Visible-light-responsive reduced graphene oxide/g-C3N4/TiO2 composite nanocoating for photoelectric stimulation of neuronal and osteoblastic differentiation.

Restoration of nerve supply in newly formed bone is critical for bone defect repair. However, nerve regeneration is often overlooked when designing bone repair biomaterials. In this study, employing graphitic carbon nitride (g-C3N4) as a visible-light-driven photocatalyst and reduced graphene oxide (rGO) as a conductive interface, an rGO/g-C3N4/TiO2 (rGO/CN/TO) ternary nanocoating with photoelectric conversion ability was fabricated on a Ti-based orthopedic implant for photoelectric stimulation of both bone and nerve repair. Compared with g-C3N4/TiO2 (CN/TO) and TiO2 nanocoatings, the ternary nanocoating exhibited stronger visible-light absorption as well as higher transient photocurrent density and open circuit potential under blue LED exposure. The improved photo-electrochemical properties of the ternary nanocoating were attributed to the enhanced separation of photogenerated carriers at the heterointerface. For the tested nanocoatings, introducing blue LED light irradiation enhanced MC3T3-E1 osteoblastic differentiation and neurite outgrowth of PC12 cells. Among them, the rGO/CN/TO nanocoating exerted the greatest enhancement. In a coculture system, PC12 cells on the ternary nanocoating released a higher amount of neurotransmitter calcitonin gene-related peptide (CGRP) under light irradiation, which in turn significantly enhanced osteoblastic differentiation. The results may provide a prospective approach for targeting nerve regeneration to stimulate osteogenesis when designing bone repair biomaterials.

Engineering Nanopatterned Structures to Orchestrate Macrophage Phenotype by Cell Shape.

Physical features on the biomaterial surface are known to affect macrophage cell shape and phenotype, providing opportunities for the design of novel "immune-instructive" topographies to modulate foreign body response. The work presented here employed nanopatterned polydimethylsiloxane substrates with well-characterized nanopillars and nanopits to assess RAW264.7 macrophage response to feature size. Macrophages responded to the small nanopillars (SNPLs) substrates (450 nm in diameter with average 300 nm edge-edge spacing), resulting in larger and well-spread cell morphology. Increasing interpillar distance to 800 nm in the large nanopillars (LNPLs) led to macrophages exhibiting morphologies similar to being cultured on the flat control. Macrophages responded to the nanopits (NPTs with 150 nm deep and average 800 nm edge-edge spacing) by a significant increase in cell elongation. Elongation and well-spread cell shape led to expression of anti-inflammatory/pro-healing (M2) phenotypic markers and downregulated expression of inflammatory cytokines. SNPLs and NPTs with high availability of integrin binding region of fibronectin facilitated integrin ß1 expression and thus stored focal adhesion formation. Increased integrin ß1 expression in macrophages on the SNPLs and NTPs was required for activation of the PI3K/Akt pathway, which promoted macrophage cell spreading and negatively regulated NF-κB activation as evidenced by similar globular cell shape and higher level of NF-κB expression after PI3K blockade. These observations suggested that alterations in macrophage cell shape from surface nanotopographies may provide vital cues to orchestrate macrophage phenotype.

Zn-doped MnO2 nanocoating with enhanced catalase-mimetic activity and cytocompatibility protects pre-osteoblasts against H2O2-induced oxidative stress.

