Enhanced Mechanical Properties, Corrosion Resistance, Cytocompatibility, Osteogenesis, and Antibacterial Performance of Biodegradable Mg-2Zn-0.5Ca-0.5Sr/Zr Alloys for Bone-Implant Application.

Magnesium (Mg) alloys are widely used in bone fixation and bone repair as biodegradable bone-implant materials. However, their clinical application is limited due to their fast corrosion rate and poor mechanical stability. Here, the development of Mg-2Zn-0.5Ca-0.5Sr (MZCS) and Mg-2Zn-0.5Ca-0.5Zr (MZCZ) alloys with improved mechanical properties, corrosion resistance, cytocompatibility, osteogenesis performance, and antibacterial capability is reported. The hot-extruded (HE) MZCZ sample exhibits the highest ultimate tensile strength of 255.8 ± 2.4 MPa and the highest yield strength of 208.4 ± 2.8 MPa and an elongation of 15.7 ± 0.5%. The HE MZCS sample shows the highest corrosion resistance, with the lowest corrosion current density of 0.2 ± 0.1 µA cm-2 and the lowest corrosion rate of 4 ± 2 µm per year obtained from electrochemical testing, and a degradation rate of 368 µm per year and hydrogen evolution rate of 0.83 ± 0.03 mL cm-2 per day obtained from immersion testing. The MZCZ sample shows the highest cell viability in relation to MC3T3-E1 cells among all alloy extracts, indicating good cytocompatibility except at 25% concentration. Furthermore, the MZCZ alloy shows good antibacterial capability against Staphylococcus aureus.

Construction of multifunctional coating with cationic amino acid-coupled peptides for osseointegration of implants.

Osseointegration is an important indicator of implant success. This process can be improved by coating modified bioactive molecules with multiple functions on the surface of implants. Herein, a simple multifunctional coating that could effectively improve osseointegration was prepared through layer-by-layer self-assembly of cationic amino acids and tannic acid (TA), a negatively charged molecule. Osteogenic growth peptide (OGP) and the arginine-glycine-aspartic acid (RGD) functional polypeptides were coupled with Lys6 (K6), the two polypeptides then self-assembled with TA layer by layer to form a composite film, (TA-OGP@RGD)n. The surface morphology and biomechanical properties of the coating were analyzed in gas and liquid phases, and the deposition process and kinetics of the two peptides onto TA were monitored using a quartz crystal microbalance. In addition, the feeding consistency and adsorption ratios of the two peptides were explored by using fluorescence visualization and quantification. The (TA-OGP@RGD)n composite membrane mediated the early migration and adhesion of cells and significantly promoted osteogenic differentiation and mineralization of the extracellular matrix in vitro. Additionally, the bifunctional peptide exhibited excellent osteogenesis and osseointegration owing to the synergistic effect of the OGP and RGD peptides in vivo. Simultaneously, the (TA-OGP@RGD)n membrane regulated the balance of reactive oxygen species in the cell growth environment, thereby influencing the complex biological process of osseointegration. Thus, the results of this study provide a novel perspective for constructing multifunctional coatings for implants and has considerable application potential in orthopedics and dentistry.

Ferroptosis Nanomedicine: Clinical Challenges and Opportunities for Modulating Tumor Metabolic and Immunological Landscape.

Ferroptosis, a type of regulated cell death driven by iron-dependent phospholipid peroxidation, has captured much attention in the field of nanomedicine since it was coined in 2012. Compared with other regulated cell death modes such as apoptosis and pyroptosis, ferroptosis has many distinct features in the molecular mechanisms and cellular morphology, representing a promising strategy for treating cancers that are resistant to conventional therapeutic modalities. Moreover, recent insights collectively reveal that ferroptosis is tightly connected to the maintenance of the tumor immune microenvironment (TIME), suggesting the potential application of ferroptosis therapies for evoking robust antitumor immunity. From a biochemical perspective, ferroptosis is intricately regulated by multiple cellular metabolic pathways, including iron metabolism, lipid metabolism, redox metabolism, etc., highlighting the importance to elucidate the relationship between tumor metabolism and ferroptosis for developing antitumor therapies. In this review, we provide a comprehensive discussion on the current understanding of ferroptosis-inducing mechanisms and thoroughly discuss the relationship between ferroptosis and various metabolic traits of tumors, which offer promising opportunities for direct tumor inhibition through a nanointegrated approach. Extending from the complex impact of ferroptosis on TIME, we also discussed those important considerations in the development of ferroptosis-based immunotherapy, highlighting the challenges and strategies to enhance the ferroptosis-enabled immunostimulatory effects while avoiding potential side effects. We envision that the insights in this study may facilitate the development and translation of ferroptosis-based nanomedicines for tumor treatment.

