Enhancing osteogenesis and angiogenesis functions for Ti-24Nb-4Zr-8Sn scaffolds with methacrylated gelatin and deferoxamine.

Repair of large bone defects remains challenge for orthopedic clinical treatment. Porous titanium alloys have been widely fabricated by the additive manufacturing, which possess the elastic modulus close to that of human cortical bone, good osteoconductivity and osteointegration. However, insufficient bone regeneration and vascularization inside the porous titanium scaffolds severely limit their capability for repair of large-size bone defects. Therefore, it is crucially important to improve the osteogenic function and vascularization of the titanium scaffolds. Herein, methacrylated gelatin (GelMA) were incorporated with the porous Ti-24Nb-4Zr-8Sn (Ti2448) scaffolds prepared by the electron beam melting (EBM) method (Ti2448-GelMA). Besides, the deferoxamine (DFO) as an angiogenic agent was doped into the Ti2448-GelMA scaffold (Ti2448-GelMA/DFO), in order to promote vascularization. The results indicate that GelMA can fully infiltrate into the pores of Ti2448 scaffolds with porous cross-linked network (average pore size: 120.2 ± 25.1 µm). Ti2448-GelMA scaffolds facilitated the differentiation of MC3T3-E1 cells by promoting the ALP expression and mineralization, with the amount of calcium contents ∼2.5 times at day 14, compared with the Ti2448 scaffolds. Impressively, the number of vascular meshes for the Ti2448-GelMA/DFO group (∼7.2/mm2) was significantly higher than the control group (∼5.3/mm2) after cultivation for 9 h, demonstrating the excellent angiogenesis ability. The Ti2448-GelMA/DFO scaffolds also exhibited sustained release of DFO, with a cumulative release of 82.3% after 28 days. Therefore, Ti2448-GelMA/DFO scaffolds likely provide a new strategy to improve the osteogenesis and angiogenesis for repair of large bone defects.

Corrosion and Biological Behaviors of Biomedical Ti-24Nb-4Zr-8Sn Alloy under an Oxidative Stress Microenvironment.

Biomaterials can induce an inflammatory response in surrounding tissues after implantation, generating and releasing reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). The excessive accumulation of ROS may create a microenvironment with high levels of oxidative stress (OS), which subsequently accelerates the degradation of the passive film on the surface of titanium (Ti) alloys and affects their biological activity. The immunomodulatory role of macrophages in biomaterial osteogenesis under OS is unknown. This study aimed to explore the corrosion behavior and bone formation of Ti implants under an OS microenvironment. In this study, the corrosion resistance and osteoinduction capabilities in normal and OS conditions of the Ti-24Nb-4Zr-8Sn (wt %, Ti2448) were assessed. Electrochemical impedance spectroscopy analysis indicated that the Ti2448 alloy exhibited superior corrosion resistance on exposure to excessive ROS compared to the Ti-6Al-4V (TC4) alloy. This can be attributed to the formation of the TiO2 and Nb2O5 passive films, which mitigated the adverse effects of OS. In vitro MC3T3-E1 cell experiments revealed that the Ti2448 alloy exhibited good biocompatibility in the OS microenvironment, whereas the osteogenic differentiation level was comparable to that of the TC4 alloy. The Ti2448 alloy significantly alleviates intercellular ROS levels, inducing a higher proportion of M2 phenotypes (52.7%) under OS. Ti2448 alloy significantly promoted the expression of the anti-inflammatory cytokine, interleukin 10 (IL-10), and osteoblast-related cytokines, bone morphogenetic protein 2 (BMP-2), which relatively increased by 26.9 and 31.4%, respectively, compared to TC4 alloy. The Ti2448 alloy provides a favorable osteoimmune environment and significantly promotes the proliferation and differentiation of osteoblasts in vitro compared to the TC4 alloy. Ultimately, the Ti2448 alloy demonstrated excellent corrosion resistance and immunomodulatory properties in an OS microenvironment, providing valuable insights into potential clinical applications as implants to repair bone tissue defects.

