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Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the composite layers is important for its wider application in orthopedics and dentistry. This study incorporated low-molecular-weight heparin (LMWH) into the FGF-2-calcium phosphate composite layers and clarified the enhancing effects of LMWH on the biological activity of FGF-2 in the composite layers in vitro. LMWH-FGF-2-calcium phosphate composite layers were successfully formed on zirconia in supersaturated calcium phosphate solutions. The composite layers comprised continuous and macroscopically homogeneous layers and particles smaller than 500 nm in size composed of amorphous calcium phosphate. The amounts of Ca and P deposited on zirconia remained almost unchanged with the addition of LMWH under the presence of FGF-2 in the supersaturated calcium phosphate solution. The LMWH in the supersaturated calcium phosphate solution increased the stability of FGF-2 in the solution and the amount of FGF-2 in the composite layers. The LMWH in the composite layers increased the mitogenic and endothelial tube-forming activities of FGF-2, and FGF-2 activity of inducing osteogenic differentiation gene expression pattern in the composite layers. Our results indicate that the enhanced biological activity of FGF-2 in the LMWH-FGF-2-calcium phosphate composite layers is attributed to an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the supersaturated calcium phosphate solution and the composite layers. The LMWH-FGF-2-calcium phosphate composite layer is a promising coating for orthopedic and dental implants. STATEMENT OF SIGNIFICANCE: Orthopedic and dental implants coated with fibroblast growth factor-2 (FGF-2)-calcium phosphate composite layers promote dermis formation, bone formation, and angiogenesis because of the biological activity of FGF-2. Enhancing the biological activity of FGF-2 in the layers is important for wider its application in orthopedics and dentistry. This study demonstrates the enhancing effects of low-molecular-weight heparin (LMWH) contained within LMWH-FGF-2-calcium phosphate composite layers on the biological activity of FGF-2 in vitro. Our results indicate that the enhanced biological activity of FGF-2 within the composite layers arises from an LMWH-mediated increase in the amount of FGF-2, which maintains its biological activity in the LMWH-FGF-2-calcium phosphate composite layers and supersaturated calcium phosphate solutions used for coating the composite layers., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2022. Published by Elsevier Ltd.)

Immune checkpoint inhibitors elicit durable tumor regression in multiple types of tumor, but may induce potential side effects with low response rates in many tumors. Herein, to increase the therapeutic efficacy of immune checkpoint inhibitors, a hollow mesoporous silica (HMS) nanosphere-based cancer vaccine was combined with an immune checkpoint inhibitor, anti-programmed death-ligand 1 (anti-PD-L1) antibody. The HMS nanospheres function as adjuvants that promote dendritic cell activation and antigen cross-presentation. Mice immunized with the HMS-based cancer vaccine show suppressed tumor growth with increased tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-2 (IL-2) levels in their spleens compared with those without HMS-based cancer vaccine. Moreover, the HMS-based cancer vaccine synergistically acts with the anti-PD-L1 antibody on the tumor. The combination of an HMS-based cancer vaccine and an antibody markedly decreases the required dose of the immune checkpoint inhibitor. Mice locally administered with the HMS-based cancer vaccine and 1/8 dose of a standard anti-PD-L1 antibody (25 µg/mouse) show comparable anti-tumor effect and significantly increased CD4 + and CD8 + T cell populations, compared with those systemically immunized with the standard anti-PD-L1 antibody done at 200 µg/mouse. Our work presents a promising cancer treatment strategy of combining an immune checkpoint inhibitor with an HMS-based cancer vaccine. STATEMENT OF SIGNIFICANCE: The clinical benefits of checkpoint blockade therapy rekindle the hope of cancer immunotherapy. However, objective response rates in checkpoint blockade therapy remain at about 10-40% owing to multiple immunosuppressive factors. To solve these problems, herein, a hollow mesoporous silica (HMS) nanosphere-based cancer vaccine was combined with an immune checkpoint inhibitor, anti-PD-L1 antibody. The HMS-based cancer vaccine synergistically acts with the anti-PD-L1 antibody on the tumor. Mice locally administered with the HMS-based cancer vaccine and 1/8 dose of a standard anti-PD-L1 antibody (25 µg/mouse) show comparable anti-tumor effect and significantly increased CD4 + and CD8 + T cell populations, compared with those systemically immunized with the standard anti-PD-L1 antibody done at 200 µg/mouse. Our work presents a promising cancer treatment strategy of combining an immune checkpoint inhibitor with an HMS-based cancer vaccine., Competing Interests: Declaration of Competing Interest Xiupeng Wang, Atsuo Ito, Yu Sogo and Tadao Ohno are inventors