[Frontiers in Bioscience E4, 2085-2100, January 1, 2012]

Histology of epiphyseal cartilage calcification and endochondral ossification

Norio Amizuka1, Tomoka Hasegawa1, Kimimitsu Oda2, Paulo Henrique Luiz de Freitas1,3, Kazuto Hoshi4, Minqi Li1, Hidehiro Ozawa5

1Departments of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan, 2Division of Biochemistry, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan, 3Oral and Maxillofacial Surgery, Mario Gatti Hospital, Campinas, Brazil, 4Division of Tissue Engineering, The University of Tokyo Hospital, Tokyo, Japan, 5Institute for Dental Science, Matsumoto Dental Collage, Shiojiri, Japan

TABLE OF CONTENTS

1. Abstract
2. Introduction
2.1. Calcification in hypertrophic zone of cartilage
3. Matrix vesicle-mediated calcification
4. Function of ALPase in cartilage calcification
5. Non-collagenous organic constitutes in cartilage calcification
5.1. Osteopontin
5.2. Osteocalcin and matrix Gla protein
5.3. Proteoglycans
6. Invasion of endothelial cells into hypertrophic zone and osteoclastic resorption of calcified cartilage matrix
7. Apoptosis of hypertrophic chondrocytes
8. Function of MMP-9, -13 and MT1-MMP in endochondral ossification
9. Concluding remarks
10. References

1. ABSTRACT

Cartilage calcification is carried out by chondrocytes as they hypertrophy and begin to secrete matrix vesicles. Calcification initiates when calcium phosphates appear inside these matrix vesicles, forming hydroxyapatite crystals that eventually break through the membrane to form calcifying globules, as in bone calcification. However, the extracellular environment in cartilage is different from that in bone: cartilage is abundant in proteoglycans but contains a small amount of osteopontin. Hypertrophic chondrocytes secrete vesicles in the cartilaginous matrix of intercolumnar septae only, forming well-calcified longitudinal septae and poorly-calcified transverse partitions. Such pattern of vesicle deposition permits the invasion of endothelial cells, which infiltrate into cartilage and induce migration of osteogenic and osteoclastic cells. Osteoclasts resorb the excess of calcified globules in the partitions, shaping calcified cartilage cores paralleling the longitudinal axis of long bones. After the formation of these calcified cartilage cores, endochondral ossification involves a series of well-defined events in which osteogenic cells deposit new bone onto the cartilage core and form primary trabecules. This review presents the histology of epiphyseal cartilage calcification and endochondral ossification.

2. INTRODUCTION

It is generally accepted that the first clue of the start of a center of ossification in a developing cartilage is hypertrophy of chondrocytes residing in the middle portion of the hyaline cartilage shaft. This finding is consistent with the enlargement of their lacunae at the expense of the intervening cartilaginous matrix. The matrix remaining in the region of the hypertrophic zone becomes calcified. The osteogenic capability of cells from the perichondrium covering the mid-portion of the shaft is subsequently activated. These cells then launch the process of intramembranous ossification. Osteoclasts invade the calcified intramembranous bone inside the mid-shaft cartilaginous anlage, accompanied by migrating blood vessels and osteogenic cells. After that, osteogenic cells differentiate into osteoblasts and start the deposition of new bone in the former hypertrophic zone. The cartilage anlage will then depict cartilaginous extremities sandwiching newly-formed bone, and will be termed epiphyseal cartilage.

The developing epiphyseal cartilage can be histologically divided into three distinct zones: reserve or resting zone, proliferative zone and hypertrophic zone (Figure 1) (1, 2). Chondrocyte precursors differentiate into chondrocytes and start secreting cartilaginous matrix abundantly, in a process known as appositional growth. Meanwhile, differentiated chondrocytes may undergo cell division and synthesize cartilaginous matrix simultaneously, in what is termed interstitial growth. The normal development and growth of endochondral bone is dependent on a balance between proliferation and differentiation of chondrocytes and the consequent appositional and interstitial growth; it also depends on effective vascular invasion allowing space and nutrition for bone matrix deposition. The spatial progression of chondrocytic differentiation is extremely important for enabling endochondral ossification (Figure 2). Chondrocytes scattered throughout the resting zone show enlarged cisterns of rough endoplasmic reticulum (rER), accumulated glycogen granules and round cell shape (Figure 1A). They will become proliferative chondrocytes, which align in the longitudinal columns known as "stacked coins" (Figure 1B). Then, proliferative chondrocytes differentiate into the hypertrophic phenotype (Figure 1C), which features an enlarged and translucent cell body and express collagen type I and X, alkaline phosphatase (ALPase), proteoglycans and osteopontin (3-7).

As chondrocytes become hypertrophic, they increase their volume five- to twelve-fold and elevate about three-fold the total amount of extracellular matrix produced (8). Cell organelles in hypertrophic chondrocytes are intact, but scattered throughout the cytoplasm. Glycogen granules, which previously accumulated in the cytoplasm of the resting and proliferative chondrocytes, are dispersed throughout the cytoplasm of hypertrophic cells. Several studies have documented the presence of a transition zone (maturation zone) between the proliferative and hypertrophic zones; in fact, this zone is composed of chondrocytes in the process of differentiation. Hypertrophic chondrocytes cannot proliferate, but acquire the ability of inducing calcification of the cartilaginous matrix. Hypertrophic chondrocytes play a key role in normal cartilage calcification, and subsequently in endochondral ossification, processes that are strictly regulated by a sequence of cellular events.

From a functional viewpoint, hypertrophic chondrocytes secrete matrix vesicles that serve to initiate calcification of the extracellular matrix (9-11). The length of the hypertrophic zone is maintained by a balance between the rate at which chondrocytes enter the hypertrophic phase and the rate at which vascular endothelial cells invade cartilage at the chondro-osseous junction (Figure 2). Hypertrophic chondrocytes are shown to synthesize vascular endothelial growth factor (VEGF), an angiogenic molecule that has been implicated in matrix metabolism and vascular invasion of the epiphyseal cartilage (12).

Cartilage calcification is known to be mediated by the matrix vesicles secreted by hypertrophic chondrocytes. While the molecular mechanism of cartilage calcification is almost same as that seen in bone, the extracellular environment and the cellular events of subsequent endochondral ossification are different from those seen in bone. In this paper, we will review the histological aspects of epiphyseal cartilage calcification and the subsequent endochondral ossification.

2.1. Calcification in hypertrophic zone of cartilage

In cartilage, biological calcification induced by hypertrophic chondrocytes is known to be initiated by small extracellular vesicles referred to as "matrix vesicles" (9-11). Using transmission electron microscopy (TEM), matrix vesicles were shown to accumulate in the longitudinal septa starting from the proliferative zone downwards, featuring "needle-like" mineral crystals in the lower hypertrophic zone (9). We also found small calcification nodules, i.e., "calcifying globules" as termed by Bonucci (11), and matrix vesicles with mineral crystals starting from the point at which chondrocytes just become hypertrophic, i.e., the maturation zone (Figure 2) (13). Therefore, calcification appears to be initiated by chondrocytes when they begin to be hypertrophic.

