Primary culture of rat growth plate chondrocytes: an in vitro model of growth plate histotype, matrix vesicle biogenesis and mineralization

During endochondral ossification (EO), cartilage is replaced by bone. Chondrocytes of growth plate undergo proliferation, maturation, hypertrophy, matrix vesicle (MV) biogenesis and programmed cell death (PCD, apoptosis). The in vitro system presented here provides a potential experimental model for studying in vitro differentiation and MV biogenesis in chondrocyte cultures. Chondrocytes were obtained from collagenase-digested tibial and femoral growth plate cartilage of 7-week-old rachitic rats. The isolated chondrocytes were plated as monolayers at a density of 0.5 × 10⁶ cells per 35-mm plate and grown for 17 days in BGJ_b medium supplemented with 10% fetal bovine serum, 50 μg/ml ascorbic acid. Light microscopy revealed Sirius red-positive, apparent bone matrix in layers at the surfaces of cartilaginous nodules that developed in the cultures. The central matrix was largely alcian blue staining thus resembling cartilage matrix. Electron microscopy revealed superficial areas of bone like matrix with large banded collagen fibrils, consistent with type I collagen. Most of the central matrix was cartilaginous, with small fibrils, randomly arranged consistent with type II collagen. The presence of peripheral type I and central type II and type X collagen was confirmed by immunohistochemical staining. Immunohistochemistry with anti-Bone morphogenetic proteins 2, 4 and 6 showed that BMP expression is associated with maturing hypertrophic central chondrocytes, many of which were TUNEL positive and undergoing cell death with plasma membrane breaks, hydropic swelling and cell fragmentation. During early mineralization, small radial clusters of hydroxyapatite-like mineral were associated with matrix vesicles. Collagenase digestion-released MVs from the cultures showed a high specific activity for alkaline phosphatase and demonstrated a pattern of AMP-stimulated nonradioactive ⁴⁰Calcium deposition comparable to that observed with native MVs. These studies confirm that primary cultures of rat growth plate chondrocytes are a reasonable in vitro model of growth plate histotype, MV biogenesis and programmed cell death.     

Cytosolic calcium concentration in bovine growth plate chondrocytes

The cytosolic free calcium ion concentration for mammalian cell systems is believed to be maintained within a narrow range compatible with cellular homeostasis. Growth plate chondrocytes have been shown to accumulate large quantities of calcium within their mitochondria, but the cytosolic free calcium concentration has not been determined. This study measures the cytosolic free ionic calcium concentration in growth plate chondrocytes using two variations of the Quin II fluorescence technique. The results indicate that in isolated growth plate chondrocytes, the cytosolic free ionic calcium concentration is similar to other nonmineralizing mammalian cell types (106–137 nM).     

Dwarfism in the Alaskan Malamute: Ultrastructural features of dwarf growth plate chondrocytes

Summary This study was performed in order to reexamine the ultrastructural morphology of the chondrocytes in the growth plates of dwarf Alaskan Malamutes and to obtain semiquantitative cytochemical data about the proteoglycans. Growth plates from age-matched dwarf and homozygous nonaffected Alaskan Malamutes were processed for routine transmission electron microscopy and also stained with ruthenium red. Chondrocytes in dwarf plates were observed to occur in clumps or cell nests. Within some of these nests, chondrocytes in the upper half of the zone of chondrocyte proliferation had bizarre shapes ranging from V-shaped to whorled or rounded. These chondrocytes contained profiles of markedly dilated rough endoplasmic reticulum (RER). Material within the RER cisternae stained positively with ruthenium red and was partially digestible with testicular hyaluronidase. The material could, therefore, represent either chondroitin sulfate or hyaluronate. The RER in these dwarf chondrocytes was not oriented parallel to the long axis of the cells; instead, it consisted of irregularly dilated cisternae. Granule counts performed on the zone of chondrocyte proliferation revealed a significant decrease in the number of ruthenium red granules in the interterritorial matrix of dwarf chondrocytes when compared to those of the homozygous nonaffected chondrocytes.     

