Spatial and temporal gene expression in chondrogenesis during fracture healing and the effects of basic fibroblast growth factor.

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 microg recombinant human bFGF into the fracture site immediately after fracture. In normal calluses, pro-alpha1(II) collagen mRNA (COL2A1) was detected in proliferative chondrocytes beginning on day 4 after the fracture, and pro-alpha1(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.     

Molecular basis for affected cartilage formation and bone union in fracture healing of the streptozotocin-induced diabetic rat

Most studies have focused on the association between diabetes mellitus (DM) and impaired osseous healing, but there is also evidence that diabetes impairs cartilage formation during fracture healing. To investigate the molecular mechanisms by which diabetes affects endochondral ossification, experiments were performed in a model of rat closed fracture healing complicated with diabetes. Diabetic rats were created by a single intravenous injection of streptozotocin (STZ), while controls were treated with vehicle alone. Fractures were made 2 weeks after STZ injection. Animals were killed at 4, 7, 10, 14, 21, 28 and 42 days following fracture, and samples were subject to radiographic, histological and molecular analyses. In the DM group, a significantly smaller cartilaginous callus was formed compared with controls throughout healing, with the cartilage area being reduced rapidly after day 14. When the bone union rate was evaluated radiographically on day 28, DM calluses exhibited a lower rate than controls. However, when evaluated on day 42, both groups showed an equivalent union rate. Cellular proliferation of chondroprogenitor cells and proliferating chondrocytes in soft calluses of the DM group was significantly reduced during early stages of healing (days 4 and 7), but no longer reduced thereafter. Moreover, expression levels of collagen type II, type X and osteopontin (OPN) were constantly low in the DM group. These results show the molecular basis for diminished cartilage formation and delayed union in fracture healing of the STZ-induced diabetic rats.     

骨痂组织冰冻切片前胶原及转化生长因子－β1基因表达的原位定位

目的观察骨痂组织中前胶原、转化生长因子－β１（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒβ１,ＴＧＦ－β１）基因表达模式,分析ＴＧＦ－β１在骨折愈合过程中的作用,探索骨组织冰冻切片原位杂交技术。方法采用不脱钙的大鼠骨痂组织冰冻切片进行原位杂交,观察骨痂组织中前胶原和ＴＧＦ－β１基因的表达,并与先前研究作对照。结果杂交信号清晰,定位良好,特异性高。骨折第１周末,成纤维细胞的Ⅲ型前胶原基因表达占主导,Ⅰ型前胶原ｍＲＮＡ阳性成骨细胞也出现于膜内化骨区。ＴＧＦ－β１在分化、增殖的成骨细胞以及接近成熟的软骨细胞有显著表达。骨折第２周末,Ⅱ型前胶原和ＴＧＦ－β１ｍＲＮＡ在成熟的软骨细胞大量表达,而Ⅰ型前胶原ｍＲＮＡ表达也明显增加。骨折第４周末,软骨骨痂基本被骨组织替代,见散在Ⅰ型前胶原ｍＲＮＡ表达阳性的成骨细胞。同时证实共有表型表达的现象存在。结论实验结果与以往有关研究结果的吻合,提示ＴＧＦ－β１在骨折愈合过程中,尤其在细胞分化、增殖中,起重要的调节作用。同时,也说明了此方法是一种快捷、灵敏、又不失特异性的骨组织原位杂交方法。     

Effects of low-dose, intermittent treatment with recombinant human parathyroid hormone (1-34) on chondrogenesis in a model of experimental fracture healing

