Growth factor regulation of human growth plate chondrocyte proliferation in vitro (original) (raw)
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In Vitro Cellular & Developmental Biology - Animal, 1992
We have developed a serum-free system to culture postembryonic growth plate chondrocytes while maintaining some important phenotypic characteristics of their tissue of origin. This serum-free medium was as effective as medium containing 10% newborn bovine serum (NBS) for recovering the cells from enzymatic isolation. Surface secretory activity of ehondrocytes cultured in monolayer, assessed through scanning electron microscopy, was also comparable to cells grown in medium containing serum. The effects of growth hormone (GH) and insulinlike growth factor-I (IGF-I) were also studied using the serum-free medium. GH had no effect on cell density and morphology of the cells compared to the control without the hormone. In contrast, ehondrocytes grown in medium containing IGF-I had a marked increase in cell density after 3 days and presented similar morphologic characteristics to cells grown in the presence of NBS. The growth factors required for proliferation of chondrocytes cultured in the serum-free medium are IGF-I and fibroblast growth factor (100 ng/ml, respectively). Addition of ascorbic acid to the serum-free medium (0 to 50 #g/ml) produced a dose-dependant decrease in cell proliferation. This medium should provide a useful tool for studying the effects of different growth factors/hormones in the regulation of longitudinal bone growth and their interactions.
Life Sciences, 1997
Growth plate width and cartilage organization are altered during skeletal unloading in growing rats. Immunohistochemical studies have identified TGF-l3 in calcified cartilage, and TGF-D is known to induce mitogenic effects on chondrocytes in vitro: On the other hand, IGF-1 was shown to be expressed in the proximal tibial growth plate and to mediate GH-induced longitudinal bone growth in rats. We therefore investigated the effect of recombinant human (rh) IGF-1 and rhTGF-82 infusion on the changes induced by unloading in the cellular organization of the growth plate in growing rats. Hindlimb unloading for 14 days induced a 13% reduction in growth cartilage height in the proximal tibia. This effect was mostly related to a 17% and 14% decrease in the proliferative zone height and chondrocyte number, respectively. In unloaded rats treated with a systemic infusion of rhTGF-82 (2ugikg/day) the number of chondrocytes in the proliferative zone was not different from those of normal loaded animals. In contrast, rhIGF-1 treatment at a 2mg/kg/day dose was not effective in counteracting the effects of unloading on growth plate height and chondrocyte number. These results show that systemic administration of rhTGF-82 prevents in large part the reduced growth of chondrocytes in the proliferative zone and the reduced epiphyseal growth plate growth induced by unloading in rats.
Growth factor responsiveness of human articular chondrocytes in aging and development
Arthritis & Rheumatism, 1995
The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor+ (TGF-P) is the most potent mitogen among a variety of factors tested. All three isoforms of TCF-P caused similar dose-dependent increases in chondrocyte proliferation. Other members of the TGF-P family, including bone morphogenetic protein 28 (BMPZB), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFCF), and insulin-like growth factor 1 (ICF-1) also stimulated proliferation but were less effective than TGF-P. In contrast to findings with other cell types, the effects of TCF-P on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines lnterleukin 1 (IL-1) and tumor necrosis factor-a (TNF-a) gave no stimulation, but IL-1 inhibited chondrocyte proliferation induced by TCF-P or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL-1 caused greater increases in proliferation than TCF-6.
Journal of Endocrinology, 2005
Linear bone growth depends upon proliferation, maturation, and apoptosis of growth plate chondrocytes, processes regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). To investigate the contribution of GH, IGF-I and apoptosis to growth plate function, the expression of GH receptor (GHR) and IGF-I receptor (IGF-IR) mRNA were evaluated by in situ hybridization in fractionated costochondral growth plates of growing rats (at 2, 4, and 7 weeks). Apoptosis was determined by TUNEL assay and morphology in histological sections. GHR mRNA was greatest in resting cells with hypertropic cells increasing GHR expression with increasing age. Hypertropic and resting cell IGF-IR mRNA declined over the ages studied. Receptor mRNA expression was altered by exposing cells to GH or IGF-I. GH and IGF significantly decreased GHR mRNA in proliferative cells. GH and IGF also decreased IGF-IR mRNA in resting cells and the 2- and 4-week-old proliferative and hypertropic cells. Treating ce...
Induction of chondrocyte growth arrest by FGF: transcriptional and cytoskeletal alterations
Journal of Cell Science, 2002
The effect of fibroblast growth factor (FGF) on mature chondrocytes, the cells responsible for axial skeletal development, is growth attenuation rather than stimulation. This singular response has been linked to signaling via FGF receptor 3 (FGFR3), partly because mutations causing chronic FGFR3 activation lead to various human disorders of bone growth. In order to study how FGF inhibits growth, we analyzed its effect on a rat chondrocyte-derived cell line. We show that the FGF-induced growth arrest occurs at the G1 phase,accompanied by profound changes in gene expression and cytoskeletal organization. Within minutes of binding, FGF induces tyrosine kinase activity in the focal substrate adhesions where it colocalizes with vinculin. Upon FGF stimulation, FGFR3 is selectively removed from the focal adhesions, which is followed by their disassembly and disruption of the organized cytoskeleton. Multiple genes are induced following FGF stimulation in chondrocytes, which has been shown b...
