Wen Song

Influence of pore size of porous titanium fabricated by vacuum diffusion bonding of titanium meshes on cell penetration and bone ingrowth.

UNLABELLEDnThe present work assesses the potential of three-dimensional (3D) porous titanium (pore size of 188-390 μm and porosity of 70%) fabricated by vacuum diffusion bonding of titanium meshes for applications in bone engineering. Rat bone marrow mesenchymal stem cells were used to investigate the proliferation and differentiation of cells on titanium scaffolds with different pore sizes at day 7, day 14 and day 21 based on DNA contents, alkaline phosphatase (ALP) activity, collagen (COL) secretion and osteogenic gene expressions including ALP, COL-1, bone morphogenetic protein-2 (BMP-2), osteopontin (OPN), runt-related transcription factor 2 (RUNX2), using smooth solid titanium plate as reference material. The rabbit models with distal femoral condyles defect were used to investigate the bone ingrowth into the porous titanium. All samples were subjected to Micro-CT and histological analysis after 4 and 12 weeks of healing. A one-way ANOVA followed by Tukey post hoc tests was used to analyze the data. It was found that the differentiation stage of cells on the porous titanium delayed compared with the smooth solid titanium plate and Ti 188 was more inclined to promote cell differentiation at the initial stage (day 14) while cell proliferation (day 1, 4, 7, 10, 14 and 21) and bone ingrowth (4 and 12 weeks) were biased to Ti 313 and Ti 390. The study indicates that the hybrid porous implant design which combines the advantages of different pore sizes may be meaningful and promising for bone defect restoration.nnnSTATEMENT OF SIGNIFICANCEnOne of the significant challenges in bone defect restoration is the integration of biomaterials and surrounding bone tissue. Porous titanium may be a promising choice for bone ingrowth and mineralization with appropriate mechanical and biological properties. In this study, based on porous titanium fabricated by vacuum diffusion bonding of titanium meshes, we have evaluated the influence of various pore sizes on rat bone marrow mesenchymal stem cells (rBMMSCs) penetration in vitro and bone ingrowth in vivo. It was interesting that we found the proliferation and differentiation abilities of rBMMSCs, as well as bone ingrowth were related to different pore sizes of such porous scaffolds. The results may provide guidance for porous titanium design for bone defect restoration.

Chitosan/siRNA functionalized titanium surface via a layer-by-layer approach for in vitro sustained gene silencing and osteogenic promotion.

Titanium surface modification is crucial to improving its bioactivity, mainly its bone binding ability in bone implant materials. In order to functionalize titanium with small interfering RNA (siRNA) for sustained gene silencing in nearby cells, the layer-by-layer (LbL) approach was applied using sodium hyaluronate and chitosan/siRNA (CS/siRNA) nanoparticles as polyanion and polycation, respectively, to build up the multilayered film on smooth titanium surfaces. The CS/siRNA nanoparticle characterization was analyzed first. Dynamic contact angle, atomic force microscopy, and scanning electron microscopy were used to monitor the layer accumulation. siRNA loaded in the film was quantitated and the release profile of film in phosphate-buffered saline was studied. In vitro knockdown effect and cytotoxicity evaluation of the film were investigated using H1299 human lung carcinoma cells expressing green fluorescent protein (GFP). The transfection of human osteoblast-like cell MG63 and H1299 were performed and the osteogenic differentiation of MG63 on LbL film was analyzed. The CS/siRNA nanoparticles exhibited nice size distribution. During formation of the film, the surface wettability, topography, and roughness were alternately changed, indicating successful adsorption of the individual layers. The scanning electron microscope images clearly demonstrated the hybrid structure between CS/siRNA nanoparticles and sodium hyaluronate polymer. The cumulated load of siRNA increased linearly with the bilayer number and, more importantly, a gradual release of the film allowed the siRNA to be maintained on the titanium surface over approximately 1 week. In vitro transfection revealed that the LbL film-associated siRNA could consistently suppress GFP expression in H1299 without showing significant cytotoxicity. The LbL film loading with osteogenic siRNA could dramatically increase the osteogenic differentiation in MG63. In conclusion, LbL technology can potentially modify titanium surfaces with specific gene-regulatory siRNAs to enhance biofunction.

In vivo osseointegration of Ti implants with a strontium-containing nanotubular coating.

Novel biomedical titanium (Ti) implants with high osteogenic ability for fast and good osseointegration under normal as well as osteoporotic conditions are urgently needed. Expanding on our previous in vitro results, we hypothesized that nanotubular, strontium-loaded (NT-Sr) structures on Ti implants would have favorable osteogenic effects and evaluated the in vivo osseointegration of these implants in rats. The structures with nanotubes of different diameters were fabricated by electrochemical anodization at 10 and 40 V, and the amounts of Sr loaded were adjusted by using two hydrothermal treatment times of 1 and 3 hours. Qualitative microcomputed tomography in two and three dimensions showed that the NT-Sr formed with an anodization voltage of 10 V and hydrothermal treatment time of 3 hours best supported bone growth in vivo. Histomorphometric examination of osseointegration also showed that more newly formed bone was found at its surface. The bone–implant contact percentage was highest (92.48%±0.76%) at 12 weeks. In conclusion, the NT-Sr formed with an anodization voltage of 10 V and hydrothermal treatment time of 3 hours showed excellent osteogenic properties, making it an attractive option for Ti surface modification with considerable clinical potential.

