Bovine Fetal Growth Plate Research Articles

Changes in Gene Expression Associated with Matrix Turnover, Chondrocyte Proliferation and Hypertrophy in the Bovine Growth Plate

The aim of the study is to investigate the interrelationships between the expression of genes for structural extracellular matrix molecules, proteinases and their inhibitors in the bovine fetal growth plate. This was analyzed by RT-PCR in microsections of the proximal tibial growth plate of bovine fetuses in relationship to expression of genes associated with chondrocyte proliferation, apoptosis, and matrix vascularization. In the resting zone the genes for extracellular matrix molecule synthesis were expressed. Extracellular matrix degrading enzymes and their inhibitors were also expressed here. Onset of proliferation involved cyclic upregulation of cell division-associated activity and reduced expression of extracellular matrix molecules. Later in the proliferative zone we noted transient expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis. With the onset of hypertrophy expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with vascularization and apoptosis were significantly upregulated. Terminal differentiation was characterized by high expression of proteinases and their inhibitors, extracellular matrix molecules, as well as activity associated with apoptosis. This study reveals the complex interrelationships of gene expression in the physis that accompany matrix assembly, resorption, chondrocyte proliferation, hypertrophy, vascularization and cell death while principal zones of the growth plate are characterized by a distinct signature profile of gene expression.

Spinal fusion with demineralized calf fetal growth plate as novel biomaterial in rat model: a preliminary study

BackgroundSpinal fusions are being performed for various pathologies of the spine such as degenerative diseases, deformities, tumors and fractures. Recently, other bone substitutes such as demineralized bone matrix (DBM) have been developed for spinal fusion. Therefore, this study was conducted to evaluate the intertransverse posterolateral fusion with the Bovine fetal growth plate (DCFGP) and compare it with commercial DBM in rat model.MethodsA total of 16 mature male rats (aged 4 months and weighing 200-300 g) were randomly divided in two groups. After a skin incision on posterolateral site, two separate fascial incisions were made 3 mm from the midline. A muscle-splitting approach was used to expose the transverse processes of L4 and L5. Group I (n = 8) underwent with implanted Bovine fetal growth plate among decorticated transverse processes. In group II (n = 8) commercial DBM was placed in the same manner. Fusion was evaluated by manual palpation, radiographical, gross and histopathological analysis.ResultsThe manual palpation, radiological, gross and histopathological findings indicate high potential of the DCFGP in spinal fusion. At the 42nd postoperative day, new bone formation as evidenced by a bridge between L4 and L5 was visualized in all rats implanted with DCFGP and commercial DBM. The newly formed bone tissue was observed in all implanted areas on the 42nd day after operation in the two groups.ConclusionsThe spinal fusion of the animals of both groups demonstrated more advanced osteogenic potential and resulted in proper fusion of the transverse process of lumbar vertebra.

Bovine fetal growth plate powder as a new osteoinduction biomaterial

Bovine fetal growth plate powder as a new osteoinduction biomaterial

Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

BackgroundRecent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.% ) of N.MethodsIn the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE:N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria®), were also investigated for comparison.ResultsResults showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE:N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels.ConclusionHypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving.

Comparison of novel xenograft (bovine fetal growth plate) and allograft effects on experimental bone defect healing in rabbit

Large bone defects resulting from trauma, tumors, osteitis, implant loosening, or corrective osteotomies require surgical therapy because spontaneous regeneration is limited to relatively small defects. Currently, transplantation of autografts or allografts, mineral bone substitutes, and callus distraction are the most commonly used techniques for skeletal reconstruction. Each method has significant limitations, e.g., availability and biological or biomechanical reasons. This study was designed to evaluate allograft and new xenograft (bovine fetal growth plate) effects on the bone healing process. Twenty male New Zealand White rabbits were used in this study. In the allograft group, the defect was filled by fresh allogeneic cortical graft; in the xenograft group, the defect was filled by a segment of bovine fetal growth plate and was fixed by cerclage wire. Radiological, histopathological, and biomechanical evaluations were performed and results were scored and analyzed statistically. Statistical tests did not support significant differences between the two groups radiographically at the 14th postoperative day (P > 0.05). There was a significant difference in bone formation at the 28th, 42nd, and 56th postoperative days. There were significant radiological differences for bone union and remodeling by the 42nd day postoperatively (P < 0.05). The xenograft was superior to the allograft by the 56th postoperative day for radiological bone formation (P < 0.03); histopathological and biomechanical evaluation revealed no significant differences between the two groups. It can be concluded that the superior bone healing process in the xenograft group was due to the presence of some osteoinduction proteins in bovine fetal growth plate.

