A novel biocomposite scaffold with antibacterial potential and the ability to promote bone repair.

Clinical treatment of bone defects caused by trauma, tumor resection and other bone diseases, especially bone defects that can lead to infection, remains a major challenge. Currently, autologous bone implantation is the gold standard for treatment of bone defects, but it is limited by secondary trauma and insufficient autologous material. Moreover, postoperative infection is an important factor affecting bone healing.AcN-RADARADARADARADA-CONH2 (RADA) is a new type of self-assembling peptide(SAP) composed of Arg,Ala,Asp and other amino acids was designed and prepared. The "RADA" self-assembling peptide hydrogels has excellent biological activity and it's completely biodegradable and non-toxic.It is also have been confirmed to promote cell proliferation, wound healing, tissue repair, and drug delivery. To promote bone regeneration and simultaneously prevent bacterial infection, we designed biocomposite scaffolds comprising RADA and calcium phosphate cement (CPC), termed RADA-CPC. The morphological features of the scaffold were characterized by scanning electron microscopy (SEM). In vitro studies demonstrated that RADA-CPC enhances osteoblast proliferation, differentiation and mineralization. In addition, the scaffold was used as a drug delivery system to treat postoperative infections by sustained release of ciprofloxacin (CIP). The RADA-CPC scaffold may have potential application prospects in orthopedics field because of its role in promoting bone repair and as a sustained-release drug carrier to prevent infections.

Additively Manufactured Continuous Cell-Size Gradient Porous Scaffolds: Pore Characteristics, Mechanical Properties and Biological Responses In Vitro.

Porous scaffolds with graded open porosity combining a morphology similar to that of bone with mechanical and biological properties are becoming an attractive candidate for bone grafts. In this work, scaffolds with a continuous cell-size gradient were studied from the aspects of pore properties, mechanical properties and bio-functional properties. Using a mathematical method named triply periodic minimal surfaces (TPMS), uniform and graded scaffolds with Gyroid and Diamond units were manufactured by selective laser melting (SLM) with Ti-6Al-4V, followed by micro-computer tomography (CT) reconstruction, mechanical testing and in vitro evaluation. It was found that gradient scaffolds were preferably replicated by SLM with continuous graded changes in surface area and pore size, but their pore size should be designed to be ≥ 450 µm to ensure good interconnectivity. Both the Gyroid and Diamond structures have superior strength compared to cancellous bones, and their elastic modulus is comparable to the bones. In comparison, Gyroid exhibits better performances than Diamond in terms of the elastic modulus, ultimate strength and ductility. In vitro cell culture experiments show that the gradients provide an ideal growth environment for osteoblast growth in which cells survive well and distribute uniformly due to biocompatibility of the Ti-6Al-4V material, interconnectivity and suitable pore size.

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.

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.

N-halamine-based multilayers on titanium substrates for antibacterial application.

Bacterial infection is one of the most severe postoperative complications leading to clinical orthopedic implants failure. To improve the antibacterial property of titanium (Ti) substrates, a bioactive coating composed of chitosan-1-(hydroxymethyl)- 5,5-dimethylhydantoin (Chi-HDH-Cl) and gelatin (Gel) was fabricated via layer-by-layer (LBL) assembly technique. The results of Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (1HNMR) and X-ray photoelectron spectroscopy (XPS) showed that Chi-HHD-Cl conjugate was successfully synthesized. Scanning electron microscopy (SEM), atomic force microscope (AFM) and water contact angle measurements were employed to monitor the morphology, roughness changes and surface wettability of Ti substrates, which proved the multilayers coating formation. Antibacterial assay against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) revealed that the Gel/Chi-HDH-Cl modified Ti substrates most efficiently inhibited the adhesion and growth of bacteria. Meanwhile, in vitro cellular tests confirmed that Gel/Chi-HDH-Cl multilayers had no obvious cytotoxicity to osteoblasts. The study thus provides a promising method to fabricate antibacterial Ti-based substrates for potential orthopedic application.

Regulation of osteogenesis by micro/nano hierarchical titanium surfaces through a Rock-Wnt5a feedback loop.

Titanium substrates with micro/nano hierarchical features could positively mediate the osteogenesis of a titanium implant; nevertheless, the underlying molecular mechanism needs to be further revealed. In this work, we fabricated a micro/nano hierarchically structured Ti (MNT) sample and attempted to evaluate its topography-mediated biological effects and potential molecular mechanisms in vitro. The results proved that MNT could not only affect cell morphology and osteogenic differentiation, but also regulate ROCK activity cell biological functions of osteoblasts involved in ROCK activation, ß-catenin accumulation, and high-Wnt5a expression in respect to topographical features. Moreover, blockade of ROCK activation resulted in significant inhibition of cell differentiation and Wnt5a expression. Furthermore, the anti-Wnt5a significantly down-regulated ROCK activity. In short, these results indicate the important role of ROCK-Wnt5a feedback loop in regulating cell differentiation by topographies.

