Evaluation of a hydroxyapatite-crosslinked fish gelatin membranes.

Background/purpose: Porcine collagen is widely used in regenerative therapies to generate membranes for bone augmentation. However, porcine or bovine gelatin or collagen is often not appropriate for patients with creed and religious beliefs or for allergic reasons. In this study, we evaluated the potential of fish gelatin to generate membranes. Materials and methods: Fish gelatin and hydroxyapatite (HAp) were used at three different ratios (2:0, 2:1, 2:1.5, and 2:2) to prepare gelatin-hydroxyapatite (G-HAp) membranes via freeze-drying and heat-crosslinking. The surface morphology and cell attachment of G-HAp membranes were observed using scanning electron microscopy and confocal laser microscopy. G-HAp membrane was placed at the bottom of a well plate, and MC3T3-E1 cells were seeded on it. Cell viability and cytotoxicity were tested after 1 and 3 days of culture. Alkaline phosphatase (ALP) and alizarin red staining was performed at 10 and 21 days, respectively. Results: Viability of cells on G-HAp membrane with the gelatin:HAp ratio of 2:1.5 was significantly higher than that on membranes with other gelatin:HAp ratios. ALP and alizarin red staining showed that ALP-positive areas and calcium deposition were the highest on G-HAp membrane with the gelatin:HAp ratio of 2:1. These membranes showed negligible cytotoxicity. Conclusion: Fish-derived G-HAp membranes have the potential to promote osteogenic differentiation of MC3T3-E1 cells with negligible cytotoxicity.

Effects of multiple firings on the translucency, crystalline phase, and mechanical strength of highly translucent zirconia.

This study aimed to clarify the effects of multiple firings on the translucency, crystal structure, and mechanical strength of highly translucent zirconia. Four types of highly translucent zirconia (LAVA Esthetic, LAVA Plus, KATANA Zirconia STML, and KATANA Zirconia HTML) were fired three times at three different temperatures, and the translucency, crystal structure, and flexural strength were evaluated before and after firing. The translucency was statistically compared using repeated-measures analysis of variance; the zirconia phase composition was assessed using X-ray diffraction followed by Rietveld analysis; and the biaxial flexural strength was assessed using Weibull analysis. The translucency of LAVA Esthetic and KATANA Zirconia HTML decreased significantly after firing, and the crystal composition of LAVA Plus and KATANA Zirconia HTML changed after multiple firings, whereas multiple firings did not affect the biaxial flexural strength of any samples. Thus, multiple firings may affect the optical properties of highly translucent zirconia.

Electrochemical Deposition of Copper on Bioactive Porous Titanium Dioxide Layer: Antibacterial and Pro-Osteogenic Activities.

Ti surfaces must exhibit antibacterial activity without cytotoxicity to promote bone reconstruction and prevent infection simultaneously. In this study, we employed a two-step electrochemical treatment process, namely, microarc oxidation (MAO) and cathodic electrochemical deposition (CED), to modify Ti surfaces. During the MAO step, a porous TiO2 (pTiO2) layer with a surface roughness of approximately 2.0 µm was generated on the Ti surface, and in the CED step, Cu was deposited onto the pTiO2 layer on the Ti surface, forming Cu@pTiO2. Cu@pTiO2 exhibited a similar structure, adhesion strength, and crystal phase to pTiO2. Moreover, X-ray photoelectron spectroscopy (XPS) confirmed the presence of Cu in Cu@pTiO2 at an approximate concentration of 1.0 atom %. Cu@pTiO2 demonstrated a sustained release of Cu ions for a minimum of 28 days in a simulated in vivo environment. In vitro experiments revealed that Cu@pTiO2 effectively eradicated approximately 99% of Staphylococcus aureus and Escherichia coli and inhibited biofilm formation, in contrast to the Ti and pTiO2 surfaces. Moreover, Cu@pTiO2 supported the proliferation of osteoblast-like cells at a rate comparable to that observed on the Ti and pTiO2 surfaces. Similar to pTiO2, Cu@pTiO2 promoted the calcification of osteoblast-like cells compared with Ti. In summary, we successfully conferred antibacterial and pro-osteogenic activities to Ti surfaces without inducing cytotoxic effects or structural and mechanical alterations in pTiO2 through the application of MAO and CED processes. Moreover, we found that the pTiO2 layer promoted bacterial growth and biofilm formation more effectively than the Ti surface, highlighting the potential drawbacks of rough and porous surfaces. Our findings provide fundamental insights into the surface design of Ti-based medical devices for bone reconstruction and infection prevention.

