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.

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.

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.

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.

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

Importance of nucleation in transformation of octacalcium phosphate to hydroxyapatite.

Octacalcium phosphate (OCP) is regarded as an in vivo precursor of hydroxyapatite (HA). It is important to understand the mechanism of transformation of OCP to HA in order to reveal the mechanism of mineralization and help in the development of artificial bone-repairing materials. Herein, we have examined the behavior of OCP in a simulated body fluid (SBF) and pure water. The OCP particles immersed in the SBF at 37 °C did not transform to HA even after 720 h of immersion, though the particles showed crystal growth. In distilled water at 60 °C, the OCP particles transformed to HA but the unreactive period was observed. Although the immersed solution became supersaturated with HA within 12h of immersion, the OCP was not transformed in the first 36 h of immersion. These results indicate that the nucleation of HA is the rate-determining step in the transformation of OCP to HA.

Promotion of normal healing of bone defects under estrogen deficiency by implantation of beta-tricalcium phosphate composed of rod-shaped particles.

We compared the healing of bone defects in ovariectomized rats implanted with beta-tricalcium phosphate (b-TCP)composed of rod-shaped particles, which were prepared using the applied hydrothermal method (HTCP), and that of bone defects implanted with conventional b-TCP composed of globular-shaped particles (CTCP), which were prepared by normal sintering. Eight week-old female Wistar rats were ovariectomized, and 2 weeks after the operation, 0.5- to 0.6-mm diameter spherical granules of each ceramic were implanted in a bone defect created in the distal end of the femur. Four, 8, and 12 weeks after implantation, the amount of newly formed bone implanted with HTCP was significantly larger than that implanted with CTCP and was equivalent to that in non-ovariectomized sham-operated rats. Without implantation, spontaneous repair of the trabecular bone was barely observed. The physiological structure of the trabecular network was maintained in the region implanted with HTCP, but that in the region implanted with CTCP was severely destroyed. Gene expression microarray analysis revealed that the expression of genes involved in interferon signaling pathways was upregulated in osteoclasts cultured on HTCP compared with that cultured on CTCP. Our results suggest that the microstructure of b-TCP affected the biological behavior of osteoclasts and regulated 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.

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.

Preparation and evaluation of spherical Ca-deficient hydroxyapatite granules with controlled surface microstructure as drug carriers.

Spherical Ca-deficient hydroxyapatite (HA) granules are expected to be useful drug carriers in bony sites because of their bone regeneration and adsorption ability. In order to control drug loading and release ability of the granules, a controlled surface microstructure was constructed. Spherical Ca-deficient granules composed of micron-sized rod-shaped particles were prepared by hydrothermal treatment of α-tricalcium phosphate (α-TCP) granules, and then, submicron HA particles were precipitated on the obtained granules by immersion in a supersaturated calcium phosphate (CP) solution. When bovine serum albumin was used as a drug model, precipitation of submicron particles causes the loading capability to increase and the release rate to decrease. The spherical Ca-deficient HA granules with the controlled surface microstructure are expected to be useful drug carriers that can act as scaffolds for bone repair.

Stimulation of Osteogenesis in Bone Defects Implanted with Biodegradable Hydroxyapatite Composed of Rod-Shaped Particles under Mechanical Unloading.

The aim of this study was to evaluate the influence of mechanical unloading on the repair of bone defects with implantation of biodegradable bone substitutes. Spherical granules of biodegradable hydroxyapatite composed of rod-shaped particles (RHA) or beta-tricalcium phosphate composed of rod-shaped particles (RTCP) were implanted into a bone defect created in the distal end of the right femur of 8-week-old Wistar rats. Two, 6, 10, and 22 weeks after implantation, part of the sciatic nerve in the thigh was resected and exposed to mechanical unloading for 2 weeks. Then, 4, 8, 12 and 24 weeks after implantation, repair of the bone defect was analyzed. As a control, the bone defect without implantation of ceramic granules was also analyzed. Both RHA and RTCP tended to be reduced, but the reduction was not obvious during the experimental period. At 12 and 24 weeks after implantation, the amount of newly formed bone in the animal implanted with RHA was significantly greater than that at 4 weeks after implantation, but that in the animal implanted with RTCP or without implantation was not significantly different. The number of osteoclasts in the region implanted with RHA was significantly larger than that of the region implanted with RTCP or without implantation at 12 and 24 weeks. The activities of alkaline phosphatase in osteoblasts and tartrate-resistant acid phosphatase in osteoclasts were remarkably increased in the bone defects with implantation compared with those in the bone defects without implantation. These results suggested that RHA stimulated osteogenesis and osteoclastogenesis even after 2 weeks of mechanical unloading, and that RHA could be expected to improve the repair of bone defects in patients under the condition of skeletal unloading.