Synergistic Amelioration of Osseointegration and Osteoimmunomodulation with a Microarc Oxidation-Treated Three-Dimensionally Printed Ti-24Nb-4Zr-8Sn Scaffold via Surface Activity and Low Elastic Modulus.

Biomaterial scaffolds, including bone substitutes, have evolved from being primarily a biologically passive structural element to one in which material properties such as surface topography and chemistry actively direct bone regeneration by influencing stem cells and the immune microenvironment. Ti-6Al-4V(Ti6Al4V) implants, with a significantly higher elastic modulus than human bone, may lead to stress shielding, necessitating improved stability at the bone-titanium alloy implant interface. Ti-24Nb-4Zr-8Sn (Ti2448), a low elastic modulus ß-type titanium alloy devoid of potentially toxic elements, was utilized in this study. We employed 3D printing technology to fabricate a porous scaffold structure to further decrease the structural stiffness of the implant to approximate that of cancellous bone. Microarc oxidation (MAO) surface modification technology is then employed to create a microporous structure and a hydrophilic oxide ceramic layer on the surface and interior of the scaffold. In vitro studies demonstrated that MAO treatment enhances the proliferation, adhesion, and osteogenesis capabilities on the scaffold surface. The chemical composition of the MAO-Ti2448 oxide layer is found to enhance the transcription and expression of osteogenic genes in bone mesenchymal stem cells (BMSCs), potentially related to the enrichment of Nb2O5 and SnO2 in the oxide layer. The MAO-Ti2448 scaffold, with its synergistic surface activity and low stiffness, significantly activates the anti-inflammatory macrophage phenotype, creating an immune microenvironment that promotes the osteogenic differentiation of BMSCs. In vivo experiments in a rabbit model demonstrated a significant improvement in the quantity and quality of the newly formed bone trabeculae within the scaffold under the contact osteogenesis pattern with a matched elastic modulus. These trabeculae exhibit robust connections to the external structure of the scaffold, accelerating the formation of an interlocking structure between the bone and implant and providing higher implantation stability. These findings suggest that the MAO-Ti2448 scaffold has significant potential as a bone defect repair material by regulating osteoimmunomodulation and osteogenesis to enhance osseointegration. This study demonstrates an optional strategy that combines the mechanism of reducing the elastic modulus with surface modification treatment, thereby extending the application scope of ß-type titanium alloy.

Physically cross-linked chitosan gel with tunable mechanics and biodegradability for tissue engineering scaffold.

Chitosan, a cationic polysaccharide, exhibits promising potential for tissue engineering applications. However, the poor mechanical properties and rapid biodegradation have been the major limitations for its applications. In this work, an effective strategy was proposed to optimize the mechanical performance and degradation rate of chitosan gel scaffolds by regulating the water content. Physical chitosan hydrogel (HG, with 93.57 % water) was prepared by temperature-controlled cross-linking, followed by dehydration to obtain xerogel (XG, with 2.84 % water) and rehydration to produce wet gel (WG, with 56.06 % water). During this process, changes of water content significantly influenced the water existence state, hydrogen bonding, and the chain entanglements of chitosan in the gel network. The mechanical compression results showed that the chitosan gel scaffolds exhibited tunable compressive strength (0.3128-139 MPa) and compressive modulus (0.2408-1094 MPa). XG could support weights exceeding 65,000 times its own mass while maintaining structural stability. Furthermore, in vitro and in vivo experiments demonstrated that XG and WG exhibited better biocompatibility and resistance to biodegradation compared with HG. Overall, this work contributes to the design and optimization of chitosan scaffolds without additional chemical crosslinkers, which has potential in tissue engineering and further clinical translation.

Improving Biological Functions of Three-Dimensional Printed Ti2448 Scaffolds by Decoration with Polydopamine and Extracellular Matrices.

