Surface modification affects human gingival epithelial cell behavior on polyetheretherketone surfaces.

Gingival epithelial attachment to the abutment is important for the prevention of peri-implantitis. Polyetheretherketone (PEEK) has recently gained attention as an alternative material to titanium; however, it is biologically inert, which is disadvantageous for obtaining soft tissue sealing of the transmucosal part of the implant abutment. Therefore, ultraviolet (UV) irradiation, argon plasma irradiation, and buffing were selected as treatments to modify the PEEK surface. None of the treatments had any effect on the material's mechanical strength. The UV and plasma treatments did not significantly affect the surface morphology. Surface elemental analysis showed a decrease in carbon content and an increase in oxygen content and wettability for all treatments. Human gingival epithelial cell adhesion, proliferation, and the expression of adhesion proteins integrin ß4 and laminin 332, were increased. Surface modification to PEEK was suggested to enhance cell activity on PEEK.

Induction of mesenchymal stem cell (MSC) differentiation from iPS cells using MSC medium.

Mesenchymal stem cells (MSCs) and induced pluripotent stem (iPS) cells have great potential as cell sources for tissue engineering and regenerative medicine. This study aimed to investigate whether iPS cells can be differentiated into MSCs using MSCGM, a commercially available MSC culture system. The cells were characterized by flow cytometry, immunostaining, and gene expression analyses. We also examined their potential to differentiate into osteoblasts and chondrocytes. Our results showed that iPS cells cultured in MSCGM (iPS-MSCGM) exhibited a fibroblast-like morphology and expressed CD73 and CD90 genes, as well as positive markers for CD73, CD90, and CD105. Moreover, iPS-MSCGM cells demonstrated the ability to differentiate into osteoblasts and chondrocytes in vitro. This study demonstrates a new and simple method for inducing the differentiation of iPS cells to MSCs using MSCGM.

Effect of Er:YAG Pulsed Laser-Deposited Hydroxyapatite Film on Titanium Implants on M2 Macrophage Polarization In Vitro and Osteogenesis In Vivo.

In a previous study, we successfully coated hydroxyapatite (HAp) onto titanium (Ti) plates using the erbium-doped yttrium aluminum garnet pulsed-laser deposition (Er:YAG-PLD) method. In this study, we performed further experiments to validate the in vitro osteogenic properties, macrophage polarization, and in vivo osseointegration activity of HAp-coated Ti (HAp-Ti) plates and screws. Briefly, we coated a HAp film onto the surfaces of Ti plates and screws via Er:YAG-PLD. The surface morphological, elemental, and crystallographic analyses confirmed the successful surface coating. The macrophage polarization and osteogenic induction were evaluated in macrophages and rat bone marrow mesenchymal stem cells, and the in vivo osteogenic properties were studied. The results showed that needle-shaped nano-HAp promoted the early expression of osteogenic and immunogenic genes in the macrophages and induced excellent M2 polarization properties. The calcium deposition and osteocalcin production were significantly higher in the HAp-Ti than in the uncoated Ti. The implantation into rat femurs revealed that the HAp-coated materials had superior osteoinductive and osseointegration activities compared with the Ti, as assessed by microcomputed tomography and histology. Thus, HAp film on sandblasted Ti plates and screws via Er:YAG-PLD enhances hard-tissue differentiation, macrophage polarization, and new bone formation in tissues surrounding implants both in vitro and in vivo.

Immunomodulatory Properties and Osteogenic Activity of Polyetheretherketone Coated with Titanate Nanonetwork Structures.

Polyetheretherketone (PEEK) is a potential substitute for conventional metallic biomedical implants owing to its superior mechanical and chemical properties, as well as biocompatibility. However, its inherent bio-inertness and poor osseointegration limit its use in clinical applications. Herein, thin titanium films were deposited on the PEEK substrate by plasma sputtering, and porous nanonetwork structures were incorporated on the PEEK surface by alkali treatment (PEEK-TNS). Changes in the physical and chemical characteristics of the PEEK surface were analyzed to establish the interactions with cell behaviors. The osteoimmunomodulatory properties were evaluated using macrophage cells and osteoblast lineage cells. The functionalized nanostructured surface of PEEK-TNS effectively promoted initial cell adhesion and proliferation, suppressed inflammatory responses, and induced macrophages to anti-inflammatory M2 polarization. Compared with PEEK, PEEK-TNS provided a more beneficial osteoimmune environment, including increased levels of osteogenic, angiogenic, and fibrogenic gene expression, and balanced osteoclast activities. Furthermore, the crosstalk between macrophages and osteoblast cells showed that PEEK-TNS could provide favorable osteoimmunodulatory environment for bone regeneration. PEEK-TNS exhibited high osteogenic activity, as indicated by alkaline phosphatase activity, osteogenic factor production, and the osteogenesis/osteoclastogenesis-related gene expression of osteoblasts. The study establishes that the fabrication of titanate nanonetwork structures on PEEK surfaces could extract an adequate immune response and favorable osteogenesis for functional bone regeneration. Furthermore, it indicates the potential of PEEK-TNS in implant applications.