Recent studies have demonstrated that intermittent administration of parathyroid hormone (PTH) enhances osteogenesis (hard callus formation) and increases mechanical strength in experimental fracture healing. Thus far, however, effects of PTH on chondrogenesis (soft callus formation) during fracture healing have not been fully elucidated. In the present study, we analyzed the underlying molecular mechanism by which exogenous PTH would affect chondrogenesis in a model of experimental fracture healing. Unilateral femoral fractures were produced in 2-month-old Sprague-Dawley rats. Daily subcutaneous injections of 10 microg/kg of recombinant human PTH(1-34) [rhPTH(1-34)] were administered over a 28-day period of fracture healing. Control animals were injected with vehicle solution (normal saline) alone. The results showed that, on day 14 after fracture, cartilage area in the PTH-treated group was significantly increased (1.4-fold) compared with the controls, but this increase was not observed at days 21 and 28. In the early stage of chondrogenesis (days 4-7), cell proliferation, expressed as the rate of proliferating cell nuclear antigen-positive cells, was increased in mesenchymal (chondroprogenitor) cells but not chondrocytes in the PTH-treated group compared with controls. In addition, gene expression of SOX-9 was up-regulated in the PTH-treated group on day 4 (1.4-fold), and this was accompanied by enhanced expression of pro-alpha1 (II) collagen (1.8-fold). After 14 days, there were no significant differences between groups in either cell proliferation or the expression levels of cartilage differentiation-related genes (SOX-9, pro-alpha1 (II) collagen, pro-alpha1 (X) collagen and osteopontin). These results suggest that intermittent treatment with low-dose rhPTH(1-34) induces a larger cartilaginous callus but does not delay chondrocyte differentiation during fracture healing.     

Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyroid hormone (1-34).

Recent reports have demonstrated that intermittent treatment with parathyroid hormone (1-34) [PTH(1-34)] increases callus formation and mechanical strength in experimental fracture healing. However, little is known about the optimal dose required for enhancement of fracture repair or the molecular mechanisms by which PTH regulates the healing process. In this study, we analyzed the underlying molecular mechanisms by which PTH affects fracture healing and tested the hypothesis that intermittent low-dose treatment with human PTH(1-34) can increase callus formation and mechanical strength. Unilateral femoral fractures were produced and a daily subcutaneous injection of 10 microg/kg of PTH(1-34) was administered during the entire healing period. Control animals were injected with vehicle solution alone. The results showed that on day 28 and day 42 after fracture, bone mineral content (BMC), bone mineral density (BMD), and ultimate load to failure of the calluses were significantly increased in the PTH-treated group compared with controls (day 28, 61, 46, and 32%; day 42, 119, 74, and 55%, respectively). The number of proliferating cell nuclear antigen (PCNA)-positive subperiosteal osteoprogenitor cells was significantly increased in the calluses of the PTH-treated group on day 2, and TRAP+ multinucleated cells were significantly increased in areas of callus cancellous bone on day 7. The levels of expression of type I collagen (COLlA1), osteonectin (ON), ALP, and osteocalcin (OC) mRNA were increased markedly in the PTH-treated group and accompanied by enhanced expression of insulin-like growth factor (IGF)-I mRNA during the early stages of healing (days 4-7). The increased expression of COL1A1, ON, ALP, and OC mRNA continued during the later stages of healing (days 14-21) despite a lack of up-regulation of IGF-I mRNA. These results suggest that treatment of fractures with intermittent low dose PTH(1-34) enhances callus formation by the early stimulation of proliferation and differentiation of osteoprogenitor cells, increases production of bone matrix proteins, and enhances osteoclastogenesis during the phase of callus remodeling. The resultant effect to increase callus mechanical strength supports the concept that clinical investigations on the ability of injectable low-dose PTH(1-34) to enhance fracture healing are indicated.     