Extracellular matrices (ECMs) provide important cues for cell proliferation and differentiation in the complex environment, which show a significant influence on cell functions. Herein, cell-derived ECMs were deposited on the polydopamine (PDA)-decorated porous Ti-24Nb-4Zr-8Sn (Ti2448) scaffolds fabricated by the electron beam melting method in order to improve biological functions. The influence of PDA-ECM coatings on cell functions was further investigated. The results demonstrated that the PDA-ECM coating facilitated adhesion, proliferation, and migration of MC3T3-E1 cells on Ti2448 scaffolds. Moreover, Ti2448-PDA-ECM scaffolds promoted osteogenesis differentiation of cells indicated by greater alkaline phosphatase activity and further mineralization, compared to the plain Ti2448 group. Meanwhile, Ti2448-PDA-ECM scaffolds enhanced bone growth after implantation for one month in rabbit femoral bone defects. Our findings suggest that the bioinspired PDA-ECM coating can be implemented on the porous Ti2448 scaffolds, which significantly improve the biological functions of orthopedic implants.

Three-Dimensionally Printed Ti2448 With Low Stiffness Enhanced Angiogenesis and Osteogenesis by Regulating Macrophage Polarization via Piezo1/YAP Signaling Axis.

Previous studies have found that the novel low-elastic-modulus Ti2448 alloy can significantly reduce stress shielding and contribute to better bone repair than the conventional Ti6Al4V alloy. In this study, the promotion of osteogenesis and angiogenesis by three-dimensionally printed Ti2448 were also observed in vivo. However, these were not significant in a series of in vitro tests. The stiffness of materials has been reported to greatly affect the response of macrophages, and the immunological regulation mediated by macrophages directly determines the fate of bone implants. Therefore, we designed more experiments to explore the role of three-dimensionally printed Ti2448 in macrophage activation and related osteogenesis and angiogenesis. As expected, we found a significant increase in the number of M2 macrophages around Ti2448 implants, as well as better osteogenesis and angiogenesis in vivo. In vitro studies also showed that macrophages pre-treated with Ti2448 alloy significantly promoted angiogenesis and osteogenic differentiation through increased PDGF-BB and BMP-2 secretion, and the polarization of M2 macrophages was enhanced. We deduced that Ti2448 promotes angiogenesis and osteogenesis through Piezo1/YAP signaling axis-mediated macrophage polarization and related cytokine secretion. This research might provide insight into the biological properties of Ti2448 and provide a powerful theoretical supplement for the future application of three-dimensionally printed Ti2448 implants in orthopaedic surgery.

Material Characterisation and Computational Thermal Modelling of Electron Beam Powder Bed Fusion Additive Manufacturing of Ti2448 Titanium Alloy.

Ti-24Nb-4Zr-8Sn (Ti2448) is a metastable ß-type titanium alloy developed for biomedical applications. In this work, cylindrical samples of Ti2448 alloy have been successfully manufactured by using the electron beam powder bed fusion (PBF-EB) technique. The thermal history and microstructure of manufactured samples are characterised using computational and experimental methods. To analyse the influence of thermal history on the microstructure of materials, the thermal process of PBF-EB has been computationally predicted using the layer-by-layer modelling method. The microstructure of the Ti2448 alloy mainly includes ß phase and a small amount of α″ phase. By comparing the experimental results of material microstructure with the computational modelling results of material thermal history, it can be seen that aging time and aging temperature lead to the variation of α″ phase content in manufactured samples. The computational modelling proves to be an effective tool that can help experimentalists to understand the influence of macroscopic processes on material microstructural evolution and hence potentially optimise the process parameters of PBF-EB to eliminate or otherwise modify such microstructural gradients.

In Vitro Study on the Piezodynamic Therapy with a BaTiO3-Coating Titanium Scaffold under Low-Intensity Pulsed Ultrasound Stimulation.

