Self-Prepared Hyaluronic Acid/Alkaline Gelatin Composite with Nano-Hydroxyapatite and Bone Morphogenetic Protein for Cranial Bone Formation.

New bone-forming substitute materials are highly useful in dental implantology. The purpose of this study was to prepare cross-linked hyaluronic acid (cHLA)/cross-linked alkaline gelatin (cAG)/nano-hydroxyapatite (nHAp)/bone morphogenic protein (BMP) constructs; and evaluate their bone-forming capabilities in rat cranial bone defects. The cHLA and cAG liquids processed with an epoxy cross-linker were blended with a 3:1 volume ratio, followed by freeze-drying. The dry composites were further infiltrated with water containing nHAp only (BMP (−)) or with water containing nHAp and BMP (BMP (+)). Prepared wet constructs (BMP (−) and BMP (+)) were implanted in rat cranial bone defects, while defects only were also made, and animals were fed for 8 weeks, followed by subsequent soft X-ray measurements and histological observations. The X-ray results showed that BMP (+) constructs disappeared, though caused inward extension of peripherical bone from defect edges with an increase in length of approximately 24%, larger than those of BMP (−) constructs and defect only with approximately 17% and 8% increments, respectively (p < 0.05). Histological observations of BMP (+) construct samples clearly indicated active bone extension consisting of an array of island-like bones. It was concluded that cHLA/cAG/nHAp/BMP could be used as novel bone-substitute materials.

Bone Formation on Murine Cranial Bone by Injectable Cross-Linked Hyaluronic Acid Containing Nano-Hydroxyapatite and Bone Morphogenetic Protein.

New injection-type bone-forming materials are desired in dental implantology. In this study, we added nano-hydroxyapatite (nHAp) and bone morphogenetic protein (BMP) to cross-linkable thiol-modified hyaluronic acid (tHyA) and evaluated its usefulness as an osteoinductive injectable material using an animal model. The sol (ux-tHyA) was changed to a gel (x-tHyA) by mixing with a cross-linker. We prepared two sol−gel (SG) material series, that is, x-tHyA + BMP with and without nHAp (SG I) and x-tHyA + nHAp with and without BMP (SG II). SG I materials in the sol stage were injected into the cranial subcutaneous connective tissues of mice, followed by in vivo gelation, while SG II materials gelled in Teflon rings were surgically placed directly on the cranial bones of rats. The animals were sacrificed 8 weeks after implantation, followed by X-ray analysis and histological examination. The results revealed that bone formation occurred at a high rate (>70%), mainly as ectopic bone in the SG I tests in mouse cranial connective tissues, and largely as bone augmentation in rat cranial bones in the SG II experiments when x-tHyA contained both nHAp and BMP. The prepared x-tHyA + nHAp + BMP SG material can be used as an injection-type osteoinductive bone-forming material. Sub-periosteum injection was expected.

Preparation of Collagen/Hydroxyapatite Composites Using the Alternate Immersion Method and Evaluation of the Cranial Bone-Forming Capability of Composites Complexed with Acidic Gelatin and b-FGF.

Bone-substitute materials are essential in dental implantology. We prepared collagen (Col)/hydroxyapatite (Hap)/acidic gelatin (AG)/basic fibroblast growth factor (b-FGF) constructs with enhanced bone-forming capability. The Col/Hap apatite composites were prepared by immersing Col sponges alternately in calcium and phosphate ion solutions five times, for 20 and 60 min, respectively. Then, the sponges were heated to 56 °C for 48 h. Scanning electron microscopy/energy-dispersive X-ray spectroscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction analyses showed that the Col/Hap composites contained poorly crystalline Hap precipitates on the Col matrix. Col/Hap composite granules were infiltrated by AG, freeze-dried, and immersed in b-FGF solution. The wet quaternary constructs were implanted in rat cranial bone defects for 8 weeks, followed by soft X-ray measurements and histological analysis. Animal studies have shown that the constructs moderately increase bone formation in cranial bone defects. We found that an alternate immersion time of 20 min led to the greatest bone formation (p < 0.05). Constructs placed inside defects slightly extend the preexisting bone from the defect edges and lead to the formation of small island-like bones inside the defect, followed by disappearance of the constructs. The combined use of Col, Hap, AG, and b-FGF might bring about novel bone-forming biomaterials.