Epidermal growth factor regulates intestinal glutamine uptake during total parenteral nutrition

Sprague Dawley rats were randomised into three groups: group I (chow) were fed rat chow and water ad libitum, group II total parenteral nutrition (TPN) received a standard formula of TPN, and group III (TPN--epidermal growth factor (EGF)) received the same TPN as group II and injections of EGF (0.1 microg/gm body weight) subcutaneously twice daily. Glutamine (GLN) concentrations in tissues and blood were measured by reversed phase high performance liquid chromatography. Gut GLN extraction was calculated by dividing the difference in GLN concentrations (Conc) between the carotid artery (ART) and portal vein (PV) by the arterial concentration [(ART Conc - PV Conc)/ART Conc]. TPN induced a marked reduction of GLN concentration in tissues and blood, and also reduction of gut GLN extraction. When EGF was administered along with TPN, gut GLN concentration did not fall and gut GLN extraction was increased by 15% (TPN - EGF 1 week, P < 0.05). Arterial blood concentration of GLN was increased when TPN and EGF were used for 1 week (P < 0.05 vs control). But EGF did not prevent the GLN concentration of other tissues decreasing during TPN. Our results suggest that EGF can regulate intestinal uptake of GLN during TPN.

Induction of osteogenic differentiation of stem cells via a lyophilized microRNA reverse transfection formulation on a tissue culture plate.

MicroRNA (miRNA) regulation is a novel approach to manipulating the fate of mesenchymal stem cells, but an easy, safe, and highly efficient method of transfection is required. In this study, we developed an miRNA reverse transfection formulation by lyophilizing Lipofectamine 2000-miRNA lipoplexes on a tissue culture plate. The lipoplexes can be immobilized on a tissue culture plate with an intact pseudospherical structure and lyophilization without any lyoprotectant. In this study, reverse transfection resulted in highly efficient cellular uptake of miRNA and enabled significant manipulation of the intracellular target miRNA level. Reverse transfection formulations containing Lipofectamine 2000 1 μL per well generated much higher transfection efficiency without obvious cytotoxicity compared with conventional and other transfection methods. Further, the transfection efficiency of the reverse transfection formulations did not deteriorate during 90 days of storage at 4°C and −20°C. We then assessed the efficiency of the miRNA reverse transfection formulation in promoting osteogenic differentiation of mesenchymal stem cells. We found that transfection with anti-miR-138 and miR-148b was efficient for enhancing osteogenic differentiation, as indicated by enhanced osteogenesis-related gene expression, amount of alkaline phosphatase present, production of collagen, and matrix mineralization. Overall, the miRNA reverse transfection formulation developed in this study is a promising approach for miRNA transfection which can control stem cell fate and is suitable for loading miRNAs onto various biomaterials.

Biofunctionalization of titanium implant with chitosan/siRNA complex through loading-controllable and time-saving cathodic electrodeposition

Although titanium (Ti) implants are already being broadly used, further improvement is still mandatory for improving its clinical performance. To this end, small interfering RNA (siRNA) biofunctionalization may present a novel strategy. In this study, siRNA biofunctionalization was realized on a titania nanotube array (TNT) on Ti by the cathodic electrodeposition (CED) of a chitosan (CS)/siRNA complex. The siRNA deposition amount was linearly correlated to the current density and the siRNA release profile was sensitive to the pH value of the surrounding solution. To confirm the activity of the cathodically electrodeposited siRNA, the siRNA targeting green fluorescent protein (siGFP) was introduced by CED, and the primary rat mesenchymal stem cells (rMSCs) encoding a GFP expression motif were seeded. High siRNA delivery efficiency and sustained target gene down-regulation were observed, which were much better than the conventional CS/siRNA nanoparticle delivery system and were related to the enhanced siRNA access into the cytoplasm. Simultaneously, good cytocompatibility was observed from the CS/siRNA biofunctionalization, as indicated by the good cell adhesion and viability. Finally, the siRNA targeting casein kinase-2 interacting protein-1 (siCkip-1), reported to have osteogenic potential, was introduced by CED to assess the potential of CS/siRNA biofunctionalization for application in bone implants. The CS/siCkip-1 biofunctionalization magnificently enhanced the osteogenic differentiation of rMSCs in terms of improved osteogenesis related gene expression, collagen secretion and calcium deposition, showing high promise for application in bone implants. The study demonstrates a new and convenient way to functionalize the biomedical metal implants with CS/siRNA for surface activation via local siRNA delivery.