Therapeutic application in prevention and treatment of bone diseases, particularly osteoporosis, has recently started to emerge for manganese dioxide (MnO2) nanoparticles and nanocoatings whereby their antioxidant catalase-mimetic property can be exploited to control oxidative stress by reducing the amount of H2O2. Doping is an efficient method to enhance the catalase-mimetic activity of MnO2, which can potentially ameliorate osteogenesis under oxidative stress. Herein, Zn2+ doped MnO2 (Zn-MnO2) nanocoating was fabricated on orthopedic titanium implant by a facile UV-photolysis reaction. The Zn-MnO2 nanocoating showed better cytocompatibility than the MnO2 nanocoating, as indicated by enhanced cell proliferation, differentiation and mineralization of MC3T3-E1 pre-osteoblasts. This was probably due to the increased surface hydrophilicity as well as the combination effect of released Zn2+ and Mn2+ from the Zn-MnO2 nanocoating. Importantly, the Zn-MnO2 nanocoating with enhanced catalase-like activity exerted greater effects to suppress the intracellular oxidation products generation and prevent the depletion of dismutase superoxide levels under H2O2-induced oxidative stress, which in turn protected MC3T3-E1 pre-osteoblast functions. Overall, surface modification of titanium implants with the Zn-MnO2 nanocoating could be utilized to ameliorate oxidative stress-inhibited osteogenesis.

Boron-incorporated micro/nano-topographical calcium silicate coating dictates osteo/angio-genesis and inflammatory response toward enhanced osseointegration.

Orthopedic implant coatings with optimal surface features to achieve favorable osteo/angio-genesis and inflammatory response would be of great importance. However, to date, few coatings are capable of fully satisfying these requirements. In this work, to take advantage of the structural complexity of micro/nano-topography and benefits of biological trace elements, two types of boron-containing nanostructures (nanoflakes and nanolamellars) were introduced onto plasma-sprayed calcium silicate (F-BCS and L-BCS) coatings via hydrothermal treatment. The C-CS coating using deionized water as hydrothermal medium served as control. Boron-incorporated CS coating stimulated osteoblastic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Specifically, the combination of ß1 integrin-vinculin-mediated cell spreading and activation of bone morphogenetic protein signaling pathway acted synergistically to cause significant upregulation of runt-related transcription factor 2 (RUNX2) protein and Runx2 gene expression in BMSCs on the F-BCS coating surface, which induced the transcription of downstream osteogenic differentiation marker genes. F-BCS coating allowed specific boron ion release, which favored angiogenesis as evidenced by the enhanced migration and tube formation of human umbilical vein endothelial cells in the coating extract. Boron-incorporated coatings significantly suppressed the expression of toll-like receptor adaptor genes in RAW264.7 macrophages and subsequently the degradation of nuclear factor-κB inhibitor α, accompanied by the inactivation of the downstream pro-inflammatory genes. In vivo experiments confirmed that F-BCS-coated Ti implant possessed enhanced osseointegration compared with L-BCS- and C-CS-coated implants. These data highlighted the synergistic effect of specific nanotopography and boron release from orthopedic implant coating on improvement of osseointegration.

Preparation and characteristics of a novel oxygen-releasing coating for improved cell responses in hypoxic environment.

Affected by environmental factors such as oxygen deficiency, the secretion of growth factor was abnormal in bone injury sites, resulting in the poor responses of osteoblasts and prolonging the healing process. Herein, in this study, we reported an in situ oxygen-releasing porous titanium coating that combines the dual degradability of poly(lactic-co-glycolic acid) with the self-releasing oxygen capacity of the CaO2 core. The resulting formulation exhibited stable oxygen-releasing capacity as well as the ability to promote proliferation and differentiation of the MC3T3 cell line under hypoxia conditions. According to these results, oxygen-releasing coatings based on improved cellular microenvironment may be a promising bone repair material that would reduce the incidence of difficult bone healing in the future.

Optimized Nanointerface Engineering of Micro/Nanostructured Titanium Implants to Enhance Cell-Nanotopography Interactions and Osseointegration.