In situ localization of procollagen gene expression on cryosections of undecalcified fracture callus

Objective:To analyze the expression of procollagen gene in fracture callus,and to search for the technique of in situ hybridization for undecalcified skeletal tissue.Methods:In situ hybridization of procollagen gene expression was performed on the undecalcified cryosections of rat fracture callus at 7,14,and 28d.Results:The hybridization signals achieved were clear and easy to be localized with high specificity.On the 7th day,the expressions of pro α1(Ⅲ) in fibroblasts and some chondrocyte-like cells were dominant;and at the end of second week high expression of type-Ⅱ procollagen mRNA was observed in chondrocytes.At the end of fourth week,the cartilaginous callus was almost all replaced by woven bone tissue,and some type-I procollagen mRNA positive osteoblasts and hypertrophic chondrocytes were found scattering in the woven bone and remnants of cartilaginous callus.Conclusions:The modified method employed in this study is easier,quicker,and more sensitive with high specificity than the conventional technique for a situ hybridization of procollagen gene expression of decalcified rat fracture callus.The phenomenon of shared phenotype expression,which was demonstrated among cells engaged in fracture healing,indicates an important approach to reveal the mechanism of the origin,differentiation,and orientation of cells.     

Diminished bone formation during diabetic fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity.

Histological and molecular analysis of fracture healing in normal and diabetic animals showed significantly enhanced removal of cartilage in diabetic animals. Increased cartilage turnover was associated with elevated osteoclast numbers, a higher expression of genes that promote osteoclastogenesis, and diminished primary bone formation. INTRODUCTION: Diminished bone formation, an increased incidence of nonunions, and delayed fracture healing have been observed in animal models and in patients with diabetes. Fracture healing is characterized by the formation of a stabilizing callus in which cartilage is formed and then resorbed and replaced by bone. To gain insight into how diabetes affects fracture healing, studies were carried out focusing on the impact of diabetes on the transition from cartilage to bone. MATERIALS AND METHODS: A low-dose treatment protocol of streptozotocin in CD-1 mice was used to induce a type 1 diabetic condition. After mice were hyperglycemic for 3 weeks, controlled closed simple transverse fractures of the tibia were induced and fixed by intramedullary pins. Histomorphometric analysis of the tibias obtained 12, 16, and 22 days after fracture was performed across the fracture callus at 0.5 mm proximal and distal increments using computer-assisted image analysis. Another group of 16-day samples were examined by microCT. RNA was isolated from a separate set of animals, and the expression of genes that reflect the formation and removal of cartilage and bone was measured by real-time PCR. RESULTS: Molecular analysis of collagen types II and X mRNA expression showed that cartilage formation was the same during the initial period of callus formation. Histomorphometric analysis of day 12 fracture calluses showed that callus size and cartilage area were also similar in normoglycemic and diabetic mice. In contrast, on day 16, callus size, cartilage tissue, and new bone area were 2.0-, 4.4-, and 1.5-fold larger, respectively, in the normoglycemic compared with the diabetic group (p < 0.05). Analysis of microCT images indicated that the bone volume in the normoglycemic animals was 38% larger than in diabetic animals. There were 78% more osteoclasts in the diabetic group compared with the normoglycemic group (p < 0.05) on day 16, consistent with the reduction in cartilage. Real-time PCR showed significantly elevated levels of mRNA expression for TNF-alpha, macrophage-colony stimulating factor, RANKL, and vascular endothelial growth factor-A in the diabetic group. Similarly, the mRNA encoding ADAMTS 4 and 5, major aggrecanases that degrade cartilage, was also elevated in diabetic animals. CONCLUSIONS: These results suggest that impaired fracture healing in diabetes is characterized by increased rates of cartilage resorption. This premature loss of cartilage leads to a reduction in callus size and contributes to decreased bone formation and mechanical strength frequently reported in diabetic fracture healing.     

Fracture healing in paraplegic rats

In paraplegic rats, histomorphometric and chemical methods were used to evaluate callus production in tibial fractures stabilized by intramedullary nails. There were no differences in the mean sizes of fracture callus between paraplegic rats and their non-weight-bearing and weight-bearing controls. However, the variance of callus size was large in paraplegic rats. The concentration of nitrogen was high in calluses of paraplegic rats during the cartilaginous stage of healing. The hydroxyproline concentration did not differ between the groups. The rate of callus ossification was more rapid in paraplegic rats than in controls, but fracture calluses of paraplegic rats showed delayed accumulation of calcium and incomplete maturation of woven new bone. The results suggest that there are both mechanical and non-mechanical factors affecting callus formation in fractures below a spinal lesion.     

