Yu Sogo

Zinc-releasing calcium phosphate for stimulating bone formation ☆

Zinc-containing tricalcium phosphate (ZnTCP) is biocompatible and bioactive, and functions as an effective zinc carrier. ZnTCP contains a maximum of 12 mol% of zinc. ZnTCP ceramics and composite ceramics of ZnTCP and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 were prepared by sintering at 1100 °C. ZnTCP/HAP continued to release zinc for more than 50 days in 0.9 wt.% sodium chloride solution. ZnTCP/HAP with a zinc content of 1.2 wt.% significantly increased osteoblastic MC3T3-E1 cell proliferation and alkaline phosphatase activity of rat stromal cells in vitro. ZnTCP/HAP with a zinc content of 0.316 wt.% increased bone formation by 51% (n=6; p=0.0509) 4 weeks after implantation in rabbit compared to the control without zinc. A zinc concentration within a noncytotoxic level of a solution does not completely block the crystal growth of apatite in the solution. When ZnTCP is added to calcium phosphate cement, the ZnTCP showed neither inhibitory nor promoting effect on the setting ability of the cement. All these findings suggest that the zinc-containing tricalcium phosphate is a biomaterial that has a pharmaceutical effect of promoting bone formation.

Synthesis and characterization of hierarchically macroporous and mesoporous CaO–MO–SiO2–P2O5 (M = Mg, Zn, Sr) bioactive glass scaffolds

Mg-, Zn- and Sr-doped hierarchically macroporous and mesoporous CaO-MO-SiO(2)-P(2)O(5) (M=Mg, Zn or Sr) bioactive glass (HMMBG) scaffolds were synthesized using the non-ionic block copolymer EO(20)PO(70)EO(20) and polyurethane sponges as cotemplates. The Mg-, Zn- or Sr-doped HMMBG scaffolds showed no distinct difference in phase composition, macroporous structure or pore volume from the HMMBG scaffolds without Mg, Zn or Sr. The Mg-, Zn- and Sr-doped HMMBG scaffolds showed no cytotoxicity. The gradual release of Ca, P, Si, Mg, Zn and Sr into the culture medium from these scaffolds contributed to the enhancement of the proliferation and ALP activity of mesenchymal stem cells (MSCs). The Mg-, Zn- and Sr-doped HMMBG scaffolds may be used as bone substitute materials.

Zinc-containing apatite layers on external fixation rods promoting cell activity

Zinc-containing apatite layers were successfully formed on commercially available anodically oxidized Ti external fixation rods using ZnCl(2)-containing supersaturated calcium phosphate solutions. With an increase in concentration of ZnCl(2) in the supersaturated calcium phosphate solutions, the amounts of zinc that precipitated on the Ti external fixation rods increased (from 0 to 0.195 + or - 0.020 microg cm(-2)); meanwhile, the amounts of calcium and phosphorus that precipitated on the Ti external fixation rods decreased (from 11.2 + or - 1.5 and 4.8 + or - 0.5 microg cm(-2) to 2.9 + or - 1.6 and 1.3 + or - 0.9 microg cm(-2), respectively). The zinc-containing apatite layers precipitated on the Ti external fixation rods caused a significant increase in fibroblastic proliferation, osteoblastic proliferation and differentiation in vitro. The Ti external fixation rods coated with zinc-containing apatite layers are expected to be more effective in accelerating the tissue regeneration around the surgical site than those coated with an apatite layer.

Calcium phosphate composite layers for surface-mediated gene transfer

In this review, the surface-mediated gene transfer system using calcium phosphate composite layers is described. Calcium phosphate ceramics are osteoconductive bioceramics used typically in orthopedic and dental applications. Additionally, calcium phosphate particles precipitated by a liquid-phase process have long been used as a safe and biocompatible transfection reagent in molecular biology. Recently, calcium phosphate composite layers immobilizing DNA were fabricated on the surfaces of base materials through a biomimetic process using supersaturated solutions. These composite layers possess useful characteristics of both osteoconductive bioceramics and transfection reagents; they thus provide a biocompatible surface to support cell adhesion and growth, and can stimulate the cell effectively via surface-mediated gene transfer. By modifying the fabrication conditions, physicochemical and biological properties of the composite layers can be varied. With such an approach, these composite layers can be designed to have improved affinity for cells and to exhibit increased gene transfer efficiency over that of conventional lipid transfection reagents. The composite layers with the increased gene transfer efficiency induced specific cell differentiation and tissue regeneration in vivo. These composite layers, given their good biocompatibility and the potential to control cell behavior on their surfaces, have great potential in tissue engineering applications.

