自组装短肽水凝胶体外三维培养兔关节软骨细胞(英文)

Self-assembling short-peptide hydrogel for three-dimensional culture of rabbit articular chondrocytes in vitro

背景:自组装短肽是人工设计合成的一类新型纳米生物材料,与软骨细胞复合培养后如能促进细胞基质的分泌,细胞分裂增殖及细胞表型的维持,将有可能成为一种较理想的细胞支架应用在软骨组织工程中。目的:观察兔关节软骨细胞在纳米自组装短肽KLD-12水凝胶中三维培养的生物学特性。设计、时间及地点:观察性实验,于2007-11/2008-04在四川大学纳米生物医学技术与膜生物学研究所实验室完成。材料:纳米短肽KLD-12序列为:Ac-KLDLKLDLKLDL-CONH2,由上海波泰生物公司合成。方法:取新西兰兔关节软骨,通过酶消化法获取软骨细胞,体外培养2代后以5×109L-1的密度接种于4g/L的自组装短肽KLD-12溶液中,用磷酸盐缓冲液诱导成胶后进行三维培养。主要观察指标:①通过倒置相差显微镜观察软骨细胞在纳米短肽水凝胶中的形态。②用甲苯胺蓝染色,免疫组化检测细胞外基质分泌情况。③反转录-聚合酶链反应检测软骨细胞黏多糖、前Ⅱ型胶原、前Ⅰ型胶原基因的表达情况。结果:①兔关节软骨消化分离的软骨细胞成活率达95%以上。②软骨细胞在纳米短肽水凝胶中体外培养时,细胞生长旺盛、增殖活跃,细胞为圆形聚集成团状或岛状,细胞团周围有类似的软骨陷窝形成。③甲苯胺蓝染色和Ⅱ型胶原免疫组化均呈阳性,细胞基质中黏多糖、Ⅱ型胶原含量随培养时间延长逐渐增加。④反转录-聚合酶链反应结果显示软骨细胞在纳米短肽水凝胶经过3周体外培养一直保持了分泌黏多糖及表达Ⅱ型胶原的能力。结论:自组装短肽KLD-12三维水凝胶能较好维持软骨细胞正常形态、功能和增殖能力。

BACKGROUND： Self-assembling short-peptide has been shown to be a kind of novel nanometer biomaterial. Following culture combined with chondrocytes, self-assembling short-peptide can promote cell matrix secretion, cell division and proliferation, and cell phenotype maintenance. For this reason, it has become a candidate for an ideal cell scaffold used for cartilage tissue engineering. OBJECTIVE： To observe the biological characteristics of rabbit articular chondrocytes cultured in three-dimensional nanoscale self-assembling short-peptide KLD-12 hydrogel scaffold. DESIGN, TIME AND SETTING： An observational experiment was performed at the laboratory of Institute for Nanobiomedical Technology and Membrane Biology, Sichuan University between November 2007 and April 2008. MATERIALS： Nanoscale short-peptide KLD-12 with a sequence of Ac-KLDLKLDLKLDL-CONH2 was synthesized by Shanghai Biotech Company, China. METHODS： Chondrocytes were harvested from New Zealand rabbit articular cartilage by enzyme digestion. Following in vitro culture, chondrocytes of passage 3 were inoculated into 4 g/L self-assembling short-peptide KLD-12 solution at a density of 5×10^9/L. Peptide gel formation was induced by phosphate buffered saline for three-dimensional culture. MAIN OUTCOME MEASURES： （1）Observation of chondrocyte morphology in the short-peptide hydrogel with inverted phase contrast microscope. （2） Detection of intracellular matrix secretion by toluidine blue staining and immunohistochemistry. （3）Measurement of mRNA expression levels of aggrecan, .type II collagen and type Ⅰ collagen in chondrocyte-seeded hydrogels by reverse transcription-polymerase chain reaction. RESULTS： （1）The viability of chondrocytes digested and isolated from rabbit articular cartilage was over 95%. （2） Chondrocytes cultured in three-dimensional short-peptide hydrogel maintained their round shape, proliferated actively, and aggregated in clusters. In addition, cartilage lacuna could be seen surrounding the clusters. （3） Both toluidine blue staining and type Ⅱ collagen immunohistochemistry results were positive. The mRNA expression levels of aggrecan and type Ⅱ collagen were increased with time. （4） Reverse transcription-polymerase chain reaction results revealed that after 3 weeks of in vitro culture in the short-peptide hydrogel, chondrocytes maintained the capability to secrete aggrecan and to express type Ⅱ collagen. CONCLUSION： Self-assembling short-peptide KLD-12 three-dimensional hydrogel can well maintain the normal cellular morphology, function and proliferative capability of chondrocytes.