The success of orthopedic implants requires rapid and complete osseointegration which relies on an implant surface with optimal features. To enhance cellular function in response to the implant surface, micro- and nanoscale topography have been suggested as essential. The aim of this study was to identify an optimized Ti nanostructure and to introduce it onto a titanium plasma-sprayed titanium implant (denoted NTPS-Ti) to confer enhanced immunomodulatory properties for optimal osseointegration. To this end, three types of titania nanostructures, namely, nanowires, nanonests, and nanoflakes, were achieved on hydrothermally prepared Ti substrates. The nanowire surface modulated protein conformation and directed integrin binding and specificity in such a way as to augment the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and induce a desirable osteoimmune response of RAW264.7 macrophages. In a coculture system, BMSCs on the optimized micro/nanosurface exerted enhanced effects on nonactivated or lipopolysaccharide-stimulated macrophages, causing them to adopt a less inflammatory macrophage profile. The enhanced immunomodulatory properties of BMSCs grown on NTPS-Ti depended on a ROCK-medicated cyclooxygenase-2 (COX2) pathway to increase prostaglandin E2 (PGE2) production, as evidenced by decreased production of PGE2 and concurrent inhibition of immunomodulatory properties after treatment with ROCK or COX2 inhibitors. In vivo evaluation showed that the NTPS-Ti implant resulted in enhanced osseointegration compared with the TPS-Ti and Ti implants. The results obtained in our study may provide a prospective approach for enhancing osseointegration and supporting the application of micro/nanostructured Ti implants.

A hydrogenated black TiO2 coating with excellent effects for photothermal therapy of bone tumor and bone regeneration.

The clinical treatment of bone tumors usually brings about residual tumor cells and large bone defects after tumor removal surgery. To solve this problem, it is imperative to develop a novel implant with bi-functions for eliminating the residual tumor cells and repairing bone defects. In this study, hydrogenated black TiO2 (H-TiO2) coating with hierarchical micro/nano-topographies is fabricated by induction suspension plasma spraying (ISPS). The fabricated H-TiO2 coating possessed excellent and controllable photothermal effect in inhibiting the tumor growth under 808 nm NIR laser irradiation in vitro and in vivo. The hierarchical hybrid micro/nano-structured surface and Ti-OH groups improved the adhesion, proliferation, differentiation and osteogenic gene expressions of rat bone mesenchymal stem cells (rBMSCs). These results demonstrate that the H-TiO2 coating may be a promising implant material for the treatment of bone tumors and bone regeneration.

The enhanced angiogenic responses to ionic dissolution products from a boron-incorporated calcium silicate coating.

Early vascularization is crucial for osteogenic repair of bone defects and plays an essential role in the fate of implanted biomaterials. Thus, there is a growing interest in the use of biomaterials to release inorganic ions that are capable of stimulating angiogenesis. Since it has been established that boron (B) may play roles in angiogenesis, the aim of our study was to investigate the in vitro angiogenic effects of the ionic dissolution products from the B-incorporated calcium silicate (Ca11Si4B2O22, B-CS) coating. The results showed that ionic products of B-CS coating extract obviously stimulated the proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as the in vitro tubule formation when compared with those of CS coating extract. In addition, the gene expression levels of pro-angiogenic growth factors (VEGF, bFGF, ANG1) and receptors (VEGFR-2, bFGFR) were significantly upregulated when stimulated with the B-CS coating extract. Moreover, VEGF and VEGFR-2 protein synthesis, eNOS, and Akt phosphorylation, as well as NO synthesized by HUVECs were increased by the B-CS coating extract. Hence, the B-CS coating offers a potential solution to enhance bone vascularization essential for successful osseointegration of orthopedic implants.

Improved osteogenesis of boron incorporated calcium silicate coatings via immunomodulatory effects.

