摘要
目的探讨软骨细胞与骨髓基质细胞（BMSC）共培养体外构建复合物体内植入形成成熟软骨组织的可行性。方法体外培养扩增猪BMSC，经绿色荧光蛋白（EGFP）基因转染标记后，与猪关节软骨细胞按1：1比例混合，以5．0×10^7／ml的细胞终浓度接种于聚羟基乙酸／聚乳酸（PGA／PLA）材料支架作为共培养组，以相同终浓度的单纯软骨细胞和单纯BMSC分别接种PGA／PLA作为阳性对照组及阴性对照组。各组标本均于体外培养2周后植于裸鼠皮下，8周后取材，通过大体观察、激光共聚焦显微镜观察、糖胺聚糖含量测定、生物力学、组织学以及免疫组织化学等方法对新生软骨组织进行全面评价。结果各组细胞均与材料黏附良好。体内植入8周后，共培养组及阳性对照组标本基本保持植入前的大小和形状，外观类似软骨组织，组织学显示两组均有连续的软骨陷窝样结构形成，免疫组织化学显示有大量Ⅱ型胶原沉积。定量测定结果显示，共培养组的糖胺聚糖含量和弹性模量均达到软骨细胞构建软骨组织的80％以上。共聚焦显微镜观察可见EGFP标记细胞形成了软骨陷窝。阴性对照组标本的体积较植入前明显缩小，组织学及免疫组织化学均未见软骨样组织形成。结论软骨细胞与BMSC共培养构建的复合物植入体内后能继续形成成熟软骨组织，这表明植入体内后软骨细胞仍能保持对BMSC的诱导作用。
Objective To explore the feasibility of in vivo chondrogenesis of bone marrow stromal cells （BMSCs） co-cultured with chondrocytes on biodegradable scaffold. Methods Porcine BMSCs were isolated, expanded and labeled with enhanced green fluorescent protein （EGFP）, and then were mixed with articular chondrocytes isolated from porcine knee joint at the ratio of 1： 1. The mixed cells were seeded onto polyglycolic acid （PGA） scaffold at the ultimate concentration of 5.0 × 10^7/ml （co-culture group）. Pure chondrocytes and BMSCs of the same ultimate concentration were seeded respectively onto the scaffold as positive control group and negative control group. After two weeks＇ culture in vitro, they were planted subcutaneously into nude mice respectively. These specimens were collected after in vivo implantation for 8 weeks to undergo microscopy. Laser co0focal microscopy was used to observe the distribution of EGFP- labeled cells in the tissue. RT-PCR was used to examine the expression of collagen type Ⅱ and aggrecan. Immunohistochemistry was used to observe the protein expression of collagen type Ⅱ. Results The cell- scaffold constructs of the co-culture group and positive control group, could maintain the original size and shape no matter in vitro or in vivo. After 8 weeks＇ in vivo implantation, the constructs in both co-culture group and positive control group formed cartilage-like tissue with typical histological structure and extracellular matrix staining similar to those of the normal cartilage. The GAG content and compressive modulus of the co-culture group reached over 80% of those of the positive control group. Confocal microscopy revealed the presence of EGFP-labeled cells in the engineered cartilage lacuna. Histological examination showed that the constructs of the negative control group shrunk gradually after in vivo implantation with no typical cartilage-like tissue formation. Conclusion In vitro co-cultured BMSC- chondrocyte-PGA contructs have the potential to form mature cartilage-like tissue in subcutaneous non- chondrogenesis environment, indicating that chondrocytes still provide enough signals for BMSC chondrogenic differentiation.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第27期1929-1933,共5页
National Medical Journal of China
基金
国家重点基础研究发展"973"计划基金(2005CB522702)
国家自然科学基金(30300353)
国家高技术发展研究计划"863"重大专项基金(2002AA205021)
