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Diabetes is a universal disease in the world. In the wounds of diabetic individuals, chronic inflammation and an inefficient fibrogenic process hinder the formation and deposition of the ECM, which delays the process of wound healing. To reconstruct the ECM of a diabetic patient's wound, in this work, we designed a pH-responsive "Double H-bonds" (hydrogen bond and hydrazone bond) hyaluronic acid-collagen hydrogel. This hydrogel can be self-gelled quickly in neutral and alkaline environments. But the weakly acidic inflammatory environment of diabetic wounds may accelerate the degradation of the hydrogel and the release of metformin. The

The blood-brain barrier (BBB) is one of the major limitations of glioblastoma therapy in the clinic. Nanodrugs have shown great potential for glioblastoma therapy. Herein, we purposefully developed a multicomponent self-assembly nanocomplex with very high drug loading content for curing orthotopic glioblastoma with synergistic chemo-photothermal therapy. The nanocomplex consisted of self-assembled pH-responsive nanodrugs derived from amino acid-conjugated camptothecin (CPT) and canine dyes (IR783) coated with peptide Angiopep-2-conjugated copolymer of Ang-PEG-g-PLL. Specifically, the carrier-free nanocomplex exhibited a high drug loading content (up to 62%), good biocompatibility, and effective glioma accumulation ability. Moreover, the nanocomplex displayed good stability and pH-responsive behavior ex vivo. Both in vitro and in vivo results revealed that the nanocomplex could effectively cross the BBB and target glioma cells. Furthermore, the combination of chemotherapy and photothermal therapy of the nanocomplex achieved a better therapeutic effect, longer survival time, and minimized toxic side effects in orthotopic glioblastoma tumor-bearing nude mice. Overall, we modified the chemotherapeutic drug CPT so that it could self-assemble with other molecules into nanoparticles, which providing an alternative for the preparation of the carrier-free nanodrugs. The results highlighted the potential of self-assembly nanodrugs as a novel platform for effective glioblastoma therapy.

Due to their simple crystal structures, one-dimensional hydroxyapatite (HA) nanostructures are easily to be applied to understand the fundamental concepts about the influences of HA dimensionality on physical, chemical, and biological properties. So, in this work, three typical HA one-dimensional nanostructures, HA nanotubes, HA nanowires, and HA nanospheres, were prepared, whose theoretical structures were built also. in vitro cytocompatibility test proved that, contrasting with TCPS, HA one-dimensional nanostructures had certain degree of cytotoxicity because HA nanostructures increase the generation of intracellular reactive oxygen species (ROS) and intracellular calcium. Theoretical simulation indicated that HA nanosphere has higher intracellular ROS generation and lower ROS storage amount than HA nanowire and HA nanotube, which were the possible reasons for its stronger cytotoxicity. Among these typical one-dimensional nanostructures, owing to higher drug storage amount and sustained delivery ability, HA nanotube was more potential application in orthopedics. The tubular structure of HA nanotubes could be used as reservoirs for small molecule drugs or growth factors. The cytocompatibility of HA nanostructures can be improved obviously when they were produced into two-dimensional structures. The prepared multilayer structure can simulate lamellar structures of Harvard system and enhance the cytocompatibility of Ti substrate. Therefore, the method used in this work is a prospective method to improve the inherently bio-inert of Ti when used in hard tissue repairing.

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.

Selective laser melting (SLM) is one of the three-dimensional (3D) printing techniques that manufacturing versatile porous scaffolds with precise architectures for potential orthopedic application. To understand how the pore sizes of porous Ti6Al4V scaffolds affect their biological performances, we designed and fabricated porous Ti6Al4V implants with straightforward pore dimensions (500, 700, and 900 µm) via SLM, termed as p500, p700, and p900 respectively. The morphological characteristics of Ti6Al4V scaffolds were assessed showing that the actual pore sizes of these scaffolds were 401 ± 26 µm, 607 ± 24 µm, 801 ± 33 µm, respectively. The mechanical properties of Ti6Al4V scaffolds were also evaluated showing that they were comparable to that of bone tissues. Meanwhile, the effect of pore size on biological responses was systematically investigated in vitro and in vivo. It was verified that 3D printing technique was able to fabricate porous Ti6Al4V implants with proper mechanical properties analogous to human bone. The in vitro results revealed that scaffolds with appropriate pore dimension were conducive to cell adhesion, proliferation and early differentiation. Furthermore, the porous Ti6Al4V scaffolds were implanted into the rabbit femur to investigate bone regeneration performance, the in vivo experiment showed the p700 sample was in favor of bone ingrowth into implant pores and bone-implant fixation stability. Taken together, the biological performance of p700 group with actual pore size of about 600 µm was superior to other two groups. The obtained findings provide basis to individually design and fabricate suitable porous Ti6Al4V with specific geometries for orthopedic application.