Osteoimmunology has revealed the importance of a favorable immune response for successful biomaterial-mediated osteogenesis. Boron-incorporated calcium silicate (Ca11 Si4 B2 O22 , B-CS) coating has been reported as a potential candidate for improving osteogenesis in orthopedic applications in vitro. However, relatively little is known about its effects on the immune response and subsequent osteogenesis. In this work, the immunomodulatory properties of the B-CS coating and its specific mechanism of action were explored. We found that the B-CS coating decreased M1 polarization and converted macrophages to the M2 phenotype via restraining the toll-like receptor signaling pathway, thus inducing a significant reduction in pro-inflammatory cytokines and an increase in anti-inflammatory cytokines. Moreover, the B-CS coating inhibited osteoclastogenesis and osteoclastic activities by downregulating osteoclastogenic genes and inhibiting the RANKL/RANK system. BMP2 and VEGF were also significantly upregulated by macrophages and bone mesenchymal stem cells, leading to activation of the BMP2 signaling pathway and subsequent upregulation of osteogenesis-associated genes, finally promoting osteogenic differentiation. These findings show that the B-CS coating could be a promising coating material for hip and knee implants. Furthermore, incorporation of the element boron into bioceramic coatings could be a good strategy in the design of bone biomaterials with beneficial immune responses. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 12-24, 2019.

Unveiling the Mechanism of Surface Hydrophilicity-Modulated Macrophage Polarization.

With inflammation increasingly recognized as a key factor that influences fracture healing, the immunologic response is considered to play a pivotal role in determining implant-mediated osteogenesis. Herein, this paper demonstrates that modification of the surface hydrophilicity of Ti surface oxides can be utilized to control immune response by steering the macrophage polarization toward pro- or anti-inflammation phenotype. Enhanced anti-inflammatory and prohealing performance of macrophages is observed on hydrophilic surfaces compared to hydrophobic ones. Further study on the detailed mechanism demonstrates that the surface hydrophilicity controls specific proteins (fibronectin and fibrinogen) adsorption and conformation, which activate different signaling pathways (PI3K and NF-κB) through selective expression of integrin ß1 or ß2 to influence the behaviors of macrophages. Thus, this study presents a mechanism of macrophage polarization modulated by surface hydrophilicity for the surface design of advanced implant materials with satisfactory anti-inflammatory and osteogenesis-promoting properties.

Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells.

Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM), X-ray diffraction (XRD) as well as transmission electron microscopy (TEM). The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs) were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta2O5 nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro.

Sr-doped nanowire modification of Ca-Si-based coatings for improved osteogenic activities and reduced inflammatory reactions.

Biomedical coatings for orthopedic implants should facilitate osseointegration and mitigate implant-induced inflammatory reactions. In our study, Ca-Si coatings with Sr-containing nanowire-like structures (NW-Sr-CS) were achieved via hydrothermal treatment. In order to identify the effect of nanowire-like topography and Sr dopant on the biological properties of Ca-Si-based coatings, the original Ca-Si coating, Ca-Si coatings modified with nanoplate (NP-CS) and similar nanowire-like structure (NW-CS) were fabricated as the control. Surface morphology, phase composition, surface area, zeta potential and ion release of these coatings were characterized. The in vitro osteogenic activities and immunomodulatory properties were evaluated with bone marrow stromal cells (BMSCs) and RAW 264.7 cells, a mouse macrophage cell line. Compared with the CS and NP-CS coatings, the NW-CS coating possessed a larger surface area and pore volume, beneficial protein adsorption, up-regulated the expression levels of integrin ß1, Vinculin and focal adhesion kinase and promoted cell spreading. Furthermore, the NW-CS coating significantly enhanced the osteogenic differentiation and mineralization as indicated by the up-regulation of ALP activity, mineralized nodule formation and osteoblastogenesis-related gene expression. With the introduction of Sr, the NW-Sr-CS coatings exerted a greater effect on the BMSC proliferation rate, calcium sensitive receptor gene expression as well as PKC and ERK1/2 phosphorylation. In addition, the Sr-doped coatings significantly up-regulated the ratio of OPG/RANKL in the BMSCs. The NW-Sr-CS coatings could modulate the polarization of macrophages towards the wound-healing M2 phenotype, reduce the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) and enhance anti-inflammatory cytokines (IL-1ra, IL-10). The Sr-doped nanowire modification may be a valuable approach to enhance osteogenic activities and reduce inflammatory reactions.

Influence of patterned titanium coatings on polarization of macrophage and osteogenic differentiation of bone marrow stem cells.

