自体骨髓提取物Buffy Coat与透明质酸复合物对软骨再生的实验研究

发布时间：2019-04-02 03:16

【摘要】：目的:探讨自体骨髓提取物Buffy Coat和透明质酸(Hyaluronic Acid,HA)复合物对兔膝关节软骨的再生的影响,为Buffy Coat修复软骨的临床应用提供实验依据。方法:将成年新西兰大白兔36只(16周龄,体重3.0kg-3.5kg)随机分为三组(A组、B组和C组),每组12只大白兔,A组软骨缺损处只注射透明质酸;B组只注射Buffy Coat;C组注射Buffy Coat与透明质酸复合物。Buffy Coat提取在大白兔髂前上棘处提取骨髓进行分离获得Buffy Coat,制备软骨缺损模型选择大白兔右膝关节的股骨髁滑车处造软骨缺损直径约5mm,深于2mm。术后第4周和第8周进行取标本,肉眼观察软骨缺损区外观并拍照保存资料。病理学观察进行常规脱钙、脱水、蜡块包埋、病理切片、潘红O染色、封片,在显微镜下观察观察再生软骨细胞形态学、染色现象、结构完整性、表面整齐性、软骨厚度、再生的软骨下骨、再生软骨和周围正常组织整合情况并拍照保存资料以及请三名相关实验人员进行ICRS评分并对三组标本评分结果统计学比较分析。结果:1.肉眼观察:术后第4周见A组缺损区保持缺陷,B组中的缺损区可见修复组织部分填充缺陷区,C组中的缺陷的填充程度比B组较宽,仍未填充满。术后第8周三组的缺损区已被填充满,A组的修复组织明显发白与周围正常软骨明显分开,而B组和C组表面光滑接近于周围正常软骨,但有少量发白。2.镜下观察:术后第4周C组潘红O染色观察染色现象轻微减少、表面中等与B组和A组比较好,A组大部分无修复软骨、软骨下骨未重建,B组和C组大部分纤维软骨修复,软骨下骨部分重建。术后第8周A组纤维组织修复,表面层较粗糙,软骨下骨部分重建。B组中的修复组织表面较整齐但仍不规则,并发现大量的胶原纤维,细胞分布由具有透明性质的柱状和簇状细胞组成,但细胞的排列较乱,重建的软骨下骨大部分重建而不规则的,也未观察到软骨组织和周围软骨之间的整合。C组中的缺陷区比B组整齐接近周围正常软骨表面,并发现大量的胶原纤维与正常软骨类似的胶原纤维的良好定向排列,细胞的排列大部分类似正常软骨细胞排列,观察到有两边的修复组织与周围组织也未能融合,重建软骨下骨大部分重建。ICRS评分及统计学分析..第4周三组比较明显有差异(P0.05),组间比较C组软骨修复效果优于B组和A组,显著统计学意义,有明显差异(P0.05),第8周ICRS评分三组比较明显有差异(P0.05),组间比较B组和C组无明显差异(P0.05),通过第4周和第8周ICRS评分可以看到每组的评分随时间的推移评分越高。结论:Buffy Coat与透明质酸复合物对兔关节软骨缺损再生有明显的效果,透明质酸凝胶状物可以与Buffy Coat混合用于解决Buffy Coat液体状难移植和固定到软骨损伤部位的难题,为Buffy Coat在临床应用提供了实验依据。
[Abstract]:Aim: to investigate the effect of autogenous bone marrow extract (Buffy Coat) and hyaluronic acid (Hyaluronic Acid,HA) complex on cartilage regeneration of rabbit knee joint in order to provide experimental basis for the clinical application of Buffy Coat in repairing cartilage. Methods: thirty-six adult New Zealand white rabbits (16 weeks old, weight 3.0kg-3.5kg) were randomly divided into three groups (group A, group B and group C) with 12 rabbits in each group. Hyaluronic acid was injected into cartilage defect of group A and Buffy Coat; was injected into group B only. Group C was injected with Buffy Coat and hyaluronic acid complex. Buffy Coat to extract bone marrow from anterior superior iliac spine of rabbits. Buffy Coat, was used to prepare cartilage defect model. The diameter of cartilage defect at trochanter of femoral condyle of rabbits was about 5 mm. Deeper than 2 mm. The specimens were taken from the 4th and 8th week after operation. The appearance of cartilage defect area was observed by naked eye and the data were photographed. Pathological observation was carried out by routine decalcification, dehydration, wax embedding, pathological section, dipyridol O staining and sealing. The morphology, staining phenomena, structural integrity, surface uniformity and cartilage thickness of regenerated chondrocytes were observed under microscope. The integration of regenerated subchondral bone, regenerated cartilage and surrounding normal tissue was recorded and the data were taken and kept. Three relevant researchers were asked to evaluate the ICRS score and compare the results of the three groups of specimens statistically. Results: 1. At the 4th week after operation, the defect area in group A remained defect, the defect area in group B was partly filled with defect area, and the filling degree of defect in group C was wider than that in group B, and the defect in group C was still not filled. At the 8th week after operation, the defect area of the three groups had been filled, and the repair tissue of group A was obviously white from the surrounding normal cartilage, while the surface of groups B and C was smooth close to that of the surrounding normal cartilage, but a small amount of white was observed. Microscopic observation: at the 4th week after operation, the staining phenomenon of dipyridamole O in group C decreased slightly, the surface of group C was better than that of group B and group A, most of the cartilage in group A had no repair, the subchondral bone had not been reconstructed, and most of fibrocartilage in group B and C had been repaired. The subchondral bone was partially reconstructed. At the 8th week after operation, the fibrous tissue of group A was repaired with rough surface layer and partial reconstruction of subchondral bone. In group B, the surface of the repaired tissue was neat but still irregular, and a large number of collagen fibers were found. The distribution of cells is composed of transparent columnar and clustered cells, but the arrangement of cells is chaotic, and the reconstructed subchondral bone is mostly reconstructed and irregular. No integration between cartilage tissue and surrounding cartilage was observed. The defect areas in group C were nearer to the surface of surrounding normal cartilage than in group B, and a large number of collagen fibers were well aligned with those similar to normal cartilage. Most of the cells were similar to those of normal chondrocytes, and no fusion was observed between the repair tissue and the surrounding tissue. ICRS score and statistical analysis were used to reconstruct the subchondral bone. At the fourth week, there was significant difference among the three groups (P0.05), and the cartilage repair effect of group C was better than that of group B and A (P0.05), and there was significant difference between group C and group A (P0.05). There was significant difference in ICRS score among the three groups at the 8th week (P0.05), but there was no significant difference between the two groups (P0.05). The ICRS scores in the 4th week and 8th week showed that the score of each group was higher with the passage of time. Conclusion the complex of: Buffy Coat and hyaluronic acid has obvious effect on the regeneration of articular cartilage defect in rabbits. Hyaluronic acid gel can be mixed with Buffy Coat to solve the difficult problem of Buffy Coat being difficult to transplant and immobilize to the injury site of cartilage. It provides experimental basis for the clinical application of Buffy Coat.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R687

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