Hypertrophic chondrocytes secrete matrix vesicles from their lateral cell membranes towards the longitudinal intercolumnar septae (Figure 1E). In the initial site of cartilage calcification, a line of matrix vesicles and calcifying globules can be seen along the lateral cell membranes of hypertrophic chondrocytes (Figure 1E-H). Consequently, the longitudinal intercolumnar septae undergo proper calcification, and would serve as scaffolds of future primary trabecules on top of which osteoblastic precursors group and start to deposit new bone matrix (Figure 2). In contrast, the transverse partitions of the cartilaginous columns are incompletely calcified, since hypertrophic chondrocytes do not secrete matrix vesicles abundantly onto this zone. Thus, the well-calcified, intercolumnar longitudinal septae and the incompletely-calcified transverse partition form the calcified cartilage cores for the primary trabeculae, parallel to the longitudinal axis of long bones (Figure 2).

3. MATRIX VESICLE-MEDIATED CALCIFICATION

Discovery of matrix vesicles was a breakthrough for further investigations in the research field of biological calcification (9-11, 14-17). They were first identified in the extracellular matrix of calcifying cartilage. In cartilage, matrix vesicles are limited by a biological membrane sized approximately 30-1000 nm in diameter (9). Hydroxyapatite has a small, ribbon-like structure approximately 25 nm wide, 10 nm high and 50 nm long (18). Calcification starts when hydroxyapatite appears inside these matrix vesicles and progresses as the crystals grow and eventually break through the membrane to form calcifying globules (Figure 3).

Some of these precipitating crystals were initially found associated with the inner leaflet of the matrix vesicle's membrane (See Figure 3A), and it seems likely that crystal nucleation occurs at this specific site. Matrix vesicle membranes are rich in acidic phospholipids such as phosphatidylserine and phosphatidylinositol, which have high affinity for Ca2+. The affinity of phosphatidyleserine to Ca2+ is particularly high, and they form a stable calcium phosphate-phospholipid complex (17). The possibility that such complexes may play an important role in crystal nucleation has been pointed out in previous studies (19, 20). Calcium phosphate crystals formed inside matrix vesicles penetrate the plasma membrane, and in the presence of physiological concentrations of extra-vesicular Ca2+, PO43− and pyrophosphate (PPi), form the stellate clusters of "needle-shaped" biological apatite that grow to fill the interstices of the longitudinal septa (21). They are identified as crystalline structures by electron diffraction, but also as non-crystalline structures containing calcium and phosphate by the freeze-substitution method or by cytochemical calcium detection such as -pyroantimonate method combined with energy-dispersive X-ray spectroscopy (22-24).

A variety of enzymes and proteins is involved in the metabolism of proteoglycans and pyrophosphate, and are also found in the matrix vesicles. Examples of the former are matrix metalloproteinase (MMP)-3 (25), ALPase (15, 24, 26-29) and ATPase (30). Among the latter are annexin II, V and VI (31), phospholipase A2, carbonic anhydrase II and lactate dehydrogenase (21). These are also believed to regulate crystal precipitation. Even though the plasma membrane of the matrix vesicles would be ripped during the process of crystal growth, calcifying globules might not lose these membrane-associated enzymes.

4. FUNCTION OF ALPASE IN CARTILAGE CALCIFICATION

One of the most important enzymes that initiate calcification in the hypertrophic zone of cartilage must be ALPase (Figure 4). ALPase can hydrolyze various phosphate esters, and is responsible for the production of inorganic phosphate: many believe it is a potent inducer of calcification. Recently, evidences based on murine knockout models point to tissue non-specific ALPase essentially as a pyrophosphatase (32). In cartilage, ALPase activity was detected on chondrocytes (3) and matrix vesicles (29, 33). These authors localized the enzyme on the chondrocyte cell membrane and in the matrix vesicles secreted by hypertrophic chondrocytes consistent with our observations (28). Indeed, as shown in Figure 4A, ALPase activity is seen along the cell membranes of all hypertrophic chondrocytes in the epiphyseal cartilage; proliferative chondrocytes, however, do not express ALPase.

Interestingly, the distribution of ALPase on cell membranes is not uniform in osteoblasts, with showing basolateral and secretory (osteoidal) domains. Takano's group reported that plasma membrane Ca2+ transport ATPase was restricted to the osteoidal domain of the osteoblastic cell membrane, while tissue non-specific ALPase was predominantly present on the basolateral domain (34). Consistently, using specific antiserum to tissue non-specific ALPase (35), we could observe relatively intense immunoreactivity and enzymatic activity for ALPase on preosteoblasts and on basolateral cell membranes of mature osteoblasts (Figure 4B,C) (36). Thus, in bone, the membranes featuring intense activity of ALPase are not identical to those that serve as the site of matrix vesicle formation. In cartilage, however, all the cell membranes of hypertrophic chondrocytes showed uniform and intense ALPase activity. Thus, the distribution pattern of ALPase related to the site of matrix vesicle formation is slightly different in osteoblasts and in hypertrophic chondrocytes.

Mice homogyzous for gene depletion of tissue non-specific ALPase have been generated (37, 38) and mimic severe hypophosphatasia, indicating that ALPase is likely involved in calcification. Tissue non-specific ALPase might act as a pyrophosphatase (32), although other pyrophosphatases may exist. ALPase-/- mice are born with an intact bone, but gradually develop growth retardation as well as skeletal deformities. Although the absence of endogenous tissue non-specific ALPase activity did not result in complete lack of mineral uptake in skeletal tissues (38), the ALPase-/- mice revealed a severe disturbance of the growth plate, suggesting abnormal endochondral ossification (39). Recently, it was shown that depletion of tissue non-specific ALPase gene results not only in hypocalcification of the skeleton, but also in a severe disorder of mineral crystal alignment in growing long bones with disordered bone matrix architecture (40). More recently, Murshed et al. (41) have reported that transgenic mice expressing tissue non-specific ALPase in the dermis showed extracellular calcification consisting of hydroxyapatite crystals. Given the evidence, it seems that ALPase activity is essential for calcification in cartilage and bone, but it is still unknown why the cell membranes with intense ALPase activity in bone are not identical to those forming matrix vesicles.