Experimental bone defect healing with xenogenic demineralized bone matrix and bovine fetal growth plate as a new xenograft: radiological, histopathological and biomechanical evaluation

The following study was designed to evaluate xenogenic bovine demineralized bone matrix (DBM) and new xenograft (Bovine fetal growth plate) effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In group I (n = 10) the defect was filled by xenogenic DBM and in group II (n = 10) the defect was filled by a segment of bovine fetal growth plate and was fixed by cercelage wire. Radiological, histopathological, and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups radiographically (P > 0.05). There was a significant difference for union at the 28th postoperative radiologically (P < 0.05). Xenograft was superior to DBM group at the 28th postoperative day for radiological union (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups. In conclusion, the results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with xenogenic bovine DBM and xenogenic bovine fetal growth plate. Complications were not identified and healing was faster in two grafting groups.

Effect of bovine fetal growth plate as a new xenograft in experimental bone defect healing: radiological, histopathological and biomechanical evaluation

Bone grafting is used to enhance healing in osteotomies, arthrodesis, and multifragmentary fractures and to replace bony loss resulting from neoplasia or cysts. They are source of osteoprogenitor cells and induce bone formation and provide mechanical support for vascular and bone ingrowth. Autografts are used commonly but quantity of retrieved bone is limit. This study was designed to evaluate autograft and new xenograft (Bovine fetal growth plate) effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In autograft group the defect was filled by fresh autogenous cortical graft, in xenograft group the defect was filled by a segment of bovine fetal growth plate and was fixed by cercelage wire. Radiological, histopathological and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups at the 14th and 28th postoperative day radiographically (P > 0.05). There was a significant difference for remodeling at the 42nd postoperative radiologically (P < 0.05). Xenograft was superior to autograft at the 56th postoperative day for radiological bone formation (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups. The results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with calf fetal growth plate. Complications were not identified and healing was faster than cortical autogenous grafting. It was concluded that the use of calf fetal growth plate as a new xenograft is an acceptable alternative to cortical autogenous graft and could reduce the morbidity associated with harvesting autogenous graft during surgery.

Action of Estradiol on Epiphyseal Growth Plate Chondrocytes

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17beta-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7-21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T3), (3) E2 concentrations ranging from 10(-13) M to 10(-7) M, or (4) a combination of T3 and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10(-7) M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T3 did not appreciably affect the response to T3 alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T3, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.

Identification, distribution and expression of osteoadherin during tooth formation.

Osteoadherin (OSAD) is a keratan sulfate-containing proteoglycan, belonging to the small leucine-rich proteoglycan (SLRP) family. In bone OSAD has been localized in primary spongiosa within the bovine fetal rib growth plate. Moreover, in situ hybridization has shown expression of OSAD in osteoblasts close to the cartilage and bone border in the growth plate of rat femur. mRNA expression has also detected OSAD in mature osteoblasts on the surface of bone trabeculae. We have identified OSAD in extracts of bovine dentin, and the identity was verified by N-terminal sequencing. Western blot analysis detected two bands in bovine bone and dentin at 85 kDa and 60 kDa. Northern blot analysis of RNA samples from 5-d-old-rat tooth and femur showed a 1.9-kb transcript for OSAD in both tissues. OSAD was located to the mineralized dentin matrix, cementum and surrounding alveolar bone by immunohistochemistry, and in situ hybridization showed OSAD to be highly expressed during early crown formation in the entire odontoblast cell layer, in the area of Hertwig's epithelial root sheath, in the cells of the newly formed mantle dentin, and in the odontoblasts at the fissures. Ultrastructural studies indicated that OSAD might be associated with collagen fibrils. Thus, OSAD may play an important role during tooth development and biomineralization of dentin.

