Hughes S S, Hicks D G, O'Keefe R J, Hurwitz S R, Crabb I D, Krasinskas A M, Loveys L, Puzas J E, Rosier R N
Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, New York, USA.
J Bone Miner Res. 1995 Apr;10(4):533-44. doi: 10.1002/jbmr.5650100405.
Endochondral ossification in fracture healing of rats at 4, 8, 11, 14, and 21 days was analyzed using immunological and molecular probes for markers of the chondrocyte and osteoblast phenotype. These markers were osteocalcin, type I and type II collagen, including the probes homologous to the alternatively spliced forms of alpha 1 type II collagen, type IIA and type IIB. Histologic examination was performed on serial sections of the same tissue blocks to correlate cellular morphology with the immunohistochemical and in situ hybridization findings. At the junction of the cartilaginous and osseous tissue, an overlap of phenotype and morphology was noted. At the 8-day time point, the cells with chondrocyte morphology expressed intracellular message for osteocalcin and type I collagen. Immunohistochemical analysis of these cells also demonstrated intracellular osteocalcin. However, high levels of the type IIA collagen mRNA, which has previously been associated with less differentiated mesenchymal precursor cells, were expressed in both chondrocytes and osteoblasts. At the later time point (21 days) there was a substantial decrease in the number of cells displaying shared phenotypic characteristics. In situ hybridization and immunohistochemistry have permitted identification of an overlapping or shared phenotype in osteoblasts and chondroblasts in fracture callus. The findings raise important questions regarding the possible plasticity of mesenchymal cell phenotypes within the dynamic environment of fracture healing. Additional examination of these issues will further define factors involved in origin, differentiation, and maturation of bone and cartilage cells.
使用针对软骨细胞和成骨细胞表型标志物的免疫和分子探针，分析了大鼠在骨折愈合第4、8、11、14和21天时的软骨内成骨情况。这些标志物包括骨钙素、I型和II型胶原蛋白，其中针对II型胶原蛋白α1链可变剪接形式的同源探针，即IIA型和IIB型。对同一组织块的连续切片进行组织学检查，以将细胞形态与免疫组织化学和原位杂交结果相关联。在软骨组织和骨组织的交界处，观察到表型和形态的重叠。在第8天时间点，具有软骨细胞形态的细胞表达了骨钙素和I型胶原蛋白的细胞内信息。对这些细胞的免疫组织化学分析也显示细胞内存在骨钙素。然而，先前与分化程度较低的间充质前体细胞相关的IIA型胶原蛋白mRNA在软骨细胞和成骨细胞中均有高表达。在后期时间点（21天），显示共同表型特征的细胞数量大幅减少。原位杂交和免疫组织化学已能够鉴定骨折痂中成骨细胞和软骨细胞的重叠或共同表型。这些发现提出了关于骨折愈合动态环境中间充质细胞表型可能可塑性的重要问题。对这些问题的进一步研究将进一步明确参与骨和软骨细胞起源、分化和成熟的因素。
