrahamsen B, Adler RA, Brown TD, Cheung AM, et al.  Atypical subtrochanteric and diaphyseal femoral fractures: second report of a task force of the American Society for Bone and Mineral Research.  J Bone Miner Res. 2014 Jan;29(1):1-23.
Bisphosphonates (BPs) and denosumab reduce the risk of spine and nonspine fractures. Studies with radiographic review consistently report significant associations between AFFs and BP use, although the strength of associations and magnitude of effect vary.  However, long-term use may be associated with higher risk (∼100 per 100,000 person- years). BPs localize in areas that are developing stress fractures; suppression of targeted intracortical remodeling at the site of an AFF could impair the processes by which stress fractures normally heal. When BPs are stopped, risk of an AFF may decline. 

Sims NA, Martin TJ. Coupling the activities of bone formation and resorption: a multitude of signals within the basic multicellular unit. Bonekey Rep. 2014;3:1–10. 

Tai N, Inoue .  Anti=Dickkopfl (Dkk1) antibody as a bone anabolic agent for the treatment of osteoporosis. Clin Calcium.  2014 Jan;24(1):75-83.  

Weaver CM, Gallant KMH.  2014. Nutrition. In: Basic and applied bone biology.  Cambridge, MA: Academic Press. P. 283-297. 

Arzate Hi, Zeichner-David M, Mercado-Celis G.  Cementum proteins: role in cementogenesis, biomineralization, periodontium formation and regeneration.  Periodontology 2000. 2015;67(1):211-233.
Destruction of the periodontium is normally associated with periodontal disease, although many other factors, such as trauma, aging, infections, orthodontic tooth movement and systemic and genetic diseases, can contribute to this process. Strategies (such as guided tissue regeneration) have been developed to guide and control regeneration using bioresorbable membranes and bone grafts. To achieve complete repair and regeneration it is necessary to recapitulate the developmental process with complete formation of cementum, bone and periodontal ligament fibers. Detailed knowledge of the biology of cementum is key for understanding how the periodontium functions.  

Balli U, Keles ZP, Avci B, Guler S, Cetinkaya BO, Keles GC.  Assessment of periostin levels in serum and gingival crevicular fluid of patients with periodontal disease.  J Periodontal Res.  2015;50:707-713.

Cobo T, Obaya A, Cal S, Solares L, Cabo R, Vega JA, et al.  Immunohistochemical localization of periostin in human gingiva.  Eur J Histochem.  2015;59:2548.

Suttie JW. Vitamin K. In: Ross AC, Caballero B, Cousins RJ, Tucker KL, Ziegler TR, eds. Modern Nutrition in Health and Disease. 11th ed. Baltimore, MD: Lippincott Williams & Wilkins; 2014:305-16.

Davis SR, Lambrinoudaki I, Lumsden M, Mishra GD, Pal L, Rees M, et al. Menopause. Nat Rev Dis Prim. 2015;1:15004. 

Holick MF, Nieves JW. 2015. Preface.  In: Holick MF, Nieves JW. Nutrition and bone health.  New York: Springer. P. 1-2.  

Huang ZB, Wan SL, Lu YJ, Ning L, Liu C, Fan SW.  Does vitamin K2 play a role in the prevention and treatment of osteoporosis for postmenopausal women:  a meta-analysis of randomized controlled trials. Osteoporos Intl. 2015 Mar;26(3):1175-86. 
We conducted this meta-analysis of 19 randomized controlled trials to identify the role of vitamin K2 for the prevention and treatment of osteoporosis.  Our results showed that vitamin K2 might play a role in maintaining the bone mineral density and in reducing the incidence of fractures for postmenopausal women with osteoporosis.  This meta-analysis seemed to support the hypothesis that vitamin K2 plays a role in the maintenance and improvement of vertebral BMD and the prevention of fractures in postmenopausal women with osteoporosis. The reduction of undercarboxylated osteocalcin and increment of osteocalcin may have some relation to the process of bone mineralization. 