Bone Regeneration of Critical-Size Calvarial Defects in Rats Using Highly Pressed Nano-Apatite/Collagen Composites.

Osteo-conductive bone substitute materials are required in dentistry. In this study, highly pressed nano-hydroxyapatite/collagen (P-nHAP/COL) composites were formed by a hydraulic press. Critical-size bone defects (Φ = 6 mm) were made in the cranial bones of 10-week-old Wistar rats, in which P-nHAP/COL and pressed collagen (P-COL) specimens were implanted. Defect-only samples (DEF) were also prepared. After the rats had been nourished for 3 days, 4 weeks, or 8 weeks, ossification of the cranial defects of the rats was evaluated by micro-computed tomography (micro-CT) (n = 6 each). Animals were sacrificed at 8 weeks, followed by histological examination. On micro-CT, the opacity of the defect significantly increased with time after P-nHAP/COL implantation (between 3 days and 8 weeks, p < 0.05) due to active bone regeneration. In contrast, with P-COL and DEF, the opacity increased only slightly with time after implantation, indicating sluggish bone regeneration. Histological inspections of the defect zone implanted with P-nHAP/COL indicated the adherence of multinucleated giant cells (osteoclasts) to the implant with phagocytosis and fragmentation of P-nHAP/COL, whereas active bone formation occurred nearby. Fluorescent double staining indicated dynamic bone-formation activities. P-nHAP/COL is strongly osteo-conductive and could serve as a useful novel bone substitute material for future dental implant treatments.

Characterization of Five Collagenous Biomaterials by SEM Observations, TG-DTA, Collagenase Dissolution Tests and Subcutaneous Implantation Tests.

Collagenous biomaterials that are clinically applied in dentistry have dermis-type and membrane-type, both of which are materials for promoting bone and soft tissue formation. The properties of materials supplied with different types could affect their biodegradation periods. The purpose of this study was to characterize five of these products by four different methods: scanning electron microscopy (SEM) observation, thermogravimetry-differential thermal analysis (TG-DTA), 0.01 wt% collagenase dissolution test, and subcutaneous implantation test in vivo. SEM micrographs revealed that both dermis and membranous materials were fibrous and porous. The membranous materials had higher specific derivative thermal gravimetry (DTG) peak temperatures in TG-DTA at around 320 °C, longer collagenase dissolution time ranging from about 300 to 500 min, and more longevity in mice exceeding 9 weeks than the dermis materials. There existed a correlation between the peak temperature in TG-DTA and the collagenase dissolution time. It was considered that higher cross-link degree among collagen fibrils of the membrane-type collagenous materials might account for these phenomena. The experimental protocol and numerical results obtained could be helpful for selection and future development of fibrous collagenous biomaterials in clinical use.

Uptake of Nanotitania by Gingival Epithelial Cells Promotes Inflammatory Response and Is Accelerated by Porphyromonas gingivalis Lipopolysaccharide: An In Vitro Study.

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonasgingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

Bone marrow-derived mesenchymal stem cells propagate immunosuppressive/anti-inflammatory macrophages in cell-to-cell contact-independent and -dependent manners under hypoxic culture.