Substance P-embedded multilayer on titanium substrates promotes local osseointegration via MSC recruitment.

In this study, the chemokine substance P (SP) was inserted into multilayered systems on titanium (Ti)-based substrates for endogenous mesenchymal stem cell (MSC) recruitment to facilitate bone healing. The multilayer was constructed with cationic chitosan (Chi), SP and anionic gelatin (Gel) via a spin-coater-assisted layer-by-layer (LBL) approach. The characterization results demonstrated that the multilayer system was successfully constructed and was capable of continuously releasing SP for almost 2 weeks. We further confirmed that MSCs grown on SP-modified Ti-based substrates showed improved migration capabilities as well as enhanced secretion of matrix metalloproteinases (MMP2, MMP9), rather than enhanced MSC proliferation and differentiation in vitro. In the CD29+/CD90+ double immunofluorescence assay, the Ti/LBL-SP group showed the highest number of MSCs migrating to the peri-implant area after implantation. Consistently, the Ti/LBL-SP implants also significantly enhanced new bone formation according to the results of micro-CT scanning analysis, H&E staining, Masson's trichrome staining and immunohistochemical staining. The obtained results reveal that SP-modified Ti-based substrates were beneficial for bone formation via recruiting endogenous MSCs.

NFAT5 directs hyperosmotic stress-induced fibrin deposition and macrophage infiltration via PAI-1 in endothelium.

Although stress can significantly promote atherosclerosis, the underlying mechanisms are still not completely understood. Here we successfully unveiled that high salt-induced nuclear factor of activated T cells 5 (NFAT5) control the endothelial-dependent fibrinolytic activity and the inflammatory adhesion-related molecules expression through regulation of plasminogen activator inhibitor-1 (PAI-1). We first observed that high salt diets instigated the expression of NFAT5 and PAI-1 in the endothelium which brought about the fibrin deposition and macrophage infiltration in the atherosclerotic arteries of ApoE-/- mice. Overexpression of NFAT5 increased PAI-1-mediated antifibrinolytic activity and activated inflammatory adhesion-related genes in endothelial cells. Knockdown of NFAT5 by siRNA inhibited the expression of PAI-1, antifibrinolytic and adhesive molecules. Moreover, chromatin immunoprecipitation assay demonstrated that high salt intake significantly promoted the binding of NFAT5 to PAI-1 promoter (TGGAATTATTT) in endothelial cells. Our study identified that NFAT5 has great potential to activate the PAI-1-mediated fibrinolytic dysfunction and inflammatory cell adhesion, thus promoting high salt-induced atherosclerosis disease.

Enzyme responsive titanium substrates with antibacterial property and osteo/angio-genic differentiation potentials.

After implantation into a host, titanium (Ti) orthopaedic materials are facing two major clinical challenges: bacterial infection and aseptic loosening, which directly determine the long-term survival of the implant. To endow Ti implant with self-defensive antibacterial properties and desirable osteo/angio-genic differentiation potentials, hyaluronic acid (HA)-gentamicin (Gen) conjugates (HA-Gen) and chitosan (Chi) polyelectrolyte multilayers were constructed on deferoxamine (DFO) loaded titania nanotubes (TNT) substrates via layer-by-layer (LBL) assembly technique, termed as TNT/DFO/HA-Gen. The HA-Gen conjugate was characterized by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (1H NMR). The physicochemical properties of the substrates were characterized by field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The on-demand DFO release was associated with the degradation of multilayers triggered by exogenous hyaluronidase, which indicated enzymatic and bacterial responsiveness. The TNT/DFO/HA-Gen substrates displayed effective antifouling and antibacterial properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), while were favourable for the adhesion, proliferation and osteo/angio-genic differentiation of mesenchymal stem cells (MSCs). The multifaceted drug-device combination (DDC) strategy showed potential applications in orthopaedic fields.