Osteogenesis of 3D printed porous Ti6Al4V implants with different pore sizes.

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 ±â€¯26 µm, 607 ±â€¯24 µm, 801 ±â€¯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.

Osteogenesis potential of different titania nanotubes in oxidative stress microenvironment.

Oxidative stress is commonly existed in bone degenerative disease (osteoarthritis, osteoporosis etc.) and some antioxidants had great potential to enhance osteogenesis. In this study, we aim to investigate the anti-oxidative properties of various TiO2 nanotubes (TNTs) so to screen the desirable size for improved osteogenesis and reveal the underlying molecular mechanism in vitro. Comparing cellular behaviors under normal and oxidative stress conditions, an interesting conclusion was obtained. In normal microenvironment, small TNTs were beneficial for adhesion and proliferation of osteoblasts, but large TNTs greatly increased osteogenic differentiation. However, after H2O2 (300 µM) treatment (mimicking oxidative stress), only large TNTs samples demonstrated superior cellular behaviors of increased osteoblasts' adhesion, survival and differentiation when comparing with those of native titanium (control). Molecular results revealed that oxidative stress resistance of large nanotubes was closely related to the high expression of integrin α5ß1 (ITG α5ß1), which further up-regulated the production of anti-apoptotic proteins (p-FAK, p-Akt, p-FoxO3a and Bcl2) and down-regulated the expression of pro-apoptotic protein (Bax). Moreover, we found that Wnt signals (Wnt3a, Wnt5a, Lrp5, Lrp6 and ß-catenin) played an important role in promoting osteogenic differentiation of osteoblasts under oxidative condition.

Surface functionalization of titanium implants with chitosan-catechol conjugate for suppression of ROS-induced cells damage and improvement of osteogenesis.

Oxidative stress induced by reactive oxygen species (ROS) overproduction would hinder bone healing process at the interface of bone/implant, yet underlying mechanism remains to be explored. To endow titanium (Ti) substrates with antioxidant activity for enhanced bone formation, multilayered structure composing of chitosan-catechol (Chi-C), gelatin (Gel) and hydroxyapatite (HA) nanofibers was constructed on Ti substrates. Surface wettability and topography of multilayer coated Ti substrates were characterized by water contact angle measurement, scanning electron microscopy and atomic force microscopy, respectively. Chi-C containing multilayer on Ti surface effectively protected osteoblasts from ROS damage, which was revealed by high level of intracellular ROS scavenging activity and reduced oxidative damage on cellular level by regulating the expression of cell adhesion related genes (integrin αv, ß3, CDH11 and CDH2). Moreover, it regulated the production of cell adhesive and anti-apoptotic related proteins (p-MYPT1, p-FAK, p-Akt and Bcl-2) and pro-apoptotic critical executioners (Bax and cleaved caspase 3). Beside, the composite multilayer of Chi-C/Gel/HA nanofibers on Ti substrates promoted osteoblasts differentiation, which was evidenced by high expression levels of alkaline phosphatase activity, collagen secretion, ECM mineralization and osteogenesis-related genes expression in vitro. The in vivo experiments of µ-CT analysis, push out test and histochemistry staining further confirmed that Chi-C multilayered implant had great potential for improved early bone healing. Overall, the study offers an effective strategy for the exploration of high quality Ti implants for orthopedic applications.

Reduced bacteria adhesion on octenidine loaded mesoporous silica nanoparticles coating on titanium substrates.

Bacterial infection is one of the most severe postoperative complications leading to implantation failure. The early bacterial stage (4-6h) was proved to be the "decisive period" for long-term bacteria-related infection. Thus, to endow potential early antibacterial capacity for a titanium (Ti) based implant, an effective antiseptic agent of octenidine dihydrochloride (OCT) was effectively loaded on the mesoporous silica nanoparticles (MSNs)-incorporated titania coating which was fabricated by an electrophoretic-enhanced micro-arc oxidation technique. The surface characteristic of the coatings were characterized by various methods (SEM, AFM, XPS, XRD, etc.), and its corrosion resistance was also examined by the potentiodynamic polarization curves. The composite coating without OCT loading not only displayed good cytocompatibility but also exhibited certain anti-bacterial property. After loading with OCT, its antibacterial efficiency of the titanium substrates with composite coating was greatly enhanced without compromising their cytocompatibility. The study provides an approach for the fabrication of anti-bacterial Ti implant for potential orthopedic application.

Alendronate-loaded hydroxyapatite-TiO2 nanotubes for improved bone formation in osteoporotic rabbits.