Katsuyama H, Fushimi S, Yamane K, Watanabe Y, Shimoya K, Okuyama T, et al. Effect of vitamin K2 on the development of stress-induced osteopenia in a growing senescence-accelerated mouse prone 6 strain. Exp Ther Med. 2015;10(3):843–50. 
Vitamin K2 (VK2) has been used as a therapeutic agent for osteoporosis, since it has been suggested to be able to reduce the frequency of fractures by improving bone quality.  In the present study, the effect of menaquinone-4 (MK-4) on bone turnover was investigated MK-4 (30 mg/kg) was injected subcutaneously 3 times a week for 4 weeks. Specifically, the Trabecular bone reduction caused by the activation of osteoclasts (Ocs), and Oc activity was suppressed by MK-4. These results indicate that MK-4 can induce recovery from the bone mineral loss caused by WRS treatment. Further studies are required to clarify the association between bone quality and MK-4.

Lombardi G, Perego S, Luzi L, Banfi G.  A four-season molecule: Osteocalcin – Updates in its physiological roles.  Endocrine. 2015 Mar;48(2):394-404.  
Aim of this review is to give a full overview of the physiological roles of OC by collecting the newest experimental findings on this intriguing molecule.  Osteocalcin (OC) is the main non-collagenous hydroxyapatite-binding protein synthesized by osteoblasts, odontoblasts, and hypertrophic chondrocytes. It has a regulatory role in mineralization and it is considered a marker of bone cell metabolism. Recent findings evidenced new extra-skeletal roles for OC, depicting it as a real hormone. OC exists in different forms based on the degree of carboxylation. Indeed, OC has three glutamic acid residues, in position 17, 21, and 24, which are subject to γ-carboxylation. The degree of carboxylation, and thus the negative charge density, determines the affinity for the calcium ions deposited in the extracellular matrix of the bone. The modulation of the carboxylation could, thus, represent the mechanism by which the body controls the circulating levels, and hence the hormonal function, of OC. There are evidences linking OC, and the bone metabolism, with a series of endocrine (glucose metabolism, energy metabolism, fertility) physiological (muscle activity) and pathological functions (ectopic calcification). 

Ma Y, Ji Y, Huang G, Ling K, Zhang X, Xu F. Bioprinting 3D cell-laden hydrogel microarray for screening human periodontal ligament stem cell response to extracellular matrix. Biofabrication 2015; 7: 044105.

Nagura N, Komatsu J, Iwase H, Hosoda H, Ohbayashi O, Nagaoka I, et al.  Effects of the combination of vitamin K and teriparatide on the bone metabolism in ovariectomized rats.  Biomed Rep. 2015 May:3(3):295-300.  
The purpose of the present study was to evaluate the combined effects of vitamin K (VK) and teriparatide (TPTD) on bone mineral density (BMD), mechanical strength and other parameters for bone metabolism using a rat ovariectomized osteoporosis model. Ovariectomized female Sprague-Dawley rats were administered with VK (an oral dose of 30 mg/kg/day), TPTD (a subcutaneous dose of 30 µg/kg, three times a week) or a combination for 8 weeks. The combination of VK and TPTD clearly increased the serum levels of Gla-OC (a specific marker for bone formation) and osteoblast surface (the number of osteoblasts attaching with the surface of cancellous bone), compared to VK or TPTD alone. Taken together, these findings suggest that the treatment with VK and TPTD may have a therapeutic advantage over VK or TPTD monotherapy for postmenopausal osteoporosis, possibly by enhancing the bone formation through the actions on OC and osteoblasts.

Poon CC, Li RW, Seto SW et al. In vitro vitamin K(2) and 1alpha,25-dihydroxyvitamin D(3) combination enhances osteoblasts anabolism of diabetic mice. European Journal of Pharmacology.  2015;767:30–40. 

Sanderson M, Sadie-Van Gijsen H, Hough S, Ferris WF. The role of MKP-1 in the anti-proliferative effects of glucocorticoids in primary rat pre-osteoblasts. PLoS One. 2015;10:e0135358. doi: 10.1371/journal.pone.0135358. 

Scaramucci T, Carvalho JC, Hara AT, Zero DT. Causes of Dental Erosion: Extrinsic Factors. Berlin: Springer International Publishing; 2015. pp. 69–96.

Shi C, Huang P, Kang H, Hu B, Qi J, Jiang M, Zhou H, Guo L, Deng L. Glucocorticoid inhibits cell proliferation in differentiating osteoblasts by microRNA-199a targeting of WNT signaling. J Mol Endocrinol. 2015;54:325–337. 

