Differentiation and mineralization of murine mesenchymal C3H10T1/2 cells in micromass culture

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4 mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4 mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. ⁴⁵Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of ⁴⁵Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1 mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.     

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10⁻¹⁰ M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-IR microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on ⁴⁵Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization. J. Cell. Biochem. 64:632–643. © 1997 Wiley-Liss, Inc.     

Analysis of cartilage maturation using micromass cultures of primary chondrocytes

A micromass culture (MM-C) system of primary immature chondrocytes for functional analysis of soluble factors involved in the maturation step of cartilage was previously developed. Ectopically expressed BMP-2 was shown to induce the expression of the Ihh and Noggin genes. Here it is demonstrated that, upon longer culture, secreted bone morphogenetic protein-2 (BMP-2) further promotes the maturation step as judged by the induction of type X collagen and BMP-6 expression, which are known to be detectable in the later phase of cartilage maturation. Induction of all of these genes by secreted BMP-2 was not inhibited by ectopic expression of parathyroid hormone-related peptide (PTHrP) induced by retrovirus vector infection, although the same virus vector showed strong inhibitory effects on the expression of type X collagen gene or alkaline phosphatase activity in mature chondrocytes. These results suggest that the maturation-promoting activity exhibited by BMP-2 is dominant over the suppressive effect of PTHrP in immature chondrocytes. When the BMP-6 gene was introduced into the same virus vector as that used for BMP-2, it induced the same sets of genes (Ihh, Noggin, type X collagen and endogenous BMP-6) as BMP-2 did. These results also suggest that BMP-6 would autonomously maintain and/or promote a later stage of chondrocytic maturation.     

Matrix GLA protein is a developmental regulator of chondrocyte mineralization and, when constitutively expressed, blocks endochondral and intramembranous ossification in the limb

Matrix GLA protein (MGP), a gamma-carboxyglutamic acid (GLA)-rich, vitamin K-dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage-dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a gamma-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. 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. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. 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.     

Type I collagen influences cartilage calcification: an immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures

Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures.     

Long-term in vitro analysis of limb cartilage development: involvement of Wnt signaling

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.     

Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.     

Progression of chondrogenesis in C3H10T1/2 cells is associated with prolonged and tight regulation of ERK1/2

Close contact of mesenchymal cells in vivo and also in super dense micromass cultures in vitro results in cellular condensation and alteration of existing cellular signaling required for initiation and progression of chondrogenesis. To investigate chondrogenesis related changes in the activity of ubiquitous cell signaling mediated by mitogen-activated protein kinases (MAP kinase), we have compared the effect of cell seeding of pluripotent C3H10T1/2 mesenchymal cells as monolayers (non-chondrogenic culture) or high density micromass cultures (chondrogenic) on the regulation and phosphorylation state of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and also on regulation of ERK1/2 nuclear targets, namely, activation protein-1 (AP-1) and serum response factor (SRF). Increasing cell density resulted in reduced DNA binding as well as activity of AP-1. SRF activity, on the other hand, was up-regulated in confluent monolayer cultures but like AP-1 was inhibited in micromass cultures. Low levels of PD 98059 (5 microM), a specific inhibitor of ERK1/2, resulted in delayed induction of AP-1 and SRF activity whereas higher concentrations of this inhibitor (10-50 microM) conferred an opposite effect. Increasing concentrations of the PD 98059 inhibitor in long term monolayer or micromass cultures (2.5 day) resulted in differential regulation of c-Fos and c-Jun protein levels as well as total expression and phosphorylation levels of ERK1/2. PD 98059 treatment of C3H10T1/2 micromass cultures also resulted in up-regulation of type IIB collagen and Sox9 gene expression. While high expression of aggrecan and type IIB collagen genes were dependent on BMP-2 signaling, ERK inhibition of BMP-2 treated micromass cultures resulted in reduced activity of both genes. Our findings show that the activity of ERK1/2 in chondrogenic cultures of C3H10T1/2 cells is tightly controlled and can cross interact with other signaling activities mediated by BMP-2 to positively regulate chondrogensis.     

Identification of superficial zone articular chondrocyte stem/progenitor cells

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.     

