Accelerated Bone Regeneration on the Metal Surface through Controllable Surface Potential.

Surface potential is rarely investigated as an independent factor in influencing tissue regeneration on the metal surface. In this work, the surface potential on the titanium (Ti) surface was designed to be tailored and adjusted independently, which arises from the ferroelectricity and piezoelectricity of poled poly(vinylidene fluoride-trifluoroethylene) (PVTF). Notably, it is found that such controllable surface potential on the metal surface significantly promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro as well as bone regeneration in vivo. In addition, the intracellular calcium ion (Ca2+) concentration measurement further proves that such controllable surface potential on the metal surface could activate the transmembrane calcium channels and allow the influx of extracellular Ca2+ into the cytoplasm. That might be the reason for improved osteogenic differentiation of BMSCs and bone regeneration. These findings reveal the potential of the metal surface with improved bioactivity for stimulation of osteogenesis and show great prospects for fabricable implantable medical devices with adjustable surface potential.

Surface Oxygen Vacancies of Rutile Nanorods Accelerate Biomineralization.

Titanium dioxide (TiO2) materials have been widely used in biomedical applications of bone tissue engineering. However, the mechanism underlying the induced biomineralization onto the TiO2 surface still remains elusive. In this study, we demonstrated that the surface oxygen vacancy defects of rutile nanorods could be gradually eliminated by the regularly used annealing treatment, which restrained the heterogeneous nucleation of hydroxyapatite (HA) onto rutile nanorods in simulated body fluids (SBFs). Moreover, we also observed that the surface oxygen vacancies upregulated the mineralization of human mesenchymal stromal cells (hMSCs) on rutile TiO2 nanorod substrates. This work therefore emphasized the importance of subtle changes of surface oxygen vacancy defective features of oxidic biomaterials during the regularly used annealing treatment on their bioactive performances and provided new insights into the fundamental understanding of interactions of materials with the biological environment.

Regulation of Macrophage Polarization on Chiral Potential Distribution of CFO/P(VDF-TrFE) Films.

Surface potentials of biomaterials have been shown to regulate cell fate commitment. However, the effects of chirality-patterned potential distribution on macrophage polarization are still only beginning to be explored. In this work, we demonstrated that the chirality-patterned potential distribution of CoFe2O4/poly(vinylidene fluoride-trifluoroethylene) (CFO/P(VDF-TrFE)) films could significantly down-regulate the M1 polarization of bone marrow-derived macrophages (BMDMs). Specifically, the dextral-patterned surface potential distribution simultaneously up-regulated the expression of M2-related markers of BMDMs. The results were attributed to the sensitive difference of integrin subunits (α5ß1 and αvß3) to the dextral- and sinistral-patterned surface potential distribution, respectively. The interaction difference between the integrin subunits and surface potential distribution altered the cell adhesion and cytoskeletal structure and thereby the polarization behavior of BMDMs. This work, therefore, emphasizes the importance of chirality of potential distribution on cell behavior and provides a new strategy to regulate the immune response of biomaterials.

Enhanced M2 Polarization of Oriented Macrophages on the P(VDF-TrFE) Film by Coupling with Electrical Stimulation.

Electrical stimulation (ES) has been considered a promising strategy in regulating intracellular communication, membrane depolarization, ion transport, etc. Meanwhile, cell topography, such as the alignment and elongation in anisotropic orientation, also plays a critical role in triggering mechanotransduction as well as the cellular fate. However, coupling of ES and cell orientation to regulate the polarization of macrophages is yet to be explored. In this work, we intended to explore the polarization of macrophages on a poly(vinylidene fluoride-trifluoroethylene [P(VDF-TrFE)] film with intrinsic microstripe roughness, which was covered on indium tin oxide planar microelectrodes. We found that mouse bone marrow-derived macrophages (BMDMs) cultured on a P(VDF-TrFE) film exhibited an elongated morphology aligned with the microstripe crystal whisker, but their polarization behavior was not affected. However, the elongated cells were susceptible to ES and upregulated their M2 polarization, as verified by the related expression of phenotype markers, cytokines, and genes, while not affecting M1 polarization. This is due to the increased expression of the M2 polarization receptor interleukin-4Rα on the surface of elongated BMDMs, while the M1 polarization receptor toll-like receptor 4 was not affected. Thus, M2 polarization was singularly enhanced after activation of polarization by ES. The combination of surface morphology and ES to promote M2 single polarization in this work provides a new perspective for regulating macrophage polarization in the field of immunotherapy.