Biomaterial surface topography plays a vital role in the osteointegration of implants by regulating the early cell responses and tissue growth-in. However, most of the previous researches focused on the effects of osteogenic cells, only a little is known about the immune cells which dominate osteogenesis after implanting. In this paper, patterned titanium coatings were fabricated and the effects of surface topography on the macrophage behaviors were investigated. On patterned titanium surface, macrophages preferred to polarize to M2, while macrophages on traditional titanium coatings presented higher M1 polarization. Nearly 70% higher expression of anti-inflammatory genes, including interleukin-4, interleukin-10, interleukin-1ra, and arginase, were detected on the patterned titanium coatings. While the pro-inflammatory genes, such as interleukin-1ß, interleukin-6, tumor necrosis factor-α, interferon-γ, and inducible nitric oxide synthase were notably depressed. Up-regulation of the osteoinductive cytokines were also detected on the patterned coatings, which indicated advantageous osteogenic microenvironment provided by macrophages. Immunomodulation effect on osteogenesis was also investigated in this study. Stimulated with RAW cells/patterned coatings conditioned medium, bone marrow stem cells presented nearly 1.5 fold higher expression of osteogenic genes and more mineralization nodules than the traditional sprayed Ti coatings. All these results suggested that modulating materials with a patterned surface might be a valuable strategy to endow the implants with favorable osteoimmunomodulatory properties.

Incorporation of Cerium Oxide into Hydroxyapatite Coating Protects Bone Marrow Stromal Cells Against H2O2-Induced Inhibition of Osteogenic Differentiation.

Oxidative stress exerts a key influence in osteoporosis in part by inhibiting osteogenic differentiation of bone marrow stromal cells (BMSCs). With their unique antioxidant properties and reported biocompatibility, cerium oxide (CeO2) ceramics exhibit promising potential for the treatment of osteoporosis resulting from oxidative stress. In this study, protective effects of CeO2-incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) on the viability and osteogenic differentiation of H2O2-treated BMSCs were examined. CeO2-incorporated HA coatings enhanced cell viability and attenuated cell apoptosis caused by H2O2. An increase in CeO2 content in HA coatings better alleviated H2O2-induced inhibition of osteogenic differentiation by increasing alkaline phosphatase (ALP) activity, calcium deposition activity, and mRNA expression levels of osteogenesis markers runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OCN) in BMSCs. Furthermore, the H2O2-induced decrease of gene and protein expressions of ß-catenin and cyclin D1 in the Wnt/ß-catenin signaling pathway was successfully rescued by the CeO2 incorporated HA coatings. Besides, the decreased expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and the increased ratio of osteoprotegerin (OPG)/RANKL in BMSCs on the CeO2-modified coatings was observed, indicating the inhibition of osteoclastogenesis. The above results were mediated by the antioxidant properties of CeO2. The CeO2-incorporated HA coatings reversed the decreased superoxide dismutase (SOD) activity, reduced reactive oxygen species (ROS) generation, and suppressed the malondiadehyde (MDA) formation. The findings suggested that CeO2-modified HA coatings may be promising coating materials for osteoporotic bone regeneration.

Vancomycin-loaded titanium coatings with an interconnected micro-patterned structure for prophylaxis of infections: an in vivo study.

