Elucidation of the mechanism of TGF-βsignaling in the repair process of full-thickness defects of rat articular cartilage.

为了在大鼠股骨内均匀地制造出全厚度的骨缺损区，我们发明了一种双刃装置。通过对修复过程中细胞增殖、N-细胞黏附分子和II型胶原β的定位观察，研究了修复过程中转化生长因子-β-1、转化生长因子-βRI和转化生长因子-RII的表达。4天时，增殖的未分化梭形细胞填满了整个缺损区，N-CAM的表达仅限于表面积以下的梭形未分化细胞。随后，这些细胞转变为多角形细胞，10天后在缺损区深处的这些细胞中检测到II型胶原mRNA。两周后，首次观察到藏红花-O所示的软骨基质，该软骨基质包裹的软骨细胞具有较高的增殖能力。术后4d，通过膜内骨化，在缺损区底部形成新的软骨下骨。4周后进展到与残余骨软骨交界处相同水平。到那时，新的软骨组织类似于残留的关节软骨再生，藏红花-O和II型胶原完全染色新合成的软骨基质。转化生长因子-β-1、转化生长因子-β-RI和转化生长因子-β-RII共同表达于渗入缺损区的梭形未分化细胞、多角形细胞以及位于深层和中层的增殖性软骨细胞。这些结果提示转化生长因子-β信号通路通过软骨形成修复过程，包括未分化细胞增殖、细胞凝聚、软骨细胞增殖和基质合成。

To create full-thickness defects in rat femoral patellar groove uniformly, we invented a twin blade device. The repair process using this device was studied histologically, and the character of cells in the reparative tissue was elucidated by observing the localization of cell proliferation, N-CAM, and type II collagen mRNA.Further, the expression of TGF-β1, TGF-βRI, and TGF-βRII during the repair processes was investigated. At four days, proliferating undifferentiated spindle-shaped cells filled the entire defect, and N-CAM expression was restricted to spindlier undifferentiated cells beneath the surface area. Subsequently, these cells changed into polygonal-shaped cells, and type II collagen mRNA was detected in these cells deep in the defects at ten days. At two weeks, the first evidence of cartilage matrix indicated by safranin-O was observed, and chondrocytes surrounded by this cartilage matrix showed a high proliferative capacity. New subchondral bone at the bottom of the defects had formed through intramembranous ossification from four days. It progressed and reached to the same level of residual osteochondral junction by four weeks. By that time, new cartilage tissue similar to residual articular cartilage regenerated, and safranin-O and type II collagen wholly stained the newly synthesized cartilage matrix. TGF-β1, TGF-βRI, and TGF-β RII were coexpressed in the spindle-shaped undifferentiated cells that infiltrated into the defects, in the polygonal-shaped cells, and in proliferating chondrocytes located in the deep and middle layers. These results suggested that TGF-β signaling was involved through the chondrogenesis pathway of repair process, which included undifferentiated cell proliferation, cellular condensation, chondrocyte proliferation, and matrix synthesis.