体外诱导成纤维细胞成骨活性表达的研究

目的探讨成纤维细胞(fibroblast,FB)体外成骨表达的诱导因素,为其能否成为骨组织工程的种子细胞提供理论依据.方法分离和纯化新西兰兔肉芽组织FB,按1×105/ml分别接种于含纤维结合蛋白(fibronectin,FN)10、20、40、60、80μg/ml条件培养液中(实验1～5组),对照组为不含FN的无血清培养液.于培养14 d后,行细胞组织学观察及钙化结节形成率、趋骨性四环素荧光标记、3H-TdR标记、骨钙素测定及3H脯氨酸标记,检测FB成骨表型表达的标志.结果组织学观察实验组发现FB由梭形逐渐趋向多突形和圆形,细胞核偏向一侧,细胞重叠成多层结构;其表面有钙盐堆积,逐渐呈云雾状物质,经不断融合和扩大形成不透光的结节.干预培养14 d钙化结节形成率:对照组15.35%±3.45%,实验1～5组分别为53.73%±9.49%、75.21%±9.80%、98.34%±15.20%、61.83%±10.04%、45.11%±8.70%,实验组与对照组比较,差异有统计学意义(P＜0.05);实验3组与其它各实验组比较,差异有统计学意义(P＜0.05).趋骨性四环素特异性标记为新生骨组织;FB增殖活性,实验3、4、5组7 d时,实验2、3、4组14 d时与对照组比较,差异有统计学意义(P＜0.05).实验1～5组FB分泌骨钙素,实验2～5组3H-脯氨酸掺入量高于对照组,7、14d时与对照组比较差异有统计学意义(P＜0.05).结论适量的FN可以促进FB增殖、胶原蛋白的合成及骨钙素分泌,FN可以诱导FB成骨表达,适宜浓度为40～60μg/ml.

FIBRONECTIN OSTEOGENIC PHENOTYPICAL EXPPESSION BY FIBROBLASTS IN VITRO

OBJECTIVE: To explore the regulator factor of osteogenesis induced by the fibroblast in vitro so as to provide enough seeding cells for the bone tissue engineering. METHODS: The fibroblasts were isolated and purified from granulation of New Zealand rabbits, and they were incubated in the media of fibronectin (FN) 10, 20, 40, 60 and 80 microg/ml, respectively, in the experimental groups 1 - 5, but there was no FN in the control group. The markers for osteogenic features were investigated by fibroblast morphogenesis, calcium nodules formation ratios, labeling of tetracycline fluorescence, labeling of 3H-TdR, determination of osteocaline, and labeling of 3H-proline within 2 weeks. RESULTS: The morphological changes of the fibroblasts were manifested as transference from a long spindle to a round or multiple form, shifted nucleus increased in number, confluenced and formed multilayered structure. There was a piling-up of calcium crystals that were gradually merged into foggy substances. The foggy substances increased and formed nodules. The calcium nodules formation ratios were as follows: 15.35% +/- 3.45% in the control group, and 53.73% +/- 9.49%, 75.21% +/- 9.80%, 98.34% +/- 15.20%, 61.83% +/- 10.04%, and 45.11% +/- 8.70% in the experimental groups 1-5, respectively. There was a significant difference between the control group and the 5 experimental groups at 14 days (P < 0.05), and a significant difference between the experimental group 3 and the other experimental groups at 14 days (P < 0.05). The histochemical study on the nodules with the specific labeling of tetracycline fluorescence indicated that the nodules were composed of new bones. CONCLUSION: Fibronectin can stimulate the fibroblast to proliferate, secrete osteocaline, and synthesize collagen fibrils. Fibronectin, in an optimal dose of 40-60 microg/ml, is capable of inducing the fibroblast to form the bone.