透明质酸改性聚乳酸支架组织工程软骨的构建

Construction of tissue engineering cartilage with modified polylactic acid scaffold using hyaluronic acid

目的利用透明质酸(hyaluronic acid,HA)改性聚乳酸(polylactic acid,PLA)支架来构建同种异体组织工程软骨,分析探讨HA改性PLA支架构建组织工程软骨的可行性。方法采用盐析法制备出高孔隙率的PLA支架,采用低浓度NaOH、碳化二亚胺和HA进行支架改性。支架预湿后接种第3代兔髁突软骨细胞悬液,体外培养1周后用于动物实验。软骨细胞-HA改性PLA支架复合体为实验组,软骨细胞-PLA支架复合体为对照组。分别于4、6、8周按照标记取出植入物,进行组织学检测。利用ImageJ软件进行免疫组化染色阳性强度的分析。结果细胞-支架复合体植入后,未产生排斥反应。HE染色结果示软骨样组织形成。Masson染色及甲苯胺蓝染色结果显示染色逐渐加深。Ⅱ型胶原免疫组化染色随时间增加阳性强度增加,实验组4、6、8周时的免疫组化染色强度差异有统计学意义(F=26.68,P=0.000),对照组4、6、8周时的免疫组化染色强度差异有统计学意义(F=20.59,P=0.000)。相同时间点实验组Ⅱ型胶原分泌强度高于对照组。结论利用HA改性PLA支架可以构建同种异体软骨,且其质量高于未改性PLA。

Objective To construct tissue engineering neocartialge based on the modified polylactic acid（PLA） scaffold with hyaluronic acid（HA）.The significance of HA in construct tissue-engineered cartilage was to be analyzed and discussed.Methods The highly porous PLA scaffold was made by salt-leaching method;Low concentration NaOH,carbodiimide and HA were used to modify the scaffold.The third passage of chondrocytes was seeded into the scaffold after prewetted.The concentration was 5×107 per milliliter.The complex was implanted into rabbits one week after vitro culture.The prepared complex was implanted into the back of the New Zealand rabbits.The complexes of chondrocyte-modified PLA scaffold were applied in the experimental group,and the complexes of chondrocyte-PLA scaffold were used in the control group.Two rabbits were sacrificed randomly at four,six,eight weeks after implantation.The tissue were taken out according to the marker made during operation at 4,6,8 weeks after operation respectively.The histological characteristics was observed by HE,Masson,toluidine blue stain and typeⅡcollagen immunohistochemical test.Results There was no immunoreactive rejection after implantation of the cell-scaffold complex.After two month two kinds of cell-scaffold appearance were similar to the articular cartilage.The results of histochemical test revealed that the tissue had been becoming better when being ompanted longer.A comparison analysis was made according to the expression intensity using Imagej.There were statistical significance between experimental and control groups（P0.05） at 6,8 weeks.The secretion cartilage matrix of modified scaffold group was better than that of the PLA group.Conclusion The allogeneic chondrocytes could be the seed of tissue-engineered cartilage.The modified PLA scaffold can be the carrier to form neocartilage in vivo.