Girotto Davide, Urbani Serena, Brun Paola, Renier Davide, Barbucci Rolando, Abatangelo Giovanni
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche, Facoltà di Medicina, Università di Padova, Viale G, Colombo 3 35121, Italy.
Biomaterials. 2003 Aug;24(19):3265-75. doi: 10.1016/s0142-9612(03)00160-1.
The re-differentiation capacities of human articular and chick embryo sternal chondrocytes were evaluated by culture on HYAFF-11 and its sulphate derivative, HYAFF-11-S, polymers derived from the benzyl esterification of hyaluronate. Initial results showed that the HYAFF-11-S material promoted the highest rate of chondrocyte proliferation. RNA isolated from human and chick embryo chondrocytes cultured in Petri dishes, HYAFF-11 or HYAFF-11-S were subjected to semi-quantitative RT-PCR analyses. Human collagen types I, II, X, human Sox9 and aggrecan, chick collagen types I, II, IX and X were analysed. Results showed that human collagen type II mRNA expression was upregulated on HYAFF-11 biomaterials. In particular, a high level of collagen type IIB expression was associated with three-dimensional culture conditions, and the HYAFF-11 material was the most supportive for human collagen type X mRNA expression. Human Sox9 mRNA levels were constantly maintained in monolayer cell culture conditions over a period of 21 days, while these were upregulated when chondrocytes were cultured on HYAFF-11 and HYAFF-11S. Furthermore, chick collagen type IIA and IIB mRNA expression was detected after only 7 days of HYAFF-11 culture. Chick collagen type IX mRNA expression decreased in scaffold cultures over time. Histochemical staining performed in engineered cartilage revealed the presence of a de novo synthesized glycosaminoglycan-rich extracellular matrix; immunohistochemistry confirmed the deposition of collagen type II. This study showed that the three-dimensional HYAFF-11 culture system is both an effective chondrocyte delivery system for the treatment of articular cartilage defects, and an excellent in vitro model for studying cartilage differentiation.
通过在HYAFF - 11及其硫酸酯衍生物HYAFF - 11 - S（由透明质酸苄酯化衍生的聚合物）上培养，评估了人关节软骨细胞和鸡胚胸骨软骨细胞的再分化能力。初步结果表明，HYAFF - 11 - S材料促进软骨细胞增殖的速率最高。从培养在培养皿、HYAFF - 11或HYAFF - 11 - S中的人和鸡胚软骨细胞中分离的RNA进行半定量RT - PCR分析。分析了人I、II、X型胶原蛋白、人Sox9和聚集蛋白聚糖，以及鸡I、II、IX和X型胶原蛋白。结果表明，在HYAFF - 11生物材料上，人II型胶原蛋白mRNA表达上调。特别是，高水平的IIB型胶原蛋白表达与三维培养条件相关，并且HYAFF - 11材料对人X型胶原蛋白mRNA表达最具支持性。在单层细胞培养条件下，人Sox9 mRNA水平在21天内持续维持，而当软骨细胞在HYAFF - 11和HYAFF - 11S上培养时，这些水平会上调。此外，在HYAFF - 11培养仅7天后就检测到鸡IIA和IIB型胶原蛋白mRNA表达。随着时间的推移，鸡IX型胶原蛋白mRNA表达在支架培养中下降。在工程化软骨中进行的组织化学染色显示存在重新合成的富含糖胺聚糖的细胞外基质；免疫组织化学证实了II型胶原蛋白的沉积。这项研究表明，三维HYAFF - 11培养系统既是治疗关节软骨缺损的有效软骨细胞递送系统，也是研究软骨分化的优秀体外模型。