Four Distinct Chondrocyte Populations in the Fetal Bovine Growth Plate: Highest Expression Levels of PTH/PTHrP Receptor, Indian Hedgehog, and MMP-13 in Hypertrophic Chondrocytes and Their Suppression by PTH (1–34) and PTHrP (1–40)

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1–34) or PTHrP (1–40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28–48) and PTH (52–84) consistently stimulated the expression of PTHR-1 by 10–20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. ( Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.

Isolation and Identification of the Major Heparan Sulfate Proteoglycans in the Developing Bovine Rib Growth Plate

Heparan sulfate proteoglycans are thought to mediate the action of growth factors. The heparan sulfate-containing proteoglycans in extracts of the bovine fetal rib growth plate were detected using the monoclonal antibody 3G10, which recognizes a neoepitope generated by heparitinase digestion (David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J., and Van den Berghe, H. (1992) J. Cell Biol. 119, 961-975). The heparan sulfate proteoglycans that react with this antibody were identified using antisera to known proteoglycans; purified using CsCl density gradient centrifugation, molecular sieve, and ion exchange chromatography; and then characterized. The major heparan sulfate proteoglycans in the growth plate had core proteins of 200 kDa and larger and were identified as perlecan and aggrecan. These two heparan sulfate proteoglycans could be effectively separated from each other by CsCl density gradient centrifugation alone. Perlecan contained 25% heparan sulfate and 75% chondroitin sulfate. The heparan sulfate chains on growth plate perlecan were considerably smaller than the chondroitin sulfate chains, and the heparan sulfate disaccharide content was different than that found for heparan sulfate from either kidney, tumor tissue, or growth plate aggrecan. Aggrecan contained only 0.1% heparan sulfate, which was localized to the CS-1 domain of aggrecan. These results indicate that perlecan and aggrecan would be the principal candidate proteoglycans involved in the action of heparan sulfate-binding proteins in the developing growth plate.

The fibroblast growth factor receptor, FGFR3, forms gradients of intact and degraded protein across the growth plate of developing bovine ribs

Point mutations in the human fibroblast growth factor (FGF) receptor 3 gene (Fgfr3) produce a constitutively active receptor, which disrupts chondrocyte differentiation in the growth plate and results in skeletal dysplasias with severe shortening of the limbs. Alternative splicing of the Fgfr3 transcript gives rise to two isoforms, IIIc and IIIb, which vary in their specificity for FGF ligands. We examined the expression of these FGFR3 isoforms in the bovine fetal rib growth plate to determine whether levels of FGFR3 expression are zone-related. Transcripts for both Fgfr3 isoforms are expressed in rib growth plate, with maximum expression in the hypertrophic region and the least expression in the reserve zone. Fgfr3 IIIc is the predominant isoform in the growth plate. Western-blot analysis revealed the presence of full-length FGFR3 (135kDa) for both isoforms in the reserve zone, a major 98kDa fragment in all zones and smaller fragments primarily in the hypertrophic zone. Immunostaining localized FGFR3 to the pericellular region of reserve chondrocytes and to the extracellular matrix in the hypertrophic zone. These results suggest that the transmembrane form of FGFR3 increasingly undergoes proteolytic cleavage towards the hypertrophic zone to produce an extracellular-domain fragment of FGFR3, which is present in large amounts in the matrix of hypertrophic cells. These findings suggest a proteolytic regulatory mechanism for FGFR3, whereby Fgfr3 fragments could control availability of FGF for the intact receptor, and by which proteolysis could inactivate the receptor.

The fibroblast growth factor receptor, FGFR3, forms gradients of intact and degraded protein across the growth plate of developing bovine ribs

