摘要
目的:观察自体骨髓间充质干细胞复合新型支架材料羟基磷灰石/聚磷酸钙纤维/左旋聚乳酸修复兔膝关节软骨全层缺损。方法:实验于2005-03/08在兰州大学骨科研究所完成。①抽取兔骨髓液经过密度梯度离心得到单个核细胞,在经过体外分离培养获得骨髓间充质干细胞,将骨髓间充质干细胞吸附于羟基磷灰石/聚磷酸钙纤维/左旋聚乳酸复合材料上,再移植于兔膝关节全层软骨缺损模型处。②36只健康青紫兰兔被随机分成3组,每组各12只。细胞材料复合物组:将骨髓间充质干细胞与羟基磷灰石/聚磷酸钙纤维/左旋聚乳酸复合物植入双侧股骨髁间窝软骨缺损处;单纯复合材料组:单纯植入羟基磷灰石/聚磷酸钙纤维/左旋聚乳酸;空白对照组:不做任何植入。③分别于术后4,8,12周各处死4只兔子,取材进行大体、组织学及免疫组织化学染色观察。大体及组织学观察结果参照O’driscoll的评分标准进行评分。结果:36只兔全部进入结果分析。①培养细胞的生物学特性观察:原代培养骨髓细胞7～10d后,多数细胞旋涡状排列。传代6h后细胞开始贴壁,5～7d铺满瓶壁。细胞形态均一呈梭形。②各组兔术后大体观察、股骨组织学检查:细胞材料复合物组术后12周大体评分明显低于单纯复合材料组和空白对照组(修复组织表面结构:0.45±0.25,1.15±0.25,2.47±0.39,P<0.05;缺损填充深度:0.35±0.15,1.27±0.27,2.63±0.27,P<0.05;与周围软骨结合情况:0.58±0.08,1.10±0.24,1.87±0.64,P<0.05)。细胞材料复合物组12周关节软骨表面光滑,光镜下可见已形成正常软骨厚度的软骨层及完整的软骨下骨,与对照组有明显差异。修复组织Ⅱ型胶原免疫组织化学染色阳性,强度与周围正常软骨无差别。软骨缺损处为透明软骨修复。组织学评分各项(缺损填充深度、与周围软骨结合情况、修复组织表面结构、细胞形态、潮线形成、甲苯胺兰染色)评分均显著低于单纯复合材料组和空白对照组。③各组兔修复组织吸光度值形态分析:修复方法在基质分泌方面优势顺序为:细胞材料复合物组>单纯复合材料组>空白对照组。结论:自体骨髓间充质干细胞复合羟基磷灰石/聚磷酸钙纤维/左旋聚乳酸材料以类透明样软骨组织完整修复缺损,是一种修复软骨缺损的行之有效的方法,羟基磷灰石/聚磷酸钙纤维/左旋聚乳酸可以做为组织工程软骨支架材料。
AIM： To observe the curative effects of homograft of mesenchymal stem cells （MSCs） seeded onto composites material of hydroxyapatite/calcium polyphosphate/poly-L-lactide （HA/CPP/PLLA） on articular cartilage defects.
METHODS： The experiment was conducted in the Institute of Orthopedics, Lanzhou Unviersity from March to August 2005. ①Mononucleareell was obtained after density gradient centrifugation from rabbit marrow, and the mesenchymal stem cells derived from bone marrow which were isolated from the nucleated cells fraction autologous bone marrow. Then the MSCs were seeded onto HA/CPP/PLLA novel scaffold composite materials, and transplanted into the full-thickness defects. ②Thirty-six healthy Qingzilan rabbits were randomly divided into 3 groups with 12 rabbits in each group： MSCs/HA/CPP/PLLA complex group： The MSCs/HA/CPP/PLLA complex were cultured and transplanted into full- thickness defects on intercondylar notch. HA/CPP/PLLA group only with HA/CPP/PLLA; and control group without any graft. ③Four rabbits in each group were killed at 4, 8 and 12 weeks after operation, and the tissue samples were evaluated grossly, histologically, immunohistochemically and graded with O＇ driscoll according to gross and histological scale.
RESULTS： All the 36 rabbits were involved in the result analysis. ① Observation of biological features of cultured cells： After 7-10 days culture, most of the primary marrow cells presented turbulence arrangement. The cells began to adhere to the bottle 6 hours after passage, and covered the bottle at 5-7 days. The cells were all fusiform shape. ② Gross and histological examination after operation： The gross scores of the MSCs/HA/CPP/PLLA group 12 weeks after operation were markedly lower than the HA/CPP/PLLA group and control group （surface structure of repaired tissue： 0.45±0.25, 1.15±0.25, 2.47±0.39, P 〈 0.05; defects filled depth： 0.35±0.15, 1.27±0.27, 2.63±0.27, P 〈 0.05; integrated condition with surrounding cartilage： 0.58±0.08, 1.10±0.24, 1.87±0.64, P 〈 0.05）. At 12 week, the cartilage surface of the MSCs/ HA/CPP/PLLA group was smooth, cartilage layer with normal thickness and complete subcartilaginous bone were observed with light microscope, which had significant differences compared with the control group. Repaired tissue type Ⅱ collage showed positive staining, and the intensity had no difference from surrounding cartilage. Cartilago hyaline was found on the defects. The scores of histology （defect filled depth, integrated condition with surrounding cartilage, surface structure of repaired tissue, cell appearance, tidal line formation, toluidine blue staining） of the MSCs/HA/ CPP/PLLA group were lower than the HA/CPP/PLLA group and control group. ③Morphologic analysis of absorbance of rabbit repaired tissue： The advantages of various repaired methods in matrix secretion： MSCs/HA/ CPP/PLLA group 〉 HA/CPP/PLLA group 〉 control group.
CONCLUSION： The full-thickness cartilage defects of rabbits are repaired with homograft of mesenchymal stem cells seeded onto HA/CPP/ PLLA composites materials, which is a promising way for the treatment of cartilage defects. The composites materials HA,/CPP/PLLA can be used as a scaffold in cartilage tissue engineering.
出处
《中国临床康复》
CSCD
北大核心
2006年第37期64-67,共4页
Chinese Journal of Clinical Rehabilitation
