磷酸钙骨水泥复合透明质酸-姜黄素对成骨细胞增殖及成骨能力影响的研究

Effects of calcium phosphate cement combined with hyaluronic acid/curcumin on the proliferation and osteogenesis of osteoblasts

目的采用透明质酸(hyaluronic acid,HA)对姜黄素(curcumin,CUR)进行改性,探讨磷酸钙骨水泥(calcium phosphate cement,CPC)复合HA/CUR后对成骨细胞增殖及成骨能力的影响。方法首先将HA与CUR成酯化共价结合制备HA/CUR,观察其性状并行红外光谱测试。然后,将HA、CUR以及HA/CUR按照5%(W/W)分别与CPC混合,制备HA-CPC、CUR-CPC、HA/CUR-CPC,行凝结时间检测、扫描电镜观察以及可注射性能、压缩强度测试;以单纯CPC作为对照。取新生SD大鼠颅骨分离培养成骨细胞,取第2代细胞分别与上述骨水泥复合培养,经扫描电镜观察、活/死细胞荧光染色、细胞计数、骨桥蛋白(osteopontin,OPN)免疫荧光染色、ALP染色以及茜素红染色,评估HA/CUR-CPC对成骨细胞增殖及成骨能力的影响。结果红外光谱测试显示HA及CUR成功共价结合。骨水泥观测显示,HA/CUR-CPC组初凝时间、终凝时间、压缩强度以及可注射率与其他组比较,差异均无统计学意义(P>0.05);扫描电镜观察示HA/CUR散在分布于CPC表面。骨水泥与成骨细胞复合培养后,扫描电镜显示成骨细胞黏附于HA/CUR-CPC且形态良好,具有成骨细胞的生长特征;HA/CURCPC组细胞存活率与其他组比较差异无统计学意义(P>0.05),细胞较其他组明显增多(P<0.05);OPN免疫荧光染色、ALP染色及茜素红染色程度均强于其他组。结论 HA/CUR-CPC具有良好的生物相容性及力学特性,能促进成骨细胞增殖并增强其成骨能力。

Objective After using hyaluronic acid(HA)to modify curcumin(CUR),the effects of calcium phosphate cement(CPC)combined with HA/CUR on the proliferation and osteogenesis of osteoblasts were investigated.Methods First,HA and CUR were esterified and covalently combined to prepare HA/CUR,and the characteristics were observed and the infrared spectrum was tested.Then,HA,CUR,and HA/CUR were mixed with CPC according to 5%(W/W)to prepare HA-CPC,CUR-CPC,and HA/CUR-CPC,respectively.Setting time detection,scanning electron microscope observation,injectable performance test,and compression strength test were conducted;and the CPC was used as a control.Osteoblasts were isolated and cultured from the skull of newborn Sprague Dawley rats,and the 2nd generation cells were cultured with the 4 types of bone cement,respectively.The effects of HA/CUR-CPC on the proliferation and osteogenesis of osteoblasts were estimated by the scanning electron microscopy observation,live/dead cell fluorescence staining,cell counting,osteopontin(OPN)immunofluorescence staining,alkaline phosphatase(ALP)staining,and alizarin red staining.Results Infrared spectroscopy test showed that HA and CUR successfully covalently combined.The HA/CUR-CPC group had no significant difference in initial setting time,final setting time,injectable rate,and compressive strength when compared with the other 3 groups(P>0.05);scanning electron microscope observation showed that HA/CUR was scattered on CPC surface.After co-culture of bone cement and osteoblasts,scanning electron microscopy observation showed that the osteoblasts,which had normal morphology and the growth characteristics of osteoblasts,clustered and adhered to HA/CUR-CPC.There was no significant difference in cell survival rate between HA/CUR-CPC group and other groups(P>0.05),and the number of cells significantly increased(P<0.05);the degrees of OPN immunofluorescence staining,ALP staining,and alizarin red staining were stronger than other groups.Conclusion HA/CUR-CPC has good biocompatibility and mechanical properties,which can promote the proliferation and osteogenesis of osteoblasts.