Immunosuppressive/anti-inflammatory macrophage (Mφ), M2-Mφ that expressed the typical M2-Mφs marker, CD206, and anti-inflammatory cytokine, interleukin (IL)-10, is beneficial and expected tool for the cytotherapy against inflammatory diseases. Here, we demonstrated that bone marrow-derived lineage-positive (Lin+) blood cells proliferated and differentiated into M2-Mφs by cooperation with the bone marrow-derived mesenchymal stem cells (MSCs) under hypoxic condition: MSCs not only promoted proliferation of undifferentiated M2-Mφs, pre-M2-Mφs, in the Lin+ fraction via a proliferative effect of the MSCs-secreted macrophage colony-stimulating factor, but also promoted M2-Mφ polarization of the pre-M2-Mφs through cell-to-cell contact with the pre-M2-Mφs. Intriguingly, an inhibitor for intercellular adhesion molecule (ICAM)-1 receptor/lymphocyte function-associated antigen (LFA)-1, Rwj50271, partially suppressed expression of CD206 in the Lin+ blood cells but an inhibitor for VCAM-1 receptor/VLA-4, BIO5192, did not, suggesting that the cell-to-cell adhesion through LFA-1 on pre-M2-Mφs and ICAM-1 on MSCs was supposed to promoted the M2-Mφ polarization. Thus, the co-culture system consisting of bone marrow-derived Lin+ blood cells and MSCs under hypoxic condition was a beneficial supplier of a number of M2-Mφs, which could be clinically applicable to inflammatory diseases.

Effect of gold deposition onto titanium on the adsorption of alkanethiols as the protein linker functionalizing the metal surface.

A new method to enhance the biofunctionalization of Ti materials was developed by the immersion of Au-sputtered titanium plates into a solution of alkanethiol with a carboxy group end (HS-C11-COOH). X-ray photoelectron spectroscopy (XPS) analysis showed that the gold content increased with the Au sputtering time and reached over 98 at% after 100 s. The content of the characteristic alkanethiol elements (S, C, and O) in the immersed Ti specimens increased with the Au sputtering time, which indicates that the binding of alkanethiol molecules is enhanced by the Au modification on the Ti surface. Qualitative analysis of XPS (Au-S, COO(-), and OH(-) species) and FTIR spectra (peaks assignable to an aliphatic carboxylic acid) for the sample after immersion also supported this. A strong positive correlation between the S and Au content confirms that Au sputtering is an effective method to control the alkanethiol treatment of Ti substrates.

Novel SCRG1/BST1 axis regulates self-renewal, migration, and osteogenic differentiation potential in mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) remodel or regenerate various tissues through several mechanisms. Here, we identified the hMSC-secreted protein SCRG1 and its receptor BST1 as a positive regulator of self-renewal, migration, and osteogenic differentiation. SCRG1 and BST1 gene expression decreased during osteogenic differentiation of hMSCs. Intriguingly, SCRG1 maintained stem cell marker expression (Oct-4 and CD271/LNGFR) and the potentials of self-renewal, migration, and osteogenic differentiation, even at high passage numbers. Thus, the novel SCRG1/BST1 axis determines the fate of hMSCs by regulating their kinetic and differentiation potentials. Our findings provide a new perspective on methods for ex vivo expansion of hMSCs that maintain native stem cell potentials for bone-forming cell therapy.

Effects of apatite particle size in two apatite/collagen composites on the osteogenic differentiation profile of osteoblastic cells.

The development of new osteoconductive bone substitute materials is expected in medicine. In this study, we attempted to produce new hydroxylapatite (HAP)/collagen (Col) composites using two HAP particles of different sizes and porcine type I collagen. The two HAP particles were either nano-sized (40 nm in average diameter; n-HAP) or had macro-pore sizes of 0.5­1.0 mm in length with fully interconnected pores (m-HAP). The aim of this study was to investigate the effects of apatite particle size in two HAP/Col composites on the osteogenic differentiation profile in osteoblast-like cells (SaOS-2). We created a collagen control sponge (Col) and two HAP/Col composite sponges (n-HAP/Col and m-HAP/Col) using freeze-drying and dehydrothermal cross-linking techniques, and then punched out samples of 6 mm in diameter and 1 mm in height. The SaOS-2 cells were cultured on three test materials for 1, 2, 3 and 4 weeks. Total RNA was extracted from the cultured cells and the expression of osteogenic differentiation-related genes was evaluated by reverse transcription PCR (RT-PCR) using primer sets of alkaline phosphatase (ALP), type 1 collagen (COL1), bone sialoprotein (BSP) and osteocalcin precursor [bone gamma-carboxyglutamate (gla) protein (BGLAP)] genes, as well as the ß-actin gene. The cells were also cultured on Col, n-HAP/Col and m-HAP/Col specimens for 1 and 4 weeks, and were then observed under a scanning electron microscope (SEM). The experimental results were as follows: RT-PCR indicated that osteogenic differentiation, particularly the gene expression of BSP, was most accelerated when the cells were cultured on n-HAP/Col specimens, followed by m-HAP/Col, whilst the weakest accelaeration was observed when the cells were cultured on Col specimens. As shown by the SEM images, the SaOS-2 cells were fibroblastic when cultured on Col specimens for up to 4 weeks; they were fibroblastic when cultured on n-HAP/Col specimens for 1 week, but appeared as spheroids, while actively phagocytizing n-HAP particles at 4 weeks; however, they appeared as deformed fibroblasts when cultured on m-HAP/Col specimens, detached from the particles. Despite limited experimental results, our study suggests that n-HAP/Col may be employed as a new osteoconductive bone substitute material.