Formation of octacalcium phosphate with incorporated dicarboxylate ions containing disulfide bonds.

Octacalcium phosphate (OCP) is a layered compound capable of incorporating carboxylate ions within its interlayer structure. In this study, we successfully synthesised OCP with incorporated 3,3'-dithiodipropionate ions. Our finding is beneficial for the development of novel OCP-based materials with dynamic properties derived from disulfide bonds.

Development of bioinspired damage-tolerant calcium phosphate bulk materials.

Improving the damage tolerance and reliability of ceramic artificial bone materials, such as sintered bodies of hydroxyapatite (HAp), that remain in vivo for long periods of time is of utmost importance. However, the intrinsic brittleness and low damage tolerance of ceramics make this challenging. This paper reports the synthesis of highly damage tolerant calcium phosphate-based materials with a bioinspired design for novel artificial bones. The heat treatment of isophthalate ion-containing octacalcium phosphate compacts in a nitrogen atmosphere at 1000°C for 24 h produced an HAp/ß-tricalcium phosphate/pyrolytic carbon composite with a brick-and-mortar structure (similar to that of the nacreous layer). This composite exhibited excellent damage tolerance, with no brittle fracture upon nailing, likely attributable to the specific mechanical properties derived from its unique microstructure. Its maximum bending stress, maximum bending strain, Young's modulus, and Vickers hardness were 11.7 MPa, 2.8 × 10‒2, 5.3 GPa, and 11.7 kgf/mm2, respectively. The material exhibited a lower Young's modulus and higher fracture strain than that of HAp-sintered bodies and sintered-body samples prepared from pure octacalcium phosphate compacts. Additionally, the apatite-forming ability of the obtained material was confirmed in vitro, using a simulated body fluid. The proposed bioinspired material design could enable the fabrication of highly damage tolerant artificial bones that remain in vivo for long durations of time.

Proliferation and differentiation of MC3T3-E1 cells on polymethyl methacrylate cements containing Fe3O4 and TiO2 for hyperthermic treatment of metastatic bone tumors.

Polymethyl methacrylate (PMMA) bone cement is widely used to relieve pain caused by metastatic bone tumors. We previously found that PMMA bone cement containing 15 mass% or more of TiO2 showed good apatite-forming ability, and 25 mass% or more of Fe3O4 generated sufficient heat for hyperthermia under an alternating current (AC) magnetic field. In this study, the cytocompatibility of PMMA bone cement with Fe3O4:TiO2 weight ratios of 25:15 (F25T15-3/2-42) and 30:15 (F30T15-3/2-42) was evaluated using osteoblastic cells (MC3T3-E1). The proliferation and differentiation of MC3T3-E1 cells were suppressed for F25T15-3/2-42 and F30T15-3/2-42 compared to PMMA bone cement without Fe3O4 and TiO2 (F0T0-3/2-42). The release of methyl methacrylate (MMA) monomers from F25T15-3/2-42 and F30T15-3/2-42 at 7 days was about 33 and 50 times higher than that from F0T0-3/2-42, respectively. The remarkable release of MMA monomers from F25T15-3/2-42 and F30T15-3/2-42 may be responsible for the suppressed proliferation and differentiation of MC3T3-E1 cells. The release of MMA monomers was not reduced when the MMA/PMMA weight ratio was decreased from 3/2 to 1/1, however, it was significantly reduced by increasing the content of benzoyl peroxide (BPO) and N, N-dimethyl-p-toluidine (DMPT) to 8 and 4 mass% against MMA, respectively. Proliferation and differentiation of MC3T3-E1 cells on PMMA-type cements containing Fe3O4 and TiO2 with increased BPO and DMPT contents need to be investigated in the future; however, our findings will be useful for designing PMMA cements for the hyperthermic treatment of metastatic bone tumors.

