羧甲基壳聚糖稳定液相矿化前体诱导胶原纤维仿生矿化

Biomimetic mineralization of collagen fibrils via carboxymethyl chitosan stabilized liquid precursor

目的通过构建二维及三维胶原模型,探讨羧甲基壳聚糖(CMC)在胶原纤维仿生矿化中的作用及优化体外仿生矿化的策略,为最终的临床应用提供实验依据。方法 CMC-磷酸钙溶液浊度实验筛选CMC诱导仿生矿化的工作浓度。在此基础上,采用Ⅰ型胶原冻干粉制备二维胶原模型,将胶原膜修剪成5 mm×5 mm×1 mm作为三维胶原支架,以含上述工作浓度CMC的饱和磷酸钙溶液作为仿生矿化诱导液,对二维及三维胶原进行矿化,不含CMC的传统矿化液作为对照组。采用透射电子显微镜观察二维胶原超微结构的变化,选区电子衍射明确矿化相,电子能谱分析矿化物的钙磷比(Ca/P)。通过热重分析(TGA)、X线衍射(XRD)分析三维胶原膜的矿化程度及晶体成分。结果 200μg/ml CMC是持续维持磷酸钙溶液澄清7 d的最低浓度,作为后续矿化的工作浓度。透射电镜结果显示,CMC诱导胶原仿生矿化3 d,可见胶原纤维内针状矿化晶体沉积,选区电子衍射证实为羟基磷灰石(HA)。TGA结果显示,仿生矿化14 d的三维胶原膜矿化物含量为18.39%,XRD结果可见HA的特征性衍射峰。结论 CMC可通过稳定液相矿化前体的方式诱导胶原纤维的体外仿生矿化,同时促进合成矿化程度相对较高的胶原-HA复合物,为胶原纤维仿生矿化的最终临床应用提供了实验依据。

Objective To test the hypothesis that the carboxymethyl chitosan（CMC）stabilized liquid precursors can induce biomimetic mineralization of collagen fibrils. Methods CMC-calcium phosphate solution turbidity assessment was used to determine the working concentration of CMC. Reconstituted 2-D typeⅠcollagen model and 3-D collagen membranes were treated with saturated calcium phosphate solution containing the above working concentration of CMC. Samples treated with traditional mineralization liquids（without CMC）were acted as control. The ultrastructural changes of the 2-D collagen were observed by transmission electron microscopy （TEM） and the mineral phase was determined by selected area electron diffraction（SAED）/energy dispersive X-ray analysis（EDXA）. The mineralization composition and degree of 3-D collagen film were analyzed by thermogravimetric（TG）and X-ray diffraction （XRD）. Results According to turbidity assessment of CMC,200 μg/ml CMC was able to stabilizing calcium phosphate liquid for 7 days and selected for further experiments. Through TEM observation and SAED analysis,needle-like crystals along the long axis of the fibrils were observed in the intrafibrillar spaces of collagen fibrils on the 3rd day. SAED analysis showed the typical diffraction ring of hydroxyapatite. EDXA revealed that the Ca/P of minerals in the collagen fibrils was 1.56. As for 3-D collagen model,the mineral content of collagen membrane treated with the biomimetic mineralization liquid for 14 days was 18.39% via TG analysis and the mineral phase was confirmed to be hydroxyapatite according to XRD curves. Conclusions Carboxymethyl chitosan can induce the biomimetic mineralization of collagen fibrils through stabilizing of liquid precursor and synthesize biomimetic collagen-hydroxyapatite complex with relatively higher degree of mineralization.