The osteogenic response to chirality-patterned surface potential distribution of CFO/P(VDF-TrFE) membranes.

Piezoelectric poly(vinylidene fluoride-trifluoroethylene) has demonstrated an ability to promote osteogenesis, and biomaterials with a chirality-patterned topological surface could enhance cellular osteogenic differentiation. In this work, we created a chirality-patterned surface potential distribution of CoFe2O4/poly(vinylidene fluoride-trifluoroethylene (CFO/P(VDF-TrFE)) membranes to explore their osteogenic response under no change in surface chemical and topology, attempting to further strengthen the ability of the membranes to promote osteogenesis. The chirality-patterned surface potential distribution was established by microdomain contact polarization with the help of sinistral/dextral-patterned ITO interdigital microelectrodes. In the in vitro evaluations, the mesenchymal stem cells showed a positive response in osteogenic differentiation to CFO/P(VDF-TrFE) membranes with both sinistral- and dextral-patterned surface potential distributions, however, the dextral-patterned distribution gave a stronger response than the sinistral-patterned one. And the in vivo evaluation showed a response tend in new bone tissue formation similar to the in vitro evaluations. The stronger response in osteogenic differentiation and osteogenesis for the CFO/P(VDF-TrFE) membrane with the dextral-patterned distributions may be attributed to that the intense interaction of the cells with the electrophysiological microenvironment appears due to a correspondingly higher expression of integrin α5ß1, which significantly up-regulates the Arp2/3 complex expression, a crucial factor for cytoskeleton reorganization, possibly increases cytoskeleton contractility, and strengthens the transduction of the osteogenesis-related signaling cascade. This work proves that the chirality-patterns in surface potential distributions could provide an osteogenic response similar to a chirality-patterned topological surface.

Improved osseointegration of strontium-modified titanium implants by regulating angiogenesis and macrophage polarization.

Strontium (Sr) has shown strong osteogenic potential and thereby been widely incorporated into dental and orthopedic implants. However, the improved osseointegration of strontium-modified titanium implants through regulation of angiogenesis and macrophage polarization is still beginning to be explored. Here, we demonstrated that the angiogenic capacity of human umbilical vein endothelial cells on the Sr-incorporated micro/nano titanium (SLA-Sr) surface was also significantly improved through the up-regulated expression of the HIF-1α protein and Erk1/2 phosphorylation. Meanwhile, SLA-Sr not only switched macrophage polarization towards the M2 phenotype, but also expressed a high level of pro-angiogenic platelet-derived growth factor. Furthermore, macrophage secretion induced by SLA-Sr was also capable of enhancing angiogenesis of human umbilical vein endothelial cells. In vivo experimental results also showed early vascularized implant osseointegration of SLA-Sr with the type H vessel formation around the SLA-Sr implant. This study emphasized the synergistic role of Sr in the regulation of macrophage polarization and angiogenesis, and therefore depicted the therapeutic potential of SLA-Sr for rapidly vascularized osseointegration.

Effects of electrical stimulation on cytokine-induced macrophage polarization.

Macrophages have two functionalized phenotypes, M1 and M2, and the regulation of M1/M2 polarization of macrophages is critical for tissue repair. Tissue-derived immune factors are considered the major drivers of macrophage polarization. Based on the main cytokine-induced polarization pathways, we tested the effect of electrical stimulation (ES) of macrophages on the regulation of M1/M2 polarization and a possible synergistic effect with the cytokines. Indium tin oxide (ITO) planar microelectrodes were used to produce ES under different voltages, frequencies and waveforms. We evaluated the influence of ES on the cytokine-induced M1/M2 polarization using mouse bone marrow-derived macrophages cultured with both lipopolysaccharide (LPS)/IFN-γ factors and IL-4 factors for M1 and M2, respectively. The results showed that ES promoted the cytokine-induced macrophage polarization. Importantly, we found that stimulation with a square waveform selectively promoted LPS/IFN-γ-induced M1 polarization, while stimulation with a sinusoidal waveform promoted both LPS/IFN-γ-induced M1, and IL-4-induced M2 polarization. Mechanistically, stimulation with a square waveform affected the intracellular ion concentration, whereas stimulation with a sinusoidal waveform promoted both the intracellular ion concentration and membrane receptors. We hereby establish an ES-mediated strategy for immunomodulation via macrophage polarization.