Visible-Light-Enhanced Antibacterial Activity of Silver and Copper Co-Doped Titania Formed on Titanium via Chemical and Thermal Treatments.

Dental implants made of titanium (Ti) are used in dentistry, but peri-implantitis is a serious associated problem. Antibacterial and osteoconductive Ti dental implants may decrease the risk of peri-implantitis. In this study, titania (TiO2) co-doped with silver (Ag) at 2.5 at.% and copper (Cu) at 4.9 at.% was formed on Ti substrates via chemical and thermal treatments. The Ag and Cu co-doped TiO2 formed apatite in a simulated body fluid, which suggests osteoconductivity. It also showed antibacterial activity against Escherichia coli, which was enhanced by visible-light irradiation. This enhancement might be caused by the synergistic effect of the release of Ag and Cu and the generation of •OH from the sample. Dental implants with such a Ag and Cu co-doped TiO2 formed on their surface may reduce the risk of peri-implantitis.

Octacalcium phosphate with incorporated carboxylate ions: a review.

Octacalcium phosphate (OCP) belongs to a family of calcium phosphate compounds. OCP has unique crystal-chemical properties; among calcium phosphate compounds, only OCP can incorporate carboxylate ions into its crystal lattice. An OCP with incorporated carboxylate ions is called an OCP carboxylate (OCPC). OCPCs are investigated for applications in novel adsorbents, electrochemical devices, and biomaterials. Several wet methods are available for the synthesis of OCPCs, and the characteristics and advantages of each method are explained. Representative characterization methods, i.e. X-ray diffraction and Fourier transform infrared spectroscopy, used for the detection of carboxylate ion incorporation into the OCP interlayers are explained. Various carboxylic acids can be incorporated into OCP, and these types of carboxylic acid are presented with reference to the latest research results. The incorporation of carboxylate ions into OCP represents a modification of the OCP crystal at the molecular level and can impart various functions. Challenging physicochemical and biomaterial applications of OCPCs are thus introduced, although they are still in the research phase. Finally, future perspectives and challenges for OCPC research are described.

Transformation behaviour of salts composed of calcium ions and phosphate esters with different linear alkyl chain structures in a simulated body fluid modified with alkaline phosphatase.

Ceramic biomaterials have been used for the treatment of bone defects and have stimulated intense research on such materials. We have previously reported that a salt composed of calcium ions and a phosphate ester (SCPE) transformed into hydroxyapatite (HAp) in a simulated body fluid (SBF) modified with alkaline phosphatase (ALP), and proposed SCPEs as a new category of ceramic biomaterials, namely bioresponsive ceramics. However, the factors that affect the transformation of SCPEs to HAp in the SBF remained unclear. Therefore, in this study, we investigated the behaviour of calcium salts of methyl phosphate (CaMeP), ethyl phosphate (CaEtP), butyl phosphate (CaBuP), and dodecyl phosphate (CaDoP) in SBF with and without ALP modification. For the standard SBF, an X-ray diffraction (XRD) analysis indicated that these SCPEs did not readily transform into calcium phosphate. However, CaMeP, CaEtP, and CaBuP were transformed into HAp and octacalcium phosphate in the SBF modified with ALP; therefore, these SCPEs can be categorised as bioresponsive ceramics. Although CaDoP did not exhibit a sufficient response to ALP to be detected by XRD, it is likely to be a bioresponsive ceramic based on the results of morphological observations. The transformation rate for the SCPEs decreased with increasing size of the linear alkyl group of the phosphate esters. The rate-determining steps for the transformation reaction of the SCPEs were changed from the dissolution of the SCPEs to the hydrolysis of the phosphate esters with increasing size of the phosphate ester alkyl groups. These findings contribute to designing novel bioresponsive ceramic biomaterials.

Synthesis of Octacalcium Phosphate Containing Glutarate Ions with a High Incorporation Fraction.