To solve the poor sustainability of electroactive stimulation in clinical therapy, a strategy of combining a piezoelectric BaTiO3-coated Ti6Al4V scaffold and low-intensity pulsed ultrasound (LIPUS) was unveiled and named here as piezodynamic therapy. Thus, cell behavior could be regulated phenomenally by force and electricity simultaneously. First, BaTiO3 was deposited uniformly on the surface of the three-dimensional (3D) printed porous Ti6Al4V scaffold, which endowed the scaffold with excellent force-electricity responsiveness under pulsed ultrasound exposure. The results of live/dead staining, cell scanning electron microscopy, and F-actin staining showed that cells had better viability, better pseudo-foot adhesion, and more muscular actin bundles when they underwent the piezodynamic effect of ultrasound and piezoelectric coating. This piezodynamic therapy activated more mitochondria at the initial stage that intervened in the cell cycle by promoting cells' proliferation and weakened the apoptotic damage. The quantitative real-time polymerase chain reaction data further confirmed that the costimulation of the ultrasound and the piezoelectric scaffolds could trigger adequate current to upregulated the expression of osteogenic-related genes. The continuous electric cues could be generated by the BaTiO3-coated scaffold and intermittent LIPUS stimulation; thereon, more efficient bone healing would be promoted by piezodynamic therapy in future treatment.

The Jieduan-Niwan (JDNW) Formula Ameliorates Hepatocyte Apoptosis: A Study of the Inhibition of E2F1-Mediated Apoptosis Signaling Pathways in Acute-on-Chronic Liver Failure (ACLF) Using Rats.

BACKGROUND: Acute-on-chronic liver failure (ACLF) is a severe, complicated human disease. E2F1-mediated apoptosis plays an important role in ACLF development. Jieduan-Niwan (JDNW) formula, a traditional Chinese medicine (TCM), has shown remarkable clinical efficacy in ACLF treatment. However, the hepatoprotective mechanisms of the formula are barely understood. PURPOSE: This study aimed to investigate the mechanisms of JDNW formula in ACLF treatment by specifically regulating E2F1-mediated apoptotic signaling pathways in rats. METHODS: The JDNW components were determined by high-performance liquid chromatography (HPLC) analysis. The ACLF rat model was established using human serum albumin immune-induced liver cirrhosis, followed by D-galactosamine and lipopolysaccharide joint acute attacks. The ACLF rat was treated with JDNW formula. Prothrombin time activity was measured to investigate the coagulation function. Liver pathological injury was observed by hematoxylin-eosin (HE) and reticular fiber staining. The hepatocyte apoptosis index and apoptosis rate were determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and flow cytometry, respectively. Additionally, the expression of key genes and proteins that regulate E2F1-mediated apoptosis was analyzed by quantitative real-time PCR and Western blot. RESULTS: Seven major components of JDNW formula were detected. The formula ameliorated the coagulation function, decreased the hepatocyte apoptosis index and apoptosis rate, and alleviated liver pathological damage in ACLF rats. The down-regulation of the expression of genes and proteins from p53-dependent and non-p53-dependent apoptosis pathways and the up-regulation of the expression of genes from blocking anti-apoptotic signaling pathways indicated that JDNW formula inhibited excessive hepatocyte apoptosis in ACLF rats via E2F1-mediated apoptosis signaling pathways. CONCLUSION: The findings indicate that JDNW formula protects livers of ACLF rats by inhibiting E2F1-mediated apoptotic signaling pathways, implying that these pathways might be a potential therapeutic target for ACLF treatment.

Improved activity of MC3T3-E1 cells by the exciting piezoelectric BaTiO3/TC4 using low-intensity pulsed ultrasound.

Developing bioactive materials for bone implants to enhance bone healing and bone growth has for years been the focus of clinical research. Barium titanate (BT) is an electroactive material that can generate electrical signals in response to applied mechanical forces. In this study, a BT piezoelectric ceramic coating was synthesized on the surface of a TC4 titanium alloy, forming a BT/TC4 material, and low-intensity pulsed ultrasound (LIPUS) was then applied as a mechanical stimulus. The combined effects on the biological responses of MC3T3-E1 cells were investigated. Results of scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction showed that an uniform nanospheres -shaped BT coating was formed on TC4 substrate. Piezoelectric behaviors were observed using piezoelectric force microscopy with the piezoelectric coefficient d33 of 0.42 pC/N. Electrochemical measures indicated that LIPUS-stimulated BT/TC4 materials could produce a microcurrent of approximately 10 µA/cm2. In vitro, the greatest osteogenesis (cell adhesion, proliferation, and osteogenic differentiation) was found in MC3T3-E1 cells when BT/TC4 was stimulated using LIPUS. Furthermore, the intracellular calcium ion concentration increased in these cells, possibly because opening of the L-type calcium ion channels was promoted and expression of the CaV1.2 protein was increased. Therefore, the piezoelectric BT/TC4 material with LIPUS loading synergistically promoted osteogenesis, rending it a potential treatment for early stage formation of reliable bone-implant contact.