Type I co-activator-associated arginine methyltransferase 1 (CARM1) and type II protein arginine methyltransferase 5 (PRMT5) are highly expressed in multiple cancers including liver cancer and their overexpression contributes to poor prognosis, thus making them promising therapeutic targets. Here, we evaluated anti-tumor activity of ribavirin in hepatocellular carcinoma (HCC). We found that ribavirin significantly inhibited the proliferation of HCC cells in a time- and dose-dependent manner. Furthermore, ribavirin suppressed the growth of subcutaneous and orthotopic xenograft of HCC in mice, decreased vascular endothelial growth factor (VEGF) and peritoneal permeability to reduce ascites production, and prolonged the survival of mice in HCC ascites tumor models. Mechanistically, ribavirin potently down-regulated global protein expression of CARM1 and PRMT5, and concurrently decreased accumulation of H3R17me2a and H3R8me2s/H4R3me2s. However, ribavirin did not affect the activity and mRNA levels of both CARM1 and PRMT5 in vivo and in vitro HCC cells. In addition, our ChIP results shown that ribavirin inhibited CARM1 which in turn decreased the H3R17me2a, binds to the eukaryotic translation initiation factor 4E (eIF4E) and VEGF promoter region, and reduced the relative mRNA expression level of eIF4E and VEGF in HCC cells. Our findings suggested a potential therapeutic strategy for patients with HCC through inhibition of the abnormal activation/expression of both CARM1 and PRMT5.

To improve the biological performance of titanium substrates, a bioactive multilayered structure of chitosan/gelatin pair, containing zinc ions, was constructed via a layer-by-layer self-assembly technique. The successful preparation of zinc ions incorporated multilayer films was demonstrated by scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle measurements, respectively. The biological behaviors of osteoblasts adhered to modified Ti substrates were investigated in vitro via cytoskeleton observation, cell viability measurement, and alkaline phosphatase activity assay. The cytocompatibility evaluation verified that the present system was capable of promoting the growth of osteoblasts. In addition, Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria were used to evaluate antibacterial property of modified Ti substrates. Bacterial adhesion and viability assay confirmed that Zn-loaded multilayer films were able to inhibit the adhesion and growth of bacteria. The approach presented here affords an alternative to reduce bacterial infection and promote osteoblast growth for titanium-based implants.

After bone tumor resection, the large bony deficits are commonly reconstructed with Ti-based metallic endoprosthesis, which provide immediate stable fixation and allow early ambulation and weight bearing. However, when used in osteosarcoma resection, Ti implant-relative infection and tumor recurrence were recognized as the two critical factors for implantation failure. Hence, in this work, a novel zinc oxide nanoparticle decorating with naringin was prepared and immobilized onto Ti substrate. The drugs delivery profiles proved that in the bacterial infection and Warburg effect of osteosarcoma-induced acidic condition, naringin and Zn2+ can be released easily from the functional Ti substrate. The anti-osteosarcoma and antibacterial assay showed the delivered naringin and Zn2+ can induce a remarkable increase of oxidative stress in bacteria (Escherichia coli and Staphylococcus aureus) and osteosarcoma (Saos-2 cells) by producing reactive oxygen species (ROS). Accumulation of ROS results in damage of bacterial biofilm and bacterial membrane, leading to the leakage of bacterial RNA and DNA. Meanwhile, the increase of ROS induces osteosarcoma cell apoptosis by activating ROS/extracellular signal-regulated kinase signaling pathway. Furthermore, in vitro cellular experiments, including cell viability, alkaline phosphatase activity, collagen secretion, extracellular matrix mineralization level, indicated that the functional Ti substrate exhibited great potential for osteoblasts proliferation and differentiation. Hence, this study provides a simple and promising strategy of developing multifunctional Ti-based implants for the reconstruction of large bony after osteosarcoma resection.

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-1β), 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.

Sclerostin is a negative regulator of the Wnt signaling pathway for osteoblast differentiation. In this study, osteoblasts were co-cultured with osteocytes (MLO-Y

The inherent bioinertness and potential bacterial infection risk are the two leading causes for Ti implant failure. To improve osseointegration and antibiosis, in this work, a novel antimicrobial osteogenic growth peptide was first synthesized by conjugating osteogenic growth peptide (OGP) and ciprofloxacin (CIP). Then, the synthetic antimicrobial peptide was immobilized onto Ti implant surface for chemoselective binding via the amide reaction. Thereafter, the capabilities of modified Ti implant on osseointegration and antibiosis were measured with cell experiments and antimicrobial activity in vitro. The results showed that antimicrobial osteogenic growth peptide (OGP-CIP) was successfully prepared and grafted onto Ti implant surface. Moreover, the antimicrobial peptide-modified Ti implants could promote osteoblasts spreading and osteodifferentiation compared with unmodified Ti substrates. Meanwhile, in vitro bacteria studies (Staphylococcus aureus and Escherichia coli) proved that the antibacterial property of antimicrobial peptide functionalized Ti implant was improved obviously. The method used in this work is a feasible and promising strategy to win the race against invading bacteria and accelerate bone integration in orthopedic implantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3021-3033, 2018.