Solubility of Mg-containing β-tricalcium phosphate at 25 °C

The equilibrium solubility of Mg-containing beta-tricalcium phosphate (betaMgTCP) with various magnesium contents was determined by immersing betaMgTCP powder for 27 months in a CH(3)COOH-CH(3)COONa buffer solution at 25 degrees C under a nitrogen gas atmosphere. The negative logarithm of the solubility product (pK(sp)) of betaMgTCP was expressed as pK(sp)=28.87432+1.40348C-0.3163C(2)+0.04218C(3)-0.00275C(4)+0.0000681659C(5), where C is the magnesium content in betaMgTCP (mol.%). The solubility of betaMgTCP decreased with increasing magnesium content owing to the increased structural stability and possible formation of a whitlockite-type phase on the surface. As a result, betaMgTCP with 10.1 mol.% magnesium had a lower solubility than that of hydroxyapatite below pH 6.0. betaMgTCP was found to be more soluble than zinc-containing beta-tricalcium phosphate given the same molar content of zinc or magnesium. The solubility of betaMgTCP and release rate of magnesium from betaMgTCP can be controlled by adjusting the Mg content by selecting the appropriate pK(sp).

Mesoporous bioactive glass coatings on stainless steel for enhanced cell activity, cytoskeletal organization and AsMg immobilization

Mesoporous bioactive glass (MBG) coatings with SiO2:CaO:P2O5 mol ratio of 100:0:0, 80:15:5 and 70:25:5 and a tunable pore size and pore structure were prepared on a stainless steel plate by spin-coating sol solutions containing a triblock copolymer and the inorganic precursors. The calcium content in the MBG coatings affected the mesoporous structure. With the increase in calcium content, the crystallinity of the MBG coatings increased and thus the Brunauer–Emmett–Teller (BET) surface area and pore volume decreased. The MBG coatings were evaluated on the basis of protein adsorption, cell attachment, cell proliferation, cell differentiation, cytoskeletal organization and L-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) immobilization for their potential in improving implant-bone integration. The results showed that the osteoblast MC3T3-E1 cells were stimulated by the mesoporous structure and chemical composition of the MBG coatings, with enhanced cell attachment, proliferation, differentiation and better developed cytoskeleton. Moreover, AsMg was successfully immobilized on the MBG coatings by using an AsMg-containing supersaturated calcium phosphate solution. The AsMg immobilized on the MBG coatings was not denatured and showed high activity enhancing the fibroblast NIH3T3 proliferation in vitro. An appropriate range of pore size and a preferred alignment of the mesochannels of the MBG coatings on stainless steel are promising to improve the implant-bone integration.

Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro

Fibronectin (Fn) and type I collagen (Col) were immobilized on a surface of a hydroxyapatite (HAP) ceramic by coprecipitation with calcium phosphate in a supersaturated calcium phosphate solution prepared by mixing clinically approved infusion fluids. These proteins and the calcium phosphate precipitate formed a composite surface layer. As a result, the proteins were immobilized firmly as not to be released completely for 3 d in a physiological salt solution. When human mesenchymal stem cells (hMSCs) were cultured on a HAP ceramic in a differentiation medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, hMSCs spread well within 1 h. The alkaline phosphatase (ALP) activity of hMSCs cultured on the Fn-calcium phosphate composite layer significantly increased compared with that of hMSCs cultured on the untreated HAP ceramic. On the other hand, Col did not increase the ALP activity of hMSCs and no synergy between Fn and Col was observed. Therefore, the Fn-calcium phosphate composite layer formed on the HAP is useful for the enhancement of the spreading and osteogenic differentiation of hMSCs in vitro.

