两种新型胶原膜引导骨组织再生的体内外性能研究

目的　研究两种新型胶原膜（鱼胶原、猪胶原）对SD大鼠骨髓间充质干细胞（rat bone marrow mesenchymal stem cells, rBMSCs）成骨分化的影响，并观察其促进SD大鼠颅顶骨缺损修复的效果。方法　利用扫描电镜（SEM）和接触角测量仪表征两种膜的表面形貌及水接触角。在两种膜上培养rBMSCs，并将细胞接种在空白孔板上作为空白对照组。1、3、5、7d时以CCK-8法检测两种膜对rBMSCs增殖的影响，并通过激光共聚焦显微镜观察24h时细胞的粘附与伸展。用碱性磷酸酶（ALP）染色和活性检测，茜素红染色和钙结节半定量测定及实时荧光定量PCR评估两种膜的体外成骨性能。体内实验中，在SD大鼠颅中缝两侧制备直径5mm的骨缺损，在左侧骨缺损区植入鱼胶原膜或猪胶原膜，右侧骨缺损区作为空白对照组，3个月后采用micro-CT检测颅顶骨缺损区骨再生情况。结果　SEM：鱼胶原膜表面致密，猪胶原膜呈疏松多孔的表面。接触角测量仪：猪胶原膜的亲水性强于鱼胶原膜(P<0.05)。CCK-8及激光共聚焦显微镜：细胞在两种膜及空白孔板上24h时铺展良好，7d内稳定增殖。体外成骨性能检测：猪胶原膜上rBMSCs在5 d时的ALP活性、14 d时细胞外基质矿化水平、7d时细胞相关成骨基因Bmp2、Col1、Runx2的表达高于鱼胶原组和空白对照组(P<0.05)。体内成骨实验（3个月）：猪胶原膜促进骨再生明显优于鱼胶原膜和空白对照组（P<0.05）, 鱼胶原膜组和空白对照组无明显差异。结论　猪胶原膜体内外促进骨再生的效果明显优于鱼胶原膜组，具有作为引导骨组织再生膜材料的潜能。

Performance study of two new collagen membranes for guiding bone tissue regeneration in vivo and in vitro

Objective To study the effects of two kinds of new collagen membranes(fish collagen and porcine collagen) on the osteogenic differentiation of SD rat bone marrow mesenchymal stem cells(rBMSCs) and observe their repair effects on the cranial parietal defects of SD rat. Methods The surface morphology and water contact angle of the two membranes were measured by scanning electron microscopy(SEM) and contact angle measuring instrument. rBMSCs were cultured on membranes, and cells were also seeded on blank well plates as a blank control group. The effects of the two membranes on the proliferation of rBMSCs at 1, 3, 5 and 7 days were examined by CCK-8 and the adhesion and extension of the cells were observed by laser confocal microscopy at 24 h. The osteogenic properties in vitro of the two membranes were evaluated by alkaline phosphatase(ALP) staining and activity assay, alizarin red staining and semi-quantitative determination of calcium nodules and real-time fluorescent quantitative PCR. In vivo experiments, bone defects with diameter of 5 mm were prepared on both sides of the cranial suture of SD rats. The left bone defect area was implanted with fish collagen membrane or porcine collagen membrane. The right bone defect area was used as a blank control group. After 3 months, micro-CT was used to detect the bone regeneration in the cranial parietal defect area. Results SEM: The surface of the fish collagen membrane was dense,and the porcine collagen membrane had a loose porous surface. Contact angle measuring instrument: The hydrophilicity of porcine collagen membrane was better than fish collagen membrane( P < 0. 05). CCK-8 and laser confocal microscopy: The cells spread well at 24 h and stably proliferated within 7 days on both membranes and blank well plates. Osteogenic properties in vitro: ALP activity of rB MSCs on porcine collagen membrane at 5 d,extracellular matrix mineralization at 14 d,and the expression of cell-associated osteogenic genes Bmp2,Col1,Runx2 at 7 d were higher than fish collagen membrane group and blank control group( P<0.05). In vivo osteogenic experiment( 3 months):Porcine collagen membrane promoted bone regeneration significantly better than fish collagen membrane and blank control group( P<0.05). There was no significant difference between fish collagen membrane group and blank control group. Conclusion The effects of porcine collagen membrane on promoting bone regeneration in vivo and in vitro are significantly better than fish collagen membrane,and it has the potential as a membrane for guiding bone tissue regeneration.