5. NON-COLLAGENOUS ORGANIC CONSTITUTES IN CARTILAGE CALCIFICATION

5.1. Osteopontin

It seems likely that the calcification process is regulated by a large number of matrix proteins and glycosaminoglycans in cartilage. Among the phosphoproteins, osteopontin is especially suited to the task of regulating calcification, because it effectively inhibits both apatite formation and growth (42, 43). Osteopontin is localized in the periphery of calcifying globules and in the lamina limitans (44-47), where it might act as a blocker of excessive calcification (48). In the calcification process, osteopontin and other organic constituents manifest themselves initially as a protein coating of the crystals, termed "crystal ghosts" by Bonucci (49), and then may coalesce to form crystal globules. In situ hybridization demonstrated the expression of osteopontin, as well as osteocalcin and matrix gla protein in the hypertrophic chondrocytes of long bones (4, 50). However, using histochemistry in cartilage, osteopontin was shown to be secreted slightly later than ALPase and formation of matrix vesicles (calcification) in the hypertrophic zone (Figure 5). Consistently, not much osteopontin coated the early calcifying globules in cartilage (Figure 6), which were relatively larger than those in bone (13). Thus, osteopontin appears to be an inhibitor of the growth of calcifying globules.

5.2. Osteocalcin and matrix Gla protein

Osteocalcin and matrix Gla protein are known for containing -carboxyglutamic acid and for their ability to bind to mineral crystals (See Figure 7B) (51-53). Recently, the unveiling of the crystal structure of porcine osteocalcin by X-ray analysis revealed a negatively charged protein surface with three Gla residues in helix 1 and conserved Aps residues in helix 2. Such charged surface could bind to Ca2+ on a hydroxyapatite crystal lattice (54). Organic components surround the calcifying globules' crystals, forming the so-called crystal ghost (49, 55, 56), where osteocalcin combines with osteopontin (57). It has been reported that calcium-binding organic materials inhibit calcification (43, 58); therefore, all these organic components that potentially bind to hydroxyapatite crystals may act on inhibiting excess accumulation of calcium and phosphate.

It is interesting that osteocalcin-deficient mice have no obvious skeletal deformity, but showed accelerated bone loss after ovariectomy (59). Boskey et al (60) have reported that ovariectomized osteocalcin-/- cortices had lower carbonate/phosphate ratios than did wild-type specimens. Complete inhibition of -carboxylated proteins must cause severe skeletal deformities. Warfarin, an inhibitor for -carboxylation of glutamine residues, induces an embryopathy consisting of nasal hypoplasia, stippled epiphyses and distal extremity hypoplasia when given to women in the first trimester of pregnancy (61, 62). Administration of warfarin to rats, which decrease the vitamin K-dependent protein of osteocalcin to only 2% of its normal state, resulted in an excessive calcification disorder characterized by complete fusion of the growth plate cartilage of the proximal tibiae, as well as cessation of longitudinal growth (63). In our observations, the administration of warfarin resulted in dispersion of numerous fragments of crystal minerals in osteoid (Figure 7) (64). Therefore, as Hunter et al (43) reported, osteocalcin and matrix Gla protein might be involved in the inhibition of nucleation of mineral crystals.

5.3. Proteoglycans

The extracellular matrices, as an ion reservoir, may constitute the adequate microenvironment for the initiation of calcification. Proteoglycans are complexes of various glycosaminoglycan (GAG) chains and core proteins. The GAG chains are highly negatively charged, which can attract free divalent cations such as Ca2+ (65, 66). It seems likely that proteoglycans significantly impact the dynamics of the extracellular fluid's mineral ionic content. Since cartilage contains abundant proteoglycans, cartilage matrices may serve as a reservoir for free divalent cations. Especially, crystal ghosts appear to be rich in sulfated GAG chains (67, 68), especially, chondroitin sulfate (69).

In cartilage, small leucine-rich proteoglycans -- decorin, biglycan, fibromodulin, lumican, and epiphycan (70, 71), also referred to as PG-Lb (72) -- are present. In situ hybridization and immunohistochemistry have verified that hypertrophic chondrocytes in developing epiphyseal growth plate cartilage express high levels of biglycan mRNA (73, 74). Immunoelectron microscopy of the growth plate revealed that the prominent immunolabelling was confined to the Golgi apparatus and cisternae of rER of hypertrophic chondrocytes, and to the early calcified cartilage matrices of the longitudinal septum of the lower hypertrophic zone (74). Therefore, proteoglycans in the extracellular matrix of the lower hypertrophic zone may be degraded by proteases and removed before calcification, and this seems to be the mechanism by which a matrix that does not possess the ability to calcify is transformed into one that has that capability. Interestingly, however, the concentration of sulfate or cartilage proteoglycans was not shown to change before and after cartilage calcification (6, 75, 76). Decorin/biglycan-double knockout mice revealed osteopenia as a result of impaired GAG-linking to decorin- and biglycan-core proteins, whereas calcification was unaffected (77). Collagen calcification based on the proposed process of removal of small proteoglycans before calcification may deserve further investigation, at least in cartilage.

According to observations of the osteoid in bone derived from the quick frozen-freeze substitution technique with electron energy loss spectroscopy (EELS), which enables elemental mapping at the molecular level, calcium was primarily localized to proteoglycans, whereas phosphate was predominantly localized to collagen fibrils (Figure 8) (28). Therefore, even if the extracellular fluid as a whole is supersaturated with Ca2+ and PO43−, it seems feasible that, in non-calcified sites, the extracellular meshwork of organic substances limits the production of hydroxyapatite and inhibits precipitation of calcified crystals by controlling the spatial distribution of Ca2+ and PO43−. However, these findings were obtained from osteoid in bone, and localization of calcium and phosphate in the cartilage matrix using TEM-EELS is still in progress.

6. INVASION OF ENDOTHELIAL CELLS INTO HYPERTROPHIC ZONE AND OSTEOCLASTIC RESORPTION OF CALCIFIED CARTILAGE MATRIX

There is an abundance of blood vessels and osteoclasts (chondroclasts) where the bottom of hypertrophic zone meets the bone tissue, the so-called erosion zone. The cellular interplay among vascular endothelial cells, osteoclasts and osteoblasts is temporarily and spatially tuned, so that longitudinal primary trabeculae can be formed (Figure 2). In the erosion zone, vascular endothelial cells invade the hypertrophic zone and penetrate the incompletely-calcified transverse partitions, making a way for the migration of osteoclastic and osteoblastic cells. As described, the longitudinal distribution of calcified cartilage matrix seems fundamental for the formation of primary trabecular bone. As vascular invasion progresses, hypertrophic chondrocytes secrete VEGF and guide angiogenesis (Figure 9).