Hydrothermally grown TiO2-nanorods on surface mechanical attrition treated Ti: Improved corrosion fatigue and osteogenesis.

Current bioactive modifications of Ti-based materials for promoting osteogenesis often decrease corrosion fatigue strength (σcf) of the resultant implants, thereby shortening their service lifespan. To solve this issue and accelerate the osteogenesis process, in the present study, a TiO2 nanorods (TNR)-arrayed coating was hydrothermally grown on optimal surface mechanical attrition treated (SMATed) titanium (S-Ti). The microstructure, bond integrity, residual stress distribution, and corrosion fatigue of TNR-coated S-Ti (TNR/S-Ti) and the response of macrophages and bone marrow-derived mesenchymal stem cells (BMSCs) to TNR/S-Ti were investigated and compared with those of mechanically polished Ti (P-Ti), S-Ti, and TNR-coated P-Ti (TNR/P-Ti). S-Ti showed a nanograined layer and an underlying grain-deformed region with residual compressive stress, which was sustained even when it was hydrothermally coated with TNR. TNR on S-Ti showed nanotopography, composition, and bond strength almost identical to those of P-Ti. While TNR/P-Ti showed a considerable decrease in σcf compared to P-Ti, TNR/S-Ti exhibited an improved σcf which was even higher than that of P-Ti. Biologically, TNR/S-Ti enhanced adhesion, differentiation, and mineralization of BMSCs, and it also promoted adhesion and M1-to-M2 transition of macrophages as compared to S-Ti and P-Ti. With rapid phenotype switch of macrophages, the level of proinflammatory cytokines decreased, while anti-inflammatory cytokines were upregulated. In co-culture conditions, the migration, differentiation, and mineralization of BMSCs were enhanced by increased level of secretion factors of macrophages on TNR/S-Ti. The modified structure accelerated bone apposition in rabbit femur and is expected to induce a favorable immune microenvironment to facilitate osseointegration earlier; it can also simultaneously improve corrosion fatigue resistance of Ti-based implants and thereby enhance their service life.

Biofunctional magnesium coated Ti6Al4V scaffold enhances osteogenesis and angiogenesis in vitro and in vivo for orthopedic application.

The insufficient osteogenesis and osseointegration of porous titanium based scaffold limit its further application. Early angiogenesis is important for scaffold survival. It is necessary to develop a multifunctional surface on titanium scaffold with both osteogenic and angiogenic properties. In this study, a biofunctional magnesium coating is deposited on porous Ti6Al4V scaffold. For osseointegration and osteogenesis analysis, in vitro studies reveal that magnesium-coated Ti6Al4V co-culture with MC3T3-E1 cells can improve cell proliferation, adhesion, extracellular matrix (ECM) mineralization and ALP activity compared with bare Ti6Al4V cocultivation. Additionally, MC3T3-E1 cells cultured with magnesium-coated Ti6Al4V show significantly higher osteogenesis-related genes expression. In vivo studies including fluorochrome labeling, micro-computerized tomography and histological examination of magnesium-coated Ti6Al4V scaffold reveal that new bone regeneration is significantly increased in rabbits after implantation. For angiogenesis studies, magnesium-coated Ti6Al4V improve HUVECs proliferation, adhesion, tube formation, wound-healing and Transwell abilities. HUVECs cultured with magnesium-coated Ti6Al4V display significantly higher angiogenesis-related genes (HIF-1α and VEGF) expression. Microangiography analysis reveal that magnesium-coated Ti6Al4V scaffold can significantly enhance the blood vessel formation. This study enlarges the application scope of magnesium and provides an optional choice to the conventional porous Ti6Al4V scaffold with enhanced osteogenesis and angiogenesis for further orthopedic applications.

