诱导和非诱导骨髓基质干细胞在关节软骨缺损处成软骨的效应比较

Comparison of the effects between induced and non-induced bone marrow stromal cells transplantation in the repair of articular cartilage defects in rabbits

目的:探讨经体外诱导和非诱导的骨髓基质干细胞复合胶原海绵修复兔膝关节全层软骨缺损成软骨效应的差异。方法:实验于2005-01/2006-06在浙江省医学科学院生物工程研究所完成。①将兔骨髓基质干细胞与载体胶原海绵共培养6h形成骨髓基质干细胞/胶原海绵复合物。②22只新西兰大白兔,分为两组。制备双膝股骨下端滑车的直径4.5mm、深3mm关节软骨缺损模型。未诱导组:一侧植入未经诱导的骨髓基质干细胞/胶原海绵复合物,作为实验侧;另一侧植入单纯胶原海绵,作为对照侧。诱导组:实验侧植入经软骨诱导的骨髓基质干细胞/胶原海绵复合物,对照侧为单纯胶原海绵。③术后未诱导组于0.5,1,2,3,5,7,9个月,诱导组于1,2,3,4,5,7个月分别处死动物,观察缺损修复情况及新生组织类型。参照Pineda标准对新生组织评分。结果:22只兔均进入结果分析:①两组兔材料移植后大体观察结果:未诱导组实验侧:移植后2个月,缺损修复处表面光滑,质地较硬,类似正常软骨,边界已不是很明显。至移植后7个月及9个月,修复组织与正常软骨基本相同。对照侧:移植后2个月,缺损处仍有较明显的凹陷,表面较粗糙,致密性差,质地较软,色泽与周围组织仍有较大差别。至移植后7个月及9个月,表面仍欠平整,质地仍欠坚韧,修复组织为白色纤维样组织,色泽与周围软骨组织仍尚有差别。诱导组大体观察情况与未诱导组相似,无明显差异。②两组兔软骨缺损区形态学观察结果:未诱导组:移植1个月,实验侧原软骨缺损区逐渐被透明软骨样组织取代,与周围软骨组织分界较明显,与周围软骨组织相比,细胞排列相对较不整齐,甲苯胺蓝染色阳性,随着时间的推移,与正常软骨组织结构相似,但并不完全一致。对照侧移植1个月以后未见明显修复,原软骨缺损区取代的主要是略带红色的纤维组织,界面明显,随着时间的推移,修复速度明显较慢,与周围软骨组织连接速度显著慢于实验侧,1个月时甲苯胺蓝染色呈阴性,3个月时呈弱阳性,5个月时异染仍不明显。③诱导组形态学观察情况与未诱导组相似,无明显差异。各阶段经改良Pineda评分的结果,未诱导组:实验侧平均评分低于对照侧(5.87±1.85,10.53±1.16,t=8.27,P<0.001);诱导组:实验侧平均评分低于对照侧(5.57±0.93,10.00±1.53,t=6.55,P<0.001)。结论:未经诱导的骨髓基质干细胞及经软骨诱导的骨髓基质干细胞移植后结果相似,提示软骨缺损处可提供诱导骨髓基质干细,胞的微环境。

AIM： To explore the difference of the effects between in vitro induced and non-induced bone marrow stromal cells （BMSCs）/collagen sponge compound in the repair of full-thickness knee articular cartilage defects in rabbits. METHODS： The experiment was performed at the Institute of Bioengineering, Zhejiang Academy of Medical Sciences from January 2005 to June 2006. ①BMSCs were cultured with collagen sponge for 6 hours to form BMSCs/collagen sponge compound. ②Totally 22 New Zealand rabbits were assigned into 2 groups. Both knees full-thickness articular cartilage defects With 4.5 mm diameter and 3 mm depth were prepared. Non-induced group： The defects were filled With BMSCs/collagen sponge compound on one side as experimental side, and filled With only collagen sponge on the other side as control side. Induced group： the experimental side was filled with cartilage-induced BMSCs/collagen sponge compound, and the control side was filled with simple collagen sponge. ③Animals were sacrificed at months 0.5, 1, 2, 3, 5, 7 and 9 in the non-induced group and at months 1, 2, 3, 4, 5 and 7 in the induced group after operation. Defect repair and type of new tissues were observed. New tissues were scored with Pineda criteria. RESULTS： Totally 22 rabbits were involved in the result analysis. ①General observational result of rabbits in the two groups after transplantation： In experimental sides of non-induced group, the defect surfaces were smooth, hard, which ware similar to the normal cartilage, and the boundary was not very significant 2 months after transplantation. Till the 7^th and 9^th months after transplantation, the repair tissues ware nearly normal morphology of cartilage. In the control side, 2 months after transplantation the defects were obvious introcession, and the surface was rough, badly compact, soft, and the color was quietly different from that of the surrounding tissues, Until the 7^th and 9^th months after transplantation, the surface was still not even, soft, and the repair tissues were white fibrous-like tissues, and the color was still different from that of the surrounding tissues. The general observation in the induced group was not significantly different from that of the non-induced group. ②Observational result of morphology of bone defects of rabbits in the two groups： One month after transplantation in the non-induced group, the original cartilage defects in the experimental side were replaced by hyaline cartilage gradually, and the boundary was very significant with the surrounding cartilage tissues： As compared with the surrounding cartilage tissues, cells arranged irregular, and positive for toluidine blue staining. With the fly of time, it had the similar morphology of cartilaginous tissue, but did not complete the same. One month after transplantation in the control side, no significant repair was seen. The original cartilage defects were replaced by erubescent fibrous tissues, with obvious boundary. With the fly of time, the repair became markedly slow, and the connect speed With surrounding cartilaginous tissues was slower than that of experimental side. At month 1, it showed negative in toluidine blue staining, weakly positive at month 3, and metachromasla was Insignificant at month 5. ③Observational result of morphology in the induced group was similar to that in the non-induced group, and there was no significant difference. The findings of Pineda score in the each phase： the average score in the non-induced group was lower in the experimental side than in the control side （5.87±1.85,10.53±1.16, t =8.27, P 〈 0.001 ）. The average score in the induced group was lower in the experimental side than in the control side （5.57±0.93,10.00＋1.53, t =6.55,P〈 0.001 ）. CONCLUSION： The results are similar between the non-induced BMSCs transplantation and cartilage-induced BMSCs transplantation, It indicates that cartilage defect region has the microenvironment of inducing BMSCs into cartilage cells.