Increased cAMP production after short-term capacitively coupled stimulation in bovine growth plate chondrocytes

Growth plate chondrocytes from newborn calf costochondral junctions grown in monolayer were subjected to a capacitive AC signal of 500 V peak to peak (P-P) at 60 kHz for 48 h and were analyzed for [3H]thymidine uptake. The stimulated chondrocytes showed a 130% greater uptake over unstimulated controls. Other newborn calf growth plate chondrocytes were stimulated at 500 V P-P at 60 kHz for 2.5, 5.0, 10.0, and 20.0 min and were analyzed for cAMP. Chondrocytes stimulated for 2.5 and 5.0 min showed a 142.8% (p less than 0.05) and 394.5% (p less than 0.01) increase over controls, respectively. The chondrocytes stimulated for 10.0 and 20.0 min showed no significant difference from the controls. It is concluded that short-term exposure of growth plate chondrocytes to an appropriate capacitively coupled field stimulates cAMP production, but longer-term application of the electrical field is ineffective.     

Demonstration of type I and type II somatomedin receptors on bovine growth plate chondrocytes

The chondrocytes of the epiphyseal growth plate are the presumed target cells for hormones regulating skeletal growth. The somatomedins, a family of low molecular weight peptides, are thought to play a stimulatory role in this regulation. The cellular actions of the somatomedins are themselves determined by binding to specific receptors on target cells. Previous studies have characterized a specific receptor for somatomedin-C (Sm-C) or insulin-like growth factor I (IGF-I) on bovine growth plate chondrocytes (GPCs). We now report the characterization of a second type of somatomedin receptor on these cells that is more specific for another class of somatomedin represented by multiplication-stimulating activity (MSA) or rat insulin-like growth factor II (rIGF-II). Binding of [125I]MSA/rIGF-II to isolated GPCs was time dependent and saturable. Unlabeled Mr 7,100 MSA/rIGF-II and Sm-C/IGF-I were approximately equipotent in competing with [125I]MSA/rIGF-II for binding, while Mr 8,600 MSA/rIGF-II was an order of magnitude less potent. Low levels of competition by insulin appeared in some studies at concentrations of 10(-7) M and higher, suggesting displacement of [125I]MSA/rIGF-II binding to the Sm-C/IGF-I receptor. In affinity-labeling studies, [125I]MSA/rIGF-I labeled a complex of Mr greater than 300,000 (unreduced) and of Mr 140,000 (reduced), consistent with a type I somatomedin receptor composed of disulfide-linked subunits. [125I]MSA/rIGF-II labeled a Mr 240,000 moiety (unreduced) and Mr 260,000 (reduced), consistent with a type II somatomedin receptor. Both affinity-labeling and kinetic data revealed cross-binding of MSA/rIGF-II and insulin with the type I receptor and of Sm-C/IGF-I with the type II receptor. In contrast, the type II receptor did not recognize insulin. These data suggest a complex pattern of graded specificity of these receptors for their ligands. These data are consistent with the hypothesis that IGF-II as well as Sm-C/IGF-I participate in the stimulation of skeletal growth.     

The effect of oxygen tension on proteoglycan synthesis and aggregation in mammalian growth plate chondrocytes

Growth plate chondrocytes isolated from the proliferative and hypertrophic zones of bovine costochondral junctions were grown in vitro in the presence of various oxygen tensions ranging from 3 to 60%. Using [35S] sulfate as an index of glycosaminoglycan synthesis, incorporation was found to be maximal at 21% O2. In contrast, proteoglycan aggregation under the same conditions was found to be maximal at 3% O2. There were no consistent differences in response between cells from the different morphologic zones even though they are exposed to different oxygen tensions in situ. These results show that proteoglycan synthesis and aggregation in growth plate chondrocytes in vitro are differentially affected by the ambient oxygen environment.     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