Shiraki M. Health benefits and demerits of calcium nutrition or supplementation in older people. Nihon Rinsho. 2015;73:1770–6. 

Southward K.  A hypothetical role for vitamin K2 in the endocrine and exocrine aspects of dental caries.  Med. Hypotheses. 2015 Mar:84(3):276-80.
There is growing awareness of oral/systemic links, especially with regard to periodontal disease, diabetes and cardiovascular disease, among others.  The process of dental caries has similar links. Bacterial and other acids in the oral environment can erode enamel and potentially initiate an inflammatory response in the dentin.  Oxidative stress emerges. The healthy tooth is nourished by a centrifugal dentinal fluid flow. This flow is controlled by signals from the hypothalamus that are relayed to the endocrine portion of the parotid gland. A systemic understanding of the actual and progression of dental caries creates opportunities for more effective approaches to preventive care.

Wu WJ, Kim MS, Ahn BY. The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption. Food Funct. 2015;6:3351-3358.

Yang J, Ye L, Hui T-Q, Yang D-M, Huang D-M, Zhou Z-D, et al.  Bone morphogenetic protein 2-induced human dental pulp cell differentiation involves p38 mitogen-activated protein kinase-activated canonical WNT pathway.  Int J Oral Science. 2015;7:95-102.

Abou NE, Aljabo A, Strange A, Ibrahim S, Coathup M, Young A, et al.  Demineralization – remineralization dynamics in teeth and bone. Int J Nanomed.  2016 Sep;11:4743-4763.
Biomineralization is a dynamic, complex, lifelong process by which living organisms control precipitations of inorganic nanocrystals within organic matrices to form unique hybrid biological tissues, for example, enamel, dentin, cementum, and bone. Understanding the process of mineral deposition is important for the development of treatments for mineralization-related diseases and also for the innovation and development of scaffolds. This review provides a thorough overview of the up-to-date information on the theories describing the possible mechanisms and the factors implicated as agonists and antagonists of mineralization. The mechanisms by which demineralization–remineralization process occurs in both teeth and bone and the new therapies/technologies that reverse demineralization or boost remineralization are also scrupulously discussed. 

AtaoÄŸlu B, Kaptan AY, Eren TK, Yapar AE, Berkay AF.  Ayptical femoral fracture following zoledronic acid treatment.  Joint Diseases and Related Surgery. 2016;27(1):54-57.
A 68-year-old female patient admitted to our clinic with right anterior thigh pain ongoing for six months and which increased in last two months.  The patients had no trauma history. The patient had been followed-up for 15 years because of osteoporosis and administrated alendronate and ibandronate treatment for 10 years.  Patient had three shots of zoledronate once a year during the last three years. Her pain was increasing when she was walking. Physical examination revealed pain in her right thigh and and MRI showed atypical fracture.  Zoledronate treatment was ended. Prophylactic surgical fixation was performed with titanium elastic nails.

Chapurlat RD, Confavreaux CB.  Novel biological markers of bone:  from bone metabolism to bone physiology.  Rheumatology (Oxford). 2016 Oct;55(10):1714-25.
Biochemical markers of bone turnover have been used for decades in the management of bone diseases, to assess the prognosis of these conditions and to monitor treatments. The new markers, however, also reflect specific physiological mechanisms in the bone or other organs. Some of the bone markers are in fact hormones produced by the bone that affect various physiological and pathological functions in other organs. Thus, osteocalcin is produced by osteoblasts and participates in the regulation of insulin sensitivity and fertility in men. 

Choi H, Ahn YH, Kim TH, Bae CH, Lee JC, You HK, et al.  TGF-β Signaling Regulates Cementum Formation through Osterix Expression.  Sci Rep. 2016 May;16(6):26046.

Dai Y, Hu S. Recent insights into the role of autophagy in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford). 2016; 55(3):403-10.

