新型双层骨引导再生壳聚糖膜修复大鼠颅骨缺损的实验研究

目的: 通过比较新型双层壳聚糖膜和Bio-Gide膜对大鼠颅骨缺损的修复效果,评估其作为引导骨再生膜的可行性。方法: 双层壳聚糖膜与MC3T3-E1细胞共培养,应用扫描电子显微镜（scanning electron microscope,SEM）观察细胞对膜的黏附效果;采用cell counting kit-8（CCK-8）法在3、7和10 d检测膜的细胞毒性;选择18只Sprague-Dawley（SD）大鼠,随机分为A、B、C组,在其颅骨中线两侧制作5 mm的极量骨缺损,左侧为实验侧,覆盖双层壳聚糖膜,右侧为对照侧覆盖Bio-Gide膜,分别于术后第2、4及8周时处死各组大鼠,通过大体观察、微计算机体层扫描技术（micro-computed tomography,micro-CT）及组织学方法,评价2种膜修复骨缺损的效果。采用SPSS20.0软件包对结果进行统计学分析。结果: 通过MC3T3-E1细胞在双层壳聚糖膜上培养2 d后的扫描电镜照片,观察到细胞较好地粘附于多孔层上。第3、7及10天时,实验组细胞相对增殖率分别为114.49%、107.17%和98.73%,细胞毒性级别为0或1级,表明膜的细胞毒性轻微。2周时,Bio-Gide膜组的骨形成量（BV）和骨体积分数（BVF、BV/TV）与实验组差异显著（P<0.05）;但在第4和8周时,2组的BV和BVF无显著差异（P>0.05）。结论: 新型双层壳聚糖的体外和体内生物学性能符合GBR技术的要求,具有作为GBR膜的潜力。

An experimental study of a novel double-layer chitosan membrane for guided bone regeneration in rat calvarial defects

PURPOSE: To compare new bone formation in rat calvarial defects using novel double-layer chitosan membranes (DLCM) or collagen (Bio-Gide) membranes, and evaluate the feasibility as guided bone regeneration (GBR) membrane. METHODS: The MC3T3-E1 cells were cultured on the DLCM for 2 days, then SEM was used to observe the attachment of the cell line to the membrane. The cytotoxicity of the DLCM was tested by CCK-8 assay at day 3, 7 and 10. Eighteen (18)SD rats were selected, and then divided into 3 groups (A,B and C). Two calvarial critical-sized defects (CSDs), 5 mm in diameter, were created symmetrically on the bilateral sides of the midline using a trephine bur. Right defect was covered with a resorbable collagen membrane (Bio-Gide) as a control and the left side was covered with DLCM. The rats were sacrificed 2, 4 and 8 weeks after surgery. The specimens were then analysed using micro-CT and histological methods,to compare new bone formation in rat calvarial defects using the two membranes. The data was analyzed for Student’s t test with SPSS20.0 software package. RESULTS: From the SEM image of MC3T3-E1 cells on the double-layer chitosan membrane after being cultured for 2 days. It was found that the cells adhered well to the porous layer. The results of cytotoxicity showed that cell proliferation rates of the experimental groups were 114.49%, 107.17% and 98.73% at day 3,7 and10, respectively, while the cytotoxicity was in grade 0 or 1, indicating that the chitosan membrane had low cytotoxicity. At 2 week, there were significant differences in bone volume (BV) and bone volume/total volume (BVF, BV/TV) (P<0.05) between the collagen group and the chitosan membrane group. But at 4 and 8 week, there was no significant difference in BV and BVF (P>0.05) between the 2 groups. CONCLUSIONS: This pilot study indicated that the in vitro and in vivo bioactivities of the novel chitosan membrane fulfilled the requirements for GBR, and has significant potential as a GBR membrane.