METHODS: The degree of a femoral osteochondral defect repair in rabbits after intra-articular injections of bone marrow-derived mesenchymal stem cells (BMSCs) that were transduced with empty adeno-associated virus (AAV) or AAV containing CRLF1 was determined by morphological, histological, and micro computer tomography (CT) analyses. The effects of CRLF1 on chondrogenic differentiation of BMSCs or catabolic events of interleukin-1beta-treated chondrocyte cell line TC28a2 were determined by alcian blue staining, gene expression levels of cartilage and catabolic marker genes using real-time PCR analysis, and immunoblot analysis of Smad2/3 and STAT3 signaling.
RESULTS: Intra-articular injections of BMSCs overexpressing CRLF1 markedly improved repair of a rabbit femoral osteochondral defect. Overexpression of CRLF1 in BMSCs resulted in the release of a homodimeric CRLF1 complex that stimulated chondrogenic differentiation of BMSCs via enhancing Smad2/3 signaling, whereas the suppression of CRLF1 expression inhibited chondrogenic differentiation. In addition, CRLF1 inhibited catabolic events in TC28a2 cells cultured in an inflammatory environment, while a heterodimeric complex of CRLF1 and cardiotrophin-like Cytokine (CLC) stimulated catabolic events via STAT3 activation.
CONCLUSIONS: A homodimeric CRLF1 complex released by BMSCs enhanced the repair of osteochondral defects via the inhibition of catabolic events in chondrocytes and the stimulation of chondrogenic differentiation of precursor cells.
方法:通过形态学测定空腺相关病毒(AAV)或含有CRLF1的AAV转导的骨髓间充质干细胞(BMSCs)关节内注射后兔股骨骨软骨缺损修复的程度。组织学,和微型计算机断层扫描(CT)分析。通过阿辛蓝染色确定CRLF1对BMSCs软骨分化或白介素-1β处理的软骨细胞系TC28a2的分解代谢事件的影响,使用实时PCR分析的软骨和分解代谢标记基因的基因表达水平,Smad2/3和STAT3信号的免疫印迹分析。
结果:关节内注射过表达CRLF1的BMSCs可显著改善兔股骨骨软骨缺损的修复。在BMSCs中CRLF1的过表达导致同型二聚体CRLF1复合物的释放,通过增强Smad2/3信号传导刺激BMSCs的软骨分化,而抑制CRLF1表达抑制软骨分化。此外,CRLF1抑制在炎症环境中培养的TC28a2细胞的分解代谢事件,而CRLF1和心肌营养素样细胞因子(CLC)的异二聚体复合物通过STAT3激活刺激分解代谢事件。
结论:BMSCs释放的同二聚体CRLF1复合物通过抑制软骨细胞的分解代谢事件和刺激前体细胞的软骨分化来增强骨软骨缺损的修复。