Early mechanical fixation between an implant and native bone is critically important for successful orthopedic implantation, especially for hosts suffering osteoporosis with reduced bone mass. To endow a titanium-based implant with a desirable local anti-osteoporosis property for enhancing its early osseointegration, alendronate-loaded hydroxyapatite-TiO2 nanotube (TNT-HA-Aln) substrates were fabricated and systematically characterized in this study. The results of Aln/Ca2+ release and Ca2+ concentration in an osteoclast medium verified that the release of Aln was significantly accelerated along with the acidity rise caused by osteoclast differentiation. Other in vitro tests, such as CCK-8, alkaline phosphatase (ALP), mineralization, gene expression (Runx2, Osterix, ALP, Col I, OPN, OC, OPG and RANKL), protein production (OPG and RANKL) and tartrate-resistant acid phosphatase (TRAP), proved that TNT-HA-Aln substrates have great potential for improving osteoblast proliferation/differentiation and inhibiting osteoclast differentiation. Moreover, in vivo tests, such as the push-out test, micro-CT and H&E staining proved that TNT-HA-Aln implants could efficiently improve local osseointegration after implantation for 3 months. The study provides an alternative to exploiting drug-device combinations to enhance early osseointegration in osteoporosis.

Influence of strontium ions incorporated into nanosheet-pore topographical titanium substrates on osteogenic differentiation of mesenchymal stem cells in vitro and on osseointegration in vivo.

Biophysical cues or biochemical cues were proved to efficiently regulate the fate of mesenchymal stem cells (MSCs), but their synergistic effects on the biological functions of MSCs remain to be further investigated. In this study, titanium (Ti) substrates were fabricated with distinct sub-micrometer nanosheet-pore topography via a vapor alkaline treatment method. Strontium (Sr) ions were then incorporated into the Ti substrates via ion exchange. Apart from the influence of biophysical cues from topography, MSCs were simultaneously affected by the biochemical cues from the continuously released Sr ions. The MSCs grown onto Ti substrates with Sr incorporated in them displayed higher (p < 0.05 or p < 0.01) cellular functions than those of pure Ti substrates, including proliferation, the genes and proteins expressions of osteogenic markers and mineralization potential when comparing them with the results of those MSCs grown onto pure Ti substrates. Furthermore, the in vivo investigations demonstrated that the Sr incorporated Ti implants promoted new bone formation. All the results indicated that the incorporated Sr ions and the nanosheet-pore topography of the Ti substrates synergistically enhanced the osteogenic differentiation of MSCs in vitro and osseointegration in vivo. This study advances the understanding of the synergistic influence of biophysical cues and biochemical cues on MSC osteogenic differentiation.

Multifunctional Fe2O3@PPy-PEG nanocomposite for combination cancer therapy with MR imaging.

In recent years, magnetic hyperthermia nanoparticles have drawn great attention for cancer therapy because they have no limitation of tissue penetration during the therapy process. In this study, cubic nanoporous Fe2O3 nanoparticles derived from cubic Prussian blue nanoparticles were used as magnetic cores to generate heat by alternating the current magnetic field (AMF) for killing cancer cells. In addition, polypyrrole (PPy) was coated on the surfaces of the cubic Fe2O3 nanoparticles to load doxorubicin hydrochloride (DOX). The PEG component was then physically adsorbed onto the surfaces of the nanoparticles, resulting in a Fe2O3@PPy-DOX-PEG nanocomposite. The nanocomposite was triggered by acid stimulus and AMF to release DOX, resulting in a remarkable combination therapeutic effect via chemotherapy and magnetic hyperthermia. Furthermore, the nanocomposite could realize magnetic resonance imaging (MRI) due to the magnetic core structure. The study provides an alternative for the development of new nanocomposites for combination cancer therapy with MR imaging in vivo.

Regulation of the biological functions of osteoblasts and bone formation by Zn-incorporated coating on microrough titanium.

To improve the biological performance of titanium implant, a series of Zn-incorporated coatings were fabricated on the microrough titanium (Micro-Ti) via sol-gel method by spin-coating technique. The successful fabrication of the coating was verified by combined techniques of scanning electron microscopy, surface profiler, X-ray diffraction, X-ray photoelectron spectroscopy, and water contact angle measurements. The incorporated zinc existed as ZnO, which released Zn ions in a sustained manner. The Zn-incorporated samples (Ti-Zn0.08, Ti-Zn0.16, and Ti-Zn0.24) efficiently inhibited the adhesion of both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria. The in vitro evaluations including cell activity, alkaline phosphatase (ALP), mineralization, osteogenic genes expressions (Runx2, ALP, OPG, Col I, OPN, and OC), and tartrate-resistant acid phosphatase, confirmed that Ti-Zn0.16 sample was the optimal one to regulate the proliferation or differentiation for both osteoblasts and osteoclasts. More importantly, in vivo evaluations including Micro-CT analysis, push-out test, and histological observations verified that Ti-Zn0.16 implants could efficiently promote new bone formation after implantation for 4 and 12 weeks, respectively. The resulting material thus has potential application in orthopedic field.

Influence of the titania nanotubes dimensions on adsorption of collagen: an experimental and computational study.

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 100nm displayed a higher adsorption amount and faster adsorption speed than that of 30nm TiNTs. Furthermore, more collagen molecules were aggregated in the tubes of 100nm 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.