Octacalcium phosphate (OCP) has received considerable attention in the field of ceramic biomaterials as an advanced functional material. It exhibits a layered structure composed of apatitic and hydrated layers and can incorporate various dicarboxylate ions into the hydrated layer. Saturated dicarboxylic acids (HOOC(CH2)nCOOH) with an odd number of methylene groups (-CH2-) exhibit lower incorporation fractions than those with an even number of methylene groups, possibly owing to a compositional dependence on the synthetic method. In this study, calcium carbonate, phosphoric acid, and various amounts of glutaric acid were used to produce glutarate-ion-incorporated OCP by a wet chemical method, which is different from the conventional synthetic strategy. While utilising 1-20 mmol of glutaric acid during synthesis did not produce the desired product, using 25 mmol of glutaric acid resulted in the formation of single-phase glutarate-ion-incorporated OCP with a Ca/P molar ratio of 1.57 and a 90% incorporation fraction of glutarate ions. This glutarate-ion-incorporation fraction is significantly higher than that reported in the previous studies (35%). Thus, the synthetic procedure proposed herein was able to produce single-phase OCP containing glutarate ions with a high incorporation fraction. Our findings can contribute to development of novel functional ceramic biomaterials in the future.

Proteomic identification of serum proteins to induce osteoconductivity of hydroxyapatite.

We performed proteomic analysis of rat serum proteins adsorbed on hydroxyapatite (HAp) and α-alumina (α-Al2O3) in order to identify proteins that specifically adsorb onto HAp and control cellular responses. Proteins with either or both molecular weight of 22-32 kDa and computed isoelectric point of 5.0-5.5 were preferentially adsorbed on HAp. In total, 182 proteins were adsorbed on both HAp and α-Al2O3, of which 14 were highly enriched on HAp, whereas 68 were adsorbed only on HAp. Therefore, 82 (14+68) proteins were further evaluated by bioinformatics and literature-based analyses. We predicted that hepatocyte growth factor and angiopoietin-like protein 3 (ANGPTL3) are candidate proteins responsible for the osteoconductivity of HAp. Although ANGPTL3 promoted the attachment and spreading of MC3T3-E1 cells, it did not promote their proliferation and differentiation. Our results suggest that specific adsorption of ANGPTL3 on HAp induced osteoconductivity by enhancing the attachment and spreading of osteoblasts.

Understanding the Steric Structures of Dicarboxylate Ions Incorporated in Octacalcium Phosphate Crystals.

Octacalcium phosphate (OCP) can incorporate various dicarboxylate ions in the interlayer spaces of its layered structure. Although not proven, these incorporated ions are believed to have a linear structure. In this study, the steric structures of twelve different dicarboxylate ions incorporated into OCP were investigated by comparing the experimentally determined interlayer distance of the OCP with the distance estimated using the molecular sizes of dicarboxylic acids calculated by considering their steric structures. The results revealed that the incorporated succinate, glutarate, adipate, pimelate, suberate, and aspartate ions possessed linear structures, whereas the incorporated azelate, sebacate, methylsuccinate, and malate ions exhibited bent structures. Further, the incorporated mercaptosuccinate ions featured linear, bent, other types of structures. Moreover, the steric structure of the incorporated malonate ion significantly differed from those of other dicarboxylate ions. The computational approach employed in this study is expected to deepen our understanding of the steric structures of dicarboxylate ions incorporated in the OCP interlayer spaces.

Time Transient of Calcium and Phosphate Ion Adsorption by Rutile Crystal Facets in Hanks' Solution Characterized by XPS.