of Patent JP-6868862-B2. This work was supported in part by the grant from Cell-Medicine, Inc., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cell morphology is related to proliferation and differentiation. We previously reported that cell attachment area of rat mesenchymal stem cells (MSCs) is negatively correlated with their osteogenic differentiation level on osteoconductive hydroxyapatite (HAp) with various microstructures. In this study, the correlation between the cell attachment area and osteogenic differentiation level was investigated on substrates without osteoconductive property using tissue culture polystyrene (TCPS), and 3 mol% yttria-stabilized zirconia (3Y-TZP) with or without surface periodic microstructures. It was found that the osteogenic differentiation level after 3 weeks of culture increased with a decrease in cell attachment area after 3 h of culture. The square of the correlation coefficient between cell attachment area and osteocalcin secretion content was 0.845 among the three types of substrates. Thus, the negative correlation between cell attachment area and differentiation level is confirmed even when cultured on substrates without osteoconductive property. These findings suggest that the correlation between the cell attachment area of rat MSCs and osteogenic differentiation level could also apply to various types of substrate, regardless of osteoconductive property., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recombinant protein-based vaccines generally show limited immunogenicity and need adjuvants to achieve robust immune responses. Herein, to combine the excellent biocompatibility of hydroxyapatite (HA) and exciting adjuvant activity of silica, Si-doped HA nanorods with Si/P molar ratio from 0 to 0.65 were hydrothermally synthesized and evaluated as immunoadjuvants. Si-doping decreases the size and increases the BET surface area of the nanorods. Si-doping in HA nanorods increases the in vitro adjuvant activity, including CD11c + CD86 + expression and cytokine secretion of bone marrow derived dendritic cells (BMDCs). Moreover, Si-doping in HA increases the ex vivo adjuvant activity as shown by the increase in both Th1 and Th2 cytokines secretion. Si-doped HA nanorods are promising as a new immunoadjuvant., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA-amorphous calcium phosphate (DNA-ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly's high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA-ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly's gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA-ACP nanocomposite spheres., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A Th1 immune response is required for modern vaccines as the most commonly used alum adjuvant has weak capacity for inducing Th1 immune response. Herein, rod-shaped hydroxyapatite (HA), magnesium-substituted HA (MgHA) and zinc-substituted HA (ZnHA) nanoparticles with irregular nanopores were synthesized and used as immunoadjuvants. Magnesium and zinc substitution in HA showed no influence on morphology, particle size, zeta potential and surface area of the nanoparticles. The rod-shaped MgHA and ZnHA nanoparticles promoted the cellular uptake of a molecular immunopotentiator, stimulated both type 1 and 2 cytokine secretion in vitro that relate to Th1 and Th2 immunity of bone marrow dentritic cells, respectively. The MgHA and ZnHA nanoparticles may be useful as immunoadjuvants for human., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A potential standard method for measuring the relative dissolution rate to estimate the resorbability of calcium-phosphate-based ceramics is proposed. Tricalcium phosphate (TCP), magnesium-substituted TCP (MgTCP) and zinc-substituted TCP (ZnTCP) were dissolved in a buffer solution free of calcium and phosphate ions at pH 4.0, 5.5 or 7.3 at nine research centers. Relative values of the initial dissolution rate (relative dissolution rates) were in good agreement among the centers. The relative dissolution rate coincided with the relative volume of resorption pits of ZnTCP in vitro. The relative dissolution rate coincided with the relative resorbed volume in vivo in the case of comparison between microporous MgTCPs with different Mg contents and similar porosity. However, the relative dissolution rate was in poor agreement with the relative resorbed volume in vivo in the case of comparison between microporous TCP and MgTCP due to the superimposition of the Mg-mediated decrease in TCP solubility on the Mg-mediated increase in the amount of resorption. An unambiguous conclusion could not be made as to whether the relative dissolution rate is predictive of the relative resorbed volume in vivo in the case of comparison between TCPs with different porosity. The relative dissolution rate may be useful for predicting the relative amount of resorption for calcium-phosphate-based ceramics having different solubility under the condition that the differences in the materials compared have little impact on the resorption process such as the number and activity of resorbing cells., Statement of Significance: