Spherical porous hydroxyapatite granules containing composites of magnetic and hydroxyapatite nanoparticles for the hyperthermia treatment of bone tumor.

Spherical porous granules of hydroxyapatite (HA) containing magnetic nanoparticles would be suitable for the hyperthermia treatment of bone tumor, because porous HA granules act as a scaffold for bone regeneration, and magnetic nanoparticles generate sufficient heat to kill tumor cells under an alternating magnetic field. Although magnetic nanoparticles are promising heat generators, their small size makes them difficult to support in porous HA ceramics. We prepared micrometer-sized composites of magnetic and HA nanoparticles, and then supported them in porous HA granules composed of rod-like particles. The spherical porous HA granules containing the composites of magnetic and HA nanoparticle were successfully prepared using a hydrothermal process without changing the crystalline phase and heat generation properties of the magnetic nanoparticles. The obtained granules generated sufficient heat for killing tumor cells under an alternating magnetic field (300 Oe at 100 kHz). The obtained granules are expected to be useful for the hyperthermia treatment of bone tumors.

Effect of silicate incorporation on in vivo responses of α-tricalcium phosphate ceramics.

In addition to calcium phosphate-based ceramics, glass-based materials have been utilized as bone substitutes, and silicate in these materials has been suggested to contribute to their ability to stimulate bone repair. In this study, a silicate-containing α-tricalcium phosphate (α-TCP) ceramic was prepared using a wet chemical process. Porous granules composed of silicate-containing α-TCP, for which the starting composition had a molar ratio of 0.05 for Si/(P + Si), and silicate-free α-TCP were prepared and evaluated in vivo. When implanted into bone defects that were created in rat femurs, α-TCP ceramics either with or without silicate were biodegraded, generating a hybrid tissue composed of residual ceramic granules and newly formed bone, which had a tissue architecture similar to physiological trabecular structures, and aided regeneration of the bone defects. Supplementation with silicate significantly promoted osteogenesis and delayed biodegradation of α-TCP. These results suggest that silicate-containing α-TCP is advantageous for initial skeletal fixation and wound regeneration in bone repair.

A bone substitute with high affinity for vitamin D-binding protein--relationship with niche of osteoclasts.

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism.

Behavior of osteoblast-like cells on calcium-deficient hydroxyapatite ceramics composed of particles with different shapes and sizes.

In designing the biomaterials, it is important to control their surface morphologies, because they affect the interactions between the materials and cells. We previously reported that porous calcium-deficient hydroxyapatite (HA) ceramics composed of rod-like particles had advantages over sintered porous HA ceramics; however, the effects of the surface morphology of calcium-deficient HA ceramics on cell behavior have remained unclear. Using a hydrothermal process, we successfully prepared porous calcium-deficient HA ceramics with different surface morphologies, composed of plate-like particles of 200-300, 500-800 nm, or 2-3 µm in width and rod-like particles of 1 or 3-5 µm in width, respectively. The effects of these surface morphologies on the behavior of osteoblast-like cells were examined. Although the numbers of cells adhered to the ceramic specimens did not differ significantly among the specimens, the proliferation rates of cells on the ceramics decreased with decreasing particle size. Our results reveal that controlling the surface morphology that is governed by particle shape and size is important for designing porous calcium-deficient HA ceramics.

Enhancement of aragonite mineralization with a chelating agent for CO2 storage and utilization at low to moderate temperatures.