To investigate the influence of magnetized hydroxyapatite on the growth and differentiation of osteoblasts, hydroxyapatite (HA) and magnetized hydroxyapatite (mHA) were synthesized and characterized. The cell viability, differentiation, and morphologies of osteoblasts were investigated in vitro, respectively. The results showed that compared to HA, cells cultured with mHA had better cell viability, and both HA and mHA were beneficial to the early differentiation of osteoblasts. Furthermore, the interaction mechanism between mHA and osteoblasts was elucidated using a molecular dynamics simulation. The simulation results indicated that when cultured with osteoblasts, HA adsorbed bovine serum protein onto its surface from the medium immediately, which was beneficial to the adhesion and proliferation of osteoblasts. The main driving force for the adsorption of bovine serum was the electronic properties of HA crystal faces. The (211) crystal face of HA had the highest electron density among its all crystal faces, thus mainly contributing to the protein adsorption of HA. Nevertheless, the (211) crystal face of mHA still had a relatively higher electron density than that of HA, thus possessing better protein adsorption than that of HA, and in turn promoting the biological functions of osteoblasts.

To improve the corrosion resistance and biological performance of commercially pure titanium (cp-Ti) substrates, potassium hydroxide was employed to modify the surfaces of titanium substrates, followed by biomimetic deposition of apatite on the substrates in a simulated body fluid. The morphologies of native and treated titanium substrates were characterized by field emission scanning electron microscopy (FE-SEM). Treatment with potassium hydroxide led to the formation of intermediate layers of potassium titanate on the surfaces of titanium substrates, while apatite was subsequently deposited onto the intermediate layer. The formation of potassium titanate and apatite was confirmed by thin-film X-ray diffraction and FE-SEM equipped with energy dispersive spectroscopy, respectively. Electrochemical impedance spectroscopy showed that the formed potassium titanate layer improved the corrosion-resistance properties of titanium substrates. The influence of modified titanium substrates on the biological behavior of mesenchymal stem cells (MSCs), including osteogenic differentiation, was investigated in vitro. Compared with cp-Ti substrates, MSCs cultured onto alkali- and heat-treated titanium substrates and apatite-deposited titanium substrates displayed significantly higher (P0.05 or P0.01) proliferation and differentiation levels of alkaline phosphatase and osteocalcin in 7 and 14day cultures, respectively. More importantly, our results suggest that the modified titanium substrates have great potential for inducing MSCs to differentiate into osteoblasts. The approach presented here may be exploited to fabricate titanium-based implants.

To enhance long-term survival of titanium implants in patients with osteoporosis, chitosan/gelatin multilayers containing bone morphogenetic protein 2(BMP2) and an antiosteoporotic agent of calcitonin (CT) are deposited on the Ti6Al4V (TC4) implants through layer-by-layer (LBL) electrostatic assembly technique. Here, the obtained titanium alloy implant (TC4/LBL/CT/BMP2) can regulate the release of loaded calcitonin and BMP2 agents in a sustaining manner to accelerate the bone formation and simultaneously inhibit bone resorption. In vitro results show that the bone-related cells on TC4/LBL/CT/BMP2 present the lowest production level of tartrate resistant acid phosphatase (TRAP) but the highest (p0.05) level of alkaline phosphatase (ALP) activity, osteocalcin production, mineralization capacity and osteoblast-related gene expression among all groups after treatment for 7 or 21 days, respectively. Besides, in vivo studies of micro-CT analysis, routine histological and immunohistochemical analysis also collectively demonstrate that the TC4/LBL/CT/BMP2 implant can dramatically promote the formation and remodeling of new bone in osteoporotic rabbits after implantation for 30 days and 90 days, respectively. In vivo push-out testing further confirms that the TC4/LBL/CT/BMP2 implant has the highest (p0.01) interfacial shear strength and favorable bone-implant osseointegration. Overall, this study establishes a simple and profound methodology to fabricate a biofunctional TC4 implant for the treatment of local osteoporotic fractures in vivo. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1437-1451, 2016.

