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 (p < 0.05) 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 (p < 0.01) 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.

To comparatively investigate the cytotoxicities of nanomaterials in circulation, in this study, three different types of nanoparticles (NPs; mesoporous SiO₂, Fe₃O₄, and TiO₂) with diameters of around 100 nm were synthesized. The morphologies, crystalline phases, and zeta potentials of those NPs were characterized by scanning electron microscopy, X-ray diffraction and zeta potential measurement, respectively. Then, we investigated the influences of different NPs on the biological functions of endothelial cells, in particular of the organelle of cells. The results indicated that different types of NPs had cytotoxic effects in a dose- and time-dependent manner, and there was no significant difference in cytotoxicity between SiO₂ and Fe₃O₄ at concentrations <0.20 mg/mL. The shape and surface charges of NPs greatly affected cellular internalization. We found that cytoskeleton and integrity of cells were destroyed by different NPs. Additionally, the production of reactive oxygen species damaged the mitochondria of cells, in turn leading to cells apoptosis and death. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 1726–1736, 2014.

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. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3598–3608, 2014.

Mesoporous silica nanoparticles (MSNs) present themselves as one of the most promising nano-carriers for drug delivery. To reduce their immunotoxicities, in this study, natural proteins of gelatin (Gel), bovine serum albumin (BSA), and lysozyme (Lys) were employed as end-caps of MSNs by using succinic anhydride as an intermediate linker, thus leading to fabrication of MSNs/protein nanocomposites, respectively. Furthermore, combined techniques of SEM, TEM, FTIR, and zeta potential instruments were utilized to monitor the construction processes of MSNs/protein nanocomposites, respectively. Finally, the immunotoxicities of those nanocomposites to macrophage cells (RAW264.7 cells) were investigated in detail, i.e., cell morphology, cell viability, nitric oxide (NO) production, reactive oxygen species (ROS), and acid phosphatase activity (ACP) as well as inflammation cytokine expressions (tumor necrosis factor-α and interleukin-1β). All results suggest that macrophages were activated after uptaking nanoparticles of SiO₂ and MSNs, which subsequently induced severe inflammation responses in vitro. In contrast, the inflammation responses of MSNs nanocomposites were reduced dramatically after end-capping with those natural proteins. Overall, this study accumulates knowledge for the development of MSNs-based drug delivery systems with reduced immunotoxicity. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3781–3794, 2014.

To mimic the extracellular matrix of natural bone, apatite/gelatin composite was deposited onto nanostructured titanium substrates via a coprecipitation method, which was pretreated by potassium hydroxide and heat treatment to generate an anticorrosive nanostructured layer. The successful formation of the apatite/gelatin nanocomposite onto titanium surfaces was revealed by Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, atomic force microscopy (AFM), and thin film X-ray diffraction (TF-XRD) measurements, respectively. The immunofluorescence staining of vinculin revealed that the apatite/gelatin nanocomposite deposited titanium substrate was favorable for cell adhesion. More importantly, bone marrow stromal cells cultured onto the apatite/gelatin nanocomposite deposited titanium substrates displayed significantly higher (p < 0.05 or p < 0.01) proliferation and differentiation levels of alkaline phosphatase, mRNA expressions of osteocalcin (OC), osteopontin (OPN), and collagen type I (Col I), and OC content after culture for 7, 14, and 21 days, respectively, which was also revealed by the immunofluorescence analysis of OC and OPN expression. The deposition of apatite/gelatin nanocomposite improved bone density (p < 0.05) and bone-implant contact rate (p < 0.05), which was reflected by microcomputed tomography analysis and histological evaluation in vivo using a rabbit model. This work provides an approach to fabricate high-performance titanium-based implants with enhanced bone osseointegration. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 101A: 653–666, 2013.