DoÄŸan GE, Demir T, LaloÄŸlu E, SaÄŸlam E, OÄŸan GE, Aksoy H, Yildirim A, et al.  Patients with dental calculus have increased saliva and gingival crevicular fluid fetuin-A levels but no association with fetuin-A polymorphisms.  Brazilian Oral Res. 2016;30(1): http://dx.doi.org/10.1590/1807-3107bor-2016.vol30.0129 
Fetuin-A is a potent inhibitor of calcium-phosphate precipitation and of the calcification process, therefore it can also be related with dental calculus. Thus, we aimed to investigate a possible relationship between fetuin-A gene polymorphism and the presence of dental calculus. A possible relationship between serum, saliva and gingival crevicular fluid (GCF) levels of fetuin-A was also investigated. Fetuin-A polymorphisms were investigated in 103 patients with or without dental calculus. However, higher GCF and saliva fetuin-A levels were detected in patients with dental calculus than in patients without dental calculus, which may result from an adaptive mechanism to inhibit mineral precipitation and eventually calculus formation.

Katagiri T, Watabe T.  Bone Morphogenetic Proteins.  Cold Spring Harb Perspect Biol.  2016 Jun 1;8(6): doi: 10.1101/cshperspect.a021899.  
Bone morphogenetic proteins (BMPs) are now known to play important roles in a wide array of processes during formation and maintenance of various organs including bone, cartilage, muscle, kidney, and blood vessels. BMPs and the related “growth and differentiation factors” (GDFs) are members of the transforming growth factor b (TGF-b) family. Because deregulation of the BMP activity at multiple steps in signal transduction is linked to a wide variety of human diseases, therapeutic use of activators and inhibitors of BMP signaling, such as matrix Gla protein, will provide potential avenues for the treatment of the human disorders.  

Okano T.  A new horizon in vitamin K research.  Yakugaku Zasshi. 2016;136(8):1141-59. 
Vitamin K is a cofactor for a variety of vitamin K-dependent proteins (VKDPs) involved in blood coagulation, bone and cartilage metabolism, signal transduction, and cell proliferation. Despite the great advances in the genetic, structural, and functional studies of VKDPs, little is known of the identity and roles of key regulators of vitamin K metabolism in mammals and humans. This review focuses on new insights into the molecular mechanisms underlying the intestinal absorption and in vivo tissue conversion of vitamin K1 to menaquinone-4 (MK-4).  

Olejnik C, Falgayrac G, During A, Cortet B, Penel G.  Doses effect of zoledronic acid on mineral apatite and collagen quality of newly-formed bone in the rat’s calvaria defect.  Bone. 2016;89:32-39.
Due to their inhibitory effects on resorption, bisphosphonates are widely used in the treatment of diseases associated to an extensive bone loss. Yet, little is known about bisphosphonates effects on newly-formed bone quality. In the present study, adult male Sprague-Dawley rats (n = 80) with a bone defect calvaria area were used and short-term effects of zoledronic acid (ZA) were studied on the healing bone area.  After ZA administration, the intrinsic bone material properties were evaluated. ZA at high doses disrupted the apatite crystal organization, suggesting that ZA may affect the early collagen organization during the bone healing.

Rathore B, Singh M, Kumar V, Misra A.  Osteocalcin: an emerging biomarker for bone turnover.  Int J Res Med Sci. 2016 Sep;4(9):3670-74.  
Osteocalcin (OC) is produced by osteoblasts during bone formation. Recent bone biology research have highlighted the importance of bone not only as a structural scaffold to support the human body, but also as a regulator of a metabolic processes that are independent of mineral metabolism. In this review, we have tried to explain different roles of osteocalcin, however further studies are required to elucidate the metabolic and hormonal role of OC in human body. 

Vanishree T, Panchmal GS, Shenoy RP, Jodalli P, Sonde L.  Caries Prevention: Vitamin Way - A Novel Approach. Int J Health Sci & Res.  2016 Jan;6(1):484-488. 
Nutrients play an important regulatory role in preserving health of the human body and of all metabolically active tissues. Micronutrients, vitamins and antioxidants play an essential role for constant regenerative processes, for coping with oxidative stress, and also for adequate immune responses. Research shows that vitamin K2 and vitamin D together result in a far greater reduction of tooth decay than does either vitamin alone. Sound nutritional habits and a sufficient supply of essential vitamins and minerals are of considerable importance for oral health. 