In-situ monitoring of the electrochemical behavior of cellular structured biomedical Ti-6Al-4V alloy fabricated by electron beam melting in simulated physiological fluid.

Ti-6Al-4V alloys with cellular structure fabricated by additive manufacturing are currently of significant interest because their modulus is comparable to bone and the cellular structure allows the cells to penetrate and exchange nutrients, promoting osseointegration. We describe here a unique simulation device that replaces the traditional steady electrochemistry approach, enabling in-situ study of variation of ion concentration and surface potential with pore depth for cellular structured Ti-6Al-4V alloys fabricated by electron beam melting (EBM) in phosphate buffered saline (PBS). This approach addresses the scientific gap on the electrochemical behavior of cellular structured titanium alloys. The study indicated that concentration of H+ and Cl- increased with the increase of pore depth, while the surface potential decreased. The exposed surface of inner cellular structure was not corroded but passivated after immersing in PBS at 37 °C for 14 days, which was independent of pore depth. Furthermore, X-ray photoelectron spectroscopy (XPS) and Mott-Schottky (M-S) studies suggested that a thinner passive film containing a greater donor density was formed on the surface of cellular structured Ti-6Al-4V alloy at the deepest pore depth. This is attributed to insufficient oxygen supply and Cl-adsorption on the surface inside the pores. STATEMENT OF SIGNIFICANCE: Porous titanium alloys are promising implants in biomedical applications. However, it is a challenge to accurately characterize the corrosion behavior of porous titanium alloys with complex pore structure using traditional electrochemical methods. In this study, we have adopted a special device to simulate the environment within the pore structure. The variation in ion concentration and surface potential of Ti-6Al-4V fabricated by EBM with pore depth was in-situ monitored. After immersing in PBS for 14 days, Ti-6Al-4V exhibited good corrosion properties and the samples with less than 60 mm pore depth were not corroded but passivated. Also, we analyzed the difference in corrosion property at different pore depth. This type of in-situ corrosion performance monitoring in EBM-produced Ti-6Al-4V has not been previously studied.

Jieduan-Niwan Formula Reduces Liver Apoptosis in a Rat Model of Acute-on-Chronic Liver Failure by Regulating the E2F1-Mediated Intrinsic Apoptosis Pathway.

Acute-on-chronic liver failure (ACLF) is a serious and complicated disease that threatens human health because its pathogenesis is unclear, and the outcome of the current therapies has been less than satisfactory. A national famous doctor of traditional Chinese medicine, Qian Ying, created the Jieduan-Niwan Formula (JDNW), based on his long-term clinical experience. However, despite the good clinical outcome, the biological mechanism by which it works is unknown. In the current study, we established an ACLF rat model by administering human serum albumin (HSA) combined with D-galactosamine (D-GalN) and lipopolysaccharide (LPS) to explore the potential mechanism of JDNW in treating ACLF. The rats were treated with JDNW by administration of the model substances and sacrificed after 4, 8, and 12 h. Then we divided the rats into normal group, model at 4 h, model at 8 h, model at 12 h, JDNW at 4 h, JDNW at 8 h, and JDNW at 12 h. Biochemical and histopathological examinations were performed to compare the rats in different groups. Compared with the ACLF model group, expression levels of alanine transaminase, aspartate aminotransferase, total bilirubin, and TNF-α and IL-6 proteins were reduced in the JDNW group at the corresponding time points, the survival rates of rats were increased, and the pathological condition of the liver was improved. In addition, JDNW treatment improved the ultrastructure of hepatocytes and mitochondria and decreased the hepatocyte apoptosis index. E2F1, P53, P73, Apaf-1, p14ARF, caspase-3, caspase-6, and caspase-7 levels in the JDNW group were distinctly lower than those in the untreated rats. Moreover, Bcl-2 and Mcl-1 levels increased. Thus, JDNW decreases ACLF-induced mortality in rats by modulating the E2F1-mediated intrinsic apoptotic pathway.

