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.

Enhanced cellular osteogenic differentiation on Zn-containing bioglass incorporated TiO2 nanorod films.

Surface nanotopography and bioactive ions have been considered to play critical roles on the interactions of biomaterials with cells. In this study, a TiO2 nanorod film incorporated with Zn-containing bioactive glass (TiO2/Zn-BG) was prepared on tantalum substrate, trying to evaluate the synergistic effects of nanotopograpgy and bioactive ions to promote cellular osteogenic differentiation activity. The expression of osteogenic-related genes, ALP as well as the ECM mineralization on TiO2/Zn-BG film were significantly upregulated compared to that of the film without TiO2 nanorod nanostructure (Zn-BG) or without Zn (TiO2/BG). Moreover, a much low Zn2+ release level on TiO2/Zn-BG film was beneficial to promote the osteogenesis, which could be ascribed to that a semi-closed space established by TiO2 nanorods with adhered cells provided an appropriate micro-environment that facilitated Zn2+ adsorption.

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.

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.

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.

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.

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.

Surface Modification by Divalent Main-Group-Elemental Ions for Improved Bone Remodeling To Instruct Implant Biofabrication.

Divalent main-group-elemental ions are widely used to improve osteogenic capacity of implants biofabricated from Ti and its alloys. However, the conclusions regarding their osseointegration and immunogenicity are always inconsistent because of the multiple bone remodeling processes as well as the distinct material surface features arising from processing. Here we successfully manufactured the porous micro/nanostructured surface topography with divalent main-group-elemental ions (Mg2+, Ca2+, Sr2+, Ba2+) on substrates through hydrothermal treatment and comprehensively evaluated the complex bone remodeling processes, including osseointegration, immunogenicity, and fibrosis of substrates and implants. We found that Sr-modified implants not only upregulated the adhesion and proliferation of mesenchymal stem cells but also the differentiation of osteogenic markers compared with those modified by other divalent main-group-elemental ions (Mg2+, Ca2+, Ba2+). More importantly, the osteoclastogenesis, immunogenicity, and fibrosis of Sr-modified implants were also significantly downregulated. In vivo, evaluations of new bone formation and histological morphology at the interface of implant and host as well as the removal torque similarly indicated the improved osseointegration of Sr-modified implants as well as the absence of immunogenicity, fibrosis, or necrosis. Our results suggested that among various divalent main-group-elemental ions, Sr2+ might be a promising one for enhancing bone remodeling, which can be used to instruct functionalization of the surfaces of biofabricated Ti-based orthopedic and dental implants in the future.

Magnetically actuated mechanical stimuli on Fe3O4/mineralized collagen coatings to enhance osteogenic differentiation of the MC3T3-E1 cells.

Mechanical stimuli at the bone-implant interface are considered to activate the mechanotransduction pathway of the cell to improve the initial osseointegration establishment and to guarantee clinical success of the implant. However, control of the mechanical stimuli at the bone-implant interface still remains a challenge. In this study, we have designed a strategy of a magnetically responsive coating on which the mechanical stimuli is controlled because of coating deformation under static magnetic field (SMF). The iron oxide nanoparticle/mineralized collagen (IOP-MC) coatings were electrochemically codeposited on titanium substrates in different quantities of IOPs and distributions; the resulting coatings were verified to possess swelling behavior with flexibility same as that of hydrogel. The relative quantity of IOP to collagen and the IOP distribution in the coatings were demonstrated to play a critical role in mediating cell behavior. The cells present on the outer layer of the distributed IOP-MC (O-IOP-MC) coating with a mass ratio of 0.67 revealed the most distinct osteogenic differentiation activity being promoted, which could be attributed to the maximized mechanical stimuli with exposure to SMF. Furthermore, the enhanced osteogenic differentiation of the stimulated MC3T3-E1 cells originated from magnetically actuated mechanotransduction signaling pathway, embodying the upregulated expression of osteogenic-related and mechanotransduction-related genes. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface and thus to promote osseointegration. STATEMENT OF SIGNIFICANCE: The magnetically actuated coating is designed to produce mechanical stimuli to cells for promoting osteogenic differentiation based on the coating deformation. Iron oxide nanoparticles (IOPs) were incorporated into the mineralized collagen coatings (MC) forming the composite coatings (IOP-MC) with spatially distributed IOPs, and the IOP-MC coatings with outer distributed IOPs (O-IOPs-MC) shows the maximized mechanical stimuli to cells with enhanced osteogenic differentiation under static magnetic field. The upregulated expression of the associated genes reveals that the enabled mechanotransduction signaling pathway is responsible for the promoted cellular osteogenic differentiation. This work therefore provides a promising strategy for implementing mechanical stimuli to activate mechanotransduction on the bone-implant interface to promote osseointegration.

Surface hydroxylation regulates cellular osteogeneses on TiO2 and Ta2O5 nanorod films.