Titanium (Ti) and its alloys are widely applied as orthopedic implants for hip and knee prostheses, fixation, and dental implants. However, Ti and its alloys are bioinert and susceptible to bacteria and biofilm formation. Thus, surface biofunctionalisation of Ti is essential for improving the biofunction of Ti. The current in vitro study indicated that calcium phosphate bone cement with vancomycin doped on micro-patterned Ti with a grid-like structure surface could preserve the property of inhibition of bacterial adhesion and biofilm formation while not affecting the osteogenic differentiation. The present study investigated whether the biological performance of the bactericidal effect is preserved in vivo. The rabbit osteomyelitis model with tibial medullary cavity placement of Ti rods was employed to analyze the antibacterial effect of vancomycin-loaded Ti coatings with interconnected micro-patterned structure (TV). Thirty female rabbits (N = 10) were used to establish the implant-associated infection. Prior to implanting the T0 and TV rods into the medullary cavity of the left tibia of the rabbits, 106 CFU mL-1 methicillin-resistant Staphylococcus aureus (MRSA) was injected into the medullary cavity of the left tibia of the rabbits. The sterile Ti rod (NT) was used as the blank control. After 3 weeks, bone pathology was evaluated using X-ray and micro-CT. The in vivo study proposed that TV has the potential for prophylaxis against MRSA infection. Thus, the interconnected micro-patterned structured Ti rods loaded with vancomycin could be applied for preventing Ti implant-associated infections.

Enhanced osteogenic activity of anatase TiO2 film: Surface hydroxyl groups induce conformational changes in fibronectin.

In this study, with an attempt to identify the effects of TiO2 crystalline phase compositions on the osteogenic properties, the anatase and rutile TiO2 thin films with similar film thickness, surface topography and hydrophilicity were prepared on Si (100) substrates by atomic layer deposition (ALD), subsequent thermal annealing and ultraviolet irradiation. The films were studied with XRD, XPS, FE-SEM, AFM, FTIR and contact angle measurements. In vitro cellular assays showed that the anatase phase led to better osteoblast compatibility in terms of adhesion, proliferation, differentiation, mineralization as well as osteogenesis-related gene expression when compared with the rutile phase. We investigated the difference between the anatase and rutile TiO2 films at the biomolecular level to explain the enhanced osteogenic activity of the anatase film. It was found that the presence of more TiOH groups on anatase surface induced more cell-binding sites of fibronectin (FN) exposed on its surface, causing a more active conformation of the adsorbed FN for subsequent osteoblast behaviors.

Sustained release vancomycin-coated titanium alloy using a novel electrostatic dry powder coating technique may be a potential strategy to reduce implant-related infection.

In order to tackle the implant-related infection, a novel way was developed in this study to coat vancomycin particles mixed with controlled release coating materials onto the surface of titanium alloy by using an electrostatic dry powder coating technique. To characterize this sustained release antibacterial coating, surface morphology, in vitro and in vivo drug release were sequentially evaluated. In vitro cytotoxicity was tested by Cell Counting Kit-8 (CCK-8) assay and cytological changes were observed by inverted microscope. The antibacterial properties against MRSA, including a bacterial growth inhibition assay and a colony-counting test by spread plate method were performed. Results indicated that the vancomycin-coated sample was biocompatible for Human osteoblast cell line MG-63 and displayed effective antibacterial ability against MRSA. The coating film was revealed uniform by scanning electron microscopy. Both the in vitro and in vivo drug release kinetics showed an initially high release rate, followed by an extended period of sustained drug release over 7 days. These results suggest that with good biocompatibility and antibacterial ability, the sustained release antibacterial coating of titanium alloy using our novel electrostatic dry powder coating process may provide a promising candidate for the treatment of orthopedic implant-related infection.

Immunomodulation effect of a hierarchical macropore/nanosurface on osteogenesis and angiogenesis.

Immune systems play pivotal roles in determining the in vivo osseointegration of bone implants. While much evidence has shown that hierarchical implant surfaces can exhibit excellent osteogenic ability, their immune response has not been well elucidated, which will preclude accurate knowledge of their osseointegration performance. In this study, the immunomodulatory properties of a hierarchical macropore/nanosurface as well as the detailed mechanism was investigated. Macrophages were found to switch to M2 phenotype on the hierarchical surface, and decreased levels of inflammatory gene expression as well as increased expression of anti-inflammatory genes were endowed, which were probably regulated by the decisive role of cytoskeleton tension induced by specific cell shape. In addition, enhanced osteogenic differentiation of bone marrow mesenchymal stem cells and angiogenesis of human umbilical vein endothelial cells could be observed when stimulated by a RAW cells/hierarchical surface conditioned medium, which were probably due to increased expression of BMP-2 and VEGF of RAW cells, respectively. These findings give comprehensive knowledge into detailed mechanism of the immunomodulatory behavior of the hierarchical surface, which will also provide insight into the surface design of advanced bone biomaterials with satisfactory immunomodulation properties.

