In vitro apatite formation on nano-crystalline titania layer aligned parallel to Ti6Al4V alloy substrates with sub-millimeter gap.

Pure titanium substrates were chemically oxidized with H2O2 and subsequent thermally oxidized at 400 °C in air to form anatase-type titania layer on their surface. The chemically and thermally oxidized titanium substrate (CHT) was aligned parallel to the counter specimen such as commercially pure titanium (cpTi), titanium alloy (Ti6Al4V) popularly used as implant materials or Al substrate with 0.3-mm gap. Then, they were soaked in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5 °C) for 7 days. XRD and SEM analysis showed that the in vitro apatite-forming ability of the contact surface of the CHT specimen decreased in the order: cpTi > Ti6Al4V > Al. EDX and XPS surface analysis showed that aluminum species were present on the contact surface of the CHT specimen aligned parallel to the counter specimen such as Ti6Al4V and Al. This result indicated that Ti6Al4V or Al specimens released the aluminum species into the SBF under the spatial gap. The released aluminum species might be positively or negatively charged in the SBF and thus can interact with calcium or phosphate species as well as titania layer, causing the suppression of the primary heterogeneous nucleation and growth of apatite on the contact surface of the CHT specimen under the spatial gap. The diffusion and adsorption of aluminum species derived from the half-sized counter specimen under the spatial gap resulted in two dimensionally area-selective deposition of apatite particles on the contact surfaces of the CHT specimen.

Calcium phosphate crystallization on titania in a flowing Kokubo solution.

Dry titania layers on air-oxidized titanium substrates have been found to be active enough to cause apatite to be deposited in Kokubo's simulated body fluid (SBF) in narrow confined spaces, such as those in narrow grooves and thin gaps. Such in vitro apatite deposition is the basis of the GRAPE(®) technique. The aim of the present study is to determine why GRAPE conditions favor apatite deposition when laminar SBF flow (at 0.01-0.3 ml/min) passes through a shallow channel (0.5 mm) between a pair of titanium substrates each with a dry layer of titania. Assessing the factors that control the heterogeneous nucleation process led to the proposal of the working hypothesis that there are nucleation pre-embryos, ion assemblies that can be stabilized to form embryos, on the titania layer but that they are removed by the SBF flow. Specimens were subjected to different combinations of processes. One combination was that titania layers were exposed to still or flowing SBF, and the other was that half of a specimen, the inlet or outlet side, was exposed to still or flowing SBF with the other half being covered. The surface morphologies of the specimens were then compared in detail. The conclusion was that exposure to still SBF for 2 days before exposure to flowing SBF was required for apatite to be deposited. Some complicated apatite deposition modes were observed, e.g., apatite was deposited even on areas unexposed to still SBF. All of the results were successfully interpreted using the working hypothesis. The conclusion was that the GRAPE(®) technique depends on the confined space holding pre-embryo and embryo assemblies.

Liquid phase deposited titania coating to enable in vitro apatite formation on Ti6Al4V alloy.

A recently developed "GRAPE(®) technology" provides titanium or titanium alloy implants with spontaneous apatite-forming ability in vitro, which requires properly designed gaps and optimum heat treatment in air. In this study, titanium alloy and commercially pure (cp) titanium substrates were thermally oxidized in air before aligning pairs of specimens in the GRAPE(®) set-up, i.e., titanium alloy and cp titanium substrates were aligned parallel to each other with optimum gap width (spatial design). A liquid phase deposition (LPD) technique was employed for titania coatings on titanium alloy substrate. Then, they were soaked in Kokubo's simulated body fluid (SBF, pH 7.4, 36.5 °C) for 7 days to confirm the in vitro apatite formation on the substrates under the specific spatial design. Anatase-type titania coatings fabricated by using LPD technique led to the deposition of apatite particles within 7 days and showed apatite X-ray diffraction. On the other hand, thermally oxidized titanium alloy substrate in air and non-treated specimens did not show any apatite X-ray diffraction. These results indicated that the heterogeneous nucleation of apatite induced on anatase-type titania coating prepared by LPD technique when it was aligned parallel to thermally oxidized cp titanium substrate with optimum gap width.

Distribution and propagation of stress and strain in cube honeycombs as trabecular bone substitutes: Finite element model analysis.

