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.

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.

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.

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.

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.

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.

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.

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.

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.

Corrosion behavior of novel Ti-24Nb-4Zr-7.9Sn alloy for dental implant applications in vitro.

Ti-24Nb-4Zr-7.9Sn (TNZS) alloy is a newly developed ß-titanium alloy considered suitable for dental implant applications due to its low elastic modulus and high strength. The aim of this study was to investigate the corrosion behavior of TNZS alloy through a static immersion test in various simulated physiological solutions, namely, artificial saliva, lactic acid solution, fluoridated saliva, and fluoridated acidified saliva for 7 days. The corrosion behavior of commercially pure titanium and Ti-6Al-4V alloy were also examined for comparison. The elemental release was measured with inductively coupled plasma mass spectroscopy, and the changes of alloy surface were observed with scanning electron microscopy (SEM). The test results showed that the quantity of each metal element released from TNZS alloy into fluoridated solutions was much higher than the solutions without fluoride ions. It was highest in fluoridated acidified saliva and lowest in artificial saliva (p < 0.01). The total elemental release from TNZS alloy was lower than commercially pure titanium and Ti-6Al-4V alloy in the same solution (p < 0.01). SEM micrographs indicated that TNZS alloy possessed better corrosion resistant performance. It can be concluded that fluoridated solutions have a negative influence on the corrosion behavior of TNZS alloy. Compared with commercially pure titanium and Ti-6Al-4V alloy, TNZS alloy demonstrates better corrosion resistance in various simulated physiological solutions, so it has greater potential for dental implant applications.