骨髓间充质干细胞复合仿生凝胶修复猪关节软骨缺损

Bone marrow mesenchymal stem cells combined with biomimetic hydrogel for repair of articular cartilage defect in porcine

目的探讨骨髓间充质干细胞（BMSCs）复合Ⅱ型胶原（ColⅡ）-透明质酸（HA）-氧化硫酸软骨素（OCS）仿生凝胶修复猪关节软骨缺损的可行性，以及移植细胞在软骨修复中所起的作用。方法以巴马香猪为实验动物，构建保留钙化层的猪膝关节软骨缺损模型，以自体BMSCs作为移植的种子细胞，并进行羟基荧光素乙酰乙酸（CFDASE）标记。按随机数字表法将动物分为三组：A组（空白对照组，缺损不做处理）、B组（无细胞仿生凝胶组，缺损内植入仿生凝胶）和C组（BMSCs复合仿生凝胶组，缺损内植入CFDASE标记的自体BMSCs复合仿生凝胶）。术后1个月处死动物并取材（于取材前24h和48h静脉注射BrdU标记液）。术后1个月，于取材前24h和48h静脉注射BrdU标记液后处死动物。取c组部分修复组织做冰冻切片，行DAPI染色及BrdU免疫荧光染色，激光共聚焦显微镜下对细胞进行观察、计数。对三组标本行大体观察、组织学染色，按照国际关节软骨修复协会（ICRS）评分方法进行大体和组织学评分。结果激光共聚焦显微镜下对C组软骨修复组织中的细胞进行观察、计数，修复组织中有（97．3±2．6）％的细胞源于植入的BMSCs，其中有增殖能力的细胞占（76．6±2．5）％。大体观察显示：C组的软骨缺损区域大部分被乳白色组织填充，而A、B组仍可见明显凹陷。组织学染色显示：C组软骨缺损区域被软骨样组织填充且与周围正常软骨整合良好，可见少量软骨陷窝，ColⅡ和糖胺聚糖含量接近邻近的正常软骨；A组缺损区域基本无修复组织；B组缺损区域有少量纤维样组织。A、B、C组的ICRS大体评分分别为（0．5±0．6）分、（2．3±0．5）分、（8．3±1．0）分，ICRS组织学评分分别为（0．5±0．6）分、（4．5±1．0）分、（10．3±2．4）分，C组与其他两组的差异均有统计学意义（P〈0．05）。结论BMSCs复合ColⅡ—HA—OCS仿生凝胶修复关节软骨缺损可取得良好的修复效果，植入的BMSCs是修复组织内细胞组成的主体，且在逐渐向软骨细胞分化。

Objective To investigate the feasibility of bone marrow mesenchymal stem cells （BMSCs） combined with type Ⅱ collagen- hyaluronie acid- oxidized chondroitin sulfate （Co1 Ⅱ-HA- OCS） biomimetic hydrogel to repair articular cartilage defect in porcine and the role of the transplanted cells played in the process of cartilage repair. Methods A articular cartilage defect model which remaining cartilage calcified zone was created in the knee of Bama minipigs, the autologous BMSCs was used as seeds for transplantation and was labeled by the 5,6-carboxyfluorescein diacetate suceinimidyl ester （CFDA SE）. Animals were randomly divided into three groups： Group A （blank group） was left untreated, group B （cell-free biomimetic hydrogel group） was filled with biomimetic hydrogel and group C （BMSCs combined with biomimetic hydrogel group） was filled with the CFDA SE labeled autologous BMSCs combined with biomimetic hydrogel. One month after the operation, BrdU labeled liquid was injected intravenously into the animals 24 h and 48 h before specimens were taken from the executed animals. Partial cartilage repaired tissue in Group C was taken, cryosectioned and stained with DAPI and BrdU immunofluorescence. Confocal laser scanning microscope was used to observe and count the cells. Specimens of the three groups were analyzed through gross observation and histological staining, and scored according to the international cartilage repair society （ICRS） gross morphological score and ICRS histological score. Results Laser scanning confocal microscopy showed （97.3 ±2.6） % of the ceils were derived from the implanted BMSCs in repaired tissue and that the ratio of these cells with proliferative capacity was （ 76.6 ±2.5 ） %. Gross observation suggested most of the cartilage defect areas in Group C were filled with ivory tissue, but those in Group A and B were still obvious depression. Histological staining showed the cartilage defect areas in Group C were filled with cartilage like tissue, which was well integrated with the surrounding normal cartilage, presented a few cartilage lacunas could be seen, and had contents of Col Ⅱ and glycosaminoglycan similar with the adjacent nornml cartilage. There was almost no filler in the defect area in Group A. There was little fibrous tissue in the defect area in Group B. ICRS gross score was （8.3 ± 1.0） points in Group C, higher than that in Group A [ （0.5 ± 0.6 ） points] and Group B [ （2.3± 0.5 ） points ] （ P 〈 0.05 ）. ICRS histological score was （ 10.3± 2.4） points in Group C, higher than that in Group A [ （0.5 ± 0.6） points ] and Group B [ （4.5 ±1.0） points ] （ P 〈 0.05 ）. Conclusions BMSCs combined with ColⅡ -HA-OCS biomimetic hydrogel for repairing porcine articular cartilage defects can achieve satisfactory results. Implanted BMSCs are the main component of the cell composition in the repaired tissue and gradually differentiated into chondrocytes.