Comparison of Osteogenic Potentials of Dental Pulp and Bone Marrow Mesenchymal Stem Cells Using the New Cell Transplantation Platform, CellSaic, in a Rat Congenital Cleft-Jaw Model.

Scaffolds stimulate cell proliferation and differentiation and play major roles in providing growth and nutrition factors in the repair of bone defects. We used the recombinant peptide Cellnest™ to prepare the three-dimensional stem cell complex, CellSaic, and evaluated whether CellSaic containing rat dental pulp stem cells (rDPSCs) was better than that containing rat bone marrow stem cells (rBMSCs). rDPSC-CellSaic or rBMSC-CellSaic, cultured with or without osteogenic induction medium, formed the experimental and control groups, respectively. Osteoblast differentiation was evaluated in vitro and transplanted into a rat model with a congenital jaw fracture. Specimens were collected and evaluated by microradiology and histological analysis. In the experimental group, the amount of calcium deposits, expression levels of bone-related genes (RUNX2, ALP, BSP, and COL1), and volume of mineralized tissue, were significantly higher than those in the control group (p < 0.05). Both differentiated and undifferentiated rDPSC-CellSaic and only the differentiated rBMSC-CellSaic could induce the formation of new bone tissue. Overall, rBMSC-CellSaic and rDPSC-CellSaic made with Cellnest™ as a scaffold, provide excellent support for promoting bone regeneration in rat mandibular congenital defects. Additionally, rDPSC-CellSaic seems a better source for craniofacial bone defect repair than rBMSC-CellSaic, suggesting the possibility of using DPSCs in bone tissue regenerative therapy.

Effect of Argon-Based Atmospheric Pressure Plasma Treatment on Hard Tissue Formation on Titanium Surface.

In this paper, we suggest that the atmospheric pressure plasma treatment of pure titanium metal may be useful for improving the ability of rat bone marrow cells (RBMCs) to induce hard tissue differentiation. Previous studies have reported that the use of argon gas induces a higher degree of hard tissue formation. Therefore, this study compares the effects of plasma treatment with argon gas on the initial adhesion ability and hard tissue differentiation-inducing ability of RBMCs. A commercially available titanium metal plate was used as the experimental material. A plate polished using water-resistant abrasive paper #1500 was used as the control, and a plate irradiated with argon mixed with atmospheric pressure plasma was used as the experimental plate. No structural change was observed on the surface of the titanium metal plate in the scanning electron microscopy results, and no change in the surface roughness was observed via scanning probe microscopy. X-ray photoelectron spectroscopy showed a decrease in the carbon peak and the formation of hydroxide in the experimental group. In the distilled water drop test, a significant decrease in the contact angle was observed for the experimental group, and the results indicated superhydrophilicity. Furthermore, the bovine serum albumin adsorption, initial adhesion of RBMCs, alkaline phosphatase activity, calcium deposition, and genetic marker expression of rat bone marrow cells were higher in the experimental group than those in the control group at all time points. Rat distal femur model are used as in vivo model. Additionally, microcomputed tomography analysis showed significantly higher results for the experimental group, indicating a large amount of the formed hard tissue. Histopathological evaluation also confirmed the presence of a prominent newly formed bone seen in the images of the experimental group. These results indicate that the atmospheric pressure plasma treatment with argon gas imparts superhydrophilicity, without changing the properties of the pure titanium plate surface. It was also clarified that it affects the initial adhesion of bone marrow cells and the induction of hard tissue differentiation.

Gas Permeability of Mold during Freezing Process Alters the Pore Distribution of Gelatin Sponge and Its Bone-Forming Ability.