The combined effects of nanotopography and Sr ion for enhanced osteogenic activity of bone marrow mesenchymal stem cells (BMSCs).

Both surface topography and chemistry have a significant influence on the biological performance of orthopedic implant coatings. In our study, a surface modification strategy embodying bioactive trace element incorporation and nanotopography construction was employed to enhance the osteogenic activity of calcium silicate (Ca-Si) coatings. We developed strontium-loaded nanolayer on plasma sprayed Ca-Si (CS) coating via hydrothermal treatment which was denoted as Sr-NT-CS. The original CS coating and the CS coating modified with similar nanotopography (NT-CS) were studied in parallel. We investigated the cellular effects of surface topography and released Sr ion on the adhesion, proliferation, differentiation, and mineralization of BMSCs and the associated molecular mechanisms. The results indicated that the nanotopography activated integrin ß1, promoted the spread of BMSCs into a polygonal osteoblastic shape, and induced higher levels of collagen secretion. The Sr incorporation stimulated osteogenic differentiation and mineralization as indicated by the increases in ALP activity and mineralized nodules formation. The examination of gene expressions revealed that Sr ion exerted the effects by interacting with extracellular calcium sensitive receptor (CaSR), and combined with the nanotopographical cue for the up-regulation of osteogenic master transcription factor Runx2. The promoted Runx2 subsequently affected osteoblast (OB) marker genes (BMP-2, BSP, OPN, and OCN), thus driving BMSCs to differentiate into OBs. Moreover, the Sr incorporation inhibited osteoclastogenesis, as indicated by the down-regulation of interleukin-6 (IL-6) and the inhibition of RANKL/RANK system. Those results suggested that our developed Sr-NT-CS coating have combined the effects of nanotopography and Sr ion for enhanced osteogenic activity of BMSCs.

The Effects of Cerium Valence States at Cerium Oxide Coatings on the Responses of Bone Mesenchymal Stem Cells and Macrophages.

Ideal orthopedic coatings should trigger good osteogenic response and limited inflammatory response. The cerium valence states in ceria are associated with their anti-oxidative activity and anti-inflammatory property. In the study, we prepared two kinds of plasma sprayed CeO2 coatings with different Ce4+ concentrations to investigate the effects of Ce valence states on the response of bone mesenchymal stem cells (BMSCs) and macrophage RAW264.7. Both the coatings (CeO2-A and CeO2-B) were characterized via XRD, SEM, and X-ray photoelectron spectroscopy. The CeO2 coatings enhanced osteogenic behaviors of BMSCs in terms of cellular proliferation, alkaline phosphatase (ALP) activity and calcium deposition activity in comparison with the Ti substrate. In particular, the CeO2-B coating (higher Ce4+ concentration) elicited greater effects than the CeO2-A coating (higher Ce3+ concentration). RT-PCR and western blot results suggested that the CeO2-B coating promoted BMSCs osteogenic differentiation through the SMAD-dependent BMP signaling pathway, which activated Runx2 expression and subsequently enhanced the expression of ALP and OCN. With respect to either CeO2-A coating or Ti substrate, the CeO2-B coating exerted greater effects on the macrophages, increasing the anti-inflammatory cytokines (IL-10 and IL-1ra) expression and suppressing the expression of the pro-inflammatory cytokines (TNF-α and IL-6) and ROS production. Furthermore, it also upregulated the expression of osteoinductive molecules (TGF-ß1 and BMP2) in the macrophages. The regulation of cerium valence states at plasma sprayed ceria coatings can be a valuable strategy to improve osteogenic properties and alleviate inflammatory response.