rhBMP-2和bFGF复合至壳聚糖-Ⅰ型胶原支架后在体外的降解和释放

目的： 探讨rhBMP-2和bFGF复合至壳聚糖-Ⅰ型胶原支架材料后在体外的释放规律。方法: 制备壳聚糖-Ⅰ型胶原支架及含rhBMP-2和bFGF的复合膜。扫描电镜观察测量支架孔径；体外观察降解情况,在不同时点收集浸出液，ELISA检测因子的释放浓度；观察浸出液对牙周膜细胞的影响。结果： 复合膜呈疏松多孔海绵状，高、中、低3种壳聚糖-Ⅰ型胶原支架平均孔径分别为（106±17）μm、（141±13）μm和（173±11）μm ；复合膜在含溶菌酶的PBS中可以降解，壳聚糖浓度越高支架降解越慢，rhBMP-2和bFGF的释放开始为“爆发性”，此后释放逐渐减慢，最后在低浓度可缓慢而持久地释放，壳聚糖浓度越高的复合支架，因子释放越慢越持久。与含溶菌酶的PBS间接接触的因子层随着其表面支架的降解而逐渐释放因子，释放延迟时间和表面支架的降解速度有关。复合bFGF因子支架的浸出液作用于HPDLCs，可以明显促进细胞的增殖。结论： 壳聚糖-Ⅰ型胶原复合因子支架体外可以降解并释放因子，降解和因子释放速度与壳聚糖的浓度密切相关；可以通过分层制作复合因子支架来控制因子有序释放，所释放的因子具有正常生物学功能。

Study on chitosan- type Ⅰ collagen composite scaffold as carrier for sustained release of rhBMP -2 and bFGF in vitro

AIM: The purpose of this study was to investigate the rule of recombinant human bone morphogenetic protein-2(rhBMP-2) and bFGF releasing in vitro after they were combined with chitosan-type I collagen composite scaffold.METHODS: High,middle and low concentration solutions of chitosan-type I collagen,dependent on the level of chitosan,were prepared,rhBMP-2 and bFGF were added in respectively.The last concentration was 20 μg/L.The scaffolds composed of several layers and each layer combined with different agent was made by emulsion freeze-dried method.For example,the scaffold was composed of rhBMP-2 layer,sodium chloride layer and bFGF layer from top to bottom.The samples were observed with scanning electron microscope and the apertures of scaffolds were measured.The samples were immerged into phosphate buffer solution (PBS) containing lysozyme and the degradation of scaffolds was observed.Leachate was collected at different time points after the samples were immerged into PBS containing lysozyme and the levels of the agents were detected by ELISA.The effects of leachate on human periodontal ligament cells(HPDLCs) growth and proliferation were studied by MTT,and the effects of leachate on HPDLCs cell cycle were studied by flow cytometry.RESULTS: Platinum sponge solid was formed after chitosan-typeⅠcollagen mixed liquor was frozen drying.The average apertures of high,middle and low concentration chitosan- typeⅠcollagen scaffolds were 106 μm,141 μm and 173 μm,respectively.The sample degradation in PBS containing lysozyme was observed,and the high concentration scaffold degradation was more slow than the middle and low concentration scaffold degradation.The agent in layers which direct contact with PBS containing lysozyme was exposed out at start,then agent was released slowly at a high level for some days,at last agent was released at a low level for a long time.The agent in high concentration scaffold released more slowly and for a longer time than that in middle and low concentration scaffolds.The agent in layers,which indirect contacted with PBS containing lysozyme,was released gradually with the degradation of super layers.The delay time of agent releasing was concerned with the degradation speed of super layers.The growth and proliferation of HPDLCs were promoted obviously by the leachate of scaffold combined with bFGF.CONCLUSION: The chitosan-type I collagen composite scaffold combined with agents can be degradated gradually and release agents slowly.The speed of degradation and agent releasing is correlated with the concentration of chitosan.The agents can be orderly released in scaffold composed of several layers,each layer combines with different agent.The agents released from scaffolds have the normal biological functions.