Proteome analyses of human macrophages exposed to low cytotoxic IC90 copper (2+) ions.

Dental noble alloys often contain copper (Cu). Eluted metal ions sometimes irritate oral tissues. The most eluted ions are Cu ions. The purpose of this study was to investigate the effects of low cytotoxic (IC90, 100 µmol/L) Cu ions on macrophages by proteome analyses consisting of two-dimensional (2D) electrophoresis and matrix-assisted laser desorption/ionization -time of flight (MALDI-TOF) mass spectrometry. The analyses revealed that stimulation with IC90 Cu ions for 1 day caused the macrophage to significantly increase five specific protein spots. Mascot peptide mass finger-print matching suggested that four of them were attributed to 70 kDa heat shock protein 1A/1B (HSP70). HSP70 expression was verified by expressions of corresponding HSPA1A and HSPA1B mRNAs of the macrophage in quantitative real-time PCR analyses. It was concluded that by producing abundant HSP70, the macrophage protected itself against intracellularly intruding cytotoxic Cu ions that might un-fold and crosslink cellular proteins.

Viscoelastic behavior of commercially available tissue conditioners under compression.

Three commercially available tissue conditioners -Tissue conditioner II (STII), Tissue conditioner (GTC), and Tissue Care (TTC)- were tested using a rheometer to evaluate their viscoelasticity under compression at a constant distortion (10%). Immediately after fabrication, STII showed the highest hardness and compressive modulus of elasticity, followed by TTC and GTC. TTC showed the highest relaxation rate, followed by GTC and STII. STII and GTC showed equivalent relaxation times, while TTC showed the shortest relaxation time. The modulus of viscosity was highest in STII, followed by GTC and TTC. The immersion of each sample in water resulted in increases in hardness, compressive modulus of elasticity, relaxation time, and modulus of viscosity and decreases in relaxation rates. The three products have different viscoelastic properties. Further, the results indicate that STII and GTC exhibit better performance when used as functional impression materials, while TTC exhibits better performance when used as a tissue conditioner.

Viscoelastic adlayers of collagen and lysozyme studied using quartz crystal microbalance with dissipation monitoring.

In this study the effects of molecular structure of proteins on their adsorption behaviour and viscoelasticity are investigated using a QCM-D method. The adsorption measurements show that spherical lysozyme is rapidly adsorbed on the gold surface as a stiff monolayer, as indicated by a sharp drop in the oscillation frequency (Deltaf) of the sensor, and by a very small increase in the energy dissipation of the adlayer (DeltaD). Fibrous calfskin collagen (CSC) is, however, adsorbed on the same surface rather slowly in two steps to form a thick and soft multilayer (large Deltaf and DeltaD) at pH 3 in salt-free conditions. The two-step adsorption is not observed for stiffly aggregated CSC. This study clearly demonstrates that the polymer structure strongly affects not only how adsorption develops but also the viscoelastic properties of the adlayer.

Microscopic observations and inflammatory cytokine productions of human macrophage phagocytising submicron titanium particles.