Titanium and tantalum have been widely used for orthopedic and dental implant applications. However, how their inherent surface features regulate cellular osteogeneses still remains elusive. In this study, we engineered two distinct TiO2 and Ta2O5 nanorod films as the two model oxidized surfaces to investigate their intrinsic osteogenic behaviors. The results indicated that the distinctive gradient on zeta potential against pH, corresponding to the deprotonation rate, but not the hydroxyl amount or hydroxylation polarity played a critical role on the cellular osteogenic performance. TiO2 nanorod film with a higher deprotonation rate significantly upregulated the expression of osteogeneses-related gene and protein, comparing to that of Ta2O5 nanorod film. These results might be attributed to that surface with higher deprotonation rateprovided more Bronsted acid-base surface sites to react with protein residues, leading to a mild change in conformation of the absorbed proteins, and subsequently facilitating to trigger the integrin-focal adhesion cytoskeleton actin transduction pathway. This study, therefore, provides a new insight into the understanding the role of material surface hydroxylation on cellular osteogenic responses.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5ß1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. STATEMENT OF SIGNIFICANCE: The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5ß1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation.

Effect of hierarchical pore structure on ALP expression of MC3T3-E1 cells on bioglass films.

Hierarchical porous bioglass films on the tantalum were designed to enhance osteointegration of metallic implants. The films were prepared by a sol-gel method using P123 as the mesopore template and polystyrene microsphere as the nanopore template. The films with 5.4nm mesopores and 100nm nanopores (MBG-100) elicited an obviously elongated morphology of the cultured MC3T3-E1 cells, as a result, a higher alkaline phosphatase level was expressed. It is suggested that the nanopores play an important role in regulating cellular behavior by initial protein adsorption through nanopore curvatures. The mesopores were proven very effective for loading rhBMP-2, and the rhBMP-2 loaded on MBG-100 films showed a better function of enhancing osteogenic differentiation, which is attributed to that the nanopore structure could expedite rhBMP-2 release and provide a microenvironment for intensifying the interaction of rhBMP-2 with the cells. Hence, the cell osteogenic differentiation can be enhanced by hierarchical porous bioglass films through both the porous structure and rhBMP-2 induction.

Mediation of cellular osteogenic differentiation through daily stimulation time based on polypyrrole planar electrodes.

In electrical stimulation (ES), daily stimulation time means the interacting duration with cells per day, and is a vital factor for mediating cellular function. In the present study, the effect of stimulation time on osteogenic differentiation of MC3T3-E1 cells was investigated under ES on polypyrrole (Ppy) planar interdigitated electrodes (IDE). The results demonstrated that only a suitable daily stimulation time supported to obviously upregulate the expression of ALP protein and osteogenesis-related genes (ALP, Col-I, Runx2 and OCN), while a short or long daily stimulation time showed no significant outcomes. These might be attributed to the mechanism that an ES induced transient change in intracellular calcium ion concentration, which was responsible for activating calcium ion signaling pathway to enhance cellular osteogenic differentiation. A shorter daily time could lead to insufficient duration for the transient change in intracellular calcium ion concentration, and a longer daily time could give rise to cellular fatigue with no transient change. This work therefore provides new insights into the fundamental understanding of cell responses to ES and will have an impact on further designing materials to mediate cell behaviors.

Surface hydroxyl groups regulate the osteogenic differentiation of mesenchymal stem cells on titanium and tantalum metals.

Titanium (Ti) and tantalum (Ta) metals have been widely used as implants for their favorable mechanical features and good biocompatibility. However, the results on their osteogenic capacity have been conflicting due to the synergistic effects of complex and multiple material surface features (such as topography, surface chemistries etc.) on cellular behaviors. Here, we directly compare the osteogenic response of mesenchymal stem cells (MSCs) to Ti and Ta metal surfaces with alterable surface hydroxyl groups. Although no difference was found on both surface topographies, cellular adhesion, proliferation, and the expression of osteogenic-related markers were upregulated with the increasing amount of surface hydroxyl groups (-OH) after ultraviolet (UV) light treatment. Moreover, Ti showed better effects in promoting osteogenic differentiation of MSCs than Ta before UV light treatment, but demonstrated the opposite after UV light treatment. These results might be attributed to the comparative quantity of the distinct type of surface hydroxyl groups (bridging-OH and terminal-OH), which regulated the conformation of the initial protein adsorption and subsequent cellular behaviors. Our results demonstrate the central role of the surface hydroxyl groups in mediating cell-material interactions and implicate this interface as helping in optimizing osteointegration of Ti and Ta based orthopaedic and dental implants.

Controlled Release of Naringin in Metal-Organic Framework-Loaded Mineralized Collagen Coating to Simultaneously Enhance Osseointegration and Antibacterial Activity.

Two important goals in orthopedic implant research are to promote osseointegration and prevent infection. However, much previous effort has been focused on the design of coatings to either enhance osseointegration while ignoring antibacterial activity or vice versa, to prevent infection while ignoring bone integration. Here, we designed a multifunctional mineralized collagen coating on titanium with the aid of metal-organic framework (MOF) nanocrystals to control the release of naringin, a Chinese herbal medicine that could promote osseointegration and prevent bacterial infection. The attachment, proliferation, osteogenic differentiation, and mineralization of mesenchymal stem cells on the coating were significantly enhanced. Meanwhile, the antibacterial abilities against Staphylococcus aureus were also promoted. Furthermore, release kinetics analysis indicated that the synergistic effect of a primary burst release stage and secondary slow release stage played a critical role in the performance and could be controlled by the relative concentrations of MOF and naringin. This work thus provides a novel strategy to engineer multifunctional orthopedic coatings that can enhance osseointegration and simultaneously inhibit microbial cell growth.