Biochemical and Biophysical Research Communications, 2002
This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant downregulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.
Regulation of Growth Plate Chondrogenesis by Bone Morphogenetic Protein-21
Endocrinology, 2001
Bone morphogenetic proteins (BMPs) regulate embryonic skeletal development. We hypothesized that BMP-2, which is expressed in the growth plate, also regulates growth plate chondrogenesis and longitudinal bone growth. To test this hypothesis, fetal rat metatarsal bones were cultured for 3 days in the presence of recombinant human BMP-2. The addition of BMP-2 caused a concentration-dependent acceleration of metatarsal longitudinal growth. As the rate of longitudinal bone growth depends primarily on the rate of growth plate chondrogenesis, we studied each of its three major components. BMP-2 stimulated chondrocyte proliferation in the epiphyseal zone of the growth plate, as assessed by [ 3 H]thymidine incorporation. BMP-2 also caused an increase in chondrocyte hypertrophy, as assessed by
Bone, 2004
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 Â 10 6 cells per 35-mm plate and grown for 17 days in BGJ b medium supplemented with 10% fetal bovine serum, 50 Ag/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 40 Calcium 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.
Journal of Bone and Mineral Research, 2009
Osteogenic protein-1 (OP-1, also called BMP-7) is a bone morphogenetic member of the TGF-P superfamily. In the present study, we examined the effect of recombinant human OP-1 on cartilage and bone formation in organ cultures of metatarsal long bones of mouse embryos and compared the OP-1 effects with those of human TGF-P, and porcine TGF-PI and P2. Cartilage formation was determined by measurement of longitudinal growth of whole bone rudiments during culture and by the incorporation of 35S04 into glycosaminoglycans. Mineralization was monitored by %a incorporation in the acid-soluble fraction and by measuring the length of the calcifying center of the rudiment. Toluidine blue-stained histologic sections were used for 'quantitative histomorphometric analysis. We found that OP-1 stimulated cartilage growth as determined by sulfate incorporation and that it increased remarkably the width of the long bones ends compared with controls. This effect was partly caused by differentiation of perichondrial cells into chondracytes, resulting in increased appositional growth. In contrast to OP-1, TGF-P, and P2 inhibited cartilage growth and reduced the length of whole bone rudiments compared with controls. In the ossifying center of the bone rudiments, both OP-1 and TGF-P inhibited cartilage hypertrophy, growth of the bone collar, and matrix mineralization. These data demonstrate that OP-1 and TGF-P exhibit opposite effects on cartilage growth but similar effects on osteogenesis in embryonic mouse long bone cultures. Since both OP-1 and TGF-P have been demonstrated in embryonic cartilage and bone, these results suggest that they act as autocrine or paracrine regulators of embryonic bone development. spine. Spine 17:S33-36. 45. Sauren YMHF, Mieremet RHP, Groot CG, Koerten HK, Scherft JP 1991 Polyethyleneimine as a contrast agent for ultrastructural
Bone, 1996
Platelet-derived growth factors (PDGFs) are potent bone cel1 mitogens which stimulate the proliferation of osteoblastic cells, may also be involved in the regulation of osteoclastic bone resorption, and indirectly induce vascular endothelial cel1 proliferation and angiogenesis. In view of the established relationship between angiogenesis and osteogenesis, the production of PDGFs by both osteoblastic and vascular endothelial cells suggests that they may play a role in bone formation during skeletal development. We have used two human models of rapid bone formation, heterotopic bone and osteophytic bone, to investigate the expression of PDGF-A mRNA and protein and the PDGF-cw receptor protein in vivo using in situ hybridization and immunohistochemistry. PDGF-A mRNA and protein were widely distributed throughout heterotopic and osteophytic bone. Within the cartilaginous tissue PDGF-A mRNA and protein were most strongly expressed by mature chondrocytes with decreased expression in the hypertrophic zone and almost no staining in the mineralizing and mineralized zones. PDGF mRNA and protein were also expressed in cells of smal1 blood vessels within fibrous and cartilaginous tissue. In contrast, PDGF-cu receptor expression was restricted to a niinority of hypertrophic chondrocytes and sites of vascular invasion. Within the bone and fibrous tissue the growth factor and the receptor were widely distributed, being detected on most cells at sites of bone formation or in remodeling sites; no receptor was detected on osteoclasts. These data demonstrate the widespread expression of PDGF-A and its receptor in forming human bone and indicate that this growth factor may exert autocrine and paracrine effects to regulate osteogenesis during skeletal development.