Zhang YL, Yin JH, Ding H, Zhang W, Zhang CQ, Gao YS.  Vitamin K2 prevents Glucocorticoid-induced osteonecrosis of the femoral head in rats.  Int J Biol Sci. 2016 Jan 28;12(4):347-58.  
Glucocorticoid medication is one of the most common causes of atraumatic osteonecrosis of the femoral head (ONFH), and vitamin K2 (VK2) has been shown to play an important and beneficial role in bone metabolism. In this study, we hypothesized that VK2 could decrease the incidence of glucocorticoid-induced ONFH in a rat model. Using in vitro studies, we demonstrated that VK2 yielded beneficial effects for subchondral bone trabecula. In conclusion, VK2 is an effective antagonist for glucocorticoid on osteogenic progenitors. The underlying mechanisms include acceleration of BMSC propagation and promotion of bone formation-associated protein expression, which combine and contribute to the prevention of glucocorticoid-induced ONFH in rats.

Zoch ML, Clemens TL, Riddle RC. New insights into the biology of osteocalcin. Bone (2016) 82:42–9.
Osteocalcin is among the most abundant proteins in bone and is produced exclusively by osteoblasts. Initially believed to be an inhibitor of bone mineralization, recent studies suggest a broader role for osteocalcin that extends to the regulation of whole body metabolism, reproduction, and cognition. Circulating undercarboxylated osteocalcin, which is regulated by insulin, acts in a feed-forward loop to increase β-cell proliferation as well as insulin production and secretion, while skeletal muscle and adipose tissue respond to osteocalcin by increasing their sensitivity to insulin. Osteocalcin also acts in the brain to increase neurotransmitter production and in the testes to stimulate testosterone production. In this review, we summarize these new discoveries, which suggest that the ability of osteocalcin to function both locally in bone and as a hormone depends on a novel post-translational mechanism that alters osteocalcin's affinity for the bone matrix and bioavailability. 

Gordeladze JA, Landin MA, Johnsen GF, Haugen HJ, Osmundsen H.  Vitamin K2 and its impact on tooth epigenetics. Published March 22, 2017.   Open access, peer-reviewed chapter.

Kuroshima S, Kaku M, Ishimoto T, Sasaki M, Nakano T, Sawase T, et al.  A paradigm shift for bone quality in dentistry: A literature review. J Prosthodont Res.  2017 Oct;61(4):353-62.
The aim of this study was to present the current concept of bone quality based on the proposal by the National Institutes of Health (NIH) and some of the cellular and molecular factors that affect bone quality, including collagen, apatite and osteoblasts and osteocytes.  In dentistry, the term "bone quality" has long been considered to be synonymous with bone mineral density (BMD) based on radiographic and sensible evaluations. In 2000, the NIH defines bone quality as comprising bone architecture, bone turnover, bone mineralization, and micro-damage accumulation. Moreover, our investigations have demonstrated that BAp, collagen, and bone cells such as osteoblasts and osteocytes play essential roles in controlling the current concept of bone quality in bone around hip and dental implants. Understanding the current concept of bone quality is required in dentistry.

Lacruz RS, Habelitz S, Wright JT, Paine ML.  Dental enamel formation and implications for oral health and disease.  Physiol Rev. 2017 Jul1;97(3):939-993.  
Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

Myneni VD, Mezey E.  Regulation of bone remodeling by vitamin K2.  Oral Dis. 2017 Nov;23(8):1021-28.
All living tissues require essential nutrients such as amino acids, fatty acids, carbohydrates, minerals, vitamins, and water. The skeleton requires nutrients for development, maintaining bone mass and density. In recent years, there has been growing interest in promotion of bone health and inhibition of vascular calcification by vitamin K2. This vitamin regulates bone remodeling, an important process necessary to maintain adult bone. Bone remodeling involves removal of old or damaged bone by osteoclasts and its replacement by new bone formed by osteoblasts. The remodeling process is tightly regulated, when the balance between bone resorption and bone formation shifts to a net bone loss results in the development of osteoporosis in both men and women. In this review, we focus on our current understanding of the effects of vitamin K2 on bone cells and its role in prevention and treatment of osteoporosis.