Freeze-drying, also known as lyophilization, is widely used in the preparation of porous biomaterials. Nevertheless, limited information is known regarding the effect of gas permeability on molds to obtain porous materials. We demonstrated that the different levels of gas permeability of molds remarkably altered the pore distribution of prepared gelatin sponges and distinct bone formation at critical-sized bone defects of the rat calvaria. Three types of molds were prepared: silicon tube (ST), which has high gas permeability; ST covered with polyvinylidene chloride (PVDC) film, which has low gas permeability, at the lateral side (STPL); and ST covered with PVDC at both the lateral and bottom sides (STPLB). The cross sections or curved surfaces of the sponges were evaluated using scanning electron microscopy and quantitative image analysis. The gelatin sponge prepared using ST mold demonstrated wider pore size and spatial distribution and larger average pore diameter (149.2 µm) compared with that prepared using STPL and STPLB. The sponges using ST demonstrated significantly poor bone formation and bone mineral density after 3 weeks. The results suggest that the gas permeability of molds critically alters the pore size and spatial pore distribution of prepared sponges during the freeze-drying process, which probably causes distinct bone formation.

Augmentation of Bone Regeneration by Depletion of Stress-Induced Senescent Cells Using Catechin and Senolytics.

Despite advances in bone regenerative medicine, the relationship between stress-induced premature senescence (SIPS) in cells and bone regeneration remains largely unknown. Herein, we demonstrated that the implantation of a lipopolysaccharide (LPS) sustained-release gelatin sponge (LS-G) increases the number of SIPS cells and that the elimination of these cells promotes bone formation in critical-sized bone defects in the rat calvaria. Histological (hematoxylin-eosin and SA-ß-gal) and immunohistological (p16 and p21 for analyzing cellular senescence and 4-HNE for oxidation) staining was used to identify SIPS cells and elucidate the underlying mechanism. Bone formation in defects were analyzed using microcomputed tomography, one and four weeks after surgery. Parallel to LS-G implantation, local epigallocatechin gallate (EGCG) administration, and systemic senolytic (dasatinib and quercetin: D+Q) administration were used to eliminate SIPS cells. After LS-G implantation, SA-ß-gal-, p16-, and p21-positive cells (SIPS cells) accumulated in the defects. However, treatment with LS-G+EGCG and LS-G+D+Q resulted in lower numbers of SIPS cells than that with LS-G in the defects, resulting in an augmentation of newly formed bone. We demonstrated that SIPS cells induced by sustained stimulation by LPS may play a deleterious role in bone formation. Controlling these cell numbers is a promising strategy to increase bone regeneration.

Releasing Behavior of Lipopolysaccharide from Gelatin Modulates Inflammation, Cellular Senescence, and Bone Formation in Critical-Sized Bone Defects in Rat Calvaria.

Lipopolysaccharide (LPS) is a well-known strong inducer of inflammation. However, there is little information regarding how LPS-release behavior affects cellular senescence at the affected area. In this paper, we demonstrate that a vacuum-heating technique (dehydrothermal treatment) can be utilized to prepare an LPS sustained-release gelatin sponge (LS-G). LPS sustained release from gelatin leads to the long-term existence of senescent cells in critical-sized bone defects in rat calvaria. Three types of gelatin sponges were prepared in this study: a medical-grade gelatin sponge with extremely low LPS levels (MG), LS-G, and a LPS rapid-release gelatin sponge (LR-G). Histological (H-E) and immunohistochemical (COX-2, p16, and p21) staining were utilized to evaluate inflammatory reactions and cellular senescence one to three weeks after surgery. Soft X-ray imaging was utilized to estimate new bone formation in the defects. The LR-G led to stronger swelling and COX-2 expression in defects compared to the MG and LS-G at 1 week. Despite a small inflammatory reaction, LS-G implantation led to the long-term existence of senescent cells and hampered bone formation compared to the MG and LR-G. These results suggest that vacuum heating is a viable technique for preparing different types of materials for releasing bacterial components, which is helpful for developing disease models for elucidating cellular senescence and bone regeneration.

Comparison of the characteristics of mesenchymal stem-like cells derived by integration-free induced pluripotent stem cells in different single-cell culture media under feeder-free conditions.

Generating mesenchymal stem-like cells (MSLCs) from induced pluripotent stem cells (iPSCs) can be a practical method for obtaining the sufficient cells for autologous tissue engineering. Single-cell culturing in specific medium and non-feeder cells is an alternative and promising strategy to overcome problems of embryo culture; however, little is known about how different culture media affect the proliferation and differentiation of MSLCs. We first derived MSLCs from iPSCs with non-integrating episomal plasmid vectors (hereafter 409B2 cells) using three different cell culture media, including single-cell culture medium in feeder-free condition: mTeSR1, DEF-CS500, or StemFit AK02N. The morphology of all MSLCs was completely altered to a fibroblastic morphology after four passages. Surface antigens CD29, CD44, CD73, CD90, but not CD34 and CD45, were expressed in all passages. RUNX2 was expressed in MSLCs cultured in all three feeder-free media, while SOX9 and PPARγ were expressed in MSLCs cultured in only DEF-CS500. MSLCs derived from DEF-CS500, which is a single-cell culture medium, grew at a slightly faster rate than those cultured in other media and expressed early-stage genes for tri-lineage differentiation. Taken together, these findings provide valuable information for generating MSLCs using single-cell culture methods.