Fracture healing in paraplegic rats

In paraplegic rats, histomorphometric and chemical methods were used to evaluate callus production in tibial fractures stabilized by intramedullary nails. There were no differences in the mean sizes of fracture callus between paraplegic rats and their non-weight-bearing and weight-bearing controls. However, the variance of callus size was large in paraplegic rats. The concentration of nitrogen was high in calluses of paraplegic rats during the cartilaginous stage of healing. The hydroxyproline concentration did not differ between the groups. The rate of callus ossification was more rapid in paraplegic rats than in controls, but fracture calluses of paraplegic rats showed delayed accumulation of calcium and incomplete maturation of woven new bone. The results suggest that there are both mechanical and non-mechanical factors affecting callus formation in fractures below a spinal lesion.     

Smoking delays chondrogenesis in a mouse model of closed tibial fracture healing.

Smoking delays the healing process and increases morbidity associated with many common musculoskeletal disorders, including long bone fracture. In the current study, a murine model of tibial fracture healing was used to test the hypothesis that smoking delays chondrogenesis after fracture. Mice were divided into two groups, a nonsmoking control group and a group exposed to cigarette smoke for 1 month prior to surgical tibial fracture. Mice were euthanized at 7, 14, and 28 days after surgery. The outcomes measured were immunohistochemical staining for type II collagen protein expression as a marker of cartilage matrix and proliferating cell nuclear antigen (PCNA) staining to measure proliferation at the site of injury. Toluidine blue staining and histomorphometry were used to quantify areas of cartilaginous and noncartilaginous fracture callus. Radiographs were analyzed for evidence of remodeling after injury. At day 7 after injury, mice exposed to cigarette smoke had a smaller fracture callus with less cartilage matrix compared to controls. Proliferation was present at high levels in both groups at this time point, but proliferating cells had a more immature morphology in the smoking group. At day 14, chondrogenesis was more active in smokers compared to controls, while a higher percentage of bone was present in the control animals. At day 28, X-ray analysis revealed a larger fracture callus remaining in the smoking animals. Together, these findings show that the chondrogenic phase of tibial fracture healing is delayed by smoking. This study represents, to our knowledge, the first analysis of molecular and cellular mechanisms of healing in a smoking mouse fracture model.     

Immature osteoblastic cells express the pro-alpha2(XI) collagen gene during bone formation in vitro and in vivo.

Type XI collagen is predominantly found in cartilage. However, expression of the pro-alpha2(XI) collagen gene (COL11A2) has recently been detected in various non-cartilaginous tissues. We identified the differentiation stage at which COL11A2 was expressed in cultured fetal rat calvarial (FRC) cells and in rat femoral fracture calluses in order to investigate the involvement of COL11A2 during bone formation in vitro and in vivo. We also studied the alternative splicing of exons 6-8 in FRC cells and fracture calluses. In FRC cells, mineralized nodules stained with von Kossa stain were observed from day 9 after confluence. COL11A2 was highly expressed on days 0 and 5, but the expression levels were rapidly decreased on day 9 by Northern blot analysis. During rat femoral fracture repair, intramembranous ossification proceeded and newly formed woven bone was observed on the cortex on day 7 after fracture. In situ hybridization showed that COL11A2 signals were detected in osteoblastic cells in the newly formed woven bone. According to the maturation and remodeling of the woven bone into the trabecular bone, the distribution of the signal for COL11A2 mRNA was limited to the superficial osteoblastic cells of the newly formed trabecular bone. These results demonstrated that COL11A2 was expressed in relatively immature osteoblastic cells during bone formation in vitro and in vivo. RT-PCR showed that the shortest band corresponding to mRNA lacking exons 6-8 was clearly detected when using RNA from soft calluses. In contrast, the largest band corresponding to mRNA with exons 6-8 was predominant when using RNA from FRC cells or from hard calluses on days 7 and 14. These results indicate that the splicing pattern of exons 6-8 in osteoblastic cells is different from the pattern in chondrocytes.     