Induction of bone by xenografts of rabbit growth plate chondrocytes in the nude mouse

Summary Subcutaneous transplantation of growth plate chondrocytes isolated enzymatically from the proximal tibia of 6-week-old rabbits into athymic (nu/nu) mice resulted in the formation of cartilaginous nodules. Calcification of the matrix was first seen after 48 hrs, and endochrondral ossification at 12 days. The mineral first occurred about hypertrophic cells. Histochemical alkaline phosphatase activity was concentrated in pericellular collars at the same location. Immunofluorescence examination with rabbit anti-mouse lymphocyte serum disclosed that the bulk of the osteoblasts was derived from the mouse. A small quantity of mouse antigen was present in the cartilage matrix at its junction with bone. It presumably diffused into the cartilaginous interface from the host, but the possibility that some chondrocytes were of murine origin has not been excluded. Five of six grafts of cells grown to confluence in monolayer culture for 10 to 14 days became ossified. The ability to induce mineralization declined in subculture. Chondrocytes killed by heating to 56° did not induce calcified cartilage or bone. Supported by grant AM17258-09 from the National Institutes of Health.     

Molybdenum-induced changes in the epiphyseal growth plate

Summary Molybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.     

组织工程软骨移植修复兔生长板缺损

目的探讨一种新的修复生长板缺损的方法,应用组织工程软骨治疗生长板缺损并发肢体畸形。方法分离收集1月龄兔关节软骨,于离心管内培养2周形成组织工程软骨。36只6周龄新西兰白兔随机分为3组,于右侧胫骨上端生长板内侧造成1/3～1/2缺损,A组即刻植入培养软骨,B组及C组分别于术后4周、术后8周再次手术,切除缺损内修复组织后再移植培养软骨。左侧胫骨经相同手术造成缺损,但无植入物充填,仅作为对照组。A组于手术后16周、B组于再手术后12周、C组于再手术后8周进行双下肢X线、组织学及免疫组织化学等检查,测量双侧胫骨长度和胫骨角,观察生长板缺损修复情况。结果培养软骨呈圆盘状,直径8mm,厚1.5mm。A、B组右侧胫骨生长显著优于左侧,内翻和短缩畸形右侧较左侧轻(P<0.01)。A、B组右侧生长板缺损经组织修复后恢复正常生长板结构和性质,Ⅱ型胶原和IGF-IRa免疫组织化学染色呈阳性;左侧胫骨生长板缺损由新生骨充填。C组双侧胫骨均发生严重畸形(P>0.05),生长板闭合或接近闭合。结论离心管培养组织工程软骨移植可以有效防止4周内生长板缺损并发早期肢体畸形,但对生长板缺损8周已形成畸形者无效。     

Lectin-binding histochemistry of intracellular and extracellular glycoconjugates of the reserve cell zone of growth plate cartilage

The distribution of intracellular and extracellular lectin-binding glycoconjugates of the reserve cell zone of growth plate cartilage was studied in the distal radial growth plate of 4-week-old Yucatan swine using a postembedment method on Epon-embedded sections. Direct comparisons were made to articular, tracheal, and auricular cartilages not involved in endochondral ossification. All patterns of lectin binding that in the growth plate were restricted to the reserve cell zone were also patterns characteristic of tracheal, articular, and auricular cartilages. These included: (a) pericellular binding with peanut agglutinin (PNA) without prior digestion with neuraminidase; (b) pericellular binding with wheat germ agglutinin (WGA) at 24 h; (c) intracellular cytoplasmic binding to concanavalin A (CON-A), Lens culinaris agglutinin (LCA), and Lotus tetragonobolus agglutinin (LTA) after periodic acid oxidation; and (d) a lack of pericellular binding with CON-A and ricin agglutinin 1 (RCA-1) after periodic acid oxidation. We conclude that reserve zone chondrocytes lack specific phenotypic markers as defined by lectin-binding affinity that are found in the cellular zones of the growth plate that undergo calcification and vascularization. The reserve zone has identical lectin-binding affinities to the three structural cartilages used as controls. One interpretation of these results is that the reserve zone may not be involved directly in endochondral ossification, but may have a structural function in growth plate cartilage.     