Point mutations in the human fibroblast growth factor (FGF) receptor 3 gene (Fgfr3) produce a constitutively active receptor, which disrupts chondrocyte differentiation in the growth plate and results in skeletal dysplasias with severe shortening of the limbs. Alternative splicing of the Fgfr3 transcript gives rise to two isoforms, IIIc and IIIb, which vary in their specificity for FGF ligands. We examined the expression of these FGFR3 isoforms in the bovine fetal rib growth plate to determine whether levels of FGFR3 expression are zone-related. Transcripts for both Fgfr3 isoforms are expressed in rib growth plate, with maximum expression in the hypertrophic region and the least expression in the reserve zone. Fgfr3 IIIc is the predominant isoform in the growth plate. Western-blot analysis revealed the presence of full-length FGFR3 (135 kDa) for both isoforms in the reserve zone, a major 98 kDa fragment in all zones and smaller fragments primarily in the hypertrophic zone. Immunostaining localized FGFR3 to the pericellular region of reserve chondrocytes and to the extracellular matrix in the hypertrophic zone. These results suggest that the transmembrane form of FGFR3 increasingly undergoes proteolytic cleavage towards the hypertrophic zone to produce an extracellular-domain fragment of FGFR3, which is present in large amounts in the matrix of hypertrophic cells. These findings suggest a proteolytic regulatory mechanism for FGFR3, whereby Fgfr3 fragments could control availability of FGF for the intact receptor, and by which proteolysis could inactivate the receptor.

Role of c-fos in the regulation of type X collagen gene expression by PTH and PTHrP: localization of a PTH/PTHrP-responsive region in the human COL10A1 enhancer.

PTH and PTHrP have been shown to inhibit maturation of growth plate chondrocytes and the expression of type X collagen. In order to examine the regulatory mechanisms involved, fetal bovine growth plate chondrocytes were incubated for 24-48 h under serum-free conditions with PTH and PTHrP and various aminoterminal, midregional, and carboxyterminal fragments of these hormones. Analysis of type X collagen mRNA levels by Northern hybridization showed a significant suppression by PTH (1-84), PTH (1-34), and PTHrP (1-40), but not by PTH (28-48) or PTH (53-84). PTH fragment (3-34) did not reduce alpha1(X) mRNA levels, while bis-indolylmaleimide, an inhibitor of the protein-kinase C pathway, did not affect alpha1(X) mRNA suppression by PTH, supporting the notion that the inhibition of type X collagen expression by PTH involves predominantly the adenylate cyclase pathway of the PTH/PTHrP-receptor. Since PTH and PTHrP have been shown to induce c-fos in osteoblasts and chondrocytes, the possibility was tested that c-fos mediated the suppressive effect of PTH/PTHrP on collagen X expression. In fetal bovine hypertrophic chondrocytes PTH (1-34), but not PTH (3-34) nor the midregional or C-terminal PTH fragments induced c-fos expression. In order to identify cis- and trans-acting elements in the COL10A1 gene involved in c-fos-mediated inhibition of collagen X expression by PTH/PTHrP, reporter gene constructs carrying various fragments of the human COL10A1 promoter coupled to the luciferase gene were transfected into hypertrophic chondrocytes. A tissue-specific, strong enhancer region, which we had previously located in the promoter of the human type X collagen gene COL10A1, was further narrowed down to a 530-bp sequence, located between - 1,870- and - 2,407 bp upstream of the transcription start site. The transcriptional activity of this enhancer element in transfected hypertrophic chondrocytes was significantly reduced after incubation with PTH (1-34) or PTHrP (1-40). Transcription of these reporter genes was also inhibited when chondrocytes were cotransfected with a c-fos expression vector. These results indicate the presence of a PTH/PTHrP responsive element in the human COL10A1 enhancer, which may be represented by multiple putative AP-1 sites located in this region.

Expression of Early and Late Differentiation Markers (Proliferating Cell Nuclear Antigen, Syndecan-3, Annexin VI, and Alkaline Phosphatase) by Human Osteoarthritic Chondrocytes

Although osteoarthritis is characterized by a progressive loss of the extracellular cartilage matrix, very little is known about the fate of articular chondrocytes during the progression of the disease. In this study we examined the expression of syndecan-3, a marker of early chondrocyte differentiation, and annexin VI, a marker of late chondrocyte differentiation, in mammalian embryonic growth plate cartilage and normal and osteoarthritic human articular cartilage. Whereas syndecan-3 was expressed in the proliferative and hypertrophic zones of growth platecartilage, immunostaining for annexin VI waspredominately found in the hypertrophic and mineralizing zones of fetal bovine growth plate cartilage. Approximately 20% of chondrocytes were immunopositive for syndecan-3 in normal human articular cartilage, the number of syndecan-3-expressing chondrocytes significantly increased during the progression of osteoarthritis with more than 80% syndecan-3-positive cells in the upper zone of severely affected osteoarthritic cartilage. Similarly, the number of annexin VI-expressing cells significantly increased in the upper cartilage zones during the progression of osteoarthritis. Furthermore, immunostaining for proliferating cell nuclear antigen, a marker for cell proliferation, was detected in chondrocytes in the upper zone of osteoarthritic cartilage. Double-labeling experiments with antibodies against syndecan-3 and annexin VI revealed chondrocytes that expressed only syndecan-3, and cells that expressed both syndecan-3 and annexin VI. These results suggest that the expression of early (proliferating cell nuclear antigen, syndecan-3) and late differentiation markers (annexin VI, alkaline phosphatase) is activated in chondrocytes of osteoarthritic cartilage.