Regulation of MSC and macrophage functions in bone healing by peptide LL-37-loaded silk fibroin nanoparticles on a titanium surface.

Titanium-based materials have been long regarded as effective bone implants for clinical use, yet the corresponding osteointegration ability needs to be optimized. This challenge can be overcome by fabricating titanium (Ti) materials with physiological functions. In this study, peptide LL-37-loaded silk fibroin nanoparticles (SFNPs) were immobilized on a titanium surface to facilitate osteointegration by regulating the physiological functions of mesenchymal stem cells (MSCs) and macrophages. According to our results, the cell viability, recruitment and paracrine responses of MSCs and macrophages were improved by the modified Ti samples. MSC differentiation was promoted by the macrophages incubated on the modified Ti samples, and the phenotype switch of macrophages was also modulated by the MSCs incubated on the modified Ti samples. In vivo studies proved that the modified Ti implant induced MSC and macrophage recruitments to injury sites and the inflammatory response was positively regulated. Moreover, better bone formation was achieved around the modified Ti implant 28 days after surgery. This suggested that the immobilization of peptide LL-37-loaded SFNPs on a titanium surface improves osteointegration via the regulation of physiological functions of MSCs and macrophages.

Matrix promote mesenchymal stromal cell migration with improved deformation via nuclear stiffness decrease.

Bone marrow derived mesenchymal stromal cells (BMSCs) migration to injury site is a prevalent event in tissue repair process after damage occurrence. The migration process is always accompanied with matrix stiffness change. In this study, sodium alginate hydrogels with different stiffness and Transwell chambers with gradient chemical factors were employed to mimic tissue repair in vivo. In this work, in the stiffness range of 1-20 kPa, BMSCs in stiffer matrix showed higher migration speed compared to those in softer matrix. Moreover, stiffer matrix decreased the nuclear stiffness of BMSCs and reduced the expression of lamin A/C, which playing a main role in the regulation of nuclear stiffness. Furthermore, it was found that BMSCs fitted environment by selecting migration strategy. This study provides a novel platform for the investigation of BMSCs migration to mimic the natural tissue repair process.

Biocompatible MoS2/PDA-RGD coating on titanium implant with antibacterial property via intrinsic ROS-independent oxidative stress and NIR irradiation.

To inhibit bacterial infection in situ and improve osseointegration are essentially important for long-term survival of an orthopedic implant, in particular for infection-associating revision surgery. Herein, we fabricate a functional molybdenum disulfide (MoS2)/polydopamine (PDA)-arginine-glycine-aspartic acid (RGD) coating on titanium (Ti) implant to address above concerns simultaneously. The coating not only improved the osteogenesis of mesenchymal stem cells (MSCs), but also endowed Ti substrates with effective antibacterial ability when exposing to near-infrared (NIR) irradiation. It accelerated glutathione (GSH) oxidation via photothermal energy and induced intrinsic ROS-independent oxidative stress damage deriving from MoS2 nanosheets. The results displayed that RGD-decorated MoS2 nanosheets significantly increased the cellular osteogenic behaviors of MSCs via up-regulating osteogenesis-related genes (ALP, Runx2, Col I and OCN) in vitro. Moreover, the functionalized Ti substrates demonstrated great antibacterial efficiency of over 92.6% inhibition for S. aureus and E. coli under NIR-irradiation. Hyperthermia induced by photothermal effect accelerated the GSH consumption and ROS-independent oxidative stress destroyed the integrity of bacteria membranes, which synergistically led to protein leakage and ATP decrease. Furthermore, co-culture experiment showed that S. aureus contamination was efficiently cleaned from MoS2/PDA-RGD surface after NIR photothermal treatment, while MSCs adhered and proliferated on the MoS2/PDA-RGD surface. In an S. aureus infection model in vivo, MoS2/PDA-RGD modified Ti rods killed bacteria with an efficiency of 94.6% under NIR irradiation, without causing damage to normal tissue. More importantly, the MoS2/PDA-RGD modified Ti implants accelerated new bone formation in comparison with TNT implants in vivo.