O’Connor EB, Durack E.  Osteocalcin: The extra-skeletal role of a vitamin K-dependent protein in glucose metabolism.  J of Nutri & Intermediary Metabolism. 2017 Mar;7:8-13.
The role of vitamin K in the body has long been associated with blood clotting and coagulation.  In more recent times, its role in a range of physiological processes has been described including the regulation of bone and soft tissue calcification, cell growth and proliferation, cognition, inflammation, various oxidative processes and fertility, where osteocalcin is thought to up-regulate the synthesis of the enzymes needed for the biosynthesis of testosterone thereby increasing male fertility. Vitamin K dependent proteins (VKDP) contain y-carboxyglutamic acid which require post-translational, gamma-glutamyl carboxylation by the vitamin K-dependent (VKD) gamma-glutamyl carboxylase enzyme for full functionality. These proteins are present both hepatically and extrahepatically. The role of bone-derived osteocalcin has many physiological roles including, maintenance of bone mass with more recent links to energy metabolism due to the role of the skeleton as an endocrine organ. This review will discuss the role of osteocalcin in relation to its role in human health, focusing specifically on glucose metabolism.  

Paldanius PM.  The role of osteocalcin in human bone metabolism and glucose homeostasis.  Helsinki:2017.

Rasouli-Ghahroudi AA, Akbari S, Najafi-Alishah M, Boholi M.  The effect of vitamin K2 on osteogenic differentiation of dental pulp stem cells:  An in vitro study. Regeneration, Reconstruction & Restoration. 2017;2(1):
Dental pulp stem cells (DPSCs) have been shown to have great capacity to differentiation toward the osteoblast lineage and they can be considered as a great cell source for bone tissue engineering. The vitamin K family, especially vitamin K2 (MK-4), have been shown to have an osteoprotective role.  In this study, we have investigated the effect of various concentrations of MK-4 on differentiation of DPSCs into osteoblast.  The addition of MK-4 at concentration of 10 µM with osteogenic medium had a significant effect on differentiation DPSCs into osteoblast (P<0.05) at 14 day.  MK-4 can promote differentiation of DPSCs into osteoblast in vitro so have a potential to be considered in improvement of cell-based bone tissue engineering therapies. 

Schwalfenberg GK. Vitamins K1 and K2: The Emerging Group of Vitamins Required for Human Health. J Nutr Metab. 2017;2017:6254836. 

Shoji-Matsunaga A, Ono T, Hayashi M, Takayanagi H, Moriyama K, Nakashima T.  Osteocyte regulation of orthodontic force-mediated tooth movement via RANKL expression.  Sci Rep. 2017 Aug 18;7(1):8753.


Ten Cates Oral Histology.  Development, Structure, and Function. 9th Edition.  Editor Antonio Nanci.  By Elsevier, published August 15, 2017.

Wang R, Dang P, Niu Z.  Expression of the matrix gla protein (MGP) gene in different developmental stages of mouse mandibular first molar root.  Biom Res. 2017;28(10):4369-4374.
This study examined the expression of the MGP gene in mouse germ tissues obtained by laser microdissection at different times during development (pre-natal and post-natal). These data show that the MGP gene expression decreased after birth, indicating a role of the MGP gene in development of the tooth root.

Zhu M, Ma J, Lu S, Zhu Y, Cui Y, Tan H, et al. Vitamin K2 analog menaquinone-7 shows osteoblastic bone formation activity in vitro.  Biomedical Research. 2017;28(3):1364-69.
An increasing number of studies have shown that vitamin K may play a critical role in metabolism of bone. The present study investigates the effect of MK-7 on osteoblastic bone formation in femoral-diaphyseal and -metaphyseal tissues of 4-week-old rats (n=18). The study results indicate that MK-7 has a stimulatory effect on bone tissue and osteoblastic SAOS-2 cells in vitro and further clinical studies are required to pave way for the use of MK-7 in bone formation.

Akbari S, Rasouli-Ghahroudi AA.  Vitamin K and Bone Metabolism: A Review of the Latest Evidence in Preclinical Studies.  Biomed Res Int. 2018 Jun 27;2018: 2018:4629383. doi: 10.1155/2018/4629383. eCollection 2018.
Bone is a metabolically active tissue that renews itself throughout one's life. Accordingly, vitamin K as a multifunctional vitamin has been recently deemed important as a topic of research as it plays a pivotal role in maintenance of the bone strength, and it has been proved to have a positive impact on the bone metabolism. Vitamin K exerts its anabolic effect on the bone turnover in different ways such as promoting osteoblast differentiation, upregulating transcription of specific genes in osteoblasts, and activating the bone-associated vitamin k dependent proteins which play critical roles in extracellular bone matrix mineralization. There is also credible evidence to support the effects of vitamin k2 on differentiation of other mesenchymal stem cells into osteoblast. The evidence could shed light on further clinical studies to improve osteogenesis in bone graft surgeries.