Bone matrix proteins: isolation and characterization of a novel cell-binding keratan sulfate proteoglycan (osteoadherin) from bovine bone.

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride-containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mof the proteoglycan was 85, 000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin alphavbeta3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts.

Ultrastructural localization of the C-propeptide released from type II procollagen in fetal bovine growth plate cartilage.

We used immunochemical and immunoelectron gold techniques to determine whether the C-propeptide previously identified in the matrix of endochondral cartilage (CPII) was still a part of the Type 11 procollagen molecule or had been released from it. Guanidinium hydrochloride extraction, followed by SDS-PAGE and Western blotting techniques and immunoelectron localization, revealed that predominantly only the released form (hereafter referred to as released CPII) was detected. The ultrastructural distribution of this CPII was examined with affinity-purified antibodies and with immunogold or immunoperoxidase localization techniques in the presence or absence of embedding resins. These methods yielded similar results. Although no significant amount of this CPII was retained in the matrix after guanidinium hydrochloride extraction, it was present in two recognizable sites under normal conditions, i.e., locally concentrated in a random association with collagen fibrils in the nonmineralized matrix and mainly concentrated in interfibrillar mineralizing sites in the mineralized matrix. These results suggest that the C-propeptide that has been released from Type II procollagen associates with collagen fibrils and then preferentially associates with mineralizing sites when these form in the endochondral cartilage. The significance of this preference for mineral is not known but may have something to do with its high affinity for hydroxyapatite.

In serum-free culture thyroid hormones can induce full expression of chondrocyte hypertrophy leading to matrix calcification.

A serum-free culture system has been developed to examine the biologic factors involved in the regulation of cellular maturation, extracellular matrix assembly, and calcification in the physis of the bovine fetal growth plate. Isolated prehypertrophic chondrocytes in high density culture undergo a process of cellular maturation whereby full expression of the hypertrophic phenotype is characterized first by type X collagen synthesis followed by matrix calcification. Using this culture system, we compared the capacity of tri-iodothyronine (T3) with thyroxine (T4) to stimulate expression of the hypertrophic phenotype and matrix calcification in three (B, C, and D) maturationally distinct prehypertrophic chondrocyte subpopulations. The B cell subpopulation was the most mature followed by C and D subpopulations in order of decreasing maturity. Comparisons were made to cultures in fetal calf serum (FCS). In Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, and selenium, both hormones (T3/T4) separately induced, in a dose-dependent manner, chondrocyte maturation to the hypertrophic phenotype characterized by increased type X collagen mRNA and induction of protein synthesis of this molecule, together with increased alkaline phosphatase activity, and eventually calcification of the extracellular matrix. Such cellular maturation to the hypertrophic phenotype was not observed in the absence of T3 or T4 with subpopulations C and D. Only in older fetuses (> 210 days) was this observed and then only in the B subpopulation. Furthermore, T3 was at least 50-fold more potent than T4. The effects of T3 were most pronounced with the most immature cells (subpopulations C and D) where, in the case of the subpopulation C, in contrast to 0.5 nM T3 50 nM T4 was unable to induce expression of the hypertrophic phenotype. Alkaline phosphatase activity was also increased in the C cell subpopulation treated with 1 nM T3 (35.5 U/micrograms of DNA) over that supplemented with 50 nM T4 (7.8 U/micrograms of DNA). Furthermore, matrix calcification, measured by the incorporation of 45Ca2+ into the cell layer, always occurred earlier in cells cultured with T3 compared with T4. Cellular maturation to the hypertrophic phenotype was not accompanied by significant changes in DNA content; this ordinarily increases during culture in the presence of serum. Compared with cells cultured in the presence of serum, either thyroid hormone more potently induced cellular maturation. This study demonstrates that the most immature chondrocytes at the prehypertrophic stage are direct targets for T3 and T4 and, to a much a lesser degree, that either hormone is able to induce full chondrocyte hypertrophy from an early maturational stage leading to matrix calcification. But T3 is much more potent than T4. These studies also offer a new serum-free chemically defined medium containing T3 or T4 for the culture of defined prehypertrophic chondrocytes that supports matrix assembly, hypertrophic expression, followed by matrix calcification.