Minocycline-incorporated multilayers on titanium substrates for simultaneous regulation of MSCs and macrophages.

Minocycline (Mino) is a well-established antibiotic, which also has osseointegration and anti-inflammatory functions. In this study, we fabricated a multilayered structure with potential osteoinduction capability on titanium via a spin-assisted layer-by-layer (LBL) assembly technique. Mino was used as the intercalated material, while gelatin and chitosan were used as the polycation and polyanion, respectively. The successful fabrication of the multilayers was validated by scanning electronic microscopy (SEM), atomic force microscopy (AFM) and water contact angle measurements. The release result shows that the sustained release of Mino might be attributed to the synergistic effect of drug diffusion and multilayer degradation. The evaluations of alkaline phosphatase (ALP), mineralization, and osteogenic gene expression consistently demonstrate that the Mino-modified samples (Ti/LBL/Mino) significantly improved the osteogenic differentiation of MSCs compared with the control group (Ti). More importantly, the results of flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and western-blot confirm that Ti/LBL/Mino substrates could indirectly promote osteogenesis through macrophage regulation, such as phenotype transformation from M1 to M2 and regulation of inflammatory/osteogenic genes. The aforementioned properties make Ti/LBL/Mino substrate highly suitable for orthopedic applications.

Fabrication of magnesium/zinc-metal organic framework on titanium implants to inhibit bacterial infection and promote bone regeneration.

The residual bacteria in the second revision surgery caused by infection would further lead to the failure of the implantation. Pathogenic bacteria adhesion to an implant surface not only interfere the functions of bone-formation related cells, but also activate the host immune system, thus resulting in inflammation and osteogenesis inhibition. Thus, to fabricate multifunctional (antibacterial, anti-inflammation and pro-osteogenesis) titanium implants is essential to address this issue. In this work, hybrid magnesium/zinc-metal organic framework (Mg/Zn-MOF74) coating was constructed on alkali-heat treated titanium (AT) surface. The hybrid Mg/Zn-MOF74 coating displayed good stability and its stability was related to the content of Zn2+. The MOF74-modified samples were sensitive to bacterial acid microenvironment and displayed strong antibacterial ability against both Escherichia coli and Staphylococcus aureus due to the degradation of MOF74 coating, leading to alkaline microenvironment (about pH 8.0) and degradation products (2,5-dihydroxyterephthalic acid and Zn2+). The coating also showed good early anti-inflammatory property to native Ti substrates. In vivo results further verified that AT-Mg/Zn3 implants had high antibacterial and anti-inflammatory properties at early stage of implantation, and greatly improved new bone formation around implants both at non-infected and infected femur sites.

Titania nanotubes promote osteogenesis via mediating crosstalk between macrophages and MSCs under oxidative stress.

Before mesenchymal stem cells (MSCs) adhere to the surface of an implant and differentiate into osteoblasts, monocytes (especially macrophages) arrive at the bone injury site and interact with the implant and subsequent MSCs. In our previous study, large titania nanotubes (TNT110) had been verified to endow superior oxidation resistance to osteoblasts. The early regulation between macrophages and MSCs by surface nanotubes under oxidative stress (OS) was evaluated further in this study. Cellular and molecular results show that TNT110 greatly increased early inflammation of macrophages by activating integrin/FAK-mediated MAPK and NFκB signals and simultaneously promoted their gene expression of SDF1, IL-8, and CCL2 (chemokines) compared with 30 nm nanotubes and titanium substrates. Co-culture results show that more MSCs were recruited by those chemokines and may promote osteogenic self-differentiation, reducing early inflammation of macrophages by accelerating their M1-to-M2 transition in the TNT110 group. All findings reveal that the early cellular behavior of macrophages and MSCs was effectively regulated by TNT110, indicating large nanotubes would be more suitable to prevent oxidative damages.

Functionalization of titanium substrate with multifunctional peptide OGP-NAC for the regulation of osteoimmunology.