Among the CaCO3 polymorphs, aragonite demonstrates a better performance as a filler material in the paper and plastic industries. Despite being ideal from the environmental protection perspective, the production of aragonite particles via CO2 mineralization of rocks is hindered by the difficulty in achieving high production efficiencies and purities, which, however, can be mitigated by exploiting the potential ability of chelating agents on metal ions extraction and carbonation controlling. Herein, chelating agent N,N-dicarboxymethyl glutamic acid (GLDA) was used to enhance the extraction of Ca from calcium silicate and facilitate the production of aragonite particles during the subsequent Ca carbonation. CO2 mineralization was promoted in the presence of 0.01-0.1 M GLDA at ≤ 80 °C, with the maximal CaCO3 production efficiency reached 308 g/kg of calcium silicate in 60 min using 0.03 M GLDA, which is 15.5 times higher than that without GLDA. In addition, GLDA showed excellent effects on promoting aragonite precipitation, e.g., the content of aragonite was only 5.1% in the absence of GLDA at 50 °C, whereas highly pure (> 90%, increased by a factor of 18) and morphologically uniform aragonite was obtained using ≥ 0.05 M GLDA under identical conditions. Aragonite particle morphologies could also be controlled by varying the GLDA concentration and carbonation temperature. This study proposed a carbon-negative aragonite production method, demonstrated the possibility of enhanced and controlled aragonite particle production during the CO2 mineralization of calcium silicates in the presence of chelating agents.

Incorporation of tetracarboxylate ions into octacalcium phosphate for the development of next-generation biofriendly materials.

Octacalcium phosphate (OCP; Ca8(HPO4)2(PO4)4 ∙ 5H2O) is a precursor of hydroxyapatite found in human bones and teeth, and is among the inorganic substances critical for hard tissue formation and regeneration in the human body. OCP has a layered structure and can incorporate carboxylate ions into its interlayers. However, studies involving the incorporation of tetracarboxylic and multivalent (pentavalent and above) carboxylic acids into OCP have not yet been reported. In this study, we investigate the incorporation of pyromellitic acid (1,2,4,5-benzenetetracarboxylic acid), a type of tetracarboxylic acid, into OCP. We established that pyromellitate ions could be incorporated into OCP by a wet chemical method using an acetate buffer solution containing pyromellitic acid. The derived OCP showed a brilliant blue emission under UV light owing to the incorporated pyromellitate ions. Incorporation of a carboxylic acid into OCP imparted new functions, which could enable the development of novel functional materials for biomedical applications.

In vitro apatite formation on organic-inorganic hybrids in the CaO-SiO(2)-PO (5/2)-poly(tetramethylene oxide) system.

The osteoconduction potential of artificial materials is usually evaluated in vitro by apatite formation in a simulated body fluid (SBF) proposed by Kokubo and his colleagues. This paper reports the compositional dependence of apatite formation on organic-inorganic hybrids in the CaO-SiO(2)-PO(5/2)-poly(tetramethylene oxide) system, initiated from tetraethoxysilane (TEOS), triethyl phosphate (OP(OEt)(3)), calcium chloride (CaCl(2)) and poly(tetramethylene oxide)(PTMO) modified with alkoxysilane. Formation of an apatite layer was observed on the surface of the organic-inorganic hybrids with molar ratios of TEOS/OP(OEt)(3) ranging from 100/0 to 20/80. The rate of apatite formation remarkably decreased when the hybrids were synthesized with TEOS/OP(OEt)(3) ratios of 40/60 or less. Hybrids without TEOS showed no apatite formation in SBF for up to 14 days. Addition of small amounts of OP(OEt)(3) to TEOS in the hybrids led to the high dissolution of calcium and silicate, while addition of large amounts of OP(OEt)(3) decreased the dissolution of calcium and silicate ions and resulted in reduced apatite formation regardless of the dissolution of phosphate ions from the hybrids.

Effects of surface carboxylic acid groups of cerasomes, morphologically stable vesicles having a silica surface, on biomimetic deposition of hydroxyapatite in body fluid conditions.

Biomimetic mineralization of supramolecular scaffolds consisting of biomolecules or their analogues has received much attention recently from the viewpoint of creation of novel biomaterials. This study investigated biomimetic deposition of hydroxyapatite (HAp) on cerasomes, morphologically stable organic-inorganic hybrid vesicles. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction studies revealed that the pristine cerasomes induced heterogeneous nucleation of HAp when they were immersed in 1.5SBF, a solution having 1.5 times higher ion concentration than that of a simulated body fluid (SBF). The HAp deposition was further accelerated when dicarboxylic and monocarboxylic acid groups were displayed on cerasome surfaces. These carboxylic acid groups are expected to enhance calcium ion binding to the cerasome surface, causing an increase of HAp nucleation sites. At lower surface concentrations on the cerasome surface, the dicarboxylic acid group is apparently more effective for HAp deposition than the monocarboxylic acid group. The resultant HAp-cerasome hybrids are useful as biocompatible materials having unique properties deriving from the lipid bilayer structure of the cerasomes.

