Fabrication of calcite blocks from gypsum blocks by compositional transformation based on dissolution-precipitation reactions in sodium carbonate solution.

Calcium carbonate (CaCO3) has been used as a bone substitute, and is a precursor for carbonate apatite, which is also a promising bone substitute. However, limited studies have been reported on the fabrication of artificial calcite blocks. In the present study, cylindrical calcite blocks (ϕ6×3mm) were fabricated by compositional transformation based on dissolution-precipitation reactions using different calcium sulfate blocks as a precursor. In the dissolution-precipitation reactions, both CaSO4·2H2O and CaSO4 transformed into calcite, a polymorph of CaCO3, while maintaining their macroscopic structure when immersed in 1mol/L Na2CO3 solution at 80°C for 1week. The diametral tensile strengths of the calcite blocks formed using CaSO4·2H2O and CaSO4 were 1.0±0.3 and 2.3±0.7MPa, respectively. The fabrication of calcite blocks using CaSO4·2H2O and CaSO4 proposed in this investigation may be a useful method to produce calcite blocks because of the self-setting ability and high temperature stability of gypsum precursors.

The roles of calcium-sensing receptor and calcium channel in osteogenic differentiation of undifferentiated periodontal ligament cells.

Elevated extracellular calcium has been shown to promote the differentiation of osteoblasts. However, the way that calcium affects the osteogenic differentiation of human periodontal ligament stem/progenitor cells (PDLSCs) remains unclear. Our aim has been to investigate the proliferation and osteogenic differentiation of a calcium-exposed human PDLSC line (cell line 1-17) that we have recently established and to elucidate the roles of the calcium-sensing receptor (CaSR) and L-type voltage-dependent calcium channel (L-VDCC) in this process. Proliferation activity was investigated by WST-1 assay, and gene and protein expression was examined by quantitative reverse transcriptase plus the polymerase chain reaction and immunostaining, respectively. Calcification assay was performed by von Kossa and Alizarin red staining. Treatment with 5 mM CaCl2 significantly induced proliferation, bone-related gene expression, and calcification in cell line 1-17. During culture with 5 mM CaCl2, this cell line up-regulated the gene expression of CaSR, which was reduced after 7 days. Simultaneous treatment with NPS2143, a CaSR inhibitor, and calcium significantly further increased bone-related gene expression and calcification as compared with CaCl2 exposure alone. The L-VDCC inhibitor, nifedipine, significantly suppressed osteogenic differentiation of cell line 1-17 treated with 5 mM CaCl2 and promoted the expression of CaSR, as compared with calcium treatment alone. Thus, elevated extracellular calcium promotes the proliferation and osteogenic differentiation of a PDLSC line. Antagonizing CaSR further enhances the effect of calcium on osteogenic differentiation, with CaSR expression being regulated by L-VDCC under extracellular calcium. Extracellular calcium might therefore modulate the osteogenic differentiation of PDLSCs through reciprocal adjustments of CaSR and L-VDCC.

Regeneration of the periodontium for preservation of the damaged tooth.

The population of the world grows every year, and life expectancy tends to increase. Thus, long-term preservation of teeth in aged individuals is an urgent issue. The main causes of tooth loss are well known to be periodontitis, caries, fractures, and orthodontic conditions. Although implant placement is a widely accepted treatment for tooth loss, most patients desire to preserve their own teeth. Many clinicians and researchers are therefore challenged to treat and preserve teeth that are irreversibly affected by deep caries, periodontitis, fractures, and trauma. Tissue engineering techniques are beneficial in addressing this issue; stem cells, signal molecules, and scaffolds are the main elements of such techniques. In this review, we describe these three elements with respect to their validation for regeneration of the periodontium and focus particularly on the potency of diverse scaffolds. In addition, we provide a short overview of the ongoing studies of 4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl-borane resin including calcium chloride or hydroxyapatite for periodontium regeneration.

Fabrication of solid and hollow carbonate apatite microspheres as bone substitutes using calcite microspheres as a precursor.