Stimulation of In Vivo Antitumor Immunity with Hollow Mesoporous Silica Nanospheres

The use of appropriate adjuvants that support the generation of robust and long-lasting antitumor immune responses is crucial for tumor immunotherapy owing to the immunosuppressive environment of the growing tumor. However, the most commonly used adjuvant, aluminum hydroxide, is ineffective for generating such immune responses and therefore not suitable for cancer immunotherapy. It is now shown that plain hollow mesoporous silica nanospheres markedly improve the antitumor immunity, the Th1 and Th2 immunity, and the CD4(+) and CD8(+) effector memory T cell population in bone marrow in vivo and may thus be used as immunoadjuvants to treat cancer in humans.

Particle-size-dependent toxicity and immunogenic activity of mesoporous silica-based adjuvants for tumor immunotherapy

Conventionally used adjuvants alone are insufficient for triggering cell-mediated immunity, although they have been successfully developed to elicit protective antibody responses in some vaccines. Here, with the aim of eliciting cell-mediated immunity, pathogen-associated molecular patterns (PAMPs) were immobilized with apatite within the pores and on the surface of mesoporous silica (MS) with particle sizes from 30 to 200nm to prepare novel MS-Ap-PAMP adjuvants, which showed cell-mediated anti-tumor immunity that was markedly improved compared to commercial alum adjuvant in vitro and in vivo. The toxicity and antitumor immunity of the MS-Ap-PAMP adjuvants were evaluated in vitro and in vivo. MS with a particle size of 200nm showed minimum in vitro cytotoxicity to NIH3T3 cells, particularly at concentrations no higher than 100μgml(-1). In particular, apatite precipitation within the pores and on the surface of MS decreased the in vitro cytotoxicity of MS particles. The MS-Ap-PAMP adjuvants showed the maximum in vitro immunogenic activity among original culture medium, PAMP and alum-PAMP. Moreover, injection of the MS-Ap-PAMP adjuvant in combination with liquid-nitrogen-treated tumor tissue (derived from Lewis lung carcinoma cells) into C57BL/6 mice markedly inhibited in vivo tumor recurrence and the development of rechallenged tumor compared to those with commercial alum adjuvant. The MS-Ap-PAMP adjuvant contributed to the elicitation of a potent systemic antitumor immunity without obvious toxicity in vivo.

Fibroblast growth factor-2-apatite composite layers on titanium screw to reduce pin tract infection rate.

Fibroblast growth factor-2 (FGF-2)-apatite composite layers were formed on anodically oxidized titanium screws to improve bone-screw interface strength and to reduce pin tract infection rate through enhanced skin tissue healing in external fixation. A calcium-containing solution supplemented with FGF-2, a phosphate-containing solution, and a sodium bicarbonate solution were mixed at a Ca/P molar ratio of 2.0 to prepare a calcium phosphate solution supersaturated with respect to calcium phosphates. Screws were individually immersed in 10 mL of the calcium phosphate solution at 37 degrees C for 2 days. Low-crystalline apatite layers incorporating FGF-2 were formed on the screw surface at FGF-2 concentrations in the supersaturated calcium phosphate solution equal to or lower than 10 mug/mL. The amounts of FGF-2 immobilized on the screws ranged from 2.3- to 2.4-mug per screw. The immobilized FGF-2 retained biological activity, as demonstrated by NIH3T3 cell proliferation. Titanium screws with the composite layer were percutaneously implanted into the bilateral proximal tibial metaphyses in rabbits for 4 weeks. The titanium screws with the composite layer formed at the optimum FGF-2 concentration showed a significantly higher bone-screw interface strength and a lower pin tract infection rate than those without the composite layer: the extraction torque and infection rates were respectively 0.230 +/- 0.073 Nm and 43.8% for the screws with the composite layer, and 0.170 +/- 0.056 Nm and 93.8% for those without the composite layer. Therefore, titanium screws with the FGF-2-apatite composite layer are useful for improving bone-screw interface strength and infection resistance in external skeletal fixation.