双层壳聚糖与HAP复合支架的初步研究

PRELIMINARY STUDY ON CHITOSAN/HAP BILAYERED SCAFFOLD

目的探讨双层壳聚糖(chitosan,CS)/HAP复合支架作为骨软骨组织工程支架的可行性,并结合兔自体BMSCs修复骨软骨缺损。方法采用冻干法和烧结法制作双层CS/HAP复合支架,检测其理化特性。取日本大耳白兔骨髓4～6mL,全骨髓培养法分离纯化BMSCs,并鉴定。调整第2代BMSCs细胞密度为2×107个/mL,应用纤维蛋白胶种植技术,接种至双层CS/HAP复合支架,体外构建细胞-支架复合物。取36只日本大耳白兔,于右侧膝关节股骨下端外侧髁负重区,作一直径4mm、深3mm的圆柱形缺损,制备兔膝关节骨软骨缺损模型。根据缺损区植入物的不同,分为A、B、C3组(n=12)。A组:植入细胞-支架复合物;B组:植入双层CS/HAP复合支架;C组:不植入任何材料,作为空白对照组。术后6、12周取材,行大体及组织学观察,采用改良Wakitani法评分。结果双层CS/HAP支架CS层孔隙率为76.00%±5.01%,孔径为200～400μm,平均300μm,孔洞相通;HAP层孔隙率为72.00%±4.23%,孔径为200～500μm,平均350μm,孔洞相通,结合部结合好。全骨髓法培养BMSCs,第7天可见集落形成,14d传代;免疫组织化学检测示CD44(+)和CD45(—)。大体观察和组织学检测显示,A组基本修复软骨缺损,骨缺损修复不良,有骨小梁长入;B、C组骨、软骨缺损修复不良,组织学检测以纤维组织或无新生组织形成,软骨及骨缺损均明显存在。术后6、12周,A组改良Wakitani评分分别为(5.17±1.17)分和(3.20±0.75)分,均优于B、C组,差异有统计学意义(P<0.05)。结论双层CS/HAP复合支架可作为骨软骨组织工程支架,复合BMSCs可修复兔关节软骨与骨缺损,重建关节解剖结构。

Objective To study repair of osteochondral defects by using composite of autologous BMSCs and chitosan/HAP （CS/HAP） bilayered scaffold in rabbits and its feasibility as osteochondral tissue engineering scaffolds. Methods CS/HAP bilayered scaffolds were produced with CS and HAP using a lyophilization and sintering method. The pore size of the scaffold was observed by scanning electron microscopy （SEM）. Anhydrous ethanol substitution method determined its porosity. BMSCs were isolated from bone marrow and cultured by general bone marrow methods. Both CD44 and CD45 on the BMSCs surface were detected with immunocytochemistry to identify BMSCs. Cell-scaffold complex was made with BMSCs as seed cells and CS/HAP bilayered scaffold as carrier by fibrin glue planting technique. The distribution of BMSCs in CS/HAP scaffold was tested by SEM. The osteochondral defect （4 mm in diameter and 3 mm in height） model was made in the right knee ioint of 36 Japanese white rabbits, which were randomly divided into 3 groups. Defects were repaired with CS/HAP and BMSCs composite （ group A, n=12） and with CS/HAP implants （group B, n=12）; defects were not treated as a control （group C, n=12）. Histological evaluation and gross observation were carried out at 6 weeks （n=6 in each group） and 12 weeks （n=6 in each group） postoperatively. Semi-quantitative histomorphological analysis was done to evaluate the repair cartilage tissue according to the modified Wakitani grading scale. Results CS/HAP bilayered scaffold possessed a porosity of 76.00% ± 5.01% and pore size of 200-400 μm （mean 300 μm ） in CS layer, and 72.00%±4.23% and 200-500 μm （mean 350μm） in HAP layer, respectively. BMSCs formed colonies within 10-14 days. Immunocytochemistry results showed BMSCs had positive CD44 expression and negative CD45 expression. At 6 and 12 weeks after operation, gross and histological observation showed that the cartilage defects were fully filled with regenerated tissue, but bone defects were partially repaired in group A; the cartilage and bone defects were partially filled with regenerated tissue in group B and group C. The modified Wakitani grading scale were 5.17 ± 1.17 and 3.20±0.75 in group A, 9.00 ±0.63 and 6.00 ± 0.89 in group B, and 10.00 ±0.89 and 9.60±0.82 in group C at 6 weeks and 12 weeks postoperatively, respectively; showing significant differences between group A and groups B, C （P 〈 0.05）. Conclusion The novel CS/HAP bilayered scaffold possesses porous structure and will possibly become a new biomaterial of osteochondral tissue engineering.