In this study, chitosan-atorvastatin (Chi-AT) conjugate was immobilized onto the surfaces of titanium substrates to reduce inflammation responses and improve cytocompatibility. Polydopamine film was initially formed onto the titanium surfaces as the intermediate layer for the successful immobilization of Chi-AT, which was confirmed by scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle measurements, respectively. Endothelial cells grown onto Chi-AT immobilized titanium substrates displayed significantly higher (p < 0.01) cell viability and statistically lower (p < 0.01) lactate dehydrogenase production than those of native titanium substrates (control) after culture for 4 days and 7 days, respectively. Furthermore, macrophages cells cultured onto Chi-AT immobilized titanium substrates demonstrated significantly lower (p < 0.01) production levels of nitric oxide, acid phosphatase, reactive oxygen species, pro-inflammatory cytokines of tumor necrosis factor α, and interleukin-1 β than those of controls. All results indicated that the immobilization of Chi-AT conjugate onto titanium substrates was beneficial for improving their cytocompatibility and inhibiting pro-inflammatory responses. The study thus presents an alternative to fabricate bio-functionalized titanium-based implants for further clinical application. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Surface topography of a biomaterial plays an essential role in the regulation of cell adhesion, proliferation, and differentiation. To investigate the influence of hierarchically micro/nano-structured substrates on the differentiation of mesenchymal stem cells (MSCs), a series of micro-, nano-, and micro/nano-structured titanium substrates were fabricated via dual acid etching and/or anodic oxidation. The morphologies of titanium substrates were characterized by field emission scanning electron microscopy (FE-SEM) and contact angle measurement, respectively. The differentiation of MSCs adhered to different substrates was investigated in vitro. Compared to native titanium, micro/nano-structured titanium substrates improved the alkaline phosphatase (ALP) production and mineralization of MSCs. More importantly, micro/nano-structured titanium substrates demonstrated great potential to induce the differentiation of MSCs, which was revealed by the expressions of osteocalcin (OCN) and osteopontin (OPN). The study provides a potential alternative to generate hierarchically micro/nano-structured titanium-based implant for enhanced osseointegration.

This study reports in situ gene delivery from gene-functionalized poly(D,L-lactic acid) (PDLLA, M_w of around 2.0 × 10⁵ g mol⁻¹) films, which were constructed via layer-by-layer (LbL) assembly technique with low molecular weight polyethylenimine-β-cyclodextrin (PEI-CD) conjugate and plasmid DNA (pDNA). PEI-CD was characterized by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR), respectively. The buildup of multilayered PEI-CD/pDNA pairs onto PDLLA films was monitored with contact angle measurements and UV–Vis spectrometer, respectively. A sustained release of pDNA from multilayered films was observed for 28 h. The mechanism of in situ gene delivery on PDLLA film was investigated in this study as well. Spherical PEI-CD/pDNA complexes were formed and released following the deconstruction of multilayered films, which was confirmed by transmission electron microscopy (TEM) and gel electrophoresis, respectively. Surface mediated in situ gene transfection was achieved when culturing hepatoma G2 (HepG2) and human embryonic kidney 293 (HEK293) onto PEI-CD/pDNA multilayered films. Furthermore, PEI-CD improved the gene transfection efficiency when compared with that of PEI. Such gene-functionalized biomaterial reported here has potential application in tissue engineering and implant technology.

In this study, we reported a smart temperature-sensitive drug release system based on titanium nanotubes. Titanium nanotubes were fabricated with an electrochemical approach. The 3-trimethoxysilyl propylmethacrylate (MPS) was initially coupling to the surfaces of titanium nanotubes, and then polymerizing with N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) to form a hydrogel covering layer. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were employed to characterize the titanium nanotubes drug delivery systems. SEM images revealed that titanium nanotubes had relatively uniform distribution in diameters (52 ± 9 nm) and around 410 nm in length. FTIR spectra suggested that MPS was immobilized and in turn triggering the formation of PNIPAAm/PAAm network onto the surfaces of titanium nanotubes. Drug release profiles indicated that a temperature-responsive controlled drug release system based on titanium nanotubes was achieved. This study provides alternative for developing novel implantable controlled drug delivery systems from titanium nanotubes.

In an effort to surface engineering of poly(D,L-lactic acid) (PDLLA), layer-by-layer (LbL) self-assembly of chitosan (Chi) and deoxyribonucleic acid (DNA) were employed to build up multilayered films. The formation of multilayers was monitored by using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), water contact-angle measurement, and atomic force microscopy (AFM), respectively. A full coverage of Chi/DNA pair film was formed only after the fifth sequential deposition (PEI/(DNA/Chi)₂), which was revealed by contact-angle measurement. Surface chemistry and topography of multilayered films were directly related to the corresponding outmost layer component. Discernable island-like structures on PEI/(DNA/Chi)₅/DNA layered PDLLA film was observed. Lysozyme-mediated multilayer degradation and DNA-releasing measurement suggested that DNA was gradually released into the incubation medium over a period of up to 32 h. The approach presented here may be exploited to develop controlled administration of functional DNA constructs from the surfaces of biomedical materials and devices in situ. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008