Yonglin Yu

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.

Strontium folic acid derivative functionalized titanium surfaces for enhanced osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo

The introduction of the bioactive strontium (Sr) element has become an attractive method in the design of bio-functional layers on titanium surfaces. However, there are still no effective solutions to some of the associated problems including the toxicity of free Sr2+ ions and the rapid and irreversible loss of the strontium element from the bio-functional layers. In this study, we successfully fabricated a bioactive layer on Ti substrates with a strontium folic acid derivative (FASr). About 3.11 at% Sr was incorporated into the Ti surface. The characterization results showed that FASr was stable over a long period of time and minimal free Sr2+ ions were detected in simulated body fluid (SBF). In the in vitro experiment, the FASr could significantly promote the cell adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) over a short period. Furthermore, it could dramatically accelerate the bone formation around the implant. In vivo, a total of 30 7-week old male Sprague Dawley (SD) rats were applied for implantation tests. The results showed that this positive stimulatory effect became more evident in the later stages of the in vivo observation. This study provides an effective strategy for designing and optimizing Ti-based implants.

Sustained raloxifene release from hyaluronan-alendronate-functionalized titanium nanotube arrays capable of enhancing osseointegration in osteoporotic rabbits

To enhance the localized bone remodeling at titanium-based implants under osteoporotic conditions, TiO2 nanotube arrays (TNT) were used as nanoreserviors for raloxifene (Ral) and then covered with the hybrid multilayered coating of chitosan and alendronate grafted hyaluronic acid (HA-Aln) via a spin-assisted layer-by-layer technique. The fabrication of this system (TNT/Ral/LBL-Aln) was characterized by field emission scanning electron microscopy (SEM), atomic force microscope (AFM) and X-ray photoelectron spectroscopy (XPS), respectively. The release test showed that the composited multilayers onto Ral-loaded TiO2 nanotube substrate (TNT/Ral) could prevent the burst release of Ral from TiO2 nanotube arrays and maintain stable Ral concentration at the implant site even after 192h. The TNT/Ral/LBL-Aln system demonstrated higher alkaline phosphatase (ALP) activity, mineralization capability in osteoblasts as well as lower tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts compared to both bare TiO2 nanotube and TNT/Ral substrate, respectively. Moreover, the in vivo tests of micro-CT, histological staining and push-out testing showed that TNT/Ral/LBL-Aln implant could efficiently enhance the formation of new bone around the implant and promote bone binding in osteoporotic rabbits. The study indicated the potential application of TNT/Ral/LBL-Aln system for bone remodeling under osteoporotic condition.

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 ± 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.

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.

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.

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, β-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.