The immune response to an orthopedic implant is closely related to the nearby bone metabolism balance. To modify titanium (Ti) substrates and accordingly regulate the balance between osteoclast activation and osteoblast differentiation, a multifunctional peptide OGP-NAC was synthesized via conjugating an osteogenic growth peptide (OGP) with N-acetylcysteine (NAC). Then, the synthesized peptide was employed to functionalize Ti substrates and the response of both osteoblasts and osteoclasts was investigated in vitro. The results showed that OGP-NAC was successfully prepared and immobilized onto Ti substrate surfaces. Thereafter, studies on introducing RAW 264.7 cells (one kind of monocyte macrophage responsible for immune responses) to osteoclasts demonstrated that the peptide modified Ti surface could inhibit RAW 264.7 cells from secreting important inflammatory cytokines (TNF-α and IL-1ß), and suppress the activation of MAPK, NF-κB and NFAT c1, which are important transcription factors for osteoclastogenesis. Meanwhile, the modified surface promoted osteoblast spreading, proliferation and differentiation. The study offers a feasible strategy to mediate the balance between osteoclast activation and osteoblast differentiation, having great potential for improving osseointegration of an orthopedic implant.

Regulation of osteoblast differentiation by osteocytes cultured on sclerostin antibody conjugated TiO2 nanotube array.

Sclerostin is a negative regulator of the Wnt signaling pathway for osteoblast differentiation. In this study, osteoblasts were co-cultured with osteocytes (MLO-Y4 cells) on the surface of sclerostin antibody-conjugated TiO2 nanotube arrays (TNTs-scl). Field emission scanning electron microscopy (SEM), contact angle measurement and confocal laser scanning microscope (CLSM) were employed to characterize the conjugation of sclerostin antibody onto the surface of TiO2 nanotube arrays. The cellular viability and morphology results displayed TNTs-scl (TNT30-scl and TNT70-scl) were beneficial to the growth of MLO-Y4 cells. There was no apparent change in sclerostin gene expression between MLO-Y4 cells grown on TNTs and TNTs-scl. However, TNTs-scl significantly reduced the amount of sclerostin in the medium. In comparison with the control groups, osteoblasts displayed higher differentiation capability when co-cultured with MLO-Y4 cells on the surface TNTs-scl, which was indicated by the ALP activity, mineralization capability as well as expression levels of key proteins in Wnt signaling. This study provides a simple strategy to engineer titanium surface for bone fracture recovery, especially in osteoporotic conditions.

The nanoparticle-facilitated autophagy inhibition of cancer stem cells for improved chemotherapeutic effects on glioblastomas.

Due to the rapid growth of a tumor, the tumor cell metabolism becomes more active, resulting in the overexpression of albumin-binding proteins for transporting albumin as an energy and amino source. In that case, making use of nutrient transporters for targeted drug delivery to the brain becomes attractive. Herein, we synthesized albumin nanoparticles by a desolvation process, modified them with folic acid to enhance blood-brain-barrier (BBB) penetration and cellular uptake, and then loaded them with the antitumor drug paclitaxel (PTX) and autophagy inhibitor chloroquine (CQ) for combination therapy. The albumin nanoparticles could cross the BBB and target glioma cells effectively, and the combination therapy of PTX and CQ induced more cell apoptosis than PTX treatment alone in vitro. The results of the role of autophagy in the sensitivity of chemotherapeutic PTX to glioma cells showed that the stemness-associating genes (SOX2, POU5F1 and NANOG) of live glioma cells increased in the presence of PTX, while they dropped sharply with the combination including CQ. More importantly, it was found that the combined delivery system FA-BSA-NPPTX/CQ exhibited the most effective cell apoptosis. Our findings demonstrated that drug-loaded albumin nanoparticles could facilitate a combination of chemotherapy and autophagy inhibition for effective glioma therapy.

Copper-nanoparticle-embedded hydrogel for killing bacteria and promoting wound healing with photothermal therapy.

