Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor
ABSTRACT Chondrogenesis is an essential component of endochondral fracture healing, though the molecular and cellular events by which it is regulated have not been fully elucidated. In this study, we used a rat model of closed fracture healing to determine the spatial and temporal expression of genes for cartilage-specific collagens. Furthermore, to determine the effects of basic fibroblast growth factor (bFGF) on chondrogenesis in fracture healing, we injected 100 μg recombinant human bFGF into the fracture site immediately after fracture.In normal calluses, pro-(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-(X) collagen mRNA (COL10A1) in hypertrophic chondrocytes beginning on day 7. In FGF-injected calluses, the cartilage enlarged in size significantly. On day 14, both COL2A1-and COL10A1-expressing cells were more widely distributed, and the amounts of COL2A1 and COL10A1 mRNAs were both approximately 2-fold increased when compared with uninjected fractures. Temporal patterns of expression for these genes were, however, identical to those found in normal calluses. The number of proliferating cell nuclear antigen-positive cells was increased in the non-cartilaginous area in the bFGF-injected calluses by day 4.The present molecular analyses demonstrate that a single injection of bFGF enhances the proliferation of chondroprogenitor cells in fracture callus, and thus contributes to the formation of a larger cartilage. However, maturation of chondrocytes and replacement of the cartilage by osseous tissue are not enhanced by exogenous bFGF, and this results in the prolonged cartilaginous callus phase. We conclude that, in the healing of closed fractures of long bones, exogenous bFGF has a capacity to enlarge the cartilaginous calluses, but not to induce more rapid healing. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.
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ABSTRACT: We characterized gene expression in the reparative callus that formed after fracture of the rat femur. The callus was divided into regions of bone formation (hard callus) and cartilage formation (soft callus), and gene expression was examined separately in each region. Expression of extracellular matrix protein genes varied with the progression of repair and differed between hard and soft calluses. Messenger ribonucleic acids (mRNAs) for osteonectin, alkaline phosphatase, and type I procollagen were detected in the hard callus at maximal levels during endochondral ossification and bone remodeling (day 15) and at 50% maximal levels during intramembranous bone formation (day 7). Messenger RNAs for these proteins in the soft callus were detected at low levels during chondrogenesis (day 9) but increased to 80% of maximal levels with chondrocyte hypertrophy and mineralization of the cartilage matrix (day 13). Messenger RNAs for type II procollagen and proteoglycan core protein were detected at maximal levels in the soft callus during chondrogenesis (day 9). Osteocalcin gene expression was detected in the hard callus during endochondral ossification and remodeling but not during intramembranous bone formation or at any time in the soft callus. Osteonectin mRNA was detected in both the hard and soft callus throughout the entire course of fracture repair. Expression of cartilage and bone-related genes correlated with the temporal sequence of histologic changes, suggesting transcriptional regulation of gene expression during repair. Differences in gene expression between hard and soft callus and in each of these regions as repair progressed suggest local regulation of gene expression during cell differentiation and matrix synthesis.Journal of Bone and Mineral Research 10/1992; 7(9):1045-55. · 6.13 Impact Factor
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ABSTRACT: The role of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I) in cartilage growth was studied in primary cultures of rat rib growth plate chondrocytes. Growth factors effects on expression of the proto-oncogene c-fos, DNA synthesis, differentiation, and morphological changes were analyzed by in situ hybridization, 3H-thymidine incorporation, and light and fluorescence microscopy. In serum-deprived cells, bFGF induced a transient expression of c-fos with a maximal effect 15-30 minutes after stimulation. After 24 h of culture it had a slightly lower stimulatory effect on DNA synthesis than IGF-I, but became a significantly more potent mitogen than IGF-I after 48 and 72 h. The stimulatory effect of bFGF on DNA synthesis coincided with a decrease in collagen type II and IGF-II expression. In contrast, IGF-I alone stimulated expression of these genes. In bFGF-treated cultures, cell morphology and the appearance of actin filaments was changed. Polygonal chondrocytes became elongated, fibroblast-like, and the smooth actin filaments were brush-like and disrupted. Addition of IGF-I reduced these changes without affecting c-fos expression induced by bFGF. Our results suggest that bFGF stimulates cell proliferation by preventing terminal differentiation of chondrocytes. This effect is mediated by induction of c-fos expression and a decrease in the steady-state levels of transcripts for collagen II and IGF-II.Journal of Bone and Mineral Research 06/1995; 10(5):735-42. · 6.13 Impact Factor
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ABSTRACT: Fibroblast growth factors are present in significant amounts in bone and several studies have suggested that they may be involved in normal fracture healing. It is well established that fibroblast growth factors have mitogenic and angiogenic activity on mesoderm and neuroectoderm derived cells. Of particular interest as a member of the fibroblast growth factor family, basic fibroblast growth factor stimulates mitogenesis, chemotaxis, differentiation, and angiogenesis. It also plays an important role in the development of vascular, nervous, and skeletal systems, promotes the maintenance and survival of certain tissues, and stimulates wound healing and tissue repair. Animal studies have shown that the direct injection of fibroblast growth factor into fresh fractures stimulates callus formation, which provides mechanical stability to the fracture, accelerates healing, and restores competence. The matrix used to present the fibroblast growth factor at the fracture site plays a critical role in the effectiveness of the treatment. The evaluation of injectable basic fibroblast growth factor in a sodium hyaluronate gel for its effectiveness in stimulating fracture healing is described. When applied directly into a freshly created fracture in the rabbit fibula, a single injection of the basic fibroblast growth factor and hyaluronan results in the stimulation of callus formation, increased bone formation, and earlier restoration of mechanical strength at the fracture site. The hyaluronan gel serves as a reservoir that sequesters the basic fibroblast growth factor at the injection site for the length of time necessary to create an environment conducive to fracture healing. It is concluded that basic fibroblast growth factor and sodium hyaluronate act synergistically to accelerate fracture healing and that the combination is suitable for clinical evaluation as a therapy in fracture treatment.Clinical Orthopaedics and Related Research 11/1998; · 2.79 Impact Factor