Spherical carbonate apatite (CO3Ap) microspheres approximately 1 mm in diameter were fabricated by granulation of calcium hydroxide around a core followed by carbonation and phosphatization through dissolution-precipitation reaction. CO3Ap microspheres with high uniformity could not be achieved without using a core. Solid CO3Ap microspheres were obtained using a calcite core whereas hollow CO3Ap microspheres were obtained using a NaCl core. The obtained microsphere was identified as B-type CO3Ap by Fourier transform infrared analysis and the carbonate content was approximately 7-8 wt% regardless of the type of core used for sample preparation. The mechanical strength of both the solid and hollow CO3Ap microspheres was sufficient for practical use as a bone substitute.

Surface modification of titanium by hydrothermal treatment in Mg-containing solution and early osteoblast responses.

Surface modification on titanium was carried out in order to improve its bioactivity. Pure titanium was hydrothermally treated in distilled water and 0.1 M MgCl(2) solutions at 200°C for 24 h. Surface morphology, roughness, wettability and chemical composition were characterized before and after treatment. Bovine serum albumin was used as model to study protein adsorption. MC3T3-E1 cells were cultured and initial cell attachment, morphology, proliferation were evaluated. After hydrothermal treatment, nano-sized precipitations were observed and samples showed superhydrophilicity. Magnesium (Mg) was immobilized into titanium surface by hydrothermal treatment. Protein adsorption was significantly increased on Mg-containing samples. Cell attachment was improved and cell spreading was enhanced on Mg-containing samples compared with untreated or those treated in distilled water. Increased early cellular attachment on the MgTi surface resulted in subsequent increase of number of proliferated cells. Hydrothermal treatment in MgCl(2) solution was expected to be an effective method to fabricate titanium implant with good bioactivity.

Lateral bone augmentation with newly developed ß-tricalcium phosphate block: an experimental study in the rabbit mandible.

OBJECTIVE: To evaluate the biological effects of newly developed ß-tricalcium phosphate (ß-TCP) to improve lateral bone augmentation. MATERIAL AND METHODS: Test samples (rod-shaped [RS]-blocks) were prepared through hydrothermal processing α-TCPs. As controls, commercially available ß-TCPs (C-blocks; Osferion) were used. The blocks were placed onto the rabbit mandibles (n=5/group, mean: 4 kg). Samples were retrieved after 6, 12 and 24 weeks. Thereafter, the sections were evaluated for histological and histomorphometric analyses. The parameters set were: BV/TV (%): [(area of newly formed bone; area-NFB)/(whole measured area; WA)] × 100, BV+Imp.V/TV (%): [(area-NFB+area of remaining ß-TCP block)/WA] × 100, N.Oc/I.Pm: (osteoclast number)/(entire ß-TCP block perimeter; 100 mm). RESULTS: BV/TV of the C-blocks (10.71 ± 3.39%) was significantly higher than the RS-blocks (3.5 ± 3.52%, P<0.05) at 6 weeks. At 24 weeks, the RS-blocks (23.66 ± 2.7%) showed significantly higher values than the C-blocks (13.23 ± 2.65%, P<0.001). The BV+Imp.V/TV of the RS-blocks was significantly higher than that of the C-block group (29.61 ± 5.84% and 19.13 ± 3.14%), maintaining high values between 6 and 24 weeks (44.16 ± 5.19% and 50.88 ± 4%, P<0.001). The N.Oc/I.Pm values were significantly greater in the RS-block group than in the C-block group throughout the observation period (P<0.05). CONCLUSIONS: The newly developed ß-TCP blocks presented subsequent replacement by newly formed bone, in conjunction with maintaining the external morphology.

Fabrication of carbonate apatite block based on internal dissolution-precipitation reaction of dicalcium phosphate and calcium carbonate.