Cancer cell migration on straight, wavy, loop and grid microfibre patterns.

Cell migration plays an important role in physiological and pathological processes where the fibrillar morphology of extracellular matrices (ECM) could regulate the migration dynamics. To mimic the morphological characteristics of fibrillar matrix structures, low-voltage continuous electrospinning was adapted to construct straight, wavy, looped and gridded fibre patterns made of polystyrene (of fibre diameter ca. 3µm). Cells were free to explore their different shapes in response to the directly-adhered fibre, as well as to the neighbouring patterns. For all the patterns studied, analysing cellular migration dynamics of MDA-MB-231 (a highly migratory breast cancer cell line) demonstrated two interesting findings: first, although cells dynamically adjust their shapes and migration trajectories in response to different fibrillar environments, their average step speed is minimally affected by the fibre global pattern; secondly, a switch in behaviour was observed when the pattern features approach the upper limit of the cell body's minor axis, reflecting that cells' ability to divert from an existing fibre track is limited by the size along the cell body's minor axis. It is therefore concluded that the upper limit of cell body's minor axis might act as a guide for the design of microfibre patterns for different purposes of cell migration.

Simultaneous acceleration of osteogenesis and angiogenesis by surface oxygen vacancies of rutile nanorods.

Advanced implants with simultaneous accelerated osteogenic and angiogenic capacities are of great importance for osteointegration. Much attention has been paid to simultaneously enhancing the osteogenesis and angiogenesis by surface decoration of bioactive molecules or ions on biomaterial surface, but the inherent physical cue of material surface down to the atomic-scale features have always been ignored. In this study, we demonstrate that regulation of surface oxygen vacancies defects of rutile nanorods are able to simultaneous accelerate the osteogenesis and angiogenesis. The concentration of surface oxygen vacancies defects of rutile nanorods can be manipulated by simple redox processing. The osteogenic differentiation of mesenchymal stem cells (MSCs), angiogenic differentiation and vessel-like tube structures of human umbilical vein endothelial cells (HUVECs) on oxygen vacancies rich surface are significantly up-regulated. This work therefore emphasizes the critical role of the inherent material atomic-scale features and provides a novel strategy to accelerate the osteogenesis and angiogenesis of Ti-based implant.

Anisotropic magneto-mechanical stimulation on collagen coatings to accelerate osteogenesis.

Mechanical stimulation has been considered to be critical to cellular response and tissue regeneration. However, harnessing the direction of mechanical stimulation during osteogenesis still remains a challenge. In this study, we designed a series of novel magnetized collagen coatings (MCCs) (randomly or parallel-oriented collagen fibers) to exert the anisotropic mechanical stimulation using oriented magnetic actuation during osteogenesis. Strikingly, we found the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were significantly up-regulated when the direction of magnetic actuation was parallel to the randomly-oriented collagen coating surface, in contrast to the down-regulated capacity under the perpendicular magnetic actuation. Moreover, further exerting a parallel mechanical stimulation along the parallel-oriented collagen coating, which cells have been oriented by the oriented collagens, were not only able to up-regulate the osteogenic differentiation of BMSCs but also promote the new bone formation during osteogenesis in vivo. We also demonstrated the anisotropic magneto-mechanical stimulation for the osteogenic differences might be attributed to the stretching or bending tensile status of collagen fibers controlled by the direction of magnetic actuation, driving the α5ß1-dependent integrin signaling cascade. This study therefore got insight of understanding the directional mechanical stimulation on osteogenesis, and also paved a way for sustaining regulation of the biomaterials-host interface.

Enhanced osteogenic differentiation of mesenchymal stem cells on P(VDF-TrFE) layer coated microelectrodes.