There is another cell-type that may invade the hypertrophic zone, the septoclast (78), which does not belong to the macrophage/osteoclast lineages and is also referred to as perivascular cell (Figure 10) (79). Septoclasts, or perivascular cells, produce cathepsin B abundantly, and thereby, could digest and penetrate into the transverse partition of cartilage matrix at the chondro-osseous junction. The relationship between vascular endothelial cells and septoclasts is, to date, unknown. Septoclasts may aid vascular invasion at the chondro-osseous junction, but further investigation on the cellular function of septoclasts is necessary for clarification of this issue.

What is the osteoclast/chondroclast function in endochondral ossification? Generally, osteoclasts resorb calcified cartilage matrix. Yet, they are consistently found behind invading endothelial cells. Osteoclasts/chondroclasts at the chondro-osseous junction do not resorb the calcified longitudinal septae of cartilage completely, but tend to resorb the excess of calcifying globules in the transverse partitions. This is consistent behavior of bone-resorbing osteoclasts, which remove the superficial layer of calcified bone matrix, but never dig deeply into trabecular bone. We postulate that osteoclasts/chondroclasts could resorb the excessive transverse accumulations of calcifying globules in order to align the calcified cartilage parallel to the longitudinal axis of the long bone, after hypertrophic chondrocytes calcify the longitudinal intercolumnar regions. This supposition is based on the observation that osteoclasts/chondroclasts do not resorb the calcified cartilage matrix completely, so that migrating osteoblasts can independently form mixed spicules of cartilage and bone.

Evidence that vascular invasion rather than osteoclastic resorption is dominant for endochondral ossification can be found in osteoclast-absent murine models such as the op/op mice, the c-fos-/- mice, the receptor activator nuclear factor kappa B ligand (RANKL) -/- mice and others. Even without osteoclasts, long bones do grow and elongate; however, there is a picture of mixed spicules with central cartilage cores and bone matrix in the periphery, forming a disorganized meshwork with intense trabecular connectivity.

Osteoclasts at the chondro-osseous junction synthesize more MMP-9 (80) than do osteoclasts located at the end of metaphyseal trabeculae (data not shown). It seems that osteoclasts may recognize the properties of cartilaginous matrix -- proteoglycan-rich or -poor components, calcified or uncalcified -- prior to resorbing it.

For endochondral ossification to happen, osteoclast-driven cartilage resorption may be finely tuned both spatially and temporally. Furthermore, the characteristics of the cartilaginous matrix and the surrounding microenvironment may be important determinants of the process. However, different cellular mechanism on the longitudinal arrangement of primary trabeculae during endochondral ossification may take part in, and therefore, more evidences appears to be necessary for preciously elucidating this issue.

7. APOPTOSIS OF HYPERTROPHIC CHONDROCYTES

It is widely accepted that, under physiological circumstances, chondrocytes in the terminal region of the hypertrophic zone undergo apoptosis as their lacunae are pierced by invading blood vessels (81, 82). Despite their rapid clearance under physiological circumstances, apoptotic chondrocytes have also been identified randomly throughout the resting and proliferative zones of the epiphyseal cartilage, often in association with viable cells in the same lacuna (83-86). These observations suggest that proliferating chondrocytes can undergo asymmetric cell division and that the surviving cell possesses a competence factor that is not expressed in the cell that dies. Consistently, histochemical investigations have confirmed that chondrocytic death happens through the apoptotic pathway (87). 

However, epiphyseal chondrocytes may be able to transdifferentiate into osteoblasts, since terminally differentiated (1, 2) chondrocytes were biosynthetically active. These cells have been shown to synthesize osteogenic markers such as type I collagen, glycosaminoglycans, osteopontin, osteocalcin and osteonectin (4, 5, 88-92). Therefore, we assumed that chondrocytic apoptosis may depend on genetic programming such as Bcl2 expression (93), but may also be influenced by other conditions, i.e., intracellular concentration of Pi (94, 95) and nitric oxide generation (96). We also reported that parathyroid hormone-related peptide deficiency may stimulate chondrocytic apoptosis (97).

In our own observations, there were apoptotic chondrocytes in the hypertrophic zone of adult mice, while 18 day-old mouse fetuses showed intact hypertrophic chondrocytes being removed from the lacunae, as shown in Figure 1D. In contrast, the developing mandibular condyle cartilage at the same fetal age, which unlike appendicular bone has no secondary ossification, showed some apoptotic chondrocytes (98). Taken together, apoptosis of chondrocytes in the hypertrophic zone might be regulated by genetic and environmental stimuli that affect cartilage growth and subsequent endochondral ossification.

8. FUNCTION OF MMP-9, -13 AND MT1-MMP IN ENDOCHONDRAL OSSIFICATION

Endochondral ossification requires proteolytic degradation by means of many proteinases, especially MMPs -9 and -13 (99, 100). These enzymes cleave collagens and aggrecan, two of the main components of cartilaginous matrix. MMP-9, a gelatinase that degrades components of the cartilaginous extracellular matrix with high specificity for degraded collagens, plays a key role in endochondral ossification, specifically capillary invasion into hypertrophic cartilage. In the erosion zone, MMP-9 was shown to be expressed by osteoclastic cells (80, 101), as well as by invading endothelial cells. This means that MMP-9-secreting osteoclasts (chondroclasts) or vascular endothelial cells can digest the incompletely-calcified transverse partitions prior to vascular invasion. However, it is still unknown which cell-type predominantly digests the cartilaginous matrix, and thereby triggers endochondral ossification. Another gelatinase, MMP-3, stromlysin deficiency has not demonstrated altered hypertrophic calcification and subsequent endochondral ossification, so far.

Proteolysis by MMP-13 (collagenase 3) was required for chondrocyte differentiation associated with matrix calcification (102). MMP-13 is expressed in the hypertrophic zone, and is most effective at cleaving type II collagen (103, 104). An inhibitor of MMP-13 was reported to suppress the expression of type X collagen, core binding factor alpha-1 (Cbfal: transcription factor for MMP-13) and MMP-13, and could also hinder calcium incorporation and degradation of type II collagen. However, MMP-13-deficient mice showed no obvious differences in cartilage calcification compared with wild-type littermates, despite the fact that endochondral ossification was dynamically affected (105). It seems that MMP-9 and -13 affect endochondral ossification but do not influence calcification in cartilage.

In contrast, mice homozygous for gene deletion of MT1-MMP (membrane type 1-MMP) referred to as MMP-14 were shown to develop dwarfism, osteopenia, arthritis and connective tissue disease (106). MT1-MMP-/-mice revealed deficient vascularization during secondary ossification of epiphyseal cartilage and growth plate development. Additionally, the authors' group demonstrated that MT1-MMP-dependent dissolution of uncalcified cartilages, coupled with apoptosis of non-hypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues (107). Therefore, MT1-MMP appears to be important for removal of uncalcified cartilage in individual growth.

As shown above, murine knockout models provide a clue for better understanding MMPs in the process of endochondral ossification, and also represent the benchmark for assessing, if not the ultimate physiological role, the dispensability of individual molecules in each envisioned process.