Bacterial infections at wound tissue sites usually delay the wound healing process and even result in severe life-threatening complications. Therefore, it is imperative to develop an efficient strategy to simultaneously enhance the antibacterial abilities and improve the wound healing process. Here, we report a composite hydrogel composed of methacrylate-modified gelatin (Gel-MA) and N,N-bis(acryloyl)cystamine (BACA)-chelated Cu nanoparticles (Cu NPs) via radical polymerization with a photoinitiator. The Cu NPs could effectively convert NIR laser irradiation (808 nm) energy into localized heat due to the localized surface plasmon resonance (LSPR) effect for effecting photothermal therapy. In vitro antimicrobial experiments revealed that the hybrid hydrogel exhibited predominant antibacterial efficacy against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, while Cu-NP-embedded hydrogel + laser group exhibited superior antibacterial capacity. The excellent antibacterial properties can be attributed to the synergistic effect of photothermal performance and rapid release of copper ions (Cu2+) because of the laser irradiation of Cu NPs. Moreover, the released Cu2+ could stimulate NIH-3T3 fibroblast proliferation without any inflammatory responses. Moreover, chronic wound healing process of S. aureus-infected model was significantly accelerated with prominent antibacterial ability, reduced inflammatory response, and promoted angiogenesis ability in vivo. In summary, Cu-NP-embedded hydrogels are a promising candidate for skin tissue regeneration and potentially valuable for clinical applications.

Peptide LL-37 coating on micro-structured titanium implants to facilitate bone formation in vivo via mesenchymal stem cell recruitment.

Titanium (Ti) and Ti-alloys were widely used in clinic orthopedics, however, the insufficient bone formation surrounding Ti-based implants still limited their biological performances. Surface modification of Ti substrates is essential to improve their interactions with bone-forming cells and bone tissue. In this study, we modified Ti substrates by coating peptide LL-37 onto micro-structured Ti substrates and aimed to (i) induce mesenchymal stem cells (MSCs) migration both in vitro and in vivo, (ii) facilitate osteogenic differentiation of MSCs and new bone formation. The surface micro-structured Ti substrates with hydroxyapatite deposition were fabricated by a two-step method including micro-arc oxidation (MAO) and hydrothermal treatment. LL-37 was loaded on micro-structured Ti substrates with the assistance of polydopamine coating. We confirmed that surface-modified Ti substrates benefited viability, adhesion, migration and osteogenic differentiation of MSCs in vitro. In a femur-defect rat model, the surface-modified Ti implants effectively induced CD29+/CD90+ positive cells migration in one week after implantation. According to the results of H&E, Masson's trichrome staining and immunohistochemical staining of OCN, OPN and collagen I, the targeted Ti implants exhibited significant new bone formation after implantation for 4 weeks. These results indicate that the surface modification of Ti samples facilitated bone formation through MSCs recruitment. STATEMENT OF SIGNIFICANCE: The inherent surface bioinertness of titanium (Ti) and Ti-alloys still limits their biological performances in clinical applications. Recently, the strategy of mesenchymal stem cells (MSCs) recruitment has been proposed to improve the osteointegration of bone implants. Herein, we reports the surface modification of Ti implants from the point of MSCs recruitment. Peptide LL-37 was coated on micro-structured Ti substrates to (i) recruit MSCs, (ii) regulate bio-physiological performance of MSCs, and (iii) facilitate bone formation in vivo. Our results improve the understanding of the interaction between Ti implants and MSCs, and provide a promising strategy of MSCs recruitment in the design of bone repair related biomaterials.

Deferoxamine loaded titania nanotubes substrates regulate osteogenic and angiogenic differentiation of MSCs via activation of HIF-1α signaling.

To develop biomaterials for inducing osteogenic and angiogenic differentiation of mesenchymal stem cells (MSCs) is crucial for bone repair. In this study, we employed titania nanotubes (TNT) as drug nanoreservoirs to load deferoxamine (DFO), and then deposited chitosan (Chi) and gelatin (Gel) multilayer as coverage structure via layer-by-layer (LBL) assembly technique, resulting in TNT-DFO-LBL substrates. Scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements were employed to characterize the physical and chemical properties of the substrates. The results proved the successful fabrication of multilayer coating on TNT array. DFO released from the TNT arrays in a sustained manner. The drug-device combination titanium (Ti) substrates positively improved the adhesion, proliferation, osteogenic/angiogenic differentiation of MSCs and mediated the growth behavior of human umbilical vein endothelial cells (HUVECs). Moreover, the TNT-DFO-LBL substrates up-regulated osteogenic and angiogenic differentiation related genes expression of MSCs by activating HIF-1α signaling pathway. The approach presents here has a potential impact on the development of high quality Ti-based orthopedic implants.