Hydroxyapatite formation on porous ceramics of alpha-tricalcium phosphate in a simulated body fluid.

Alpha-tricalcium phosphate (alpha-TCP) ceramic is a bioresorbable material that degrades in bone tissue after implantation, since it exhibits higher solubility than beta-tricalcium phosphate (beta-TCP) ceramics. The high solubility of alpha-TCP in an aqueous solution causes its transformation into hydroxyapatite (HAp) through hydrolysis. While one expects the formation of hydroxyapatite after exposure to an aqueous solution mimicking a body environment, we occasionally find variation in HAp formation in the simulated body fluid (SBF). In the present study, HAp formation resulting from exposure to SBF was investigated for some types of alpha-TCP ceramics with different porosities and specific surface area. Reduced porosity and large surface area of porous specimens may increase the local density of Ca2+ in the surrounding SBF to increase the degree of supersaturation with respect to HAp. Thus, the porosity and specific surface area are significant parameters for determining not only bioabsorbability but also the ability to form HAp.

Effect of preparation conditions on the properties of bioactive glasses for testing SBF.

A simulated body fluid (SBF) with ion concentrations similar to body fluid, proposed by Kokubo et al., is widely used to evaluate bone-bonding potential through the formation of an apatite layer. To be confident of the evaluation of the potential for the apatite formation in SBF, standard substrates are required. Although Na(2)O-CaO-SiO(2) glasses have been focused upon as candidate standard substrates, it has not been clarified whether the preparation conditions of the glasses affect their apatite formation potential in SBF. In this study, Na(2)O-CaO-SiO(2) glasses were prepared by a conventional melting-quenching method with different melting periods and annealing processes to examine their properties, including apatite formation in SBF. The Na(2)O-CaO-SiO(2) glasses show reproducible apatite-forming ability when prepared using moderate melting and annealing processes, and can be useful substrates to test the reproducibility of SBF.

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.

Adhesion Behavior of Microorganisms Isolated from Soil on Hydroxyapatite and Other Materials.

The adhesion behavior of microorganisms on different materials was examined to obtain basic knowledge for designing support materials for microorganisms. The microorganisms were isolated from soil, and their adhesion behavior on hydroxyapatite (HA), carbon-coated HA (Carbon), poly (vinyl chloride) (PVC), and polyurethane (PU) pellets was investigated. The total metabolic activity on and adherence of microorganisms to the tested materials were in the following order: HA > Carbon > PVC > PU. This order was consistent with the extent of hydrophilicity of the materials. Morphological examination and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) analysis of microorganisms adhered to the materials revealed that the activities and states of microorganisms were affected by the composition of the pellets. PCR-DGGE analysis revealed various species of microorganisms adhered to the HA pellet. HA ceramics are expected to be one of the most suitable materials for supporting microorganisms.

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.

Review paper: behavior of ceramic biomaterials derived from tricalcium phosphate in physiological condition.

Various calcium phosphates are used for bone repair. Although hydroxyapatite (HA) sintered ceramics are widely used due to their osteoconductivity, its bioresorbability is so low that HA remains in the body for a long time after implantation. In contrast, tricalcium phosphate (TCP) ceramics show resorbable characters during bone regeneration, and can be completely substituted for the bone tissue after stimulation of bone formation. Therefore, much attention is paid to TCP ceramics for scaffold materials for supporting bone regeneration. This paper reviews bioresorbable properties of calcium phosphate ceramics derived from beta-TCP and alpha-TCP.

Adhesion behaviors of Escherichia coli on hydroxyapatite.