Electrical stimulation has been proved to be critical to regulate cell behavior. But, cell behavior is also susceptible to multiple parameters of the adverse interferences such as surface current, electrochemical reaction products, and non-uniform compositions, which often occur during direct electric stimulation. To effectively prevent the adverse interferences, a novel piezoelectric poly(vinylidene fluoride-trfluoroethylene)(P(VDF-TrFE)) layer was designed to coat onto the indium tin oxide (ITO) planar microelectrode. We found the electrical stimulation was able to regulate the osteogenic differentiation of mesenchymal stem cells (MSCs) through calcium-mediated PKC signaling pathway. Meanwhile, the surface charge of the designed P(VDF-TrFE) coating layer could be easily controlled by the pre-polarization process, which was demonstrated to trigger integrin-mediated FAK signaling pathway, finally up-regulating the osteogenic differentiation of MSCs. Strikingly, the crosstalk in the downstream of the two signaling cascades further strengthened the ERK pathway activation for osteogenic differentiation of MSCs. This P(VDF-TrFE) layer coated electrical stimulation microelectrodes therefore provide a distinct strategy to manipulate multiple-elements of biomaterial surface to regulate stem cell fate commitment.

Accelerated Neurite Outgrowth and Neurogenesis of PC12 Cells on an Fe-doped TiO2 Nanorod Film Triggered by Visible Light.

Acceleration of neurite outgrowth and neuronal differentiation of neural cells are critical for effective neural tissue regeneration. In addition to biochemical cues, biomaterials have proven to be a valuable tool for engineering neural cellular physiological processes. However, strategies with convenient potential spatiotemporal control are still desirable. We here design a novel Fe-doped TiO2 nanorod film using hemoglobin as the Fe source to endow it with visible-light-responsive regulated surface hydroxyl groups (-OH), which was demonstrated as the central role in mediating cell-material interactions in our previous study. The acceleration of neurite outgrowth and neuronal differentiation of PC12 cells might be attributed to the upregulated distinct terminal hydroxyl groups triggered by visible light. We also demonstrate that the actin cytoskeletal system plays a pivotal role during these processes, approved by the inhibition experiment results. This study therefore sheds light on the regulation of neurite outgrowth and neuronal differentiation of neural cells using a convenient spatiotemporal controllable strategy.

Polarization behavior of bone marrow-derived macrophages on charged P(VDF-TrFE) coatings.

The immune response of bone implants is closely related to the interaction between macrophages and biomaterial surfaces. In this work, the polarization behavior of mouse bone marrow-derived macrophages (BMDMs), including their morphology and expression of phenotypic markers, genes and cytokines, on charged surfaces with different potential intensities was systematically explored. We found that the charged surface could effectively promote BMDM polarization, and a higher potential intensity was conducive to the upregulation of the polarization of BMDMs into the M2 phenotype. Based on the analysis of the signaling pathways involved in integrins (αMß2 and α5ß1) and the potassium ion channel (Kv1.3), a possible underlying mechanism revealed that the integrin originated signaling pathways could more dominantly regulate macrophage polarization to the M2 phenotype. The present work therefore demonstrates that the surface charge, as a physicochemical property of material surfaces, could effectively regulate macrophage polarizations, which may provide an immunoregulation view for the surface design of biomaterials.

M2 macrophage-derived exosomes promote the c-KIT phenotype of vascular smooth muscle cells during vascular tissue repair after intravascular stent implantation.

Rationale: For intravascular stent implantation to be successful, the processes of vascular tissue repair and therapy are considered to be critical. However, the mechanisms underlying the eventual fate of vascular smooth muscle cells (VSMCs) during vascular tissue repair remains elusive. In this study, we hypothesized that M2 macrophage-derived exosomes to mediate cell-to-cell crosstalk and induce dedifferentiation phenotypes in VSMCs. Methods:In vivo, 316L bare metal stents (BMS) were implanted from the left iliac artery into the abdominal aorta of 12-week-old male Sprague-Dawley (SD) rats for 7 and 28 days. Hematoxylin and eosin (HE) were used to stain the neointimal lesions. En-face immunofluorescence staining of smooth muscle 22 alpha (SM22α) and CD68 showed the rat aorta smooth muscle cells (RASMCs) and macrophages. Immunohistochemical staining of total galactose-specific lectin 3 (MAC-2) and total chitinase 3-like 3 (YM-1) showed the total macrophages and M2 macrophages. In vitro, exosomes derived from IL-4+IL-13-treated macrophages (M2Es) were isolated by ultracentrifugation and characterized based on their specific morphology. Ki-67 staining was conducted to assess the effects of the M2Es on the proliferation of RASMCs. An atomic force microscope (AFM) was used to detect the stiffness of the VSMCs. GW4869 was used to inhibit exosome release. RNA-seq was performed to determine the mRNA profiles of the RASMCs and M2Es-treated RASMCs. Quantitative real-time PCR (qRT-PCR) analysis was conducted to detect the expression levels of the mRNAs. Western blotting was used to detect the candidate protein expression levels. T-5224 was used to inhibit the DNA binding activity of AP-1 in RASMCs. Results: M2Es promote c-KIT expression and softening of nearby VSMCs, hence accelerating the vascular tissue repair process. VSMCs co-cultured in vitro with M2 macrophages presented an increased capacity for de-differentiation and softening, which was exosome dependent. In addition, the isolated M2Es helped to promote VSMC dedifferentiation and softening. Furthermore, the M2Es enhanced vascular tissue repair potency by upregulation of VSMCs c-KIT expression via activation of the c-Jun/activator protein 1 (AP-1) signaling pathway.Conclusions: The findings of this study emphasize the prominent role of M2Es during VSMC dedifferentiation and vascular tissue repair via activation of the c-Jun/AP-1 signaling pathway, which has a profound impact on the therapeutic strategies of coronary stenting techniques.