In this study, we investigated a novel method for fabrication of carbonate apatite block without ionic movement between precursor and solution by using precursor that includes all constituent ions of carbonate apatite. A powder mixture prepared from dicalcium phosphate anhydrous and calcite at appropriate Ca/P ratios (1.5, 1.67, and 1.8) was used as starting material. For preparation of specimens, the slurry made from the powder mixture and distilled water was packed in a split stainless steel mold and heat - treated, ranging from 60 degrees C to 100 degrees C up to 48 hours at 100% humidity. It appeared that carbonate apatite could be obtained above 70 degrees C and monophasic carbonate apatite could be obtained from the powder mixture at Ca/P ratio of 1.67. Carbonate content of the specimen was about 5-7%. Diametral tensile strength of the carbonate apatite blocks slightly decreased with increasing treatment temperature. The decrease in diametral tensile strength is thought to be related to the crystal size of the carbonate apatite formed.

Sensing of protein adsorption with a porous bulk composite comprising silver nanoparticles deposited on hydroxyapatite.

Porous bulk composites were produced by depositing silver nanoparticles of diameter 11.0 +/- 3.2 nm on hydroxyapatite of micrometer sizes. Adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) on the composite material was observed in 2 and 10 mol m(-3) phosphate buffer solutions. More BSA than LSZ was adsorbed in 2 mol m(-3) phosphate buffer and this was attributed to a larger a-face surface area present in the plate- and rod-shaped hydroxyapatite compared with the c-face surface area. Peak shifts in localized surface plasmon resonance (LSPR) spectra were clearly related to adsorbed amounts of BSA and LSZ after exposure of the porous bulk composites to protein solutions. The sensing capability of the porous bulk composite results from changes in the dielectric constant of the surface fluid surrounding the silver nanoparticles. Adsorption/desorption cycles of BSA were applied to the porous bulk composite, confirming the reversibility of the sensing capability.

Stimulatory effect of hydrothermally synthesized biodegradable hydroxyapatite granules on osteogenesis and direct association with osteoclasts.

Calcium-deficient hydroxyapatite (HA) granules with a unique spherical shape were prepared using an applied hydrothermal method. Spherical stoichiometric HA granules were also prepared by normal sintering and both granules were used for implantation into rat tibiae to compare the biological responses to each implant. Twelve and 24 weeks after implantation, the volume of calcium-deficient HA granules was significantly less than that of stoichiometric HA granules, and the biodegradability of calcium-deficient HA granules was confirmed. The larger number of osteoclasts, larger osteoblast surface and larger bone volume in the implanted area of calcium-deficient HA than those of stoichiometric HA suggested that osteoclastic resorption of calcium-deficient HA affected osteogenesis in that area. To analyze the direct contribution of osteoclasts to osteogenesis, C2C12 multipotent myoblastic cells, which have the potential to differentiate into osteoblasts in the presence of bone morphogenetic protein 2, were cultured with supernatants of osteoclasts cultured on calcium-deficient HA, stoichiometric HA, beta-tricalcium phosphate disks or plastic dishes, or bone marrow macrophages cultured on plastic dishes. Supernatants of osteoclasts but not bone marrow macrophages stimulated the expression of Runx2 and osteocalcin in C2C12 cells in concert with bone morphogenetic protein 2. The expression of alkaline phosphatase was stimulated with supernatants of osteoclasts cultured on ceramic disks. These results suggested that osteoclasts produced certain soluble factors which stimulated osteoblastic differentiation and they were thought to be associated with the induction of a larger osteoblast surface and bone volume in the animals implanted with calcium-deficient HA granules.

The slow resorption with replacement by bone of a hydrothermally synthesized pure calcium-deficient hydroxyapatite.