Expression of metalloproteinase-13 (Collagenase-3) is induced during fracture healing in mice.

In fracture healing, a large amount of cartilage is formed, then rapidly replaced by osseous tissue. This process requires the transition of extracellular matrix component from type II to type I collagen. We investigated the expression of matrix metalloproteinase-13 (MMP-13), which has a high potential to cleave type II as well as type I collagen, during fracture repair in mouse ribs. In situ hybridization demonstrated that MMP-13 mRNA was present throughout the healing process. It was detected in the cells of the periosteum at day 1. As fracture callus grew, strong MMP-13 mRNA signals were detected in cells of the cartilaginous callus. In the reparative and remodeling phases, both hypertrophic chondrocytes and immature osteoblastic cells in the fracture callus expressed MMP-13 mRNA strongly. These cells were located adjacent to tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts at the sites of cartilage/bone transition. In osteoclasts, MMP-13 expression was not detected. The level of MMP-13 mRNA peaked at day 14 postfracture by northern blotting. Immunohistochemical staining showed that MMP-13 was detected primarily in hypertrophic chondrocytes. These results indicate that MMP-13 is induced during fracture healing. The site- and cell-specific expression of MMP-13 and its enzymatic property suggest that MMP-13 initiates the degradation of cartilage matrix, resulting in resorption and remodeling of the callus. In conclusion, MMP-13 plays an important role in the healing process of fractured bone in mice.     

Programmed removal of chondrocytes during endochondral fracture healing

This investigation tested the hypothesis that the removal of chondrocytes during endochondral fracture healing involves an ordered process of programmed cell death. To accomplish this, unilateral closed fractures were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for expression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL) technique. To determine the potential for trans–differentiation of chondrocytes to osteoblasts calluses were also hybridized to detect expression of osteocal in mRNA. Cell proliferation was assessed by an immunohistochemical detection method for proliferating cell nuclear antigen. A separate group of four rats was killed on day 28 to represent the later stage of the endochondral ossification, and the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time and space of the activities of cellular proliferation and programmed cell death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14) although TUNEL expression was apparent in hypertrophic chondrocytes on day 14 after fracture and persisted until day 28. In the later stages of fracture healing (days 14 through 28), proliferating cell nuclear antigen was no longer synthesized in hard callus (intramembranous bone) and cell removal was the dominant activity in soft callus chondrocytes. Expression of osteocalcin mRNA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hypothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferation, maturation, and apoptotic cell death.     

Low-intensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression.

We evaluated the effect of low intensity-pulsed ultrasound stimulation on rat chondrocytes in vitro using two different 1.0-MHz ultrasound signals with spatial and temporal average intensities of 50 or 120 mW/cm2. The pulses had a duration of 200 microseconds and were repeated every millisecond, with corresponding average peak-pressure amplitudes of 230 or 360 kPa, respectively. Cells were stimulated one, three, or five times for 10 minutes each day starting the third day after plating. One group of cells was exposed to sham ultrasound as a control. The cultures were evaluated for cell proliferation (by [3H]thymidine incorporation and DNA measurement), steady-state mRNA levels of alpha1(I) and alpha1(II) procollagens and aggrecan (by Northern blotting), and proteoglycan synthesis (by [35S]sulfate incorporation). The results revealed that ultrasound causes increases in the level of aggrecan mRNA (p < 0.05) and in proteoglycan synthesis (p < 0.03) after three and five treatments. Expression of mRNA for alpha1(II) procollagen increased over time, but ultrasound had no stimulatory effect. Expression of mRNA for alpha1(I) procollagen was initially low and remained unchanged with time. Although cell proliferation increased with time in both groups, there was no statistically significant difference between the cultures treated with ultrasound and the controls (p = 0.1). The in vitro results support our previous in vivo findings that low-intensity ultrasound stimulates aggrecan mRNA expression and proteoglycan synthesis by chondrocytes, which may explain the role of ultrasound in advancing endochondral ossification, increasing the mechanical strength of fractures, and facilitating fracture repair.     

