兔自体细胞-载体复合物修复关节软骨缺损的实验研究

EXPERIMENTAL RESEARCH ON REPAIR OF RABBIT ARTICULAR CARTILAGE DEFFECTS WITH COMPOSITE OF AUTOLOGOUS CELL-CARRIERS

目的研究兔MSCs向软骨细胞方向诱导后复合自体双相骨基质明胶(bone matrix gelatin,BMG)对膝关节软骨缺损的修复作用。方法健康成年新西兰大白兔24只,雌雄不限,体重2～3kg,随机分为A、B、C组,每组8只。取A组骨髓行原代培养,胰酶消化按1∶2比例传至第5代,倒置相差显微镜观察细胞形态。取1、3、5代细胞绘制生长曲线。于第3代MSCs生长密集时加入TGF-β110ng/mL、IGF-110ng/mL、维生素C50ng/mL诱导8d后倒置相差显微镜下观察,爬片行免疫组织化学及Mallory染色。取A组髂骨按Urist方法制备双相BMG,将第3代经诱导8d得到的种子细胞以5×106/mL密度接种于自体BMG制备细胞-载体复合物,体外培养3d后扫描电镜观察。制作膝关节软骨直径3mm,深3mm的缺损模型,A组植入自体细胞-载体复合物,B组植入自体BMG,C组不作处理,第8、12周取材行大体观察、HE染色、免疫组织化学染色观察,Wakitani法组织学评分。结果倒置相差显微镜观察MSCs原代细胞呈短梭形;传代细胞呈长梭形。传代细胞1～3d增殖缓慢,3d后增殖旺盛,7～8d进入平台期,第3代增殖最为旺盛。诱导后MSCs细胞呈椭圆形,Ⅱ型胶原、S-100免疫组织化学染色阳性,Mallory染色胞浆蓝染。细胞-载体复合物扫描电镜观察:BMG结构符合细胞载体要求,细胞种植于BMG后生长良好。动物实验:大体观察A组术后8、12周缺损处均由透明软骨样组织修复,8周较12周表面稍凹陷;B、C组术后8、12周均为纤维样组织修复,表面凹凸不平。组织学观察HE染色A组术后8、12周修复组织与周围软骨组织结合良好,以圆形、椭圆形透明软骨样细胞为主,12周较8周细胞数多且大;B、C组均为纤维样组织修复,12周较8周纤维样组织增厚。Ⅱ型胶原免疫组织化学染色A组术后8周呈阳性、12周呈强阳性;B、C组均无表达。Wakitani评分术后8周A、B、C组分别为(3.50±1.51)、(10.00±1.41)、(12.00±0.93)分,术后12周分别为(1.13±0.99)、(8.38±1.30)、(10.13±1.64)分,A组优于B、C组,B组优于C组,差异均有统计学意义(P<0.05)。结论自体MSCs向软骨细胞方向诱导后,复合自体BMG对关节软骨缺损有较好的修复作用。

Objective To study the effect of the repair of rabbit articular cartilage defects by the composite of chondrogenic induction of autologous MSCs and autologous ＂two-phase＂ bone matrix gelatin （BMG）. Methods Twentyfour healthy adult New Zealand rabbits weighing 2 to 3 kg were divided into group A, B and C with8 in each. Autologous MSCs derived from group A were cultured in vitro and observed under inverted phase contrast microscope when enoughcells through trypsinization transferring in vitro were obtained. Then the growthcurves of 1, 3 and 5 passage culture of MSCs were drawn. The 3rd passage MSCs were induced into chondrogenic differentiation by adding TGF-β1 （10 ng/mL）, IGF-1 （10 ng/mL） and vitamin C （50 ng/mL） in vitro. At 8 days after induction, the features of chondrocytes were observed under inverted phase contrast microscope,and immunohistochemical staining and Mallory staining were made. Getting out part of the ilium of group A and B, according to the method of Urist, the ＂two-phase＂ BMG was acquired. Chondrogenic induction of autologous MSCs was inoculated into the corresponding BMG to set up a composite of cell-carrier, and then it was observed through scanning electric microscope after 3 days of culture. The model of articular cartilage defects of rabbits was made： in group A,autologous cell-carriers were implanted; in group B, there only existed autologous BMG; in group C,there was nothing. At 8, 12 weeks after operation, the gross, HE staining and immunohistochemical staining were made, and grading scales were evaluated according to Wakitani histological grading method. Results Features of MSCs were as follows： the shape of primary cells was shotspindled and of passage cells was long. As to the growth curves of 1, 3 and 5 passage culture of MSCs,passage cells grew slowly for 3 days after being passaged and went into log-growth during the 3rd and the 7th days and into plateau later, but the 3rd passage cells grew best. Observation of MSCs after chondrogenic induction was performed： the shape of cells was elliptical and the effect of induction was verified by the positive results of collagen type Ⅱ, S-100 and Mallory staining. Under scanning electric microscope,the structure of BMG was good and cells were observed growing in it well. As far as repair of articular cartilage defects are concerned at 8, 12 weeks after transplantation, the defects in group A were repaired by the hyline-like tissue and the structures of the cartilage surface and normal cartilage were in integrity, and immunohistochemical staining of collagen type Ⅱ was positive, while those in group B and C were repaired by the fibrous-like tissues and the surfaces were irregular. In Wakitanni histological score, at 8 weeks after operation, group A was （3.50 ± 1.51） points, group B was （10.00 ± 1.41） points and group C was （12.00 ± 0.93） points; at 12 weeks, group A was （1.13± 0.99） points, group B was （8.38±1.30） points, and group C was （10.13 ±1.64） points. At different time points,group A was significantly better than group B and C, showing significant differences （P 〈 0.05）. Conclusion Induced autologous MSCs and the composite with autologous ＂two-phase＂ BMG have the function to repair articular cartilage defects,and they are better than autologous BMG transplanted only or nothing transplanted.