Optimum design of support materials for microorganisms is required for the construction of bioreactors. However, the effects of support materials on microorganisms are still unclear. In this study, we investigated the adhesion behavior of Escherichia coli (E. coli) on hydroxyapatite (HA), polyurethane (PU), poly(vinyl chloride) (PVC), and carbon (Carbon) to obtain basic knowledge for the design of support materials. The total metabolic activity and number of E. coli adhering on the samples followed the order of HA ≈ Carbon>PVC>PU. On the other hand, the water contact angle of the pellet surfaces followed the order of HA

Diversity of multinucleated giant cells by microstructures of hydroxyapatite and plasma components in extraskeletal implantation model.

UNLABELLED: Foreign body giant cells (FBGCs) and osteoclasts are multinucleated giant cells (MNGCs), both of which are formed by the fusion of macrophage-derived mononuclear cells. Osteoclasts are distinct from FBGCs due to their bone resorption ability; however, not only morphological, but also functional similarities may exist between these cells. The characterization and diversity of FBGCs that appear in an in vivo foreign body reaction currently remain incomplete. In the present study, we investigated an in vivo foreign body reaction using an extraskeletal implantation model of hydroxyapatite (HA) with different microstructures. The implantation of HA granules in rat subcutaneous tissue induced a foreign body reaction that was accompanied by various MNGCs. HA granules composed of rod-shaped particles predominantly induced cathepsin K (CTSK)-positive FBGCs, whereas HA granules composed of globular-shaped particles predominantly induced CTSK-negative FBGCs. Plasma, which was used as the binder of ceramic granules, stimulated the induction of CTSK-positive FBGCs more strongly than purified fibrin. Furthermore, the implantation of HA composed of rod-shaped particles with plasma induced tartrate-resistant acid phosphatase (TRAP)-positive MNGCs in contrast to HA composed of globular-shaped particles with purified fibrin, which predominantly induced CTSK-negative and TRAP-negative typical FBGCs. These results suggest that CTSK-positive, TRAP-positive, and CTSK- and TRAP-negative MNGCs are induced in this subcutaneous implantation model in a manner that is dependent on the microstructure of HA and presence or absence of plasma. STATEMENT OF SIGNIFICANCE: We attempted to elucidate the mechanisms responsible for the foreign body reaction induced by the implantation of hydroxyapatite granules with different microstructures in rat subcutaneous tissue with or without plasma components as the binder of ceramic granules. By analyzing the expression of two reliable osteoclast markers, we detected tartrate-resistant acid phosphatase-positive multinucleated giant cells, cathepsin K-positive multinucleated giant cells, and tartrate-resistant acid phosphatase- and cathepsin K-negative multinucleated giant cells. The induction of tartrate-resistant acid phosphatase-positive multinucleated giant cells was plasma component-dependent while the induction of cathepsin K-positive multinucleated giant cells was influenced by the microstructure of hydroxyapatite. This is the first study to show the conditions dividing the three kinds of multinucleated giant cells in the foreign body reaction.

Regulation and Biological Significance of Formation of Osteoclasts and Foreign Body Giant Cells in an Extraskeletal Implantation Model.

The implantation of biomaterials induces a granulomatous reaction accompanied by foreign body giant cells (FBGCs). The characterization of multinucleated giant cells (MNGCs) around bone substitutes implanted in bone defects is more complicated because of healing with bone admixed with residual bone substitutes and their hybrid, and the appearance of two kinds of MNGCs, osteoclasts and FBGCs. Furthermore, the clinical significance of osteoclasts and FBGCs in the healing of implanted regions remains unclear. The aim of the present study was to characterize MNGCs around bone substitutes using an extraskeletal implantation model and evaluate the clinical significance of osteoclasts and FBGCs. Beta-tricalcium phosphate (ß-TCP) granules were implanted into rat subcutaneous tissue with or without bone marrow mesenchymal cells (BMMCs), which include osteogenic progenitor cells. We also compared the biological significance of plasma and purified fibrin, which were used as binders for implants. Twelve weeks after implantation, osteogenesis was only detected in specimens implanted with BMMCs. The expression of two typical osteoclast markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin-K (CTSK), was analyzed, and TRAP-positive and CTSK-positive osteoclasts were only detected beside bone. In contrast, most of the MNGCs in specimens without the implantation of BMMCs were FBGCs that were negative for TRAP, whereas the degradation of ß-TCP was detected. In the region implanted with ß-TCP granules with plasma, FBGCs tested positive for CTSK, and when ß-TCP granules were implanted with purified fibrin, FBGCs tested negative for CTSK. These results showed that osteogenesis was essential to osteoclastogenesis, two kinds of FBGCs, CTSK-positive and CTSK-negative, were induced, and the expression of CTSK was plasma-dependent. In addition, the implantation of BMMCs was suggested to contribute to osteogenesis and the replacement of implanted ß-TCP granules to bone.