Ishida Y, Kawai S. Comparative efficacy of hormone replacement therapy, etidronate, calcitonin, alfacalcidol, and vitamin K in postmenopausal women with osteoporosis: the yamaguchi osteoporosis prevention study. Am J Med. (2018) 117:549–55. 

Li Y, Jacox LA, Little SH, Ko C-C.  Orthodontic tooth movement: the biology and clinical implications.  The Kaosiumg J of Med Sci. 2018 Apr;34(4):207-214.

Ni S-L, Zhang J, Liu A, Li X-W, Sun Y-J, Zhang X, et al.  Effects of human bone morphogenetic protein 2 (hBMP2) on tertiary dentin formation.  Am J Transl Res. 2018;10(9):2868-76. 

Malik M, Alexiou B, Hallgrimsson.  Bone Morphogenetic Protein 2 Coordinates Early Tooth Mineralization.  J Dent Res. 2018 Jul;97(7):835-43. 
Formation of highly organized dental hard tissues is a complex process involving sequential and ordered deposition of an extracellular scaffold, followed by its mineralization. Similar to early tooth development, various Bmps are expressed during this process.  Here, we investigated the role of odontoblast-derived Bmp2 for tooth mineralization using Bmp2 conditional knockout mice. They established that Bmp2 provides an early temporal, nonredundant signal for directed and organized tooth mineralization.

Poso M, Claret M.  Hypothalamaic control of systemic glucose homeostasis:  the pancreas connection. Trends Endocrinol Metab. 2018 Aug 1:29(8):581-94.
Maintenance of glucose homeostasis is mandatory for organismal survival. It is accomplished by complex and coordinated interplay between glucose detection mechanisms and multiple effector systems. The brain, in particular homeostatic regions such as the hypothalamus, plays a crucial role in orchestrating such a highly integral response. We review here current understanding of how the hypothalamus senses glucose availability and participates in systemic glucose homeostasis. 

Simon P, Gruner D, Worch H, Pompe W, Lichte H, et al.  First evidence of ostacalcium phosphate@osteocalcin nanocomplex as skeletal bone component directing collagen triple-helix nanofibril mineralization.  Scientific Reports. Open access. Published: 12 Sept 2018.

Wen L, Chen J, Duan L, Li S.  Vitamin K-dependent proteins involved in bone and cardiovascular health.  Mol Med Rep. 2018 Jul:18(1):3-15.
In postmenopausal women and elderly men, bone density decreases with age and vascular calcification is aggravated. This condition is closely associated with vitamin K2 deficiency. A total of 17 different vitamin K-dependent proteins have been identified to date. Vitamin K-dependent proteins are located within the bone, heart and blood vessels. For instance, carboxylated osteocalcin is beneficial for bone and aids the deposition of calcium into the bone matrix. Carboxylated matrix Gla protein effectively protects blood vessels and may prevent calcification within the vascular wall. Furthermore, carboxylated Gla-rich protein has been reported to act as an inhibitor in the calcification of the cardiovascular system, while growth arrest-specific protein-6 protects endothelial cells and vascular smooth muscle cells, resists apoptosis and inhibits the calcification of blood vessels by inhibiting the apoptosis of vascular smooth muscle cells. These vitamin K-dependent proteins may exert their functions following γ-carboxylation with vitamin K, and different vitamin K-dependent proteins may exhibit synergistic effects or antagonistic effects on each other. This review describes and briefly discusses several important vitamin K-dependent proteins that serve an important role in bone and the cardiovascular system. The results of the review suggest that the vascular calcification and osteogenic differentiation of vascular smooth muscle cells may be associated with the location of the bone and cardiovascular system during embryonic development.