This study was performed to microscopically observe and measure inflammatory cytokine production by human macrophages phagocytosing submicron titanium (Ti) particles. Observations with secondary electron microscopy (SEM), SEM/electron probe microanalysis (EPMA) and transmission electron microscopy (TEM) indicated that macrophages [phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells] at 24 h in culture actively phagocytosed and accumulated submicron Ti particles in intracellular phagosomes, in which refinement of Ti particles occurred. The macrophages were also cultured for 24 h in four media with and without submicron Ti particles and lipopolysaccharide (LPS; components of bacteria). Whilst neither stimulus reduced cell viability, submicron Ti particles and LPS activation independently and synergistically caused the macrophages to produce three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) at high levels in the culture supernatants. The inflammatory and osteolysis conditions caused by macrophages phagocytosing submicron Ti particles would be worsened by challenge with LPS in patients wearing Ti prostheses.

Gene expression analyses of human macrophage phagocytizing sub-micro titanium particles by allergy DNA chip (Genopal).

The purpose of this study was to examine gene expressions of macrophage phagocytizing sub-micro Ti particles by a DNA chip. Human monocytic cell line THP-1 was differentiated into macrophages by culturing for two days in medium supplemented with 200 nM phorbol ester (PMA). The macrophages were then cultured in four media: medium without PMA (control); medium with suspended sub-micro Ti particles (0.5 wt%); medium with 1.0 microg/ml lipopolysaccharide (LPS); and medium with LPS and Ti particles. After 6 hours' culture, total RNA were extracted and gene expressions were evaluated by DNA allergy chip with 205 allergy and inflammation related gene spots. We found that phagocytosis of sub-micro Ti particles and LPS independently and synergistically up-regulated 17 inflammation-related genes more than two-fold. The extensive expressions of four genes (CCL1, IL1B, IL6 and IL8) were further confirmed by real-time quantitative PCR. It turned out that dual stimulation of LPS and Ti particles most up-regulated three genes (IL1B, IL6 and IL8), followed by LPS while Ti particles moderately but least increased, suggesting that phagocytosis of sub-micro Ti particles induces moderate inflammation with its degree less than LPS, but phagocytosis of sub-micro Ti particles has the potential to worsen inflammation caused by LPS-stimulated macrophages.

Preparation and in vivo evaluation of apatite/collagen packed composite by alternate immersion method and Newton press.

Further development of bio-compatible, bio-absorbable, and osteo-conductive bio-materials is desired for bone grafts in dental and medical clinics. One candidate material might be a high-density apatite/collagen composite, which cures relatively large bone defects. To produce such a composite, we freeze-dried type I collagen solution, cross-linked the formed sponge by 2 wt % glutaraldehyde, immersed the insoluble sponge in CaCl(2) and Na(2)HPO(4) solutions alternately five times, and compacted the sponge by Newton press at 5000 kgf. For comparison, cross-linked collagen without alternate immersion was also pressed. SEM/EPMA, XRD, and FTIR analyses clarified that alternate immersion successfully coated the collagen sponge with hydroxyapatite. Packed apatite/collagen composite and collagen disks 6 mm in diameter and 0.5 mm in height were implanted in the subperiostea of rabbit tibiae for 2, 4, 8, and 12 weeks to assess bio-compatibility, bio-absorbability, and osteo-conductivity. Histological observations showed that the packed apatite/collagen composite was biocompatible, osteo-conductive for up to 8 weeks, and largely bio-absorbed at 12 weeks, while the packed collagen sponge caused an undesirable foreign body reaction, which worsened with the implantation period. The overall findings suggest that this packed apatite/collagen composite might be used as a new bio-absorbable bone graft material.

Effect of pH and addition of salt on the adsorption behavior of lysozyme on gold, silica, and titania surfaces observed by quartz crystal microbalance with dissipation monitoring.

The adsorption behaviors of lysozyme on dentally related Au, SiO2, and TiO2 surfaces were investigated by a quartz crystal microbalance with dissipation monitoring (QCM-D) method. Frequency shifts indicated that while lysozyme (pI 11) was fairly adsorbed on the SiO2 (pI 1.9) surface at both pH 3 and 7, it was adsorbed on TiO2 (pI 6.3) surface only at pH 7. However, adsorption was disturbed by 50 mM NaCl. These results strongly suggested an electrostatic nature of the adsorption behavior. Though a large-scale adsorption of the lysozyme on Au sensor was pH-insensitive, softness of the adlayer as seen from the dissipation profile was pH-dependent, indicating an interaction of another type. With all the surfaces, the small dissipation change indicated a stiff lysozyme adlayer. Results of this study revealed that the controlled electrostatic interaction between the material surface and lysozyme might be a useful method for imparting antibacterial property to the dental materials.