9. CONCLUDING REMARKS

Matrix vesicle-mediated calcification in cartilage, which is achieved by regular cartilage calcification with well-calcified longitudinal intercolumnar septa and poorly-calcified transverse partitions, leads to a series of orchestrated cellular events that result in endochondral ossification. Histological examinations on why hypertrophic chondrocytes secrete matrix vesicles from their lateral cell membranes, what are the functions of septoclasts, and how do proliferative chondrocytes hypertrophy at the same time despite the absence of cellular connections among them may provide clues for better understanding of the mechanisms of calcification in cartilage.

10. REFERENCES

1. Adams CS and Shapiro IM. The fate of the terminally-differentiated cghondrocytes: Evidence for microenvironmental regulation of chondrocyte apoptosis. Crit Rev. Oral Biol med. 13(6): 465-473, 2002.
doi:10.1177/154411130201300604

2. Ali SY, Sajdera SW, Anderson HC. Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage. Proc Natl Acad Sci USA. 67(3):1513-1520, 1970.
doi:10.1073/pnas.67.3.1513

3. Amling M, Neff L, Tanaka S, Inoue D, Kuida K, Weir E, Philbrick WM, Broadus AE, Baron R. Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J Cell Biol. 136(1):205-213, 1997.
doi:10.1083/jcb.136.1.205
PMid:9008714    PMCid:2132464

4. Amizuka N, Davidson D, Liu H, Valverde-Franco G, Chai S, Maeda T, Ozawa H, Hammond V, Ornitz DM, Goltzman D, Henderson JE. Signalling by fibroblast growth factor receptor 3 and parathyroid hormone-related peptide coordinate cartilage and bone development. Bone 34(1):13-25, 2004.
doi:10.1016/j.bone.2003.08.009
PMid:14751559

5. Amizuka N, Li M, Hara K, Kobayashi M, de Freitas PH, Ubaidus S, Oda K, Akiyama Y. Warfarin administration disrupts the assembly of mineralized nodules in the osteoid. J Electron Microsc. 58(2):55-65, 2009.
doi:10.1093/jmicro/dfp008
PMid:19225034

6. Anderson HC. Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol. 41(1):59-72, 1969.
doi:10.1083/jcb.41.1.59
PMid:5775794    PMCid:2107736

7. Anderson HC. Molecular biology of matrix vesicles. Clin. Orthop. Relat. Res. 314: 266-280, 1995.
PMid:7634645

8. Anderson HC, Sipe JB, Hessle L, Dhanyamraju R, Atti E, Camacho NP, Millan JL. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am J Pathol. 164(3):841-847, 2004.
doi:10.1016/S0002-9440(10)63172-0

9. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat. Cell Biol. 2: 737-744, 2000.
doi:10.1038/35036374
PMid:11025665    PMCid:2852586

10. Bianco, P., Cancedda, F., Riminucci, M. and Cancedda, R. Bone formation by cartilage models: the "borderline" chondrocyte. Matrix Biol. 17:185-192, 1998.
doi:10.1016/S0945-053X(98)90057-9

11. Bianco P, Fisher LW, Young MF, Termine JD, Robey PG. Expression and localization of the two small proteoglycans biglycan and decorin in developing human skeletal and non-skeletal tissues. J Histochem Cytochem. 38(11):1549-1563, 1990.
PMid:2212616

12. Bianco P, Hayashi Y, Silvestrini G, Termine JD, Bonucci E. Osteonectin and Gla-protein in calf bone: ultrastructural immunohistochemical localization using the Protein A-gold method. Calcif Tissue Int. 37(6):684-686, 1985.
doi:10.1007/BF02554931
PMid:2418932

13. Bonucci E. Fine structure of early cartilage calcification. J Ultrastruct Res. 20(1):33-50, 1967.
doi:10.1016/S0022-5320(67)80034-0

14. Bonucci E. Fine structure and histochemistry of "calcifying globules" in epiphyseal cartilage. Z Zellforsch Mikrosk Anat. 103(2):192-217, 1970.
doi:10.1007/BF00337312

15. Bonucci E. The locus of initial calcification in cartilage and bone. Clin Orthop Relat Res. 78:108-139, 1971.
doi:10.1097/00003086-197107000-00010
PMid:4939008

16. Bonucci E and Gherardi G. Histochemical and electron microscopy investigations on medullary bone. Cell Tissue Res. 163(1):81-97, 1975.
PMid:1102107

17. Bonucci E and Silvestrini G. Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage. Eur J Histochem. 36(4):407-422, 1992.
PMid:1283829

18. Boskey AL and Posner AS. The role of synthetic and bone extracted Ca-phospholipid-PO4 complexes in hydroxyapatite formation. Calcif Tissue Res. 23:251-258, 1977.
doi:10.1007/BF02012794
PMid:902143

19. Bosky AL, Maresca M, Ullrich W, Doty SB, Butler WT, Prince CW. Osteopontin-hydroxyapatite interactions in vitro: Inhibition of hydroxyapatite formation and growth in a gelatin-gel. Bone Miner. 22:147-159, 1993.
doi:10.1016/S0169-6009(08)80225-5

20. 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. 23(3):187-196, 1998.
doi:10.1016/S8756-3282(98)00092-1

21. Boyan BD, Schwartz Z, Swain LD, Khare A. Role of lipids in calcification of cartilage. Anat Rec. 224:211-219, 1989.
doi:10.1002/ar.1092240210
PMid:2672885

22. Chen JK, Shapiro HS, Wrana JL, Reimers S, Heersche JN, Sodek J. Localization of bone sialoprotein (BSP) expression to sites of mineralized tissue formation in fetal rat tissues by in situ hybridization. Matrix. 11(2):133-143, 1991.
PMid:2072878

23. Corsi A, Xu T, Chen XD, Boyde A, Liang J, Mankani M, Sommer B, Iozzo RV, Eichstetter I, Robey PG, Bianco P, Young MF. Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues. J Bone Miner Res. 17(7):1180-1189, 2002.
doi:10.1359/jbmr.2002.17.7.1180
PMid:12102052

24. de Bernard B, Stagni N, Colautti I, Vittur F, Bonucci E. Glycosaminoglycans and endochondral calcification. Clin Orthop Relat Res. 126:285-291, 1977.
PMid:598133

25. de Bernard B, Bianco P, Bonucci E, Costantini M, Lunazzi GC, Martinuzzi P, Modricky C, Moro L., Panfili E, Pollesello P., Stagni N, Vittur F: Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2+-binding glycoprotein. J Cell Biol 103:1615-1623, 1986.
doi:10.1083/jcb.103.4.1615
PMid:3771650

26. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, and Karsenty G. Increased bone formation in osteocalcin-deficient mice. Nature 382:448-452, 1996.
doi:10.1038/382448a0
PMid:8684484

27. Egeblad M. New roles for the matrix metalloproteinases in the progression of cancer. Nat. Rev. Cancer 2:163-176, 2002.
doi:10.1038/nrc745
PMid:11990853

28. Embery G, Rees S, Hall R, Rose K, Waddington R, Shellis P. Calcium- and hydroxyapatite-binding properties of glucuronic acid-rich and iduronic acid-rich glycosaminoglycans and proteoglycans. Eur J Oral Sci. 106 Suppl 1:267-273, 1998.
PMid:9541236

29. Farnum C and Wilson N. Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine. Calcif Tiss Int. 52:110-119, 1993.
doi:10.1007/BF00308319
PMid:8443686

30. Farnum CE and Wilsman NJ. Condensation of hypertrophic chondrocytes at the chondro-osseous junction of growth plate cartilage in Yucatan swine: relationship to long bone growth. Am J Anat. 186: 346-358, 1989.
doi:10.1002/aja.1001860404
PMid:2589219

31. Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Weinstein RS, Waymire K, Narisawa S, Millan JL, MacGregor GR, Whyte MP. Alkaline phosphatase knock-out mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 14:2015-2026, 1999.
doi:10.1359/jbmr.1999.14.12.2015
PMid:10620060    PMCid:3049802

32. Fleish H and Neuman WF. Mechanisms of calcification: role of collagen, polyphosphates, and phosphatase. Am J Physiol. 200:1296-1300, 1961.
PMid:13700202

33. Fleish H and Neuman W. The role of phosphatase and polyphosphates in calcification of collagen. Helv Physiol Pharmacol Acta. 19:C17-18, 1961.

34. Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation. Nat Med. 5(6):623-628, 1999.
PMid:10371499

35. Greenspan JS and Blackwood HJ. Histochemical studies of chondrocyte function in the cartilage of the mandibular codyle of the rat. J Anat. 100(Pt 3):615-626, 1966.
PMid:4290576    PMCid:1270801

36. Hale JE, Fraser JD, Price PA. The identification of matrix Gla protein in cartilage. J Biol Chem. 263(12):5820-5824, 1988.
PMid:3258600

37. Hall JG, Pauli RM, and Wilson KM. Maternal and fetal sequelae of anti-coagulation during pregnancy. Am J Med. 68:122-140, 1980.
doi:10.1016/0002-9343(80)90181-3

38. Hauschka PV, Lian JB, Gallop PM. Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue. Proc Natl Acad Sci USA. 72(10):3925-3929, 1975.
doi:10.1073/pnas.72.10.3925

39. Heinegård D and Oldberg A. Structure and biology of cartilage and bone matrix noncollagenous macromolecules. FASEB J. 3(9):2042-2051, 1989.

40. Hoang QQ, Sicheri F, Howard AJ, Yang DS. Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature 425: 977-980, 2003.
doi:10.1038/nature02079
PMid:14586470

41. Holmbeck K, Bianco P, Caterina J, Yamada S, Kromer M, Kuznetsov SA, Mankani M, Robey PG, Poole AR, Pidoux I, Ward JM, Birkedal-Hansen H.MT1-MMP-deficient mice develop dwarfism, osteopenia, arthritis, and connective tissue disease due to inadequate collagen turnover. Cell. 99(1):81-92, 1999.
doi:10.1016/S0092-8674(00)80064-1

42. Holmbeck K, Bianco P, Chrysovergis K, Yamada S, Birkedal-Hansen H. MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth. J Cell Biol. 163(3):661-671, 2003.
doi:10.1083/jcb.200307061
PMid:14610065    PMCid:2173657

43. Hoshi K, Amizuka N, Oda K, Ikehara Y, Ozawa H. Immunolocalization of tissue non-specific alkaline phosphatase in mice. Histochem Cell Biol. 107(3):183-191, 1997.
doi:10.1007/s004180050103
PMid:9105889

44. Hoshi K, Ejiri S, Ozawa H. Localizational alterations of calcium, phosphorus, and calcification-related organics such as proteoglycans and alkaline phosphatase during bone calcification. J Bone Miner Res. 16: 289-298, 2001.
doi:10.1359/jbmr.2001.16.2.289
PMid:11204429

45. Hossain KS, Amizuka N, Ikeda N, Nozawa-Inoue K, Suzuki A, Li M, Takeuchi K, Aita M, Kawano Y, Hoshino M, Oda K, Takagi R, Maeda T. Histochemical evidences on the chronological alterations of the hypertrophic zone of mandibular condylar cartilage. Microsc Res Tech. 67(6):325-335, 2005.
doi:10.1002/jemt.20211
PMid:16173089

46. Hunter GK, Hauschka PV, Poole AR, Rosenberg LC, Goldberg HA. Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J. 317: 59-64, 1996.
PMid:8694787    PMCid:1217486

47. Hunziker EB. Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Micro Res Tech 28:505-519, 1994.
doi:10.1002/jemt.1070280606
PMid:7949396

48. Ikeda T, Nomura S, Yamaguchi A, Suda T, Yoshiki S. In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections. J Histochem Cytochem. 40(8):1079-1088, 1992.
PMid:1619274

49. Iozzo RV and Murdoch AD. Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J. 10(5):598-614, 1996.
PMid:8621059

50. Kirsch T, Nah HD, Shapiro IM, Pacifici M. Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes. J Cell Biol. 137:1149-1160, 1997.
doi:10.1083/jcb.137.5.1149
PMid:9166414    PMCid:2136219


51. Knäuper VC, Lopez-Otin C, Smith B, Knight G, Murphy G. Biochemical characterization of human collagenase-3. J Biol Chem 271:1544-1550, 1996.
doi:10.1074/jbc.271.3.1544

52. Lee ER, Lamplugh L, Shepard NL, Mort JS. The septoclast, a cathepsin B-rich cell involved in the resorption of growth plate cartilage. J Histochem Cytochem. 43(5):525-536, 1995.
PMid:7730591

53. Mark MP, Butler WT, Prince CW, Finkleman RD, Ruch J-V. Developmental expression of 44-kDa phosphoprotein (osteopontin) and bone-carboxyglutamic acid (Gla)-containing protein (osteocalcin) in calcifying tissues of rat. Differentiation 37: 123-136, 1988.
doi:10.1111/j.1432-0436.1988.tb00804.x
PMid:3260880

54. Mansfield K, Rajpurohit R, Shapiro IM. Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. J Cell Physiol. 179(3):276-286, 1999.
doi:10.1002/(SICI)1097-4652(199906)179:3<276::AID-JCP5>3.0.CO;2-#

