He Chuan, Deng Lian-fu, Yang Qing-ming, Shen Wei, Feng Wei, Zhang Yue, Zhu Ya-ping
Shanghai Institute of Traumatology and Orthopaedics, Shanghai 200025, China.
Zhonghua Wai Ke Za Zhi. 2006 Feb 15;44(4):271-4.
To study the feasibility of osteogenic phenotype expression by human skin fibroblasts induced in polyglycolic acid (PGA) foams and the effect of tumor necrosis factor-alpha (TNF-alpha) on the expression of bone morphogenetic protein (BMP) receptors.
The fibroblasts were isolated, purified from human skin. (1) Fibroblasts were seeded onto PGA foams. The cell-PGA complexes were cultured in RCCS for 6 weeks, in the media of TNF-alpha (50 U/ml) and BMP-2 (0.1 microg/ml). 1 d, 3 and 6 weeks later, cells and extracellular matrix were investigated by electron microscopic and histochemistry observation respectively. Secretion of osteogenic markers were analyzed by biochemical methods. (2) Fibroblasts were seeded on the glass fragments or culture flasks and treated with TNF-alpha (50 U/ml) in different usage (one-time, all-time). The RT-PCR method and the immunohistochemistry fluorescence staining were used to examine the influence of TNF-alpha on the mRNA expression and the protein expression of the type I BMP receptors at 2, 4, 6, 8 d after treatment.
Fibroblasts seeded on the PGA foams formed 3-dimensional matrix 3 weeks after seeding, which was demonstrated as osteo-tissue by tetracycline labeling and ARS staining. Cells secreted much more bone-specific alkaline phosphatase (B-AKP) and osteocalcin (OCN) into supernatant than the cells that were cultured in the media without TNF-a and BMP2. Eight days after all-time usage, the TNF-alpha (50 U/ml) increased the expression of the mRNA and protein of the type IB BMP receptor.
Fibroblasts on 3-D cell-foam structures can express osteoblastic phenotype under certain inducing conditions. The numerous fibroblasts in body would be a promising resource for cell seeds candidate of tissue- engineered bone. TNF-alpha provides the essential condition for BMP2's target effect on fibroblasts, and combined use of TNF-alpha and BMP2 is one of the regulating factors.
研究人皮肤成纤维细胞在聚乙醇酸（PGA）泡沫中诱导成骨表型表达的可行性以及肿瘤坏死因子-α（TNF-α）对骨形态发生蛋白（BMP）受体表达的影响。
从人皮肤中分离、纯化成纤维细胞。（1）将成纤维细胞接种到PGA泡沫上。细胞-PGA复合物在旋转细胞培养系统（RCCS）中培养6周，培养介质中含有TNF-α（50 U/ml）和BMP-2（0.1 μg/ml）。1天、3周和6周后，分别通过电子显微镜和组织化学观察对细胞和细胞外基质进行研究。采用生化方法分析成骨标志物的分泌情况。（2）将成纤维细胞接种在玻璃碎片或培养瓶上，用不同方式（一次性、全程）给予TNF-α（50 U/ml）处理。采用逆转录聚合酶链反应（RT-PCR）法和免疫组织化学荧光染色法，检测处理后2、4、6、8天TNF-α对Ⅰ型BMP受体mRNA表达和蛋白表达的影响。
接种在PGA泡沫上的成纤维细胞在接种3周后形成三维基质，通过四环素标记和茜素红染色证明为骨组织。与在不含TNF-α和BMP2的培养基中培养的细胞相比，细胞向上清液中分泌了更多的骨特异性碱性磷酸酶（B-AKP）和骨钙素（OCN）。全程使用TNF-α（50 U/ml）8天后，ⅠB型BMP受体的mRNA和蛋白表达增加。
三维细胞-泡沫结构上的成纤维细胞在一定诱导条件下可表达成骨细胞表型。体内大量的成纤维细胞将是组织工程骨细胞种子候选的有前途的来源。TNF-α为BMP2对成纤维细胞的靶向作用提供了必要条件，TNF-α与BMP2联合使用是调节因子之一。