Zhao B, Zhao W, Wang Y, Zhao Z, Zhao C, Wang S, et al.  Prior administration of vitamin K2 improves the therapeutic effects of zoledronic acid in ovariectomized rats by antagonizing zoledronic acid-induced inhibition of osteoblasts proliferation and mineralization.  2018;PLoS ONE 13(8):e0202269. https://doi.org/10.1371/journal.pone.0202269
Zoledronic acid (ZA) exerts complex influence on bone by suppressing bone resorption, mostly due to the direct osteoclasts inhibition and uncertain influence on osteoblasts. Vitamin K2 (VK2, Menaquinone-4) as an anabolic agent stimulates bone formation via anti-apoptosis in osteoblasts and mild osteoclasts inhibition. Based on these knowledge, the therapeutic effect of the combined or sequential therapy of VK2 and ZA depends on the influence on the osteoblasts, since both cases exert similar inhibitory effect on osteoclasts. In a series of in vitro studies, we confirmed the protective effect of VK2 in osteoblasts culture, especially when followed by exposure to ZA, and the proliferation and mineralization inhibition induced by ZA towards osteoblasts. These findings suggested that pretreatment with VK2 before ZA therapy might serve a new long-term therapy protocol for osteoporosis.

Azuma K, Inoue S.  Multiple Modes of Vitamin K Actions in Aging-Related Musculoskeletal Disorders.  Biosensors (Basel). 2019;Jun;20(11):2844. 

Halder M, Petsophonsakul P, Akbulut AC, Pavlic A, Bohan F, et al.  Vitamin K: Double Bonds beyond Coagulation Insights into Differences between Vitamin K1 and K2 in Health and Disease.  Int J Mol Sci. 2019 Feb;20(4):896.

Ilea A, Andrei V, Feurdean CN, Babtan A-M, Petrescu NB, Campian RD, et al.  Saliva, a Magic Biofluid Available for Multilevel Assessment and a Mirror of General Health—A Systematic Review.  Biosensors (Basel). 2019 Mar;9(1):27. 

Li W, Zhang S, Liu J, Liu Y, Liang Q. Vitamin K2 stimulates MC3T3-E1 osteoblast differentiation and mineralization through autophagy induction.  Mol Med Rep. 2019 May; 19(5):3676-3684.
Vitamin K2 likely exerts its protective effects during osteoporosis by promoting osteoblast differentiation and mineralization. However, the precise mechanism remains to be fully elucidated. Autophagy maintains cell homeostasis by breaking down and eliminating damaged proteins and organelles. Increasing evidence in recent years has implicated autophagy in the development of osteoporosis. The present study confirmed that VK2 stimulated autophagy in MC3T3 cells to promote differentiation and mineralization, which may be a potential therapeutic target for osteoporosis treatment.

Wasilewski (GB, Vervloet MG, Schurgers LJ.  The bone-vasculature axis: Calcium supplementation and the role of vitamin K.  Front Cardiovasc Med. 2019;6:6.
Calcium supplements are broadly prescribed to treat osteoporosis either as monotherapy or together with vitamin D to enhance calcium absorption. It is still unclear whether calcium supplementation significantly contributes to the reduction of bone fragility and fracture risk. Data suggest that supplementing post-menopausal women with high doses of calcium has a detrimental impact on cardiovascular morbidity and mortality. Chronic kidney disease (CKD) patients are prone to vascular calcification in part due to impaired phosphate excretion. Calcium-based phosphate binders further increase risk of vascular calcification progression. In both bone and vascular tissue, vitamin K-dependent processes play an important role in calcium homeostasis and it is tempting to speculate that vitamin K supplementation might protect from the potentially untoward effects of calcium supplementation. This review provides an update on current literature on calcium supplementation among post-menopausal women and CKD patients and discusses underlying molecular mechanisms of vascular calcification. We propose therapeutic strategies with vitamin K2 treatment to prevent or hold progression of vascular calcification as a consequence of excessive calcium intake.

Yang C, Shi X, Xia H, Yang X, Liu H, Pan D, et al.  The Evidence and Controversy Between Dietary Calcium Intake and Calcium Supplementation and the Risk of Cardiovascular Disease: A Systematic Review and Meta-Analysis of Cohort Studies and Randomized Controlled Trials.  J Am Coll Nutr. 2019 Oct 18;1-19.
Our objective was to synthesize both trial and observational studies and undertake a meta-analysis to explore the associations between calcium from dietary and supplemental intakes and cardiovascular disease (CVD) risks.  We concluded that calcium intake from dietary sources do not adequately increase the risk of CVD including CHD and stroke, while calcium supplements might raise CHD risk, especially MI.