Cellular and matrix changes before and at the time of calcification in the growth plate studied in vitro: arrest of type X collagen synthesis and net loss of collagen when calcification is initiated.

To understand the growth, maturation, and regulation of growth plate chondrocytes, it is necessary to isolate the different chondrocytes into distinct subpopulations of maturational development. Five subpopulations (A-E) of bovine fetal growth plate chondrocytes were separated by discontinuous gradient centrifugation. Four subpopulations (B, C, D, and E, from low to high density) with good viability were cultured at high density in microwells for up to 30 days. They all established an extensive extracellular matrix composed of proteoglycan and collagen. The largest and last dense cells in subpopulation B were the first to synthesize (at days 5-6) type X collagen and to calcify this matrix. Matrix calcification (formation of hydroxyapatite in the presence of sodium beta-glycerophosphate) always followed the initiation of type X synthesis. All the other subpopulations synthesized type X collagen and calcified their extracellular matrix. Although these events occurred in the same order, they were delayed according to the order of increasing cell size. These observations indicate that these subpopulations represent different stages in cellular maturation that lead to expression of the hypertrophic phenotype. Once mineral formation was well established, there was an increase in the matrix content of the C-propeptide of type II collagen (which is known to bind to hydroxyapatite and accumulate in calcifying extracellular matrix). This was accompanied by a reduction in the total collagen content, which accompanied an abrupt reduction in type X collagen synthesis, whereas type II collagen synthesis was largely maintained. These reductions in collagen content and type II collagen synthesis were not observed in the absence of calcification (beta-glycerophosphate omitted from culture). This new culture system recreates many of the sequential cellular and extracellular changes exhibited in situ during the development of the physis and provides new information about cellular and extracellular matrix changes that occur before and at the time of calcification.

A Cartilage Explant System for Studies on Aggrecan Structure, Biosynthesis and Catabolism in Discrete Zones of the Mammalian Growth Plate

The structure, biosynthesis and catabolism of aggrecan has been studied in the bovine fetal rib growth plate. Comparative analyses were made on six 1-mm transverse slices which represent the resting zone (slice 6), proliferative zone (slices 5 and 4), upper hypertrophic zone (slice 3), middle hypertrophic zone (slice 2) and lower hypertrophic zone (slice 1). Aggrecan was abundant and exhibited very high aggregability in all zones. The aggrecan monomer was similar in structure in the resting and proliferative zones but showed a marked increase in hydrodynamic size in the lower hypertrophic zone; this was apparently due to an increase in the size of substituent glycosaminoglycans and an increase in core protein size as indicated by peptide analysis for G3 domain abundance. Biosynthetic studies with [35S]-sulfate showed the rate of synthesis per cell to be highest in the upper hypertrophic zone, and the structure of the newly synthesised molecules to be similar to the resident population in all zones.During explant culture in basal medium both aggregating and non-aggregating forms of aggrecan were released slowly from all zones. Addition of 10 nM retinoic acid to explants stimulated the release of both these forms of aggrecan whereas higher concentrations of retinoic acid (100 nM and 1000 nM) preferentially stimulated the release of the degraded forms. In this regard hypertrophic cells were the most responsive and resting cells were the least responsive. Analysis of the degraded fragments by polyacrylamide gel electrophoresis and by N-terminal sequencing indicated that aggrecan catabolism in all zones of the growth plate is due to the action of aggrecanase, a novel cartilage proteinase which is also active in normal and osteoarthritic articular cartilages (Sandy et al., 1992). These observations are discussed in terms of the role of aggrecan in the extensive matrix remodelling which accompanies chondrocyte hypertrophy in the growth plate.