Mitochondrial granules in chondrocytes

Rat and mice epiphyseal growth plates were studied with the electron microscope. A gradient of mitochondrial electron-dense granules was found. Chondrocytes in the proliferative zone had few granules, while those of the succeeding zones showed a gradual increase in number and density until the zone of provisional calcification was reached. This zone showed a peripheral distribution of mitochondria and a decrease in the number and density of mitochondrial granules. Isotopic⁴⁷calcium was used autoradiographically to determine the location of calcium in these cells. Grains were found over the endoplasmic reticulum membranes and over most mitochondria. The demonstration of a gradient of these granules and their spatial relation to the mineralization front suggests a possible involvement of mitochondria in the onset of matrix calcification.This work was supported in part by a grant No. D.E. 224-01 from the National Institutes of Health, United States Public Health Service.     

大鼠胫骨生长板软骨细胞的分离与培养鉴定

目的 探讨高效生长板软骨细胞分离方法 和体外培养条件.方法 采用二步酶消化法对6只SD大鼠胫骨生长板的软骨细胞进行分离,采用含炭吸附过的胎牛血清培养基培养,按5×105个/瓶的密度接种细胞并对软骨细胞进行形态学观察和鉴定,描绘原代软骨细胞在无激素培养基中的生长曲线.结果 软骨细胞贴壁较慢,12 h后开始附壁,第8天时90%融合,互相连接成"铺路石"样结构.原代软骨细胞胞质Ⅱ型胶原免疫着色强阳性,传代后染色减弱.结论 本研究所采用的方法 能高效快速获得原代软骨细胞,原代软骨细胞最接近体内生理状态,最适合进行实验研究.     

Alterations of the growth plate in chronic renal failure

Chronic renal failure modifies the morphology and dynamics of the growth plate (GP) of long bones. In young uremic rats, the height of cartilage columns of GP may vary markedly. The reasons for this variation are unknown, although the severity and duration of renal failure and the type of renal osteodystrophy have been shown to influence the height of GP cartilage. Expansion of GP cartilage is associated with that of the hypertrophic stratum. The interference of uremia with the process of chondrocyte differentiation is suggested by some morphological features. However, analysis by immunohistochemistry and/or in situ hybridization of markers of chondrocyte maturation in the GP of uremic rats has yielded conflicting results. Thus, there have been reported normal and reduced mRNA levels for collagen X, parathyroid hormone/parathyroid hormone-related peptide receptor, and matrix metalloproteinase 9, as well as normal mRNA and protein expression for vascular endothelial growth factor and chondromodulin I, peptides related to the control of angiogenesis. In addition, a decreased immunohistochemical signal for growth hormone receptor and low insulin-like growth factor I mRNA in the proliferative zone of uremic GP are supportive of reduced chondrocyte proliferation. Growth hormone treatment improves chondrocyte maturation and activates bone metabolism in the primary spongiosa.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine

Summary Postnatal bone growth occurs by the process of endochondral ossification in cartilaginous growth plates at the ends of long bones. The rate and extent of long bone growth is determined by a combination of chondrocytic proliferation, matrix production, and increase in chondrocytic height in the direction of growth during cellular enlargement. In this study, single pulse and/or repeated pulse labeling with the thymidine analog bromodeoxyuridine (BrdU) was used to study the role of cellular proliferation in controlling long bone growth. Variables studied included progression of the label over time following a pulse, and patterns and progression of the label over time following repeated pulse labeling for 24 and 48 hours. Examination was made of the proliferative characteristics of chondrocytes, the spatial pattern of cellular proliferation, and cell cycle kinetics. With respect to the spatial pattern of proliferative chondrocytes, results suggest that chondrocytes within a column are more synchronized with each other than are chondrocytes in different columns. This is consistent with the concept that each column represents a clonal expansion of a stem cell, which may proceed independently from adjacent columns. Despite this apparent heterogeneity, all chondrocytes in the proliferative zone complete at least one cell cycle in 24–28 hours. This estimate of the cell cycle time is significantly shorter than previous estimates of cell cycle times in similar growth plates. Our results also suggest that chondrocytes entering the cell cycle in the proximal part of the growth plate spend an average of 4 days in the proliferative cell zone, representing approximately four cellular divisions. After leaving the cell cycle, an additional 48 hours is required for the label to reach the terminal chondrocyte, which represents the time required to complete hypertrophy. These data are important when considering hypotheses concerning both the role of controls on proliferation in the determination of overall rate of long bone growth, as well as the interplay between proliferation and hypertrophy in regulating the overall amount of growth achieved by a given growth plate.     

Interaction of basic fibroblast growth factor with bovine growth plate chondrocytes.

The basic fibroblast growth factor (bFGF) family of peptides influences a wide range of cellular actions. To better understand the possible role of bFGF in the growth plate, we have characterized the interaction of this growth factor with isolated bovine growth plate chondrocytes. Basic FGF interacts with two classes of binding sites on these cells. One is consistent with high-affinity bFGF receptors and the other with low-affinity heparin-like binding sites on the chondrocyte surface. Radiolabeled bFGF binding studies revealed approximately 4 x 10(6) binding sites per cell, with a Kd of approximately 42 nM. Graded concentrations of heparin or NaCl competed with [125I]-labeled bFGF in a dose-dependent fashion, reducing [125I]-labeled bFGF binding by 75 and 97%, respectively. The data suggest the presence of a high-capacity, low-affinity class of binding sites with the properties of a heparin-like moiety. Affinity cross-linking of [125I]-labeled bFGF to chondrocytes labeled two principal species with apparent molecular masses of 135 and 160 kDa. Labeled bFGF was specifically displaced from both species by subnanomolar concentrations of unlabeled bFGF. These high-affinity, low-capacity binding sites are characteristic of classical bFGF receptors. Binding of [125I]-labeled bFGF to these sites was also influenced by heparin, consistent with coregulation of binding to the two classes of binding sites. The data suggest that bFGF participates in the regulation of skeletal growth at the growth plate and that this regulation may involve bFGF interaction with at least two distinct classes of binding sites.     

Intramembranous ossification mechanism for bone bridge formation at the growth plate cartilage injury site.

Salter's type III and type IV growth plate injuries often induce bone bridge formation at the injury site. To understand the cellular mechanisms, this study characterized proximal tibial transphyseal injury in rats. Histologically, bony bridge trabeculae appeared on day 7, increased on day 10, and became well-constructed on day 14 with marrow. Prior to and during bone bridging, there was no cartilage proteoglycan metachromatic staining and no collagen-X immunostaining at the injury site, nor was there any up-regulation of BrdU-labelled chondrocyte proliferation at the adjacent physeal cartilage, suggesting no new cartilage formation at the injury site. However, infiltration of vimentin-immunopositive mesenchymal cells from metaphysis and epiphysis was apparent on day 3, with the mesenchymal population being prominent on days 7 and 10 and subsided on day 14. Among these infiltrates were osteoprogenitor precursors expressing osteoblast differentiation factor (cbf-alpha1) on day 3, along with some cbf-alpha1+ osteoblast-like cells lining bone trabeculae on days 7 and 10. Some mesenchymal cells and trabecula-lining cells were also alkaline phosphatase-immunopositive, further suggesting their osteoblast differentiation. From day 7 onwards, some trabecula-lining cells became osteocalcin-producing mature osteoblasts. These results suggest that bone bridge formation after growth plate injury occurs directly via intramembranous ossification through recruitment of marrow-derived osteoprogenitor cells.