骨髓基质细胞源性软骨细胞复合聚乳酸/聚羟基乙酸共聚物修复兔关节软骨缺损(英文)

Repairing articular cartilage defects in rabbits using bone marrow stromal cell-derived chondrocytes compounded with poly(lactic-co-glycolic acid)

背景:作为软骨组织工程的基质材料在体内降解过快或过慢,影响组织再生及塑形改建,是长期困扰学者们的难题之一。目的:在体外将骨髓基质细胞扩增、诱导为软骨细胞后,探讨以聚乳酸/聚羟基乙酸共聚物为载体修复兔关节软骨缺损的可行性。设计、时间及地点:同体对比观察实验,于2002-06/2008-06分别在解放军第四军医大学全军骨科研究所及解放军空军总医院中心实验室完成。材料:选取2月龄新西兰兔36只,自双侧股骨转子处抽取骨髓4～6mL,行原代及传代培养,传代培养时培养液中含骨形态发生蛋白2(100μg/L),培养瓶底预涂高分子透明质酸诱导为软骨细胞。调整第3代细胞浓度为2.0×1010L-1,与聚乳酸/聚羟基乙酸共聚物共培养24h,即制成聚乳酸/聚羟基乙酸共聚物-细胞复合物。方法:在36只兔髌股关节股骨髁部造成直径4mm,深达髓腔的缺损,在右侧36个膝关节植入聚乳酸/聚羟基乙酸共聚物-细胞复合物,为实验组,左侧18膝植入聚乳酸/聚羟基乙酸共聚物,为单纯载体组,另18膝造成缺损后作为空白对照。主要观察指标:术后4,8,12,24周取材,行大体及组织学观察,组织学评分。结果:单纯载体组与空白对照组在各时间点大体及组织学表现相似,故一并描述,统称为对照组。24周时实验组缺损内充填白色半透明新生软骨组织,色泽与正常软骨相似,质韧,表面平整,与正常软骨界限消失,表面细胞平行于关节面,深层细胞排列紊乱,有柱状排列的趋势,基质异染广泛,软骨下骨形成及潮线恢复正常,与周围正常软骨连接良好。对照组缺损边缘细胞呈团块状增生,底部为纤维组织。组织学评分经统计学分析,24,12周分别与8,4周时比较差异具有显著性意义(P<0.01),各时间点实验组与对照组比较差异具有显著性意义(P<0.01)。结论:用骨形态发生蛋白2和高分子透明质酸成功地在体外将骨髓基质细胞诱导为软骨细胞;聚乳酸/聚羟基乙酸共聚物在新生软骨形成的同时,逐渐降解吸收,是组织工程修复关节软骨缺损适宜的支架材料。

BACKGROUND：Matrix material for cartilage tissue engineering exhibits too fast or too slow degradation in vivo,affecting tissue regeneration and shaping reconstruction,which has troubled scholars.OBJECTIVE：To amply bone marrow stromal cells （BMSCs） and induce them to chondrocytes in vitro,so as to study the feasibility of repairing articular cartilage defects in rabbits using poly（lactic-co-glycolic acid） （PLGA） loaded with BMSC-derived chondrocytes.DESIGN,TIME AND SETTING：Comparative experiment was performed at the Institute of Orthopaedics in the Fourth Military Medical University of Chinese PLA and the Center Laboratory of the Airforce General Hospital of Chinese PLA between June 2002 and June 2008.MATERIALS：A total of 36 two-month-old New Zealand white rabbits were used,and 4-6 mL bone marrow was aspirated from bilateral femoral trochanters in each animal.Primary culture and subcultures were done.In subcultures,the medium contained bone morphogenetic protein-2 （100 μg/L）,and high polymer hyaluronic acid was spread on bottom of the culture flasks in advance.In this way,the BMSCs were induced into chondrocytes and the third passage of cells at the adjusted density of 2.0×1010/L were co-cultured with PLGA for 24 hours,then PLGA-cell composites were prepared.METHODS：A defect of 4-mm in diameter and reaching medullary cavity were created in femoral condyles of 36 rabbits,and 36 right knees were treated with PLGA-cell composites,serving as experimental group,while 18 left knees with PLGA only as material group,and the other 18 knees remained untreated,as blank control group.MAIN OUTCOME MEASURES：At 4,8,12,24 weeks after operation,the animals were euthanized and the newly formed tissues were observed macroscopically and microscopically,graded histologically,and analyzed statistically.RESULTS：Material group and blank control group shared identical outcomes of gross and histological observation,thus assigning into a control group.In the experimental group at 24 weeks,the defects were filled with white translucent cartilage tissue which appeared smooth and tenacious.The color and the luster were similar to that of normal cartilage,and was ill-demarcated from the surrounding normal cartilage.The cells on the surface paralleled to joint surface.Though the cells in the deep layer arranged disorderly,they tended to align vertically.The matrix was extensively stained.The subchondral bone formed.The tide mark basically recovered,and the new cartilage integrated with normal cartilage finely.In the control group,chondrocytes proliferated in the border,but in the bottom,there were mainly fibrous tissues.The histological grade of 12 and 24 weeks was different significantly from that of 4 and 8 weeks （P 0.01）.There were also significant differences between experimental group and control group at each time intervals after operation （P 0.01）.CONCLUSION：BMSCs were successfully induced into chondroncytes by use of bone morphogenetic protein-2 and high polymer hyaluronic acid.PLGA can be degraded and absorbed gradually while new cartilage tissues form.It can be used as a suitable scaffold material for repairing articular cartilage defects in tissue engineering.