Multilayered coating of titanium implants promotes coupled osteogenesis and angiogenesis in vitro and in vivo.

We used surface-modified titanium (Ti) substrates with a multilayered structure composed of chitosan-catechol (Chi-C), gelatin (Gel) and hydroxyapatite (HA) nanofibers, which were previously shown to improve osteogenesis, as a platform to investigate the interaction of osteogenesis and angiogenesis during bone healing. Combined techniques of Transwell co-culture, wound healing assay, enzyme linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemical staining were used to evaluate adhesion, morphology and migration of adipose-derived mesenchymal stem cells (Ad-MSCs) and human umbilical vein endothelial cells (HUVECs) grown on different Ti substrates. We investigated the effect of substrates on the osteogenic differentiation of Ad-MSCs and reciprocal paracrine effects of Ad-MSCs on HUVECs or vice versa. The multilayered Ti substrates directly regulated the cellular functions of Ad-MSCs and angiogenic HUVECs and mediated communication between them by enhancing paracrine effects via cell-matrix interactions in vitro. The in vivo results showed that the change of microenvironment induced by surface-modified Ti implants promoted the adhesion, recruitment and proliferation of MSCs and facilitated coupled osteogenesis and angiogenesis in bone healing. The study proved that multilayer-film-coated Ti substrates positively mediated cellular biological function in vitro and improved bone healing in vivo. STATEMENT OF SIGNIFICANCE: Recent studies have revealed that osteogenesis and angiogenesis are coupled, and that communication between osteoblasts and endothelial cells is essential for bone healing and remodeling processes; however, these conclusions only result from in vitro studies or in vivo studies using transgenic murine models. Relatively little is known about the communication between osteoblasts and endothelial cells in peri-implants during bone healing processes. Our results revealed the cellular/molecular mechanism of how multilayered Ti substrates mediate reciprocal paracrine effects between adipose-derived mesenchymal stem cells and human umbilical vein endothelial cells; moreover, the interactions between the cell-matrix and peri-implant was proven in vivo with enhanced bone healing. This study contributes to our understanding of the fundamental mechanisms of angiogenesis and osteogenesis that affect peri-implantation, and thus, provides new insights into the design of future high-quality orthopedic implants.

Polydopamine-Assisted Hydroxyapatite and Lactoferrin Multilayer on Titanium for Regulating Bone Balance and Enhancing Antibacterial Property.

Maintaining the balance between bone formation and bone resorption as well as reducing bacterial infection are two major challenges for titanium (Ti) when it is used as the implant in orthopedic surgery. Because of its excellent properties, including anti-inflammatory, antimicrobial, promoting osteoblasts, and inhibiting osteoclasts growth, lactoferrin (LF) is a potential bioactive molecule for surface modification of Ti implants. Inspired by the highly hierarchical structure of natural bone tissue, in this work, a polydopamine-assisted hydroxyapatite and lactoferrin multilayer structure (PDA-HA-LF) was prepared onto the Ti substrate surface by a biomimetic approach and spin-assisted layer-by-layer (LBL) assembly technique. Meanwhile, its capabilities on regulation of bone balance and antibacterial properties were measured with cell experiments and antimicrobial activity in vitro. Furthermore, the regulation theory was investigated by qPCR and theoretical simulation. The results showed that the biological properties of LF are highly correlated with its concentration. High concentration of LF was toxic to osteoblasts but has obvious effects on inhibition of osteoclasts and bacteria (S. aureus and E. coli). However, through polydopamine-assisted hydroxyapatite deposition, cytotoxicity of LF could be improved. The modified Ti implant could greatly improve the proliferation and differentiation of osteoblasts. Meanwhile, the activity of osteoclasts was somewhat inhibited, which indicated that the modified Ti implant could efficiently regulate the balance between bone resorption and bone formation, as well as have a certain antibacterial effect.