纯钛微纳米复合形貌对成骨细胞生物学行为的影响

目的对比研究纯钛表面不同微纳米图案对成骨细胞生物学活性的影响，探讨微米与纳米结构在影响细胞行为当中的不同作用。 方法制备4组纯钛微纳米复合表面形貌：喷砂（S）、喷砂-酸蚀（SLA）、喷砂-碱热（SAH）及喷砂-阳极氧化（SAN）。检测各组材料表面理化性能，扫描电镜观察各组材料表面形貌及细胞黏附形态，CCK-8法测定细胞在材料表面增殖能力，碱性磷酸酶（ALP）活性检测细胞在材料表面分化能力。利用单因素方差分析进行统计分析，最小有意义差异（LSD）t检验对同时间点不同组的CCK-8及ALP结果进行比较。 结果（1）粗糙度结果示：SLA组粗糙度大于其余3组，差异有统计学意义（F_Ra = 38.449，P_Ra<0.001；F_Rq = 29.564，P_Rq<0.001）。（2）扫描电镜示：S组仅形成弹坑状一级微米结构，黏附细胞伪足短小，SLA、SAH及SAN组分别修饰沟壑状、网状及管状二级纳米多孔图案，细胞于SAH、SAN组表面伪足更为伸展，其中SAH组表面细胞伪足生长进入孔隙形成机械锁结。（3）CCK-8结果示：第5天，SAH和SAN组细胞增殖A值（1.546和1.528）显著高于S组（1.31），差异有统计学意义（F = 3.229，P = 0.042）；第7天时，SAH和SAN组细胞增殖A值（2.646和2.57）显著高于S组（2.24），差异有统计学意义（F = 3.51，P = 0.035）。（4）细胞分化检测示：接种7 d后，SAH组ALP活性（77.656）显著高于S、SLA及SAN组（53.132、51.052和62.207），差异有统计学意义（F = 29.734，P<0.001）；接种14 d后，SAH与SAN组ALP活性（104.107和109.963）显著高于S与SLA两组（82.885和73.303），差异有统计学意义（F = 46.052，P<0.001）。 结论钛片表面微纳米图案影响细胞伪足形态，促进细胞增殖及ALP活性，其中多孔形貌显著增加细胞活性，碱热处理表面早期ALP活性显著增加，且形成纳米网比纳米管更有利于形成机械锁结。

The influence of different hybrid micro/nano hierarchical titanium topographies on osteoblast biological functions

ObjectiveTo compare different effects of cells on titanium surfaces with different micro/nano structures, and to evaluate different functions of micro- and nano-features, respectively. MethodsAll the titanium disc samples were divided into four groups and accepted the following treatments: (1) sandblasted only (S) ; (2) sandblasted followed by acid-etching (SLA) ; (3) sandblasted followed by alkali-heat (SAH) ; (4) sandblasted followed by anodizing (SAN) . The surface topography, elemental component and roughness of samples were examined. Surface biocompatibility and osteogenic capability were evaluated by cell counting kit (CCK-8) and alkaline phosphate activity (ALP) in vitro. The level of significance was determined by a one-way ANOVA followed by a least significant difference (LSD) t-test for a multiple comparison procedure. ResultsSurface of group S were showed only microtopography of pits, while the other three groups exhibited primary micro-structure modified by secondary nano-features of ditches (SLA) , nano-porous network layers (SAH) and nanotubes (SAN) , respectively. There was no significant difference of cell adhesion and proliferation in the 1st and 3rd day. Group SAH and SAN (1.546 and 1.528) enhanced cell proliferation significantly than Group S (1.31) in the 5th day (F = 3.229, P = 0.042) , in the 7th day, Group SAH and SAN (2.646 and 2.57) also significantly enhanced cell proliferation than Group S (2.24) (F = 3.51, P = 0.035) and induced affluent and stretched cell pseudopods. Moreover, pseudopods on the surface of group SAH grow into the network and wrap around the net to link to each other. In comparison, group SAH (77.656) significantly enhanced ALP activity in the 7th day than Group S, SLA and SAN (53.132, 51.052 and 62.207, respectively) (F = 29.734, P<0.001) , though the ALP activity of group SAN depressed in the early stage but enhanced significantly than Group S and SLA (82.885 and 73.303) in the 14th day (F = 46.052, P<0.001) . ConclusionsMicrostructures decided the roughness while nanostructures, especially nano-porous functioned in enhancing cell proliferation and differentiation. The forms of nano-porous induced cell pseudopods stretch, for which nano-porous network allowed cells to grow into and form extensive interlocking.