The evaluation and subsequent optimization of the resorbability of calcium phosphate are crucial in the use of resorbable calcium phosphates. Although the resorbability of calcium phosphates has usually been evaluated in vivo, establishment of a standard in vitro method that can predict in vivo resorption is beneficial for accelerating development and commercialization of new resorbable calcium phosphate materials as well as reducing use of animals. However, there are only a few studies to propose such an in vitro method within which direct comparison was carried out between in vitro and in vivo resorption. We propose here an in vitro method based on measuring dissolution rate. The efficacy and limitations of the method were evaluated by international round-robin tests as well as comparison with in vivo resorption studies for future standardization. This study was carried out as one of Versailles Projects on Advanced Materials and Standards (VAMAS)., (Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Surface-mediated nonviral gene transfer systems using biocompatible apatite-based composite layers have potential use in tissue engineering applications. Herein, we investigated a relatively efficient system based on a DNA-lipid-apatite composite layer (DLp-Ap layer): an apatite (Ap) layer with immobilized DNA and lipid (Lp) complexes (DLp complexes). DLp-Ap layers were fabricated on substrates using supersaturated calcium phosphate coprecipitation solutions supplemented with DLp complexes, and the molecular compositions of the DLp-Ap layers were controlled by varying the net DNA concentrations and Lp/DNA ratios in the coprecipitation solutions. Increases in both the DNA concentration and Lp/DNA ratio in the coprecipitation solution increased the DLp complex content of the resulting DLp-Ap layer. However, a higher DLp complex content did not always provide increased gene transfer efficiency to the CHO-K1 cells, because there was a threshold content of approximately 10μg/cm(2). In addition, DLp-Ap layers with similar DLp complex contents exhibited different gene transfer efficiencies, most likely due to the different Lp/DNA ratios in the layers. Notably, the optimized Lp/DNA ratios in the coprecipitation solutions for maximizing the gene transfer efficiency were lower than those of the conventional particle-mediated lipofection systems. These findings will serve as a useful design guide for the preparation of DLp-Ap layers with high gene transfer efficiency., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Although fibroblast growth factor 2 (FGF2) is a promising agent for treating brain infarction, current methods of FGF2 administration are associated with a short circulating half-life. An FGF2 apatite coating was developed as a slow-releasing drug delivery system (DDS) by forming an FGF2/calcium phosphate composite layer. Hydroxyapatite was coated with high or low doses of FGF2, denoted as FGF-high and FGF-low. This study investigated the efficacy of the coating as angiogenesis therapy for brain infarction. Rats were subjected to permanent occlusion of the middle cerebral artery, an FGF2 apatite-coated implant was inserted, and the rat brains were removed 2 weeks after implantation. Rats in groups treated with FGF-high had significantly smaller areas of brain infarction, particularly in the external capsule and the lateral side of the putamen, and better capillary density than rats in groups treated with non-FGF2 apatite-coated implants. Histologic analysis indicated that the new vessels were larger and had thicker walls in the FGF2 apatite-coated groups than in the non-FGF2 groups. Fluorescence immunohistochemistry of the peri-infarction region showed that FGF2 released from FGF2 apatite-coated implants might have biological activity. Moreover, fluorescence immunohistochemistry showed that released FGF2 influenced microglia cells. This new FGF2 DDS involving an FGF2 apatite coating can prevent infarction of the penumbra through the multipotential effects of FGF2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The initial attachment of mesenchymal stem cells (MSCs) to substrates and osteogenic differentiation are supported by culture on a hydroxyapatite substrate. Cell attachment areas of rat MSCs after 2 h of culture on hydroxyapatite substrates with various microstructures and the osteogenic differentiation activity thereafter were measured. The perceived outcome was that, after 2 h of culture, rat MSCs with a small attachment area would have a high osteogenic differentiation activity, whereas those with a large attachment area would have a low osteogenic differentiation activity. Furthermore, rat MSCs with a small attachment area had many cytoplasmic processes, while those with a large attachment area revealed clear stress fibers and focal contacts. These results suggest that cell attachment area of rat MSCs after 2 h of culture has a strong effect on the osteogenic differentiation of rat MSCs. Thus, the measurement of cell attachment area after 2 h of culture could become valuable for estimating the osteogenic differentiation activity of rat MSCs