Platelet concentrate injection improves Achilles tendon repair in rats

Background and purpose We have previously shown that early fracture callus of rat rib has viscoelastic and contractile properties resembling those of smooth muscle. The cells responsible for this contractility have been hypothesized to be myofibroblast-like in nature. In soft-tissue healing, force generated by contraction of myofibroblasts promotes healing. Accordingly, we tried to identify myofibroblast-like cells in early fibrous callus.

Animals and methods Calluses from rat rib fractures were removed 7, 14, and 21 days after fracture and unfractured ribs acted as controls. All tissues were analyzed using qPCR and immunohistochemistry. We analyzed expression of smooth muscle- and myofibroblast-associated genes and proteins including alpha smooth muscle actin (αSMA), non-muscle myosin, fibronectin extra domain A variant (EDA-fibronectin), OB-cadherin, connexin-43, basic calponin (h1CaP), and h-caldesmon.

Results In calluses at 7 days post-fracture, there were statistically significant increases in expression of αSMA mRNA (2.5 fold), h1CaP mRNA (2.1 fold), EDA-fibronectin mRNA (14 fold), and connexin-43 mRNA (1.8 fold) compared to unfractured ribs, and by 21 days post-fracture mRNA expression in calluses had decreased to levels approaching those in unfractured rib. Immunohistochemistry of 7 day fibrous callus localized calponin, EDA-fibronectin and co-immunolabeling of OB-cadherin and αSMA (thus confirming a myofibroblastic phenotype) within various cell populations.

Interpretation This study provides further evidence that early rat rib callus is not only smooth muscle-like in nature but also contains a notable population of cells that have a distinct myofibroblastic phenotype. The presence of these cells indicates that in vivo contraction of early callus is a mechanism that may occur in fractures so as to facilitate healing, as it does in soft tissue wound repair.     

Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I.

The addition of insulin-like growth factor-I to cartilage cultures is known to stimulate the synthesis of proteoglycan and type-II collagen in explant and monolayer studies. The purpose of this study was to determine the effects of long-term supplementation with insulin-like growth factor-I in chondrocytes cultured in fibrin discs as a preliminary investigation to in vivo application of chondrocyte/insulin-like growth factor-I/fibrin grafts to articular-cartilage repair procedures. Chondrocyte-fibrin cultures were maintained for 14 days, with insulin-like growth factor-I added at varying concentrations of 0, 10, 50, or 100 ng/ml medium. Cultures supplemented with 50 or 100 ng of growth factor/ml had increased levels of aggrecan and type-IIB procollagen mRNA, and translation to aggrecan and type-IIB collagen was confirmed by dye-binding assay of total proteoglycan, type-II collagen immunohistochemistry, and determination of collagen content by high-performance liquid chromatography. Maintenance of the chondrocyte phenotype during the 14 days of culture was confirmed by round cell morphology on routine staining, expression of type-II procollagen mRNA on in situ hybridization, evidence of production of pericellular type-II collagen on immunocytochemistry, synthesis of large-molecular-size aggrecan monomer on CL-2B column chromatography, and lack of appreciable message expression for type I or IIA collagen on Northern blot hybridization. Dose-response effects of insulin-like growth factor-I on the expression of chondrocyte matrix constituents were most pronounced at 50 and 100 ng of growth factor per milliliter of medium. These data confirm that (a) culture of chondrocytes for extended periods in three-dimensional cultures of fibrin maintains the chondrocyte phenotype and (b) supplementation with increasing concentrations of insulin-like growth factor-I enhances chondrocyte matrix synthesis and may provide a means to enhance chondrocyte phenotypic stability and function during transplantation grafting procedures.