Effect of Microarc Oxidation-Treated Ti6Al4V Scaffold Following Low-Intensity Pulsed Ultrasound Stimulation on Osteogenic Cells in Vitro.

The porous Ti6Al4V alloy has emerged to solve the biomechanical mismatch between implant and bone as its tunable mechanical properties. Cell-surface interaction is related to numerous factors-the surface's chemical composition, morphological structure, and external effect. The microarc oxidation (MAO) method was employed in this study to improve the surface properties of scaffolds produced by Electron Beam Melting (EBM), and low-intensity pulse ultrasound (LIPUS) provides physical stimulation for cells in vitro. Although MAO-treated and untreated scaffolds shared the same three-dimensional (3D) structures, the former recreated a more affinity surface than the latter in the 3D room, which could stimulate cell adhesion, proliferation, and differentiation. Therefore, MG63 cells were represented with a stereoscopic cytoskeleton structure on the MAO-treated scaffold as numerous cellular filopodia/lamellipodia with rich extracellular matrix secretion, while flat and sheetlike cells were observed on the untreated scaffold. The expression of ALP, OCN, BMP2, Bmpr1a, and Runx2 were up-regulated by the MAO-treated scaffold; in addition, LIPUS stimulation effectively promoted cell proliferation and osteogenesis differentiation. In the future, the EBM-MAO strategy can be applied to prepare 3D hierarchical macro-/microstructure titanium implants for bone grafts, and LIPUS stimulation can be used as a therapeutic method simultaneously.

A unique hybrid-structured surface produced by rapid electrochemical anodization enhances bio-corrosion resistance and bone cell responses of ß-type Ti-24Nb-4Zr-8Sn alloy.

Ti-24Nb-4Zr-8Sn (Ti2448), a new ß-type Ti alloy, consists of nontoxic elements and exhibits a low uniaxial tensile elastic modulus of approximately 45 GPa for biomedical implant applications. Nevertheless, the bio-corrosion resistance and biocompatibility of Ti2448 alloys must be improved for long-term clinical use. In this study, a rapid electrochemical anodization treatment was used on Ti2448 alloys to enhance the bio-corrosion resistance and bone cell responses by altering the surface characteristics. The proposed anodization process produces a unique hybrid oxide layer (thickness 50-120 nm) comprising a mesoporous outer section and a dense inner section. Experiment results show that the dense inner section enhances the bio-corrosion resistance. Moreover, the mesoporous surface topography, which is on a similar scale as various biological species, improves the wettability, protein adsorption, focal adhesion complex formation and bone cell differentiation. Outside-in signals can be triggered through the interaction of integrins with the mesoporous topography to form the focal adhesion complex and to further induce osteogenic differentiation pathway. These results demonstrate that the proposed electrochemical anodization process for Ti2448 alloys with a low uniaxial tensile elastic modulus has the potential for biomedical implant applications.

Effect of low-intensity pulsed ultrasound on the biological behavior of osteoblasts on porous titanium alloy scaffolds: An in vitro and in vivo study.

Low-intensity pulsed ultrasound (LIPUS) has been used in patients with fresh fractures, delayed union and non-union to enhance bone healing and improve functional outcome. However, there were few studies concerning the effects of LIPUS on the biological behavior of osteoblasts on porous scaffolds. This study aimed to evaluate the effects of LIPUS on the biological behavior of osteoblasts on porous titanium-6aluminum-4vanadium (Ti6Al4V) alloy scaffolds in vitro and in vivo. Scaffolds were randomly divided into an ultrasound group and a control group. Mouse pre-osteoblast cells were cultured with porous Ti6Al4V scaffolds in vitro. The effects of LIPUS on the biological behavior of osteoblasts were evaluated by observing the adhesion, proliferation, differentiation and ingrowth depth on porous Ti6Al4V scaffolds. In addition, scaffolds were implanted into rabbit mandibular defects in vivo. The effects of LIPUS on bone regeneration were evaluated via micro-CT, fluorescent staining and toluidine blue staining. The results revealed that osteoblast adhered well to the scaffolds, and there was no significant difference in the methyl thiazolyl tetrazolium value between the ultrasound group and the control group (p>0.05). Compared with the control group, ultrasound promoted the alkaline phosphatase activity, osteocalcin levels and ingrowth depth of the cells on the scaffolds (p<0.05). In addition, micro-CT and histomorphological analysis showed that the volume and amount of new bone formation were increased and that bone maturity was improved in the ultrasound group compared to the control group. These results indicate that LIPUS promotes osteoblast differentiation as well as enhances bone ingrowth in porous Ti6Al4V scaffolds, and promotes bone formation and maturity in porous Ti6Al4V scaffolds.