Synthesis of nanosized porous hydroxyapatite granules in hydrogel by electrophoresis.

Nanosized porous hydroxyapatite (HA) granules are expected to be useful as DNA and drug carriers for cells. We attempted to synthesize nanosized HA granules in an agarose gel by electrophoresis of calcium and phosphate ions. Wells were prepared on an agarose gel, and a CaCl(2) solution was placed in the wells on the positive side and a Na(2)HPO(4) solution was placed in the wells on the negative side. After a potential was applied, white precipitates appeared in the gel. The precipitates were calcium-deficient HA granules of approximately 300-600 nm in diameter. These granules were composed of fiber-like particles and had a porous structure. The diameters of the granules were decreased by an increase in the gel concentration. Hence, we successfully synthesized nanosized porous HA granules to be used as DNA and drug carriers.

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.

Preparation and biological evaluation of a fibroblast growth factor-2-apatite composite layer on polymeric material.

A polymeric percutaneous device with good biocompatibility and resistance to bacterial infection is required clinically. In this study, a fibroblast growth factor-2 (FGF-2)-hydroxyapatite (HAp) composite layer (FHAp layer) was formed on the surfaces of ethylene-vinyl alcohol copolymer (EVOH) specimens using a coating process in a supersaturated calcium phosphate solution supplemented with FGF-2. FGF-2 in the FHAp layer retained its biological activity to promote proliferation of fibroblasts. The EVOH specimens coated with HAp and FHAp layers were percutaneously implanted in the scalp of rats. Not only the HAp layer but also the FHAp layer showed good biocompatibility, and FGF-2 showed no harmful effects on the skin tissue responses to the implanted specimen as long as 14 d. No significantly higher infection resistance was verified for the FHAp layer over the HAp layer, although an FHAp layer coated on a metallic percutaneous device for bone fixation demonstrated higher resistance to bacterial infection over an HAp layer in the previous study. The efficacy of FHAp layers coated on percutaneous implants in resistance to bacterial infection depends on physical factors including fixation condition, stiffness and movement of implants.

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.

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.

Observation and quantitative analysis of rat bone marrow stromal cells cultured in vitro on newly formed transparent beta-tricalcium phosphate.

To observe living cell morphology on ceramics by light microscopy, we fabricated a new material-transparent beta - tricalcium phosphate (t-beta TCP) ceramic-for the purpose of serving as a tissue culture substrate. Bone marrow stromal cells (BMSCs) were obtained from rat femora and cultured on both t-beta TCP ceramic disks and culture grade polystyrene (PS) dishes in an osteogenic medium. After 1 day of culture, cell attachment and spreading on both the t-beta TCP and PS substrata were equally and clearly detected by ordinary light microscopy. After 14 days of culture, extensive cell growth, alkaline phosphatase (ALP) staining, and bone mineral deposition could be detected on both substrata. In addition, quantitative biochemical analyses revealed high DNA content, ALP activity, and osteocalcin content of these cultures. This experiment is significant in that all of the results were similarly observed on both the t-beta TCP and PS substrata, indicating the excellent properties of beta TCP ceramics for BMSCs culture towards osteogenic differentiation.

Observation of osteogenic differentiation cascade of living mesenchymal stem cells on transparent hydroxyapatite ceramics.

The use of bioceramics and cultured cells for tissue engineering is a novel approach, which is available in a wide variety of clinical situations. The approach requires apparent verification of the cellular functions occurring on the ceramic surface, and these functions could be monitored by microscopic observation of the cultured living cells on the ceramic material. However, such observation is difficult due to the opaque nature of ordinary ceramics. To overcome this drawback, we used transparent hydroxyapatite (tHA) ceramics as a culture substrate and a transgenic rat having an enhanced green fluorescent protein (EGFP)-expressing gene as the cell source. Marrow mesenchymal stem cells (MSC) were obtained from the rat and cultured on both tHA ceramics and a tissue culture polystyrene (TCPS) dish. One hour after the cell seeding, many MSC had attached and showed initial cell spreading. The attachment and spreading were more obvious 5h after the seeding. Following the culture in the osteogenic condition, the cells differentiated into osteoblasts, which fabricated bone matrix on the culture substrate. The phenomena were similarly observed on both the tHA ceramics and TCPS substrata. These results confirm the excellent properties of tHA ceramics, which support cell attachment, proliferation, and differentiation. Transparent materials make us know the biological usefulness of ceramics in tissue-engineering field.