Functions of transforming growth factor-beta family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes

We investigated the effects of bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta superfamily, on the regulation of the chondrocyte phenotype, and we identified signaling molecules involved in this regulation. BMP-2 triggers three concomitant responses in mouse primary chondrocytes and chondrocytic MC615 cells. First, BMP-2 stimulates expression or synthesis of type II collagen. Second, BMP-2 induces expression of molecular markers characteristic of pre- and hypertrophic chondrocytes, such as Indian hedgehog, parathyroid hormone/parathyroid hormone-related peptide receptor, type X collagen, and alkaline phosphatase. Third, BMP-2 induces osteocalcin expression, a specific trait of osteoblasts. Constitutively active forms of transforming growth factor-beta family type I receptors and Smad proteins were overexpressed to address their role in this process. Activin receptor-like kinase (ALK)-1, ALK-2, ALK-3, and ALK-6 were able to reproduce the hypertrophic maturation of chondrocytes induced by BMP-2. In addition, ALK-2 mimicked further the osteoblastic differentiation of chondrocytes induced by BMP-2. In the presence of BMP-2, Smad1, Smad5, and Smad8 potentiated the hypertrophic maturation of chondrocytes, but failed to induce osteocalcin expression. Smad6 and Smad7 impaired chondrocytic expression and osteoblastic differentiation induced by BMP-2. Thus, our results indicate that Smad-mediated pathways are essential for the regulation of the different steps of chondrocyte and osteoblast differentiation and suggest that additional Smad-independent pathways might be activated by ALK-2.     

Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.     

Combination of osteoinductive bone proteins differentiates mesenchymal C3H/10T1/2 cells specifically to the cartilage lineage

During embryonic development, cartilage formation involves the condensation of mesenchymal stem cells and a series of maturation steps that ultimately results in the mineralized hypertrophic chondrocyte. The embryonic, murine, mesenchymal stem cell line, C3H/10T1/2, is pluripotent; exposure to azacytidine or to bone morphogenetic protein-2 or -4 results in low rates of differentiation to three mesengenic lineages. In contrast to previous studies, we report conditions for 10T1/2 differentiation specifically to the cartilage lineage and at high yields. These conditions include high cell density micromass cultures, a purified mixture of osteoinductive proteins (BP; Intermedics Orthopedics, Denver, CO), a serum substitute, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate. The cartilagenous fate was confirmed by 1) histological detection of sulfated proteoglycans, 2) electron microscopic detection of proteoglycan and rounded cells separated by extracellular matrix containing short, disorganized collagen fibrils, 3) morphological detection of a chondrocytes surrounded by a territorial matrix and encompassed within a distinct perichondrium, and 4) immunocytochemical detection of type II collagen and link protein. After 4 weeks in culture, mature although unmineralized cartilage was observed, as indicated by hypertrophic morphology, immunocytochemical detection of osteocalcin, and histological detection of lacunae. These conditions promote overt chondrogenesis for most of the treated cells and preclude lineage determination to the fat, muscle, and bone lineages, as assayed by electron microscopy and histomorphology. The faithful recapitulation of cartilage differentiation that we have established in vitro provides a versatile alternative to the use of chondrocyte and limb bud explant cultures. We propose this as a model system to study the factors that regulate commitment to the chondrogenic lineage, exclusion to related mesengenic pathways, and maturation during chondrogenesis. J. Cell. Biochem. 65:325–339. © 1997 Wiley-Liss, Inc.     

Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes

Epiphyseal chondrocytes end their life cycle through apoptosis. While this event provides a mechanism for the removal of terminally differentiated cells from cartilage, agents that promote this physiological process have not been defined. To address this issue, using a cell culture technique that models events that take place in the growth plate, we asked the following questions: Can agents that promote chondrocyte maturation and cartilage mineralization serve as specific triggers for cell death? Are chondrocytes susceptible to apoptogens at a singular developmental stage? Treatment of embryonic tibial chondrocytes with inorganic phosphate (Pi) induced death in a dose- and time-dependent manner. Within 48 hr, 3 mM Pi increased chondrocyte death by 30%; lower concentrations of Pi induced death after 48 hr. To ascertain if death was due to apoptosis, we evaluated Pi-induced death by a number of different methods and compared the results to those induced by the apoptogen, staurosporine. Analysis of the death process indicated that cartilage cells shared many of the common biological features of the apoptotic process. Thus, there was DNA fragmentation, terminal deoxynucleotidyl transferase (TUNEL) labeling, an increase in cells in the sub-G1 fraction of the cell cycle, and morphological evidence of apoptosis. To explore the specificity of the Pi effect, the experiment was repeated using embryonic sternal cephalic and caudal chondrocytes, cells that are at an earlier developmental stage than the terminally differentiated tibial cells. We noted that these cells remained vital despite a major increase in the medium Pi content. Results of this study suggest that Pi is a stage-specific inducer of apoptosis in maturing chondrocytes and that this role may be linked to chondrocyte maturation and mineralization of the extracellular matrix.     

Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival

Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: Evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca²⁺ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca²⁺ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca²⁺- and Pi-rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5–2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca²⁺-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca²⁺ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca²⁺ and Pi is directly involved in endochondral calcification.     

Indian hedgehog in the late-phase differentiation in mouse chondrogenic EC cells, ATDC5: upregulation of type X collagen and osteoprotegerin ligand mRNAs

Endochondral bone formation includes a cascade of cellular events such as proliferation, maturation, hypertrophic conversion and calcification of chondrocytes and the cartilage replacement by bone. During these processes, hypertrophic conversion and calcification of chondrocytes (the late-phase differentiation) is a crucial process of chondrogenic differentiation. Indian hedgehog (Ihh), a secreted protein expressed in early hypertrophic chondrocytes, is thought to be involved in regulation of hypertrophic conversion via a feedback loop through the perichondrium. In the present study, we showed by Northern analysis and in situ hybridization that Smoothened (Smo), a key component in hedgehog signal transduction, was expressed in chondrocytes in both adult mice and mouse embryos at 16 days post-coitum in vivo, suggesting that Ihh directly acts on chondrocytes. We previously reported that Ihh, Patched and Smo were all expressed in differentiated ATDC5 cells. Exogenously administered mouse recombinant N-terminal protein of Ihh (mrIhh-N) upregulated the gene expression of type X collagen, a phenotypic marker of hypertrophic chondrocytes, as well as osteoprotegerin ligand (OPGL), a potent stimulator of osteoclastogenesis and osteoclast activity, while it did not modulate the expression of Ihh itself, bone morphogenetic protein (BMP)-4, BMP-6, transforming growth factor (TGF)-beta1 and TGF-beta2 in differentiated ATDC5 cells. Moreover, when added to the osteoclast cultures, mrIhh-N markedly stimulated the formation of resorption pits on dentine slices. Our data support the hypothesis that Ihh stimulated the late-phase chondrogenic differentiation in differentiated ATDC5 cells and upregulated the gene expression of OPGL in these cells.     

Cell proliferation, extracellular matrix mineralization, and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast-like cells

Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage- dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, F., C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblast-like cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.     

Transport of inorganic phosphate in primary cultures of chondrocytes isolated from the tibial growth plate of normal adolescent chickens

This report describes Pi transport activity in chondrocytes isolated from the growth plate (GP) of normal adolescent chickens grown in primary cell culture. Our recent work showed that Pi transport in matrix vesicles (MV) isolated from normal GP cartilage was not strictly Na+-dependent, whereas previously characterized Pi transport from rachitic GP cartilage MV was. This Na+-dependent Pi transporter (NaPiT), a member of the Type III Glvr-1 gene family, is expressed only transiently during early differentiation of GP cartilage, is enhanced by Pi-deficiency, and is most active at pH 6.8. Since GP mineralization requires abundant Pi and occurs under slightly alkaline conditions, it seemed unlikely that this type of Pi transporter was solely responsible for Pi uptake during normal GP development. Therefore we asked whether the lack of strict Na+-dependency in Pi transport seen in normal MV was also evident in normal GP chondrocytes. In fact, cellular Pi transport was found not to be strictly Na+-dependent, except for a brief period early in the culture. Choline could equally serve as a Na+ substitute. Activity of choline-supported Pi transport was optimum at pH 7.6-8.0. In addition, prior exposure of the cells to elevated extracellular Pi (2-3 mM) strongly enhanced subsequent Pi uptake, which appeared to depend on prior loading of the cells with mineral ions. Prevention of Pi loading by pretreatment with Pi transport inhibitors not only inhibited subsequent cellular Pi uptake, it also blocked mineral formation. Treatment with elevated extracellular Pi did not induce apoptosis in these GP chondrocytes.