Enhancing osteogenic differentiation of BMSCs on high magnetoelectric response films.

High performance of biomaterial surfaces provides a sound basis to mediate cellular growth behavior. In this work, we attempted to incorporate both positive and negative magnetostriction particles of CoFe2O4 (CFO) and TbxDy1-xFe2 alloy (TD) into piezoelectric poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) for forming high magnetoelectric effect films, on which osteogenic differentiation could be dynamically mediated by a magnetic-field-induced surface potential (φME).The negatively poled film with TD/CFO volume ratio of 1:4 (1T4C) showed a highest magnetoelectric effect with φME of -171 mV at 2800 Oe. Compared with CFO/P(VDF-TrFE) and TD/P(VDF-TrFE) films, the φME increased about 213% and 173%, respectively. This could result from that P(VDF-TrFE) dipole domains receive a larger off-axial stress caused by the distribution characteristic of CFO and TD in P(VDF-TrFE), consequently to facilitate P(VDF-TrFE) dipole domain rearrangement. When MSCs were cultured on 1T4C film for 7 or 14 days, the magnetic actuation was setup to begin at the 4th or 8th day after the culture. The 7-day osteogenic differentiation was hardly affected for magnetic actuation at 4th day, moreover, the 14-day differentiation was significantly enhanced for magnetic actuation at 8th day. The enhancement appears just at a relatively late period of the cell growth, probably because the cells need a steady change in cell membrane potential to disassociate pairs of ß-catenin and E-cadherin and activate osteogenic-related signaling pathway. This work could provide an alternative way to promote performance for magnetoelectric materials, and get insight into understanding of interactions of surface potential with cells.

Ultraviolet Radiant Energy-Dependent Functionalization Regulates Cellular Behavior on Titanium Dioxide Nanodots.

Titanium dioxide (TiO2) photofunctionalization has been demonstrated as an effective surface modification method for the osseointegration of implants. However, the insufficient understanding of the mechanism underlying photofunctionalization limits its clinical applications. Here, we report an ultraviolet (UV) radiant energy-dependent functionalization on TiO2 nanodots (TN) surfaces. We found the cell adhesion, proliferation, and osteogenic differentiation gradually increased with the accumulation of UV radiant energy (URE). The optimal functionalizing treatment energy was found to be 2000 mJ/cm2, which could regulate cell-specific behaviors on TN surfaces. The enhanced cell behaviors were regulated by the adsorption and functional site exposure of the extracellular matrix (ECM) proteins, which were the result of the surface physicochemical changes induced by the URE. The correlation between the URE and the reconstruction of surface hydroxyl groups was considered as an alternative mechanism of this energy-dependent functionalization. We also demonstrated the synergistic effects of FAK-RHOA and ERK1/2 signaling pathways on mediating the URE-dependent cell behaviors. Overall, this study provides a novel insight into the mechanisms of photofunctionalization, guiding the design of implants and the clinical practice of photofunctionalization.

Novel Platform for Surface-Mediated Gene Delivery Assisted with Visible-Light Illumination.