Heterogeneous nucleation of hydroxyapatite on protein: structural effect of silk sericin.

Acidic proteins play an important role during mineral formation in biological systems, but the mechanism of mineral formation is far from understood. In this paper, we report on the relationship between the structure of a protein and hydroxyapatite deposition under biomimetic conditions. Sericin, a type of silk protein, was adopted as a suitable protein for studying structural effect on hydroxyapatite deposition, since it forms a hydroxyapatite layer on its surface in a metastable calcium phosphate solution, and its structure has been reported. Sericin effectively induced hydroxyapatite nucleation when it has high molecular weight and a beta sheet structure. This indicates that the specific structure of a protein can effectively induce heterogeneous nucleation of hydroxyapatite in a biomimetic solution, i.e. a metastable calcium phosphate solution. This finding is useful in understanding biomineralization, as well as for the design of organic polymers that can effectively induce hydroxyapatite nucleation.

A composite of hydroxyapatite with electrospun biodegradable nanofibers as a tissue engineering material.

Biodegradable and biocompatible poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a copolymer of microbial polyester, was fabricated as a nanofibrous film by electrospinning and composited with hydroxyapatite (HAp) by soaking in simulated body fluid. Compared with a PHBV cast (flat) film, the electrospun PHBV nanofibrous film was hydrophobic. However, after HAp deposition, both of the surfaces were extremely hydrophilic. The degradation rate of HAp/PHBV nanofibrous films in the presence of polyhydroxybutyrate depolymerase was very fast. Nanofiber formation increased the specific surface area and HAp enhanced the invasion of enzyme into the film by increasing surface hydrophilicity. The surface of the nanofibrous film showed enhanced cell adhesion over that of the flat film, although cell adhesion was not significantly affected by the combination with HAp.

Continuous expansion of the interplanar spacing of octacalcium phosphate by incorporation of dicarboxylate ions with a side chain.

Octacalcium phosphate (OCP) is composed of apatitic and hydrated layers, and can incorporate dicarboxylate ions in its hydrated layers by substitution of HPO4(2-). The (100) interplanar spacing of OCP is increased by incorporation of dicarboxylate ions. Herein, we report continuous expansion of the interplanar spacing of OCP by incorporation of dicarboxylate ions with a side chain. We synthesized OCP with incorporated succinic acid (Suc) and mercaptosuccinic acid (Msuc) by a wet chemical process. The (100) interplanar spacing of OCP synthesized with Suc increased non-continuously as the amount of Suc used in the synthesis increased. In contrast, the (100) interplanar spacing of OCP synthesized with Msuc increased continuously with the amount of Msuc used during synthesis, and thus in the OCP. The values of the (100) interplanar spacing of OCP synthesized with Msuc first increased and then became constant as the amount of Msuc increased. OCP with incorporated Msuc formed a continuous solid solution. The expansion of the (100) interplanar spacing of OCP with incorporated Suc and Msuc depended on the chemical structure of the incorporated dicarboxylate ions. The only structural difference between Suc and Msuc is a mercapto (R-SH) side chain. Therefore, continuous solid solution formation was most likely induced by the R-SH side chain of Msuc. These results suggest that incorporation of dicarboxylic acids with a side chain is one approach to obtain OCP with arbitrary interlayer distance.