For the elucidation of the mechanism of calcium phosphate formation on commercially pure titanium (CP Ti) in the human body, rutile TiO2 single crystal plates with (001), (110), and (111) facets, namely, TiO2(001), TiO2(110), and TiO2(111), and polycrystalline plates (TiO2(poly)) were immersed in a simulated body fluid, Hanks' solution (Hanks), for 100-105 s, and the adsorption of calcium and phosphate ions was precisely characterized employing X-ray photoelectron spectroscopy (XPS). Previously published CP Ti data were used for comparison. Prior to immersion in Hanks, oxygen content was more than twice as high as that of titanium due to the existence of hydroxyl groups and water on the oxides. After immersion in Hanks, the composition and chemical state of the TiO2 substrates remained unchanged. Among the electrolytes contained in Hanks, only calcium and phosphate ions were adsorbed by and incorporated onto TiO2 surfaces. Adsorption of calcium ions onto rutile did not exhibit any systematic increase of calcium with immersion time except TiO2(poly). Adsorption of phosphate ions was initially constant, followed by an increase with the logarithm of immersion time. The adsorption rate of phosphate ions decreased in the following order: TiO2(001), TiO2(poly), TiO2(111), CP Ti, and TiO2(110). The coordination number and band gap of each crystal facet of rutile is important for the adsorption and incorporation of phosphate ions. Regular calcium phosphate formation on CP Ti is possibly enabled by the surface oxide film, which consists chiefly of amorphous TiO2. However, calcium phosphate formation kinetics on CP Ti differed from those on the TiO2 crystalline phase. These findings may further the understanding of CP Ti hard tissue compatibility.

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.

Apatite formation and bacterial growth on raw silk fabric heated in argon gas.

Raw silk has the potential to be a flexible, osteoconductive material because it forms bone-like apatite on its surface in acellular simulated body fluid with ion concentrations nearly 1.5 times greater than that of human plasma (1.5SBF). It has been reported that silk-which has many similarities to raw silk-develops antibacterial properties when heated in inert gas, which may be advantageous in preventing bacterial infection. Hence, raw silk heated in inert gas may be a flexible, osteoconductive material with antibacterial activity. Thus, we examined the effect of the heat treatment of raw silk fabric on its apatite-forming ability in 1.5SBF and on the growth of Escherichia coli. Raw silk fabric was heated in argon gas at several temperatures, to a maximum of 500 °C. The results of soaking tests in 1.5SBF indicate that the apatite-forming ability of raw silk decreases with increasing temperature. This may be because favourable structures for apatite formation, such as carboxyl groups, are thermally decomposed. The results of bacterial tests indicate that raw silk fabrics heated to 300 °C or 500 °C exhibit reduced bacterial growth compared to those that were not heated or were heated only to 100 °C. This might be because hydrophobic surfaces inhibit bacterial adhesion, or because the thermal decomposition of sericin-a component of raw silk-leads to a lack of available nutrients for the bacteria. Although this study did not demonstrate the expected material properties needed for clinical applications, this research contributes to a better understanding of silk biomaterials.

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.

Density functional study of the phase stability and Raman spectra of Yb2O3, Yb2SiO5 and Yb2Si2O7 under pressure.

The phase stability and Raman spectra of Yb2O3, Yb2SiO5 and Yb2Si2O7 under hydrostatic pressure are investigated using density functional theory calculations. The calculated energies of polymorphs of each compound show that the stable phases at zero pressure, viz., C-type Yb2O3, X2-Yb2SiO5 and ß-Yb2Si2O7, exhibit a pressure-induced phase transition as compressive pressure increases, which is consistent with available experimental data. The theoretical Raman spectra at zero pressure are in good agreement with experimental results for the stable phases and can be used to identify each polymorph. Although the calculated pressure dependence of Raman peak positions of C-type Yb2O3 is overestimated compared to available experimental data, piezospectroscopic coefficients extracted from Raman peaks of X2-Yb2SiO5 and ß-Yb2Si2O7 suggest that Raman spectroscopy can be used to measure stresses and strains in Yb silicates. Normal mode analyses reveal that characteristic Raman peaks of Yb silicates at frequencies above 600 cm-1 are strongly associated with vibrations of Si-O bonds in SixOy tetrahedral units.

Enantioselective incorporation of dicarboxylate guests by octacalcium phosphate.

Enantioselectivity by octacalcium phosphate (OCP) is revealed through the incorporation of (S)-(-)-methylsuccinic acid (MeSuc) into its crystal lattice, with hardly any (R)-(+)-MeSuc incorporated. This phenomenon clearly indicates that OCP recognizes the steric structures of guest molecules, extending chiral recognition in inorganic materials to three-dimensional crystal growth.

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.

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.