Hayes Anthony J, Hall Amanda, Brown Liesbeth, Tubo Ross, Caterson Bruce
Connective Tissue Biology Laboratory and Cardiff Institute of Tissue Engineering and Repair, Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3US, Wales, UK.
J Histochem Cytochem. 2007 Aug;55(8):853-66. doi: 10.1369/jhc.7A7210.2007. Epub 2007 May 3.
Recent advances in tissue engineering offer considerable promise for the repair of focal lesions in articular cartilage. Here we describe (1) the macromolecular organization of tissue-engineered neocartilage grafts at light and electron microscopic levels, (2) their in vitro development, and (3) the effect of chondrocyte dedifferentiation, induced by monolayer expansion, on their resultant structure. We show that grafts produced from primary cultures of chondrocytes are hyaline in appearance with identifiable zonal strata as evidenced by cell morphology, matrix organization, and immunohistochemical composition. Like native articular cartilage, their surface zone contains type I collagen, surface zone proteoglycan, biglycan and decorin with type II collagen, aggrecan, chondroitin sulfate, chondroitin-4-sulfate, and keratan sulfate, becoming more prominent with depth. Assessment of cell viability by Live/Dead staining and cell-cycle analysis with BrDU suggest that the in vitro tissue has a high cellular turnover and develops through both appositional and interstitial growth mechanisms. Meanwhile, cell-tracker studies with CMFDA (5-chloromethyl-fluorescein diacetate) demonstrate that cell sorting in vitro is not involved in their zonal organization. Finally, passage expansion of chondrocytes in monolayer culture causes progressive reductions in graft thickness, loss of zonal architecture, and a more fibrocartilaginous tissue histology, consistent with a dedifferentiating chondrocyte phenotype.
组织工程学的最新进展为修复关节软骨局灶性损伤带来了巨大希望。在此，我们描述了：（1）组织工程化新软骨移植物在光镜和电镜水平下的大分子组织；（2）它们的体外发育过程；（3）单层扩增诱导的软骨细胞去分化对其最终结构的影响。我们发现，由软骨细胞原代培养产生的移植物外观呈透明软骨样，具有可识别的分层结构，这可通过细胞形态、基质组织和免疫组织化学组成得以证明。与天然关节软骨一样，其表面层含有I型胶原、表面层蛋白聚糖、双糖链蛋白聚糖和核心蛋白聚糖，以及II型胶原、聚集蛋白聚糖、硫酸软骨素、硫酸软骨素-4-硫酸酯和硫酸角质素，且随着深度增加而更加显著。通过活/死染色评估细胞活力以及用溴脱氧尿苷进行细胞周期分析表明，体外组织具有较高的细胞更新率，并通过表面沉积和间质生长机制发育。同时，用CMFDA（5-氯甲基荧光素二乙酸酯）进行的细胞追踪研究表明，体外细胞分选与它们的分层结构无关。最后，软骨细胞在单层培养中的传代扩增导致移植物厚度逐渐减小、分层结构丧失以及组织学上更偏向纤维软骨，这与软骨细胞去分化表型一致。