thereafter., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA-apatite composite layer (D-Ap layer) and DNA-lipid-apatite composite layer (DLp-Ap layer) were prepared on ceramic hydroxyapatite disk and scaffold using supersaturated calcium phosphate solutions supplemented with 0.5-5 μg/mL plasmid and 0-10 μL/mL lipid transfection reagent FuGENE®. Both in vitro and in vivo studies were carried out using mesenchymal stem cells (MSCs) and two kinds of gene (luciferase and bone morphogenetic protein (BMP)-2) for demonstrating potential application of the gene transfer system using the D-Ap and DLp-Ap layers in bone tissue engineering. In the in vitro study using luciferase gene, the DLp-Ap layers showed 1-2 orders of magnitudes higher gene transfer efficiency to MSCs than the D-Ap layer. In the in vivo study using BMP-2 gene, DLp-Ap layer slightly increased BMP-2 protein concentration than D-Ap layer, thereby enhancing their osteogenic differentiation than D-Ap layer. The present gene transfer system using the DLp-Ap layers, with the advantages of good biocompatibility, bone-bonding ability, and efficacy in in vitro and in vivo gene transfer to MSCs, would be useful in bone tissue engineering., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Artificial materials coated with a protein-apatite composite layer have great potential in clinical applications as a third generation biomaterial. Such composite materials can be prepared by immersing a surface modified substrate into a supersaturated calcium phosphate solution (CP solution: 142 mM NaCl, 3.75 mM CaCl(2), 1.5mM K(2)HPO(4)·3H(2)O, buffered at pH 7.4 at 25 °C with tris(hydroxymethyl)aminomethane and HCl) supplemented with a protein. In the present study proteins of various molecular weights (MW) and isoelectric points (pI) were used to form a protein-apatite composite layer on a polymeric material to determine how the molecular properties of the protein affect the efficiency of protein immobilization (i.e. the amount of immobilized protein in the apatite layer as a percentage of the total amount of protein in solution). The results indicated that the efficiency of protein immobilization did not correlate with the MW of the protein. In contrast, the efficiency of protein immobilization was strongly related to the pI of the protein. As the pI decreased the efficiency of protein immobilization increased due to the high adsorption affinity of negatively charged acidic proteins for positively charged apatite crystals and/or apatite precursors in the CP solution. Thus, the use of acidic rather than basic proteins improves the immobilization efficiency in the present coating process., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

To improve the biocompatibility, inter-connective pore structure, and drug delivery ability of self-setting apatite/collagen composite cement (ACC), a three-dimensionally perforated porous apatite/collagen composite cement (TPPACC) containing 3% indomethacin (IMC) was obtained in an arranged multi-cross with 20, 40, and 60 stainless steel needlelike male dies, and stored and hardened at 37 degrees C and 100% relative humidity for 24 h. The mean radius of micro-pores of the TPPACC was evaluated to be 0.125 microm by mercury porosimetry. X-ray powder diffraction and FT-IR spectroscopy suggested that TPPACC consisted of carbonated apatite and had a structure similar to that of natural rat bone. The IMC release rates from a TPPACC block containing the drug were measured in simulated body fluid. The rate of release increased with the number of macro-pores that from planar surface matrix systems followed the Higuchi equation. The relationship between the Higuchi constant and surface area of TPPACC showed a straight line with K = 0.2123 and R(2) = 0.9892. These results indicated that the rate of drug release from TPPACC could be controlled by the number of macro-pores for bone cells.

Ascorbate-apatite and ascorbate-fibroblast growth factor-2 (FGF-2)-apatite composite layers were successfully formed on anodically oxidized Ti rods clinically used for external fixation by a one-step procedure at 25 degrees C, using a metastable supersaturated calcium phosphate solution supplemented with l-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) and FGF-2. The AsMg-apatite and AsMg-FGF-2-apatite composite layers were evaluated in vitro using fibroblastic NIH3T3 and osteoblastic MC3T3-E1 cells. The AsMg-FGF-2-apatite composite layer markedly enhanced the NIH3T3 cell proliferation and procollagen type capital I, Ukrainian gene expression. Without FGF-2, the AsMg-apatite composite layer whose ascorbate content was 3.64+/-1.27microgcm(-2) obviously enhanced osteoblastic proliferation and differentiation. However, the AsMg-FGF-2-apatite composite layers whose FGF-2 contents were from 0.15+/-0.03 to 0.31+/-0.04microgcm(-2) inhibited osteoblastic differentiation in vitro. Thus, the AsMg-FGF-2-apatite composite layer should be precipitated on the surface of external fixators attached to skin and soft tissue. On the other hand, the AsMg-apatite composite layer should be precipitated at the part attached to bone tissue.

