氧化石墨烯/壳聚糖复合涂层影响成骨细胞的生物学行为

Graphene oxide-chitosan composite coating affects the biological behavior of osteoblasts

背景:壳聚糖作为医用金属表面的涂层材料可以有效改善其生物学性能,但单纯壳聚糖存在自身生物活性与成骨诱导能力不足的问题;氧化石墨烯能够提高多种聚合物材料的机械性能和生物相容性,并具有促进成骨分化的作用,将两者结合得到的涂层材料可能具有更好的生物性能和成骨活性。目的:构建氧化石墨烯改性的壳聚糖复合涂层材料,分析涂层材料的表面形貌、化学组成和物理性质,并进一步分析涂层材料对成骨细胞增殖、黏附和成骨分化行为的影响。方法:使用3-氨丙基三乙氧基硅烷对钛片表面进行处理,在钛片表面形成硅烷基团,然后利用戊二醛使壳聚糖与硅烷基团形成交联,分别制备单纯壳聚糖涂层与氧化石墨烯/壳聚糖复合涂层。通过原子力显微镜、扫描电镜、傅里叶变换红外光谱仪、接触角测量系统表征涂层的表面形貌、化学结构及亲水性能。将大鼠成骨细胞直接接种于两种涂层材料表面,通过CCK-8实验、扫描电镜和半定量PCR分析材料对大鼠成骨细胞增殖、细胞铺展与成骨分化的影响。结果与结论:(1)通过原子力显微镜测得氧化石墨烯片层的厚度为2 nm左右;扫描电镜下可见,两种涂层表面均光滑致密,其中壳聚糖分子紧密排列,氧化石墨烯能够均匀分布于壳聚糖中,同时由于石墨烯的加入,壳聚糖表面出现褶皱样形貌;氧化石墨烯-壳聚糖复合涂层的接触角小于单纯壳聚糖涂层(P<0.05);(2)CCK-8实验结果显示,相较于单纯壳聚糖涂层,氧化石墨烯/壳聚糖复合涂层可促进成骨细胞的增殖;(3)扫描电镜下可见,单纯壳聚糖涂层表面的成骨细胞铺展性较差,氧化石墨烯/壳聚糖复合涂层表面的成骨细胞表现出更好的铺展状态;(4)半定量PCR检测显示,氧化石墨烯/壳聚糖复合涂层组碱性磷酸酶、Runx2 mRNA表达量多于单纯壳聚糖涂层组(P<0.05);(5)结果表明,氧化石墨烯/壳聚糖复合涂层具有良好的理化性能、生物相容性和成骨诱导性能。

BACKGROUND:As a coating material,chitosan can effectively improve biological properties of medical metal surfaces due to its excellent film-forming and metal binding abilities.However,the biological activity and osteoinductive ability of chitosan are insufficient.Graphene oxide can improve the mechanical properties and biocompatibility of various polymer materials,as well as promote osteogenic differentiation.The combination of chitosan and graphene oxide may have better biological properties and osteogenic activity.OBJECTIVE:To construct the graphene oxide-modified chitosan composite coating material,analyze the surface morphology,chemical composition,and physical properties of the coating material,as well as the effects of the coating materials on proliferation,adhesion,and osteogenic differentiation of osteoblasts.METHODS:The titanium surface was treated with 3-aminopropyltriethoxysilane to form silane groups on the surface,and then glutaraldehyde was used to cross-link chitosan with silane groups to prepare simple chitosan coating and graphene oxide/chitosan composite coating,respectively.The surface morphology,chemical structure and hydrophilic properties of the coatings were characterized by atomic force microscopy,scanning electron microscopy,Fourier transform infrared spectroscopy,and contact angle measurement system.Rat osteoblasts were seeded on the surface of the two materials,and the proliferation,spreading,and osteogenic differentiation of rat osteoblasts were analyzed by CCK-8 assay,scanning electron microscopy,and reverse transcription-polymerase chain reaction analysis.RESULTS AND CONCLUSION:(1)The thickness of the graphene oxide sheets was about 2 nm as measured by atomic force microscopy;the scanning electron microscopy showed that the surfaces of both coatings were smooth and dense,in which the chitosan molecules were closely arranged and the graphene oxide was uniformly distributed in the chitosan,while wrinkles appeared on the surface of chitosan due to the addition of grapheme.The contact angle of the graphene oxide-chitosan composite coating was significantly smaller than the chitosan coating alone(P<0.05).(2)CCK-8 assay showed that the graphene oxide-chitosan composite coating better promoted the proliferation of osteoblasts compared with the chitosan coating alone.(3)The scanning electron microscopy showed that better spreading was found in osteoblasts on the surface of the graphene oxide-chitosan composite coating compared with the chitosan coating alone.(4)Reverse transcription-polymerase chain reaction analysis showed that the mRNA expression of alkaline phosphatase and Runx2 was significantly increased in the graphene oxide/chitosan composite coating group compared with in the chitosan coating alone group(P<0.05).(5)These findings suggest that graphene oxide/chitosan composite coating had good biocompatibility,physicochemical,and osteoinductive properties.