Dose-dependent effects of Ni (II) ions on production of three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6), superoxide dismutase (SOD) and free radical NO by murine macrophage-like RAW264 cells with or without LPS-stimulation.

The effect of Ni (II) ions on macrophages is not well understood. The purpose of this study was to examine the dose-dependent effects of Ni (II) ions up to 1,000 micromol/L on production of three inflammatory cytokines (TNF-alpha, IL-1beta and IL-6), superoxide dismutase (SOD) and nitric oxide (NO) by murine macrophage-like RAW264 cells with (+) or without (-) lipopolysaccharide (LPS) -stimulation. Ni (II) ions caused LPS (-) RAW264 cells to slightly increase production of TNF-alpha and IL-6, proportionally to the Ni (II) ion concentration while IL-1beta was not produced, and to slightly increase production of SOD and NO. It can be concluded that Ni (II) ions dose-dependently increased the inflammatory and oxidative stress conditions of LPS (-) RAW264 cells. LPS-stimulation caused RAW264 cells to produce in abundance the three inflammatory cytokines, SOD and NO. Ni (II) ions dose-dependently reduced the three cytokine quantities and NO amounts in LPS (+) RAW264 cells, while dose-independently increasing SOD amounts. It was noted that Ni (II) ions dose-dependently reduce the resistance power against bacteria of LPS (+) macrophages, because the production of volatile NO--bacteria killer is diminished proportionally to the Ni (II) ion concentration.

New index for the stability of a type I collagen affected by hydrophobic environment.

Effects of hydrophobic environment adjusted by various alcohols on the structural stability of calfskin collagen (CSC) were studied to elucidate the nature of collagen-monomer interaction in adhesion. The stability of CSC in aqueous alcohol solutions was represented by its denaturation temperature, Td, measured by DSC. The hydrophobicity of the alcohol solutions was quantified with their specific dielectric constants, epsilon(r), calculated from their concentrations. The effect of each alcohol to stabilize or destabilize CSC was evaluated by the initial slope of each Td vs. epsilon(r) plot, denoted as -(dTd/d epsilon(r))ini and termed as stabilization power. Results showed that a hydrophobic environment with a smaller epsilon(r) lowered the stabilization power. Stabilization power ranged from -3 (strong destabilization) for phenol (epsilon(r) =12) to +0.3 (weak stabilization) for glycerol (epsilon(r)=47). In view of the encouraging results obtained in this study, the new index was therefore helpful in predicting the effects of new dental materials of known epsilon(r) values on the stability of dentinal collagen.

Fabrication of calcium phosphate films for coating on titanium substrates heated up to 773 K by RF magnetron sputtering and their evaluations.

Calcium phosphate films were fabricated on titanium substrates heated up to 773 K using radiofrequency (RF) magnetron sputtering. The deposition rate, phase and preferred orientation of the calcium phosphate films were studied. Immersion tests for the films were conducted using Hanks' solution and PBS(-), and the surface reactions on the specimens coated with the calcium phosphate films were investigated. The bonding strength between the coating films and the titanium substrates before and after the immersion tests was evaluated; the bonding strength decreased after the immersion tests. The alkaline phosphatase (ALP) activity of SaOS-2 cells on a titanium plate coated with a calcium phosphate film was examined by conducting a culture test. Calcium phosphate coating increased the ALP activity of SaOS-2 cells cultured for 3 and 7 days. Titanium cylinders were coated with an amorphous calcium phosphate film and implanted into the mandibles of beagle dogs. An increase in the extent of bone-implant contact for the coated titanium cylinders was confirmed 8 to 12 weeks after implantation and compared with the case for uncoated titanium cylinders.