For designing trabecular (Tb) bone substitutes suffering from osteoporosis, finite element model (FEM) simulations were conducted on honeycombs (HCs) of 8 × 8 × 1 (2D) and 8 × 8 × 8 (3D) assemblies of cube cellular units consisting of 0.9 mm long Nylon® 66 (PA, Young's modulus E: 2.83 GPa) and polyethylene (PE, E: 1.1 GPa) right square prisms. Osteoporotic damage to the Tb bone was simulated by removing the inner vertical struts (pillars; the number of removed pillars: Δn ≤ 300) and by thinning the strut (thickness, d: 0.4-0.1 mm), while the six facade lattices were kept flawless. Uniform and uniaxial compressive loads on the HCs induced elastic deformation of the struts. The pillars held almost all the load, while the horizontal struts (beams) shared little. E for PA 3D HCs of all d smoothly decreased with Δn. PA 3D HCs of 0.2 mm struts deserved to be the substitutes for Tb bone, while PE 3D HCs of 0.05 mm struts were only for the Tb bone of the poorest bone quality. For the PA 3D HCs, the maximum von Mises stress (σM) first rapidly increased with Δn and showed a break at Δñ50, then gradually approached the yield stress of PA (50 MPa). Moreover, small portions of the stress were transferred from the façade pillars to the adjacent inner beams, especially those near the lost-pillar sites, denoted as X defects. The floor beams of thinner struts associated with the X-defects were lifted, and similar lifting effects in smaller amounts were propagated to the other floors. The 3DHCs of the thicker struts showed no such flexural deformations. The concept of force percolation through the remaining struts was proposed to interpret those mechanical behaviors of the HCs.

Sol-gel-derived silicate nano-hybrids for biomedical applications.

Organic-inorganic hybrids of poly(dimethyl siloxane), gelatin, and chitosan with such silanes as tetraethoxysilane and 3-glycidoxytriethoxysilane are derived via the sol-gel routes. Their biomedical applications are discussed from biomimetic deposition of bone-like apatite, cell culture, and in vivo behavior.

Does mirabegron deteriorate spermatogenesis? A lesson from spinal cord injury cases.

OBJECTIVES: To evaluate whether the long-term usage of mirabegron, which was reported to have potential side effects on male reproductive organs in animal studies, was harmful to spermatogenesis in human testis. METHODS: Thirty consecutive patients with spinal cord injury (20-48 years old) who performed clean intermittent catheterization were involved in this study. Ten patients were treated with mirabegron (50 mg/d) for more than 2 years and refrained from using an antimuscarinic agent due to the side effects of constipation and dry mouth. Twenty patients were treated with neither anticholinergic agents nor mirabegron. All underwent conventional testicular sperm extraction. The sperm recovery rate and histopathologic findings of the retrieved testicular tissue were compared between both groups. RESULTS: We found no difference in the sperm recovery rate (P = .083) between both groups. Spinal cord injury patients treated with mirabegron had better spermatogenesis than those not treated with mirabegron (P < .05). CONCLUSIONS: From these data, we conclude that the therapeutic dose of mirabegron had no harmful effect on spermatogenesis in spinal cord injury patients of reproductive age.

Synthesis of bioactive HEMA-MPS-CaCl2 hybrid gels: effects of catalysts in the sol-gel processing on mechanical properties and in vitro hydroxyapatite formation in a simulated body fluid.

We investigated synthetic conditions for the fabrication of bioactive hybrid gels from monomers of 2-hydroxyethylmethacrylate (HEMA) and 3-methacryloxypropyltrimethoxysilane (MPS) in combination with CaCl(2), at a starting molar ratio of HEMA: MPS : CaCl(2) of 9 : 1 : 1. Hydroxyapatite formation, essential to show bone bonding, was observed on the HEMA- MPS-CaCl( 2) hybrid gels with the added catalysts NH(3) or HCl with a molar ratio to MPS of 0.1, but not on the hybrid gel with HCl at a molar ratio to MPS of 1. The mechanical properties of the gels were dependent on the catalysts, which may affect the microstructures that develop during sol-gel processing.

Novel fabrication of nano-rod array structures on titanium and in vitro cell responses.