The final aim of our study is to develop a novel calcium phosphate cement based on zinc-containing α-tricalcium phosphate (αZnTCP) and evaluate its potential as bonegraft material in vivo. In the present study, in vivo efficacy of zinc in hardened bodies of αZnTCP was explored. The hardened bodies prepared from αZnTCP with zinc content of 0.00, 0.04, 0.08, 0.11 and 0.19 wt % were prepared by mixing pure αTCP or αZnTCP powder with 12 wt% sodium succinate solution at a solid-to-liquid ratio of 2.0. Due to the release of zinc ions into the physiological salt solution during curing, the zinc content in the hardened bodies was calculated to be 0.00, 0.03, 0.06, 0.10 and 0.18 wt%, respectively. The hardened bodies were implanted in the femora and tibia of white rabbits for 4 weeks. Histological and histomorphometric evaluation showed that the hardened body containing 0.03 wt% zinc, significantly promoted more new bone formation without evoking adverse tissue reactions than that without zinc. The hardened bodies containing 0.06 and 0.10 wt% zinc also resulted in the increase in numbers of active osteoblasts surrounding the new bone but caused inflammation at the implant sites. Results of this study indicate that the hardened body prepared with αZnTCP is superior to that prepared with αTCP in promoting new bone formation due to the release of zinc ions. This study also indicates that the optimum amount of zinc in the hardened body is about 0.03 wt % to avoid inflammatory reaction.

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).

An artificial material coated with a laminin-apatite composite layer on its surface would be useful as a percutaneous device with cell-adhesion properties and good biocompatibility. The present authors recently prepared such a composite by a liquid-phase coating process in which a surface-modified material was immersed in a laminin-containing calcium phosphate solution (LCP solution) supersaturated with respect to apatite. As a result of this coating process, however, the material increased in surface roughness, which may have an adverse effect on the prevention of bacterial infection. In the present study, a reduction of the surface roughness of the laminin-apatite composite layer was attempted by adding MgSO(4) to the LCP solution as an inhibitor of apatite crystal growth. The surface roughness, Ra, of the resulting laminin-apatite composite layer decreased from approximately 0.43 to 0.31mum upon increasing the MgSO(4) concentration in the LCP solution from 0 to 1.5mM, while retaining its cell adhesion characteristics. The thus-obtained material, having a laminin-apatite composite coating with reduced surface roughness, has potential as a material for percutaneous devices with improved resistance to bacterial infection through the interface between the device and the skin.

The purpose of this study was to evaluate the therapeutic efficacy of a new calcium phosphate (CaP)-based formulation in improving the bone mineral deficiency in ovariectomized (OVX) rats. The ions release experiments for CaP preparations (G2: 0.46% Mg, 5.78% Zn, and 2.5% F; G3:3.1% Mg, 0.03% Zn, and 3.01% F; G4: 1.25% Mg, 1.77% Zn, 1.35% F) and of a Zn-TCP (G1: 6.17% Zn) powders, the initial Mg and Zn ion release rates of MZF-CaPs were performed in acetate buffer at pH 4.5 (37 degrees C). Wistar rats were divided into six groups including a normal (not OVX) group (GN) and a control, OVX group (GC). Rats in groups GC, G1, G2, G3, G4 were OVX. Suspensions consisting of CaP preparations (G2, G3, G4) and of a Zn-TCP (G1) powders were injected in the right thighs of OVX rats in all groups except for GN and GC, once a week for 4 weeks. GN and GC rats were injected with saline solutions. Plasma was analyzed for Zn land alkaline phosphatase levels. The bone mineral density (BMD) was measured using DEXA and the bone (femur) strength determined using three-point-bending analysis. G1 and G2 groups showed high plasma Zn levels. The area under the curve of plasma Zn was significantly greater in the G1, G2, and GN groups than in the G3, G4, and GC groups (p < 0.05). The BMD and bone mechanical strength of the right femur were significantly higher in the G1, G2, G3, and G4 groups than GC group on day 28. The right femur had significantly greater BMD and bone mechanical strength than the left femur in G1, G2, G3, and G4 groups. However, there was no significant difference in the BMD of the right femur between the G1, G2, G3, and G4 groups. Results indicate that the new injectable CaP formulations are effective in improving bone properties of OVX rats and may be useful in osteoporosis therapy., ((c) 2007 Wiley-Liss, Inc.)