In Vitro and In Vivo Osteogenic Activity of Titanium Implants Coated by Pulsed Laser Deposition with a Thin Film of Fluoridated Hydroxyapatite.

To enhance biocompatibility, osteogenesis, and osseointegration, we coated titanium implants, by krypton fluoride (KrF) pulsed laser deposition, with a thin film of fluoridated hydroxyapatite (FHA). Coating was confirmed by scanning electron microscopy (SEM) and scanning probe microscopy (SPM), while physicochemical properties were evaluated by attenuated reflectance Fourier transform infrared spectroscopy (ATR-FTIR). Calcium deposition, osteocalcin production, and expression of osteoblast genes were significantly higher in rat bone marrow mesenchymal stem cells seeded on FHA-coated titanium than in cells seeded on uncoated titanium. Implantation into rat femurs also showed that the FHA-coated material had superior osteoinductive and osseointegration activity in comparison with that of traditional implants, as assessed by microcomputed tomography and histology. Thus, titanium coated with FHA holds promise as a dental implant material.

Application of fluoridated hydroxyapatite thin film coatings using KrF pulsed laser deposition.

Fluoridated hydroxyapatite (FHA) was investigated for application as an implant coating for titanium bone substitute materials in dental implants. A KrF pulsed excimer deposition technique was used for film preparation on a titanium plate. The compacts were ablated by laser irradiation at an energy density of 1 J/cm2 on an area 1×1 mm2 with the substrate at room temparature. Energydispersive spectrometric analysis of the FHA film revealed peaks of fluorine in addition to calcium and phosphorus. X-ray diffraction revealed the presence of crystalline FHA on the FHA film after a 10 h post annealing treatment at 450°C. The FHA film coating exhibited significant dissolution resistance to sodium phosphate buffer for up to 21 days, and favorable cell attachment of human mesenchymal stem cells compared with HA film. The results of this study suggest that FHA coatings are suitable for real-world implantation applications.

In vivo bioactivity of porous polyetheretherketone with a foamed surface.

The in vivo bioactivity of porous polyetheretherketone (PEEK) with a foamed surface was evaluated using rabbit femoral bone. Cylindrical porous PEEK scaffolds, with pore diameter of 550 µm and porosity of 70%, were first prepared and immersed in 98% sulfuric acid, and then washed and immersed in 3 M potassium carbonate solution used as a foaming reagent. Numerous open pores of various sizes, as well as new functional groups, were visualized on the treated PEEK surface by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Micro computed tomography (micro-CT) showed that the volumetric density of treated PEEK was higher than that of bare PEEK at 8 weeks after surgery (p<0.05). Additionally, von Kossa staining indicated ingrowth of mature new bone tissue at 4 weeks relative to the bare PEEK group. Our data indicate that surface-treated PEEK exhibited improved bioactivity in vivo.

Interferon-γ enhances the efficacy of autogenous bone grafts by inhibiting postoperative bone resorption in rat calvarial defects.

PURPOSE: Interferon (IFN)-γ is a major cytokine produced by immune cells that plays diverse roles in modulating both the immune system and bone metabolism, but its role in autogenous bone grafting remains unknown. Here, we present that local IFN-γ administration improved the efficacy of autogenous bone graft treatment in an experimental rat model. METHODS: An autogenous bone graft model was prepared with critically sized rat calvariae defects. Four weeks (w) after bone graft implantation, rats were treated locally with IFN-γ or were not treated. The effect of IFN-γ on bone formation was evaluated for up to 8w with micro-computed tomography, quantitative histomorphometry, and Von Kossa staining. Osteoclastogenesis was assessed by tartrate-resistant acid phosphatase staining. Immunohistochemistry staining or quantitative polymerase chain reactions were used to estimate the expression of osteoclast differentiation factor and inflammatory cytokines including tumor necrosis factor (TNF)-α, a well-known stimulant of osteoclastogenesis and an inhibitor of osteoblast activity, in defects. RESULTS: Newly formed bone gradually replaced the autogenous bone grafts within 4w, although severe bone resorption with osteoclastogenesis and TNF-α expression occurred after 6w in the absence of IFN-γ administration. IFN-γ administration markedly attenuated bone loss, osteoclastogenesis, and TNF-α expression, while it enhanced bone formation at 8w. CONCLUSION: Local IFN-γ administration promoted bone formation in autogenous bone grafts possibly via regulating osteoclastogenesis and TNF-α expression. The data provide insights into the potential roles of IFN-γ in autogenous bone grafting.

Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro.

Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25-10 µM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 µM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 µM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy.

The Effect of Interferon-γ and Zoledronate Treatment on Alpha-Tricalcium Phosphate/Collagen Sponge-Mediated Bone-Tissue Engineering.

Inflammatory responses are frequently associated with the expression of inflammatory cytokines and severe osteoclastogenesis, which significantly affect the efficacy of biomaterials. Recent findings have suggested that interferon (IFN)-γ and zoledronate (Zol) are effective inhibitors of osteoclastogenesis. However, little is known regarding the utility of IFN-γ and Zol in bone tissue engineering. In this study, we generated rat models by generating critically sized defects in calvarias implanted with an alpha-tricalcium phosphate/collagen sponge (α-TCP/CS). At four weeks post-implantation, the rats were divided into IFN-γ, Zol, and control (no treatment) groups. Compared with the control group, the IFN-γ and Zol groups showed remarkable attenuation of severe osteoclastogenesis, leading to a significant enhancement in bone mass. Histomorphometric data and mRNA expression patterns in IFN-γ and Zol-injected rats reflected high bone-turnover with increased bone formation, a reduction in osteoclast numbers, and tumor necrosis factor-α expression. Our results demonstrated that the administration of IFN-γ and Zol enhanced bone regeneration of α-TCP/CS implants by enhancing bone formation, while hampering excess bone resorption.

Local Controlled Release of Polyphenol Conjugated with Gelatin Facilitates Bone Formation.

Catechins are extensively used in health care treatments. Nevertheless, there is scarce information about the feasibility of local administration with polyphenols for bone regeneration therapy, possibly due to lack of effective delivery systems. Here we demonstrated that the epigallocatechin-3-gallate-conjugated gelatin (EGCG/Gel) prepared by an aqueous chemical synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-morpholinium chloride (DMT-MM) gradually disintegrated with time and facilitated bone formation in a critical size defect of a mouse calvaria. Conjugation of EGCG with the Gel generated cross-linking between the two molecules, thereby leading to a retardation of the degradation of the EGCG/Gel and to a delayed release of EGCG. The prepared EGCG/Gels represented significant osteogenic capability compared with that of the uncross-linked Gel and the cross-linked Gel with uncombined-EGCG. In vitro experiments disclosed that the EGCG/Gel induced osteoblastogenesis of a mouse mesenchymal stem cell line (D1 cells) within 14 days. Using fluorescently-labeled EGCG/Gel, we found that the fraction of EGCG/Gel adsorbed onto the cell membrane of the D1 cells possibly via a Gel-cell interaction. The interaction might confer the long-term effects of EGCG on the cells, resulting in a potent osteogenic capability of the EGCG/Gel in vivo. These results should provide insight into local controlled release of polyphenols for bone therapy.

Human Gingival Integration-Free iPSCs; a Source for MSC-Like Cells.

Mesenchymal stem cells (MSCs) are considered a potential autologous therapy for tissue engineering. The available procedures for MSC retrieval from patients are invasive, and their limited in vitro proliferation restricts their use in the treatment of damaged tissues. Therefore, it is important to establish an alternative and safe source of MSCs. The objective of this study was to demonstrate induced pluripotent stem cell (iPSC) generation from a combination of an accessible source tissue and an integration-free method; we also attempted the differentiation of iPSCs into MSC-like cells (MSLCs) for future autologous tissue engineering. iPSCs were derived from human gingival tissues, which are easily accessible in the field of dentistry, via the use of non-integrating episomal plasmids. Established iPSCs expressed embryonic stem (ES) cell-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratoma formation were formed from iPSCs, showing their capacity to differentiate into three germ layers. Furthermore, we were successful in differentiating iPSCs into MSLCs. They tested positively for their capacity of trilineage differentiation. Our results demonstrate that human gingival integration-free iPSCs, readily accessible stem cells generated using episomal plasmid vectors, are a promising source of MSLCs, which can be used in tissue regeneration.

A novel membrane-type apatite scaffold engineered by pulsed laser ablation.

Cell sheet technology is a scaffold-free method for tissue reconstruction. A sheet-shaped scaffold would be suitable for the regeneration of periodontal membrane. We designed a stem cell sheet combining human mesenchymal