Nano-scale rod arrays of titania were fabricated on titanium surface by a glass phase topotaxy growth (GPT) method, which was featured by an interfacial reaction between sodium tetraborate coating and the preheated metallic titanium at elevated temperature. The samples were characterized by thin-film X-ray diffraction (XRD), scanning electron microscope (SEM), profilometer and contact angle measurement. Thin-film XRD analysis indicated that the nano-rod arrays were composed of pure rutile titania phase. SEM images showed that these rutile rods were 100-200 nm wide and 1-2 microm long. The nano-rod arrays had significantly higher average roughness (P < 0.05) and greater hydrophilicity (P < 0.05) compared to the control. Human embryonic palatal mesenchymal (HEPM) cells were grown to evaluate in vitro cell responses to the nano-rod array structures in terms of cell attachment and proliferation. An equivalent high attachment rate of 94% was observed after 4-h incubation, but a lower proliferation rate was observed on the nano-rod array after 12-day culture compared to the control (P < 0.05).

Vascular endothelial growth factor promotes brain tissue regeneration with a novel biomaterial polydimethylsiloxane-tetraethoxysilane.

In the brain after infarction or trauma, the tissue eventually becomes pannecrotic and forms a cavity. In such situations, a scaffold is necessary for the implanted or migrated cells to produce new tissue. In this present study, therefore, we attempted to restore brain tissue using a novel biomaterial, polydimethylsiloxane-tetraethoxysilane (PDMS-TEOS) hybrid with or without vascular endothelial growth factor (VEGF), which is crucial for new vessel formation. When PDMS-TEOS scaffold was implanted into the artificial brain defect, it remained at the implanted site and kept the integrity of the brain shape. At 30 days after the implantation, the marginal territory of PDMS-TEOS scaffold became occupied by newly formed tissue. Immunohistochemical analysis revealed that the new tissue was constituted by astrocytes and endothelial cells. Addition of VEGF increased the newly produced tissue volume, and the immunohistochemical analysis showed that the numbers of astrocytes and endothelial cells were increased. Double staining with proliferation maker Ki67 demonstrated that VEGF significantly increased newly formed astrocytes and endothelial cells, indicating that addition of VEGF accelerated tissue restoration and angiogenesis. These findings show that implantation of PDMS-TEOS scaffold with VEGF might be effective for treating old brain infarction or trauma.

Implantation of a new porous gelatin-siloxane hybrid into a brain lesion as a potential scaffold for tissue regeneration.

For brain tissue regeneration, any scaffold for migrated or transplanted stem cells with supportive angiogenesis is important once necrotic brain tissue has formed a cavity after injury such as cerebral ischemia. In this study, a new porous gelatin-siloxane hybrid derived from the integration of gelatin and 3-(glycidoxypropyl) trimethoxysilane was implanted as a three-dimensional scaffold into a defect of the cerebral cortex. The porous hybrid implanted into the lesion remained at the same site for 60 days, kept integrity of the brain shape, and attached well to the surrounding brain tissues. Marginal cavities of the scaffolds were occupied by newly formed tissue in the brain, where newly produced vascular endothelial, astroglial, and microglial cells were found with bromodeoxyuridine double positivity, and the numbers of those cells were dose-dependently increased with the addition of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Extension of dendrites was also found from the surrounding cerebral cortex to the newly formed tissue, especially with the addition of bFGF and EGF. The present study showed that a new porous gelatin-siloxane hybrid had biocompatibility after implantation into a lesion of the central nervous system, and thus provided a potential scaffold for cell migration, angiogenesis and dendrite elongation with dose-dependent effects of additive bFGF and EGF.

Selective protein adsorption property and characterization of nano-crystalline zinc-containing hydroxyapatite.

Nano-crystalline Zn-containing hydroxyapatite (ZnHAp) was prepared by the wet-chemical method and the selective adsorption of essential proteins was examined, taking bovine serum albumin (BSA) and pathogenic protein such as beta(2)-microglobulin (beta(2)-MG) as model proteins. Transmission electron microscopy observation and X-ray diffraction analysis indicated that the increase of Zn content led to smaller crystallites and their specific surface area of ZnHAps increased with increasing Zn content, accordingly. Furthermore, the amounts of BSA adsorption on ZnHAp particles decreased with increasing Zn content in spite of the increase in the specific surface area. It is thus concluded that nano-crystalline ZnHAps had a highly selective adsorption property with regard to beta(2)-MG.

