双相生物钙磷陶瓷生物相容性及异位骨诱导的实验研究

Compatibility and ectopic bone formation of biphasic calcium phosphate ceramic

目的检测运用羟基磷灰石及β-磷酸三钙制备的双相钙磷陶瓷的生物相容性及其异位骨诱导效率。方法采用化学共沉淀法及过氧化氢发泡法,将羟基磷灰石及β-磷酸三钙以6∶4的比例在1 100 ℃条件下烧结3 h获得双相钙磷陶瓷,利用X线衍射评估材料组成成分。分离SD大鼠骨髓间充质干细胞,接种于双相钙磷陶瓷,通过扫描电镜、鬼笔环肽及DAPI染色观察细胞的黏附,CCK8法评估细胞增殖,碱性磷酸酶测定法评估骨髓间充质干细胞的碱性磷酸酶表达活性。将不含骨髓间充质干细胞的双相钙磷陶瓷置入比格犬竖脊肌内,于4、8、12周对样本行大体检测、组织染色,测算新骨生成率,从而评估双相钙磷陶瓷的异位骨诱导效率。结果成功制备双相钙磷陶瓷,X线衍射分析可见羟基磷灰石及β-磷酸三钙特异性的衍射峰。扫描电镜可见双相钙磷陶瓷表面广泛分布大孔及连通孔,孔壁粗糙不平,孔内可见均匀分布的微孔。鬼笔环肽及DAPI染色显示,骨髓充质干细胞在材料表面伸展黏附,共培养后逐渐从不规则形转变为均一的长梭形。CCK8法提示共培养后第1天,细胞活力降低,而第3、4、5、7天,细胞增殖活力逐渐增加。碱性磷酸酶活性检测提示,与对照组相比,共培养后第1、7天,双相钙磷陶瓷组的骨髓间充质干细胞可分泌更多的碱性磷酸酶。双相钙磷陶瓷顺利置入比格犬竖脊肌内,术后8周材料孔隙内可见骨样组织沉积,术后12周大孔成骨比例为0.77±0.11,孔内成骨面积比例为0.71±0.14。结论双相钙磷陶瓷具有良好的生物相容性及异位骨诱导效率。

Objective To manufacture one kind of biphasic calcium phosphate(BCP)ceramic by hydroxyapatite(HA)andβ-tricalciumphosphate(β-TCP),and to further investigate its compatibility and its efficacy of ectopic bone formation.Methods BCP was prepared with the ratio of HA andβ-TCP at 6/4 using precipitation and H2O2 foaming method and then sintered at 1100℃for 3 hours.The chemical composition of BCP was investigated by X-ray diffraction(XRD).BMSCs were isolated from the bone marrow of Sprague-Dawley(SD)rat and seeded on BCP.The adhesion and morphology of BMSCs on BCP was observed under scanning electron microscope(SEM)and special staining.Cell proliferation was quantified by cell counting kit-8(CCK8)assay.Alkaline phosphatase(ALP)activity of BMSCs was measured by ALP assay kit.For further confirmation,the intramuscularly ectopic implantation models of Beagle were used,general observation,histological,and histomorphometric analyses were conducted at 4,8,12 weeks after implantation.Results BCPs were successfully manufactured.XRD analysis showed the specific diffraction peaks of HA andβ-TCP.SEM showed that the surface of the BCP ceramics was widely distributed with macropores and connections,and the pore walls were rough,and the micropores were evenly distributed in the macropores.Phalloidin and DAPI staining showed that the BMSCs extended and adhered to the surface of the material,and the shape gradually changed from irregularity to uniform long spindle.CCK8 method showed that although the cell viability decreased on the first day after coculture,on the third,fourth,fifth and seventh days,the cell viability gradually increased.The assay of alkaline phosphatase activity indicated that BMSCs cultured on the BCP could secrete more alkaline phosphatase on day 1 and 7 compared with the control group.BCP implanted in the muscle could generate osteoid/bone tissue at 8 weeks and 12 weeks,the number of osteoid/bone filled pores were 0.77±0.11,the percentage of osteoid/bone tissue inside the pores were 0.71±0.14.Conclusions The BCP had a good biocompatibility and favorable efficacy of ectopic osteoinduction.