A newly developed calcium-deficient hydroxyapatite composed of rod-shaped particles synthesized by the hydrothermal method (HHA) and stoichiometric hydroxyapatite (SHA) synthesized by the sintering method was used for in vivo implantation and in vitro culture systems to compare these biological responses. In the rabbit femur, implanted HHA was slowly resorbed and about 80% of the implant remained 24 weeks after implantation; however, up to 72 weeks after implantation, most of the implanted HHA was resorbed. The implanted SHA was unresorbed throughout the experimental period, but degradation by the invasion of newly formed bone was seen at 72 weeks after implantation. Bone histomorphometry showed that the volume of newly formed bone and the number of osteoclasts in the implanted region were significantly higher in HHA than in SHA 24 weeks after implantation. In vitro culture of C2C12 cells with the induction of osteoblastic phenotypes using recombinant bone morphogenetic protein-2 showed similar cell density and the induction of alkaline phosphatase activity between the cells on HHA and SHA discs. In vitro osteoclastogenesis of HHA and SHA discs using bone marrow macrophages and recombinant receptor activator of nuclear factor-kappaB ligand showed higher TRAP activity of osteoclasts cultured on HHA discs. These results showed that slow biodegradability did not always correlate to final replaceability in bone tissue, and suggested that the activity of osteoclasts correlated to the bone-forming activity of osteoblasts.

Relationship between apatite-forming ability and mechanical properties of bioactive PMMA-based bone cement modified with calcium salts and alkoxysilane.

Polymethylmethacrylate (PMMA)-based bone cement is used for the fixation of artificial joints in orthopaedics. However, the fixation is liable to loosen in the body, because the cement does not bond to living bone. So-called bioactive ceramics bond directly to living bone through the apatite layer formed on their surfaces in the body. We previously revealed that modification using gamma-methacryloxypropyltrimethoxysilane (MPS) and water-soluble calcium salts such as calcium acetate and calcium hydroxide was effective for providing the PMMA-based bone cement with apatite-forming ability in a simulated body fluid (SBF, Kokubo solution) that closely reproduces the body environment. However, the effect of the chemical reaction forming the apatite on the mechanical properties of the cements has not been clarified. The present work aimed to investigate this issue from the viewpoint of the interface structure between the apatite and the cement. The surface of the cement modified with calcium acetate and MPS was fully covered with newly formed apatite after soaking in Kokubo solution within 7 days, while half of the surface area of the cement modified with calcium hydroxide and MPS was covered with the apatite. The bending strength of the modified cements decreased after soaking in Kokubo solution. Porous structure was observed in the region about 50-100 microm in depth from the top surface because of release of the Ca2+ ions by both modified cements after soaking in Kokubo solution. The decrease in bending strength of the modified cements could be attributed to the formation of the pores. In addition, the pores on the top surfaces of the cements were filled with the newly formed apatite. The apatite formation would be effective not only for bioactivity but also for decreasing the reduction of mechanical strength.

The effect of the microstructure of beta-tricalcium phosphate on the metabolism of subsequently formed bone tissue.

The response of bone cells to a newly developed porous beta-tricalcium phosphate composed of rod-shaped particles (RSbeta-TCP), beta-TCP composed of conventional non-rod-shaped particles (Cbeta-TCP), and hydroxyapatite (HA) was analyzed using in vivo implantation and in vitro osteoclastogenesis systems. Implantation of the materials into the rabbit femur showed that RSbeta-TCP and Cbeta-TCP were bioresorbable, but HA was not. Up to 12 weeks after the implantation, bioresorption of RSbeta-TCP and Cbeta-TCP accompanied by the formation of new bone occurred satisfactorily. At 24 weeks post-implantation, most of the RSbeta-TCP had been absorbed, and active osteogenesis was preserved in the region. However, in the specimens implanted with Cbeta-TCP, the amount of not only the implanted Cbeta-TCP but also the newly formed bone tissue decreased, and bone marrow dominated the region. The implanted HA was unbioresorbable throughout the experimental period. When osteoclasts were generated on RSbeta-TCP, Cbeta-TCP, or HA disks, apparent resorption lacunae were formed on the RSbeta-TCP and Cbeta-TCP, but not HA disks. Quantitation of the calcium concentration in the culture media showed an earlier and more constant release of calcium from RSbeta-TCP than Cbeta-TCP. These results showed that the microstructure of beta-TCP affects the activity of bone cells and subsequent bone replacement.