Osseointegration is essential for the long-term survival of orthopedic implants. Inspired by the hierarchical structure of natural bone, we fabricated a hierarchical structure with osteoinduction potential on titanium alloy (Ti6Al7Nb) substrates via a spin-assisted layer-by-layer assembly technique, with hydroxyapatite nanofibers as the intercalated materials and gelatin and chitosan as the polycation and polyanion, respectively. The as-synthesized hydroxyapatite nanofibers were characterized using scanning electron microscopy (SEM), transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. The change of water contact angle corresponding to different layers indicated the formation of a multilayered structure, since different components have their inherent wettability natures. The multilayered lamellar structure was revealed by the cross-sectional view of SEM, suggesting that the film was successfully deposited onto Ti6Al7Nb substrates. Osteoblasts cultured on the hierarchical structure deposited Ti alloy substrates displayed significantly higher cell viability (P < 0.01) and better adhesion, a higher production level of alkaline phosphatase, mineralization, genes expressions of osteocalcin and osteopontin (P < 0.01 or P < 0.05) compared to those of native Ti6Al7Nb substrates after culture for 4, 7 or 14 days. These results indicated that the lamellar structure was beneficial for the biological functions of osteoblasts, establishing the basis for osseointegration of a titanium alloy implant.

To investigate the influence of the titanium nanotube (TiNT) diameters on the adsorption of collagen type I (Col-I), TiNTs with different diameters were prepared with anodization. The adsorption amount of Col-I on the different TiNTs substrates was evaluated by spectrophotometric measurement and immunofluorescence staining, respectively. The results showed that the diameters of TiNTs played a key role in the adsorption process of Col-I. TiNTs with diameters around 100 nm displayed a higher adsorption amount and faster adsorption speed than that of 30 nm TiNTs. Furthermore, more collagen molecules were aggregated in the tubes of 100 nm TiNTs. Molecular dynamics simulation was performed to elucidate the adsorption mechanism. The simulation results confirmed that physical adsorption was the main driving force, including van der Waals force and hydrogen bond between Col-I molecules and TiNTs. The calculated interaction energies indicated that the TiNTs with bigger dimensions had higher interaction energies, thus leading to the higher collagen adsorption.

In this study, we report the influence of titania nanotubes (TiNTs) dimensions on the adsorption of collagen (COL) and fibronectin (FN), and its subsequent effect on the growth of osteoblasts. TiNTs with different diameters of around 30 and 100 nm were prepared with anodization. The adsorption profiles of proteins and cell behaviors were evaluated using spectrophotometric measurement, immunofluorescence staining, cell viability, and cytoskeleton morphology, respectively. The results showed that although the growth of osteoblasts was highly sensitive to the dimensions TiNTs, the preadsorbed COL and FN could reduce the difference. Molecular dynamics (MD) simulation results confirmed that the main driving force for protein adsorption was the physical adsorption. The TiNTs with bigger dimensions had higher interaction energies, and thus leading to higher proteins (COL and FN) adsorption and obvious influences on cell behaviors. MD simulation revealed that the orientation and conformation of proteins adsorbed onto surfaces of TiNTs was critical for cell integrins to recognize specific sites. When FN molecules adsorbed onto the surfaces of TiNTs, their RGD (Arg-Gly-Asp) sites were easily exposed to outside and more likely to bond with the fibronectin receptors, in turn regulating the cellular behaviors.

Bacteria-related inflammation is a common postoperative complication in orthopedic implantation. In this study, cecropin B (CecB), a cationic peptide, was immobilized onto the surfaces of titanium substrates to improve their cytocompatibility and reduce inflammation responses. Polydopamine film was coated onto the surfaces of titanium substrates as an intermediate layer for the further immobilization of the CecB, which was confirmed by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and water contact angle measurement, respectively. Osteoblasts grown onto the CecB-immobilized titanium substrates displayed significantly higher (p

The extracellular microenvironment plays a key role in the regulation of cellular behavior. To mimic the natural extracellular microenvironment, TiO(2) nanotube (TNT) arrays as drug nanoreservoirs for loading of bone morphogenetic protein 2 (BMP2) were constructed on titanium substrates and then covered with multilayered coatings of gelatin/chitosan (Gel/Chi) for controlled drug release. The multilayered coatings were constructed via a spin-assisted layer-by-layer assembly technique. The successful fabrication of this system was monitored by field emission scanning electron microscopy, atomic force microscopy, X-ray photoelectron spectroscopy and contact angle measurements. Multilayered coating with Gel/Chi retained the drug bioactivity and release properties, which were revealed by superoxide dismutase activity measurement. In addition, cytoskeleton observation and wound healing assay confirmed that BMP2-loaded and multilayer-coated TNT arrays were able to stimulate motogenic responses of mesenchymal stem cells (MSCs). More importantly, the system demonstrated that it was capable of promoting the osteoblastic differentiation of MSCs. This study may have potential impact on the development of bone implants for enhanced bone osseointegration.