Role of the unfolded protein response in topography-induced osteogenic differentiation in rat bone marrow mesenchymal stem cells

The topography of biomaterials can significantly influence the osteogenic differentiation of cells. Understanding topographical signal transduction is critical for developing biofunctional surfaces, but the current knowledge is insufficient. Recently, numerous reports have suggested that the unfolded protein response (UPR) and osteogenic differentiation are inter-linked. Therefore, we hypothesize that the UPR pathway may be involved in the topography-induced osteogenesis. In the present study, different surface topographies were fabricated on pure titanium foils and the endoplasmic reticulum (ER) stress and UPR pathway were systematically investigated. We found that ER stress and the PERK-eIF2α-ATF4 pathway were activated in a time- and topography-dependent manner. Additionally, the activation of the PERK-eIF2α-ATF4 pathway by different topographies was in line with their osteogenic induction capability. More specifically, the osteogenic differentiation could be enhanced or weakened when the PERK-eIF2α-ATF4 pathway was promoted or inhibited, respectively. Furthermore, tuning of the degree of ER stress with different concentrations of thapsigargin revealed that mild ER stress promotes osteogenic differentiation, whereas excessive ER stress inhibits osteogenic differentiation and causes apoptosis. Taken together, our findings suggest that the UPR may play a critical role in topography-induced osteogenic differentiation, which may help to provide new insights into topographical signal transduction.nnnSTATEMENT OF SIGNIFICANCEnSuitable implant surface topography can effectively improve bioactivity and eventual bone affinity. However, the mechanism of topographical signaling transduction is unclear and criteria for designation of an appropriate implant surface topography is lacking. This study shows that the ER stress and PERK-eIF2α-ATF4 pathway were activated by micro- and micro/nano-topographies, which is corresponding to the osteogenic induction abilities of these topographies. Furthermore, we have found that mild ER stress improves osteogenic differentiation, whereas excessive ER stress inhibits osteogenic differentiation and causes apoptosis. Our findings demonstrate that the UPR plays a critical role in the topography induced osteogenic differentiation, which may help to provide new insights into the topographical signaling transduction.

The influence of nanotopography on organelle organization and communication

Cellular differentiation can be affected by the extracellular environment, particularly extracellular substrates. The nanotopography of the substrate may be involved in the mechanisms of cellular differentiation in vivo. Organelles are major players in various cellular functions; however, the influence of nanotopography on organelles has not yet been elucidated. In the present study, a micropit-nanotube topography (MNT) was fabricated on the titanium surface, and organelle-specific fluorescent probes were used to detect the intracellular organelle organization of MG63 cells. Communication between organelles, identified by organelle-specific GTPase expression, was evaluated by quantitative polymerase chain reaction and western blotting. Transmission electron microscopy was performed to evaluate the organelle structure. There were no significant differences in organelle distribution or number between the MNT and flat surface. However, organelle-specific GTPases on the MNT were dramatically downregulated. In addition, obvious endoplasmic reticulum lumen dilation was observed on the MNT surface, and the unfolded protein response (UPR) was also initiated. Regarding the relationships among organelle trafficking, UPR, and osteogenic differentiation, our findings may provide important insights into the signal transduction induced by nanotopography.

Effect of arginine on gastrointestinal immunity during total parenteral nutrition

It has been well recognized that the prolonged use of total parenteral nutrition (TPN) leads to intestinal immunodeficiency and bacterial translocation (BT). Arginine (ARG) is known to have immunostimulatory effects. But its effects on gut immunity are unknown. This experiment was designed to evaluate the effects of arginine on gut immunity during TPN. Male Sprague Dowley rats were randomized to three groups: group I (chow) was fed rat chow and water ad libitum, group II (TPN) received a standard formula of TPN and group III (TPN-ARG) received the same formula of TPN as group II with the amino acid composition containing 0.5% arginine. With the duration of TPN, the rates of BT increased and interleukin 2 (IL-2) production decreased in TPN group. The results in TPN-ARG group were partly reversed. When TPN was administered for 2 weeks, the rate of BT decreased significantly (P < 0.05) and IL-2 production increased markedly (P < 0.01) in the TPN-ARG group compared with those in the TPN group. Our results suggest that arginine can decrease BT and increase IL-2 production in rats during prolonged TPN.

Improved bone formation and ingrowth for additively manufactured porous Ti6Al4V bone implants with strontium laden nanotube array coating

A Ti implant with an interconnected porous structure may be a better choice for bone defect restoration; an important issue is to improve its bone formation and ingrowth abilities. In this study, a porous Ti6Al4V implant is fabricated via electron beam melting (EBM) technology with a precisely controlled pore shape and size as well as good interconnectivity. Anodization treatment and further Sr incorporation give rise to an even distribution of the titania nanotube array (NT) and a strontium-laden NT (NTSr) coating on the outer and inner surface of the porous implant, which significantly enhance its hydrophilicity. The NT and NTSr coatings, especially NTSr, significantly improve the in vitro infiltration and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) as well as the in vivo bone formation and ingrowth abilities of the porous implant. The data also show that the pore size differentially influences the biological effect of the porous implant, especially after surface modification. The smaller pores (600 μm) favor in vitro BMMSC proliferation and osteogenic differentiation and in vivo new bone mass formation, while the larger pores (800 μm) favor in vitro cell ingrowth and in vivo bone ingrowth. Our study suggests that the NTSr coating is very promising for porous implant applications to improve their biological performance and also uncover the differential effect of the pore size on the biological effect of the porous implant.