Role of hierarchical topography of titanium surface on mesenchymal stem cells adhesion and differentiation behaviours in vitro

摘要

Introduction: The topography of a substrate material surface plays an important role for attachment, proliferation, and differentiation of cells on the substrate. In particular, the initial cellular adhesion behavior would have effect on the consequent cellular functions. In this study, to elucidate the effect of hierarchical topography surface on cellular initial cellular adhesion and consequent cellular differentiation of human mesenchymal stem cells (hMSCs), three kinds of surface topographies of titanium (Ti) were fabricated by a femtosecond laser. An initial cellular attachment and accompanied multilineage differentiation were evaluated by fluorescent staining and target gene expression detection through real time RT-PCR, respectively. Materials and Methods: Ti (grade 2) was mirror-polished and the surface was scanned by a femtosecond laser. As a result, 3 types surfaces with one-directional grooves ware obtained: micron groove (Micron, circuler polarization, fluence: 0.7J•cm~(-2)), submicron groove (Submicron, linear polarization, fluence: 0.8J•m~(-2)), and micron/submicron hierarchical grooves (Hybrid: scanned by both polarization beams) grooves. The Initial adhered cellular morphology was detected by fluorescent immunochemistty staining. After hMSCs reached to 100% confluence, the growth medium was changed into the differentiation mediums to induce the adipogenic, osteogenic, neurogenic and chondrogenic differentiation, respectively. The multilineage differentiation of hMSCs response to different surface topographies was detected by real time RT-PCR and fluorescent antibody staining. Results and Discussion: A highly orientated and extended cellular morphology of hMSCs were showed on specimens with surface topographies (Fig. 1). A larger cellular elongation was obtained by cells on Ti surfaces with Submicron or Hybrd. The fluorescent images indicated that the Micron had effect on the cellular alignment through the cellular initial polarization distribution, while, the Submicron had effect on the cellular extension. A superposition cellular effects were obtained by hierarchical topography of titanium surface, Hybrid. A highly aligned and highly elongated cellular morphology was observed by hMSCs on Hybrid. This enhancement of orientated and extended cellular morphology was expected to control the specific cellular function. The gene expression detectine through real time RT-PCR showed that different surface features had different effects on the differentiation of hMSCs. In particular, the hMSCs cultured on Hybrid were promoted the osteogenic differentiation and chondrogenic differentiation. Conclusion: In this work, three kinds of surface topographies of Ti were fabricated by a femtosecond laser micron, submicron, and hybrid (micron/submicron hierarchical grooves). A highly orientated cellular morphology and a larger cellular elongation was obtained by cells on Ti surfaces with hierarchical surface topographies, Hybrid. In addition, hMSCs cultered on this Hybrid speicmens were strongly promoted the osteogenic differentiation and chondrogenic differentiation. This surface modification with hierachical topography is expected to contribute to promote the novel biomaterials design and use of those biomaterials for orthopedic/dental applications. Figure 1. The conception of this work and the fluorescent images of the hMSCs on Ti specimens with different surface topographies. The cellular orientation direction is in agreement with the direction of the grooves.