Surface-mediated gene delivery has attracted more and more attentions in biomedical research and applications because of its characteristics of low toxicity and localized delivery. Herein, a novel visible-light-regulated, surface-mediated gene-delivery platform is exhibited, arising from the photoinduced surface-charge accumulation on silicon. Silicon with a pn junction is used and tested subsequently for the behavior of surface-mediated gene delivery under visible-light illumination. It is found that positive-charge accumulation under light illumination changes the surface potential and then facilitates the delivery of gene-loaded carriers. As a result, the gene-expression efficiency shows a significant improvement from 6% to 28% under a 10 min visible-light illumination. Such improvement is ascribed to the increase in surface potential caused by light illumination, which promotes both the release of gene-loaded carriers and the cellular uptake. This work suggests that silicon with photovoltaic effect could offer a new strategy for surface-mediated, gene-delivery-related biomedical research and applications.

KLF2+ stemness maintains human mesenchymal stem cells in bone regeneration.

Mesenchymal stem cells (MSCs), which are undifferentiated stem cells with the property of stemness and the potential to differentiate into multiple lineages, including osteoblasts, have attracted a great deal of attention in bone tissue engineering. Consistent with the heterogeneity of MSCs, various surface markers have been used. However, it is still unclear which markers of MSCs are best for cell amplification in vitro and later bone regeneration in vivo. Krüppel-like Factor 2 (KLF2) is an important indicator of the stemness of human MSCs (hMSCs) and as early vascularization is also critical for bone regeneration, we used KLF2 as a novel in vitro marker for MSCs and investigated the angiogenesis and osteogenesis between KLF2+ MSCs and endothelial cells (ECs). We found a synergistic interaction between hMSCs and human umbilical vein ECs (HUVECs) in that KLF2+ stemness-maintained hMSCs initially promoted the angiogenesis of HUVECs, which in turn more efficiently stimulated the osteogenesis of hMSCs. In fact, KLF2+ hMSCs secreted angiogenic factors initially, with some of the cells then differentiating into pericytes through the PDGF-BB/PDGFR-ß signaling pathway, which improved blood vessel formation. The matured HUVECs in turn synergistically enhanced the osteogenesis of KLF2+ hMSCs through upregulated vascular endothelial growth factor. A three-dimensional coculture model using cell-laden gelatin methacrylate (GelMA) hydrogel further confirmed these results. This study provides insight into the stemness-directed synergistic interaction between hMSCs and HUVECs, and our results will have a profound impact on further strategies involving the application of KLF2+ hMSC/HUVEC-laden GelMA hydrogel in vascular network bioengineering and bone regeneration.

Controlled Release of Naringin in GelMA-Incorporated Rutile Nanorod Films to Regulate Osteogenic Differentiation of Mesenchymal Stem Cells.

Naringin, a Chinese herbal medicine, has been demonstrated to concentration-dependently promote osteogenic differentiation of mesenchymal stem cells (MSCs). However, it remains a challenge to load naringin on coatings for osteogenesis and further control the release kinetics. Here, we demonstrated that the release behavior of naringin on rutile nanorod films could be controlled by either mixing naringin with gelatin methacryloyl (GelMA) before spinning onto the films or soaking the obtained GelMA-incorporated films with the naringin solution to achieve the distinct degradation-type release and diffusion-type release, respectively. We further revealed that the naringin-loaded coatings facilitated adhesion, proliferation and late differentiation, and mineralization of MSCs. Our findings provided a novel strategy to engineer the coatings with controlled release of naringin and emphasized the bioactivity of naringin for the osteogenic differentiation of MSCs.

Enhanced osteogenesis of quasi-three-dimensional hierarchical topography.

Natural extracellular matrices (ECMs) are three-dimensional (3D) and multi-scale hierarchical structure. However, coatings used as ECM-mimicking structures for osteogenesis are typically two-dimensional or single-scaled. Here, we design a distinct quasi-three-dimensional hierarchical topography integrated of density-controlled titania nanodots and nanorods. We find cellular pseudopods preferred to anchor deeply across the distinct 3D topography, dependently of the relative density of nanorods, which promote the osteogenic differentiation of osteoblast but not the viability of fibroblast. The in vivo experimental results further indicate that the new bone formation, the relative bone-implant contact as well as the push-put strength, are significantly enhanced on the 3D hierarchical topography. We also show that the exposures of HFN7.1 and mAb1937 critical functional motifs of fibronectin for cellular anchorage are up-regulated on the 3D hierarchical topography, which might synergistically promote the osteogenesis. Our findings suggest the multi-dimensions and multi-scales as vital characteristic of cell-ECM interactions and as an important design parameter for bone implant coatings.