55. Mansfield K, Teixeira CC, Adams CS, Shapiro IM. Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. Bone. 28(1):1-8, 2001.
doi:10.1016/S8756-3282(00)00409-9

56. Matsuzawa T and Anderson HC. Phosphatases of epiphyseal cartilage studied by electron microscopic cytochemical methods. J Histochem Cytochem. 19:801-808, 1971.
PMid:4335252

57. McKee MD, Farach-Carson MC, Butler WT, Hauschka PV, Nanci A. Ultrastructural immunolocalization of noncollagenous (osteopontin and osteocalcin) and plasma (albumin and α2HS-glycoprotein) proteins in rat bone. J.Bone Miner Res 8: 485-496, 1993.
doi:10.1002/jbmr.5650080413
PMid:8475798

58. McKee MD and Nanci A. Post-embedding colloidal-gold immunocytochemistry of noncollagenous extracellualr matrix proteins in mineralized tissues. Microsc Res Tech 31: 44-62, 1995.
doi:10.1002/jemt.1070310105
PMid:7626799

59. McKee MD and Nanci A. Osteopontin at mineralized tisue interfaces in bone teeth and osteointegrated implants: Ultrastructural distribution and implications for mineralized tissue formation, turnover and repair. Microsc Res Tech 33: 141-164, 1996.
doi:10.1002/(SICI)1097-0029(19960201)33:2<141::AID-JEMT5>3.0.CO;2-W

60. Mitchell PG, Magna HA, Reeves LM, Lopresti-Morrow LL, Yocum SA, Rosner PE, Geoghegan KF, Hambor JE. Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage. J Clin Invest 97:761-768, 1996.
doi:10.1172/JCI118475
PMid:8609233    PMCid:507114

61. Murshed M, Harmey D, Millan JL, McKee MD, Karsenty G. Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 19(9):1093-1104, 2005.
doi:10.1101/gad.1276205

62. Nakamura H and Ozawa H. Ultrastructural, enzyme-, lectin, and immunohistochemical studies of the erosion zone in rat tibiae. J Bone Miner Res. 11(8):1158-1164, 1996.
PMid:8854252

63. Nakano Y, Beertsen W, van den Bos T, Kawamoto T, Oda K, Takano Y. Site-specific localization of two distinct phosphatases along the osteoblast plasma membrane: tissue non-specific alkaline phosphatase and plasma membrane calcium ATPase. Bone. 35(5):1077-1085, 2004.
doi:10.1016/j.bone.2004.07.009
PMid:15542032

64. Narisawa S, Fröhlander N, Millian JL Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn 208:432-446, 1997.
doi:10.1002/(SICI)1097-0177(199703)208:3<432::AID-AJA13>3.0.CO;2-1

65. Oda K, Amaya Y, Fukushi-Irie M, Kinameri Y, Ohsuye K, Kubota I, Fujimura S, Kobayashi J. A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase. J Biochem. 126: 694-699, 1999.
PMid:10502677

66. Oshima O, Leboy PS, McDonald SA, Tuan RS, Shapiro IM. Developmental expression of genes in chick growth cartilage detected by in situ hybridization. Calcif Tissue Int. 45(3):182-192, 1989.
doi:10.1007/BF02556062
PMid:2505910

67. Ozawa H, Yamada M, Yajima T. The ultrastructural and cytochemical aspects of matrix vesicles and calcification processes. In Formation and Calcification of Hard Tissues, eds: R.V.Talmage and H.Ozawa. pp9-57, Shakai Hoken Pub. Tokyo, 1978.

68. Ozawa H, Yamada M, Yamamoto T. Ultrastructural observations on the location of lead and calcium in the mineralizing dentine of rat incisor. In Matrix Vesicles, eds: Ascenzi A,Bonucci E, de Bernard B, pp179 -187, Wiching Editore srl, Milano, 1981.

69. Ozawa H. Current concepts of the morphophysiology of matrix vesicle calcification. Connective Tissue. 15: 1-12, 1983.

70. Ozawa H. Ultrastructural aspects on the biological calcification with special reference to freeze-substitution at liquid helium temperature. In Proc. XIth Int. Cong. on Electron Microscopy, Kyoto. pp57- 60, 1986.

71. Pauli RM, Lian JB, Mosher DF, Suttie JW. Association of congenital deficiency of multiple vitamin K-dependent coagulation factors and the phenotype of the warfarin embryopathy: Cues to the mechanism of teratogenicity of coumarin derivatives. 41: 566-583, 1987.

72. Poole AR, Pidoux I, Rosenberg L. Role of proteoglycans in endochondral ossification: immunofluorescent localization of link protein and proteoglycan monomer in bovine fetal epiphyseal growth plate. J Cell Biol. 92(2):249-260, 1982.
doi:10.1083/jcb.92.2.249
PMid:7037793

73. Poole A.R. The growth plate: cellular physiology, cartilage assembly and mineralization. In Cartilage: Molecular Aspects, eds: B.K. Hall and S.A. Newman, Boca Raton: CRC Press, pp 179-211, 1991.

74. 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. 73(5):1447-1451, 1976.
doi:10.1073/pnas.73.5.1447

75. Price PA, Williamson MK, Haba T, Dell RB, Jee WS. Excessive mineralization with growth plate closure in rats on chronic warfarin treatment. Proc Natl Acad Sci US A. 79(24):7734-7738, 1982.
doi:10.1073/pnas.79.24.7734

76. Ritter NM, Farach-Carson MC, Butler WT. Evidence for the formation of a complex between osteopontin and osteocalcin. J Bone Miner Res. 7(8):877-885, 1992.
PMid:1442202

77. Roach H, Erenpriesa J, Aigner T. Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis. J Cell Biol 131: 483-494, 1995.
doi:10.1083/jcb.131.2.483
PMid:7593173

78. Roach H. New aspects of endochondral ossification in the chick: chondrocyte apoptosis, bone formation by former chondrocytes and acid phosphatase activity in the endochondral bone matrix. J Bone Miner Res 12:795-805, 1997.
doi:10.1359/jbmr.1997.12.5.795
PMid:9144346

79. Romberg RW, Werness PG, Riggs BL, Mann KG. Inhibition of hydroxyapatite crystal growth by bone-specific and other calcium-binding proteins. Biochemistry. 25(5):1176-1180, 1986.
doi:10.1021/bi00353a035
PMid:3008822

80. Sasaki T, Amizuka N, Irie K, Ejiri S, Ozawa H. Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae. Arch Histol Cytol. 63(3):271-284, 2000.
doi:10.1679/aohc.63.271
PMid:10989939

81. Scherft JP. The lamina limitans of the organic matrix of calcified cartilage and bone. J Ultrastruct Res. 38(3):318-331, 1972.
doi:10.1016/S0022-5320(72)90008-1