An organic-inorganic hybrid scaffold for the culture of HepG2 cells in a bioreactor.

Much interest has recently been shown in the potential utility of bioartificial liver (BAL) as a bridge support for patients and as a module for experimental purposes. A radial-flow bioreactor (RFB), one of the perfused bed/scaffold-type bioreactors, enables a highly functional three-dimensional culture as BAL. The functional capacity of bioreactors depends not only on their mechanistic structures but also on scaffolds packed in them. In the present study, we examined the possible utility of a new porous organic-inorganic-hybrid scaffold in an RFB. The scaffold was made from tetraethoxysilane (TEOS) and polydimethylsiloxane (PDMS) by a sol-gel method using sieved sucrose particles as a porogen. In the porous TEOS-PDMS hybrid scaffold, human hepatocellular carcinoma cells (HepG2) proliferated actively and formed cell clusters more efficiently than they did in a polyvinyl-alcohol scaffold. When cultivated in PDMS-TEOS, HepG2 cells secreted a approximately three-fold greater amount of albumin than that secreted in a monolayer culture. For potential application of BAL to pharmacological studies and future clinical use, it is essential to develop a method to propagate liver cells that maintain highly specific functions. The present results indicate that PDMS-TEOS may be a promising scaffold for developing such functional culture methods.

In vitro cytocompatibility of MG63 cells on chitosan-organosiloxane hybrid membranes.

Chitosan-silicate hybrids were synthesized using gamma-glycidoxypropyltrimethoxysilane (GPSM) as the agent for cross-linking the chitosan chains. CaCl2 was introduced in the hybrids in expectation that it would improve cell adhesion and differentiation of the hybrid surfaces. Fourier-transform infrared (FT-IR) spectroscopy and 29Si CP-MAS NMR spectroscopy were used to analyze the structures of the hybrids. Cytocompatibility of the hybrids was investigated in terms of proliferation of an osteoblastic cell line, MG63. The adhesion and proliferation of the osteoblastic cells cultured on the surface of a chitosan-GPSM hybrid without calcium were similar to those on a control culture plate, and were better than those on a chitosan membrane. The ALP activity of the cells cultured on this hybrid was higher than that on the chitosan membrane. Contrary to expectations, the incorporation of calcium ions into the hybrids did not improve cell attachment and proliferation on their surfaces.

In vitro apatite formation on organic polymers modified with a silane coupling reagent.

Gamma-methacryloxypropyltrimethoxysilane (gamma-MPS) was grafted to high-density polyethylene, polyamide and silicone rubber substrates by the emulsion polymerization procedure in order to provide these organic polymers with in vitro apatite-forming ability. The contact angles towards distilled water of the gamma-MPS-grafted specimens were lower than those of the original organic polymer specimens, indicating that the grafted substrates were more hydrophilic. The in vitro apatite formation in a simulated body fluid (Kokubo solution) was confirmed for several of the gamma-MPS-grafted specimens.

Preparation of Porous Chitosan-Siloxane Hybrids Coated with Hydroxyapatite Particles.

This paper describes the apatite deposition of chitosan-silicate porous hybrids derived from chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) in alkaline phosphate solution. The preparation of porous hybrids with needle-like apatite on their surfaces is described. Following apatite deposition the porous hybrids maintained high porosity. The enzymatic degradation rate was low even after 6 months and the porous hybrids were very flexible and cut easily using surgical scissors.

Preparation and in vitro cytocompatibility of chitosan-siloxane hybrid hydrogels.

Injectable systems can be used in minimally invasive surgical applications. Although chitosan-glycerophosphate hydrogel systems are biodegradable and biocompatible, the long periods of time required for their effective gelation have severely limited their clinical application. The challenges currently facing researchers in this field are therefore focused on shortening the gelation time and biocompatibility of these materials to develop hydrogels suitable for clinical application. Chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) hybrids have recently demonstrated good cytocompatibility with respect to human osteoblastic cells (MG63) and human bone marrow cells. Although these precursor sols could form gels under physiological conditions, they required neutralization with a sodium hydroxide solution. In this study, the chitosan-GPTMS hybrid systems were neutralized with glycerophosphate to prepare injectable hydrogels. The results revealed that the gelation time of the hydrogels could be controlled by the amount of GPTMS in the precursor sols. The in vitro cytocompatibility of the hydrogels were evaluated in terms of the proliferation of MG63 cells cultured either directly onto the hydrogels or indirectly onto the cell culture plate under a hydrogel insert. In the former case, the cells showed good attachment and proliferated for up to 7 days. Similar results were observed in the in direct culture. These results suggest that this new chitosan-GPTMS hydrogel could potentially be used as an injectable biomaterial in clinical applications.

