纳米羟基磷灰石-壳聚糖骨组织工程支架的研究

A STUDY ON NANO-HYDROXYAPATITE-CHITOSAN SCAFFOLD FOR BONE TISSUE ENGINEERING

目的以一种简单、有效的方法制备多孔的纳米羟基磷灰石（nano hydroxyapatite，nano—HA）-壳聚糖（chitosan，CS）复合支架，并评价其理化性能及与细胞相容性。方法采用原位复合一冷冻干燥方法，制备多孔nano—HA—CS支架。通过扫描电镜、透射电镜、X线衍射和傅立叶红外光谱分析支架的微观形貌及材料的组成。分离初生Wistar大鼠的成骨细胞，取传代培养第3代细胞分别与nano—HA—CS支架和纯CS支架共培养2、4、6、8h，各时间点各取4个样品，测定细胞在支架上的黏附率，并通过组织化学染色、扫描电镜观察细胞形态。结果nano—HA—CS复合支架具有多孔结构，孔径为100～500弘m，大多数孔径为400～500μm。具有很高的孔隙率，随CS和HA含量的增加，孔隙率明显降低，密度升高。扫描电镜和透射电镜观察显示合成的HA晶体，晶粒大小为纳米级，在支架孔壁上均匀、连续分布如“铺路石”样。X线衍射和红外光谱分析表明合成的HA是含CO3^2-弱结晶纳米晶体。细胞相容性实验显示，成骨细胞在支架上黏附、增殖，并分泌纤维状细胞外基质；在复合支架上的黏附率明显高于纯CS支架。结论采用原位复合与冷冻干燥法结合制备的nano—HA—CS复合支架具有良好的理化性质和细胞相容性，有望应用于组织工程骨的构建。

Objective To fabricate a nano-hydroxyapatite-chitosan （nano-HA-CS） scaffold with high porosity by a simple and effective technique and to evaluate the physical and chemical properties and the cytocompatibility of the composite scaffold. Methods The three-dimensional nano-HA-CS scaffolds with high porosity were prepared by the in situ hybridization-freeze-drying method. The microscopic morphology and components of the composite scaffolds were analyzed by the scanning electron microscopy （SEM）, the transmission electron microscopy （TEM）, the X-ray diffraction（XRD）examination, and the Fourier transformed infrared spectroscopy （FTIR）. The calvarial osteoblasts were isolated from the neonatal Wistar rats. The serial subcultured cells （3rd passage） were respectively seeded onto the nano-HA-CS scaffold and the CS scaffold, and then were co-cultured for 2, 4, 6 and 8 hours. At each time point, four specimens from each matrix were taken to determine the cell-adhesion rate. The cell morphology was observed by the histological staining and SEM. Results The macroporous nano-HA-CS scaffolds had a feature of high porosity with a pore diameter from 100 to 500μm （mostly 400-500 μm）. The scaffolds had a high interval porosity; however, the interval porosity was obviously decreased and the scaffold density was increased with an increase in the contents of CS and HA. The SEM and TEM results showed that the nano-sized HA was synthesized and was distributed on the pore walls homogeneously and continuously. The XRD and FTIR results showed that the HA crystals were carbonatesubstituded and not well-crystallized. The cytocompatibility test showed that the seeded osteoblasts could adhere the scaffolds, proliferating and producing the extracellular matrix on the scaffolds. The adherence rate for the nano-HA-CS scaffolds was obviously higher than that for the pure CS scaffolds. Conclusion The nano-HA-CS scaffolds fabricated by the in situ hybridization-freeze-drying method have a good physical and chemical properties and a good cytocompatibility; therefore, this kind of scaffolds may be successfully used in the bone tissue engineering.