82. Scherft JP and Moskalewski S. The amount of proteoglycans in cartilage matrix and the onset of mineralization. Metab Bone Dis Relat Res. 5(4):195-203, 1984.
doi:10.1016/0221-8747(84)90030-4

83. Schmid TM and Linsenmayer TF. Immunohistochemical localization of short chain cartilage collagen (type X) in avian tissues. J Cell Biol. 100(2):598-605, 1985.
doi:10.1083/jcb.100.2.598
PMid:2578471

84. Schmitz JP, Schwartz Z, Sylvia VL, Dean DD, Calderon F, Boyan BD. Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C. J Cell Physiol. 168:570-579, 1996
doi:10.1002/(SICI)1097-4652(199609)168:3<570::AID-JCP9>3.0.CO;2-M

85. Shepard N and Mitchell N. Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification. A comparative light and electron microscopic study. Histochemistry. 70(2):107-114, 1981.
doi:10.1007/BF00493202
PMid:7216829

86. Shinomura T and Kimata K. Proteoglycan-Lb, a small dermatan sulfate proteoglycan expressed in embryonic chick epiphyseal cartilage, is structurally related to osteoinductive factor. J Biol Chem. 267(2):1265-70, 1992.
PMid:1730648

87. Silbermann M and Frommer J. Vitality of chondrocytes in the mandibular condyle as revealed by collagen formation. An autoradiographic study with 3 H-Proline. Am J Anat. 135(3):359-369, 1972.
doi:10.1002/aja.1001350305
PMid:4562788

88. Silbermann M and Frommer J. Ultrastructure of developing cartilage in the mandibular condyle of the mouse. Acta Anat (Basel). 90(3):330-346, 1974.
doi:10.1159/000144342

89. Stickens D, Behonick DJ, Ortega N, Heyer B, Hartenstein B, Yu Y, Fosang AJ, Schorpp-Kistner M, Angel P, Werb Z. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development. 131(23):5883-5895, 2004.
doi:10.1242/dev.01461
PMid:15539485    PMCid:2771178

90. Strauss PG, Closs EI, Schmidt J, Erfle V. Gene expression during osteogenic differentiation in mandibular condyles in vitro. J Cell Biol. 110(4):1369-1378, 1990.
doi:10.1083/jcb.110.4.1369
PMid:1691190

91. Suda N, Shibata S, Yamazaki K, Kuroda T, Senior P, Beck F, Hammond V. Parathyroid hormone related protein regulates proliferation of condylar hypertrophic chondrocytes. J Bone Miner Res 14:1838-1847, 1999.
doi:10.1359/jbmr.1999.14.11.1838
PMid:10571683

92. Takagi M, Kamiya N, Urushizaki T, Tada Y, Tanaka H. Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage. Histochemical J. 32: 175-186, 2000
doi:10.1023/A:1004099324169
PMid:10841312

93. Takano Y, Ozawa H, Crenshaw MA. Ca-ATPase and ALPase activities at the initial calcification sites of dentine and enamel in the rat incisor. Cell Tissue Res. 243:91-99, 1986.
doi:10.1007/BF00221856
PMid:2935260

94. Teixeira CC, Mansfield K, Hertkorn C, Ischiropoulos H, Shapiro IM. Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation. Am J Physiol Cell Physiol. 281(3):C833-839, 2001.

95. Tesch W, Vandenbos T, Roschgr P, Fratzl-Zelman N, Klaushofer K, Beertsen W, Fratzl P. Orientation of mineral crystallites and mineral density during skeletal development in mice deficient in tissue nonspecific alkaline phosphatase. J Bone Miner Res. 18(1):117-125, 2003.
doi:10.1359/jbmr.2003.18.1.117
PMid:12510812

96. von der Mark K, von der Mark H. The role of three genetically distinct collagen types in endochondral ossification and calcification of cartilage. J Bone Joint Surg Br. 59-B(4):458-464, 1977.
PMid:72756

97. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D, Shapiro SD, Senior RM, Werb Z. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93: 411-422, 1998.
doi:10.1016/S0092-8674(00)81169-1

98. Vu TH. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev. 14: 2123-2133, 2000.
doi:10.1101/gad.815400

99. Wallis GA. Bone growth: coordinating chondrocyte differentiation. Curr Biol. 6(12):1577-1580, 1996.
doi:10.1016/S0960-9822(02)70776-8

100. Waymire KG, Mahuren JD, Jaje JM, Guilarte TR, Coburn SP, MacGregor GR. Mice lacking tissue non-specific alkaline phosphatase die from seizures due to defective metabolism of vitamin B-6. Nat Genet 11:45-51, 1995.
doi:10.1038/ng0995-45
PMid:7550313

101. Weatherell JA and Weidmann SM. The distribution of organically bound sulphate in bone and cartilage during calcification. Biochem J. 89:265-267, 1963.
PMid:14084612    PMCid:1202356

102. Weiner S. Organization of extracellularly mineralized tissues: a comparative study of biological crystal growth. CRC Crit Rev Biochem. 20(4):365-408, 1986.
doi:10.3109/10409238609081998

103. Werner A and Grunder W. Calcium-induced structural changes of cartilage proteoglycans studied by HNMR relaxometry and diffusion measurements. Magn Reson Med. 41:43-50, 1999.
doi:10.1002/(SICI)1522-2594(199901)41:1<43::AID-MRM8>3.0.CO;2-E

104. Wu CW, Tchetina EV, Mwale F, Hasty K, Pidoux I, Reiner A, Chen J, Van Wart HE, Poole AR. Proteolysis involving matrix metalloproteinase 13 (collagenase-3) is required for chondrocyte differentiation that is associated with matrix mineralization. J Bone Miner Res. 17(4):639-651, 2002.
doi:10.1359/jbmr.2002.17.4.639
PMid:11918221

105. Wuthier RE. Lipid composition of isolated epiphyseal cartilage cells, membranes and matrix vesicles. Biochim Biophys Acta. 409(1):128-143, 1975.
PMid:1182191

106. Yamada M. Ultrastractural and cytochemical studies on the calcification of the tendon-bone joint. Arch. Histol. Jap. 39:347- 378, 1976.
PMid:66047

107. Yasui N, Ono K, Konomi H, Nagai Y. Transitions in collagen types during endochondral ossification in human growth cartilage. Clin Orthop Relat Res. 183:215-218, 1984.
PMid:6365388

Key Words: Cartilage, Osteogenesis, Physiologic calcification, Chondrocytes, Osteoclasts, Review

Send correspondence to: Norio Amizuka, Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine, Hokkaido University, Kita 13, Nishi 7, Kita-Ku, Sapporo, Hokkaido, 060-8586, Japan, Tel: 81-11-706-4223, Fax: 81-11-706-4225, E-mail:amizuka@den.hokudai.ac.jp