Novel approach to fabricate porous gelatin-siloxane hybrids for bone tissue engineering.

Porous and bioactive gelatin-siloxane hybrids were successfully synthesized by using a combined sol-gel processing, post-gelation soaking, and freeze-drying process to provide a novel kind of materials in the developments and optimization of bone tissue engineering. The pore sizes of the hybrids can be well controlled by varying the freezing temperature. The scaffolds were soaked in a simulated body fluid (SBF) up to 14 days to evaluate the in vitro bioactivity. The Ca(2+)-containing scaffolds showed in vitro bioactivity as they biomimetically deposited apatite, but the Ca(2+)-free scaffolds failed. Cytotoxicity and cytocompatibility of those scaffolds and their extracts were monitored by the MC3T3-E1 cell responses, including the cell proliferation and the alkaline phosphatase activity. It was demonstrated that appropriate incorporation of Ca(2+) ions stimulated osteoblast proliferation and differentiation in vitro.

Preparation of alginic acid layers on stainless-steel substrates for biomedical applications.

This study is concerned with the blood compatibility of alginic acid layers immobilized on gamma-aminopropyltriethoxysilane (gamma-APS)-grafted stainless-steel (SUS316L). The surfaces were characterized with contact angle measurement and X-ray photoelectron spectroscopy (XPS). The blood compatibility was evaluated in terms of platelet adhesion and blood clotting time. An in vitro platelet adhesion assay indicated that only a small number of platelets adhered to substrate surfaces modified with gamma-APS and subsequently with alginic acid. Moreover, alginic-acid-immobilized SUS316L substrates had little effect on the blood clotting time. This indicated that alginic-acid-immobilized SUS316L substrates do not adsorb some blood-clotting proteins or factors, or stimulate them.

The chitosan prepared from crab tendons: II. The chitosan/apatite composites and their application to nerve regeneration.

The chitosan tubes derived from crab tendons form a hollow tube structure, which is useful for nerve regeneration. However, in order to use the chitosan tubes effectively for nerve regeneration, there remain two problems to be solved. First, the mechanical strength of the tubes is quite high along the longitudinal axis, but is somewhat low for a pressure from side. Second, the chitosan tube walls swell to reduce the inner space of the tubes in vivo. These two problems limit the clinical use of the chitosan tubes. In this study, to solve the problems, apatite was made to react with the chitosan tubes to enhance the mechanical strength of the tube walls. Transmission electron microscopy showed that apatite crystals were formed in the walls of the chitosan tubes. The c-axis of the crystals aligned well in parallel with chitosan molecules. These results indicate that the apatite crystals grow in the tubes starting from the nucleation sites of the chitosan molecules, probably by forming complexes with amino groups of chitosan and calcium ions. Further, the tubes were thermally annealed at 120 degrees C to prevent from swelling, and simultaneously formed into a triangular shape to enhance the stabilization of the tube structure. By these treatments, the hollow tubes could keep their shape even in vivo after implantation. Animal tests using SD rats further showed that the chitosan tubes effectively induced the regeneration of nerve tissue, and were gradually degraded and absorbed in vivo.

Bioactive titania-gel layers formed by chemical treatment of Ti substrate with a H2O2/HCl solution.

An amorphous titania gel layer was formed on the titanium surface after the titanium specimen was treated with a H2O2/0.1 M HCl solution at 80 degrees C. The thickness of the gel layer increased almost linearly with the period of the treatment. A subsequent heat treatment above 300 degrees C transformed gradually the amorphous gel to the anatase crystal structure and the rutile started to appear after heat treatment at 600 degrees C. Meanwhile, the densification of the gel occurred significantly after heat treatment above 700 degrees C. Similar to the sol-gel derived titania gel coatings. titania gel layers obtained in the present study exhibited in vitro apatite deposition ability after the gel layers exceeded a minimum thickness (0.2 microm) and was subsequently heated in a proper temperature range (400-600 degrees C).