Porous titanium-6 aluminum-4 vanadium cage has better osseointegration and less micromotion than a poly-ether-ether-ketone cage in sheep vertebral fusion.

Interbody fusion cages made of poly-ether-ether-ketone (PEEK) have been widely used in clinics for spinal disorders treatment; however, they do not integrate well with surrounding bone tissue. Ti-6Al-4V (Ti) has demonstrated greater osteoconductivity than PEEK, but the traditional Ti cage is generally limited by its much greater elastic modulus (110 GPa) than natural bone (0.05-30 GPa). In this study, we developed a porous Ti cage using electron beam melting (EBM) technique to reduce its elastic modulus and compared its spinal fusion efficacy with a PEEK cage in a preclinical sheep anterior cervical fusion model. A porous Ti cage possesses a fully interconnected porous structure (porosity: 68 ± 5.3%; pore size: 710 ± 42 µm) and a similar Young's modulus as natural bone (2.5 ± 0.2 GPa). When implanted in vivo, the porous Ti cage promoted fast bone ingrowth, achieving similar bone volume fraction at 6 months as the PEEK cage without autograft transplantation. Moreover, it promoted better osteointegration with higher degree (2-10x) of bone-material binding, demonstrated by histomorphometrical analysis, and significantly higher mechanical stability (P < 0.01), shown by biomechanical testing. The porous Ti cage fabricated by EBM could achieve fast bone ingrowth. In addition, it had better osseointegration and superior mechanical stability than the conventional PEEK cage, demonstrating great potential for clinical application.

Effects of the surface characteristics of nanoporous titanium oxide films on Ti-24Nb-4Zr-8Sn alloy on the initial adhesion of osteoblast-like MG-63 cells.

The aim of the present study was to investigate the effects of the surface characteristics of nanoporous titanium oxide films, formed by anodization on Ti-24Nb-4Zr-8Sn (Ti2448) alloy, on the early adhesion of osteoblast-like MG-63 cells. Nanoporous titanium oxide films with two different pore sizes (30 and 90 nm) were formed by anodization in NH4F solution on Ti2448 alloy. The surface roughness of the nanoporous titanium oxide films was determined using a Surftest Formtracer and field emission scanning electron microscopy (FESEM). Cell viability was evaluated at different time points using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. To investigate the regulatory mechanisms involved in the focal adhesion of osteoblasts to Ti2448 alloy, we quantified the expression levels of integrin ß1 and paxillin mRNAs on the nanoporous titanium oxide films during early osteoblast adhesion using real-time RT-PCR. Samples with a 30-nm nanoporous film exhibited a greater number of overlapping microporous structures with microprojections compared with the 90-nm nanoporous film samples. The MTT assay indicated that cell viability on the 30-nm nanoporous surface following 24 and 48 h of cell culture was higher than those observed on the unanodized control and 90-nm nanoporous surfaces. Integrin ß1 mRNA expression levels on the 30-nm nanoporous surface following cell culture for 48 h were also significantly higher compared with those on the unanodized control and 90-nm nanoporous surfaces. The results demonstrated that a 30-nm nanoporous titanium oxide film on Ti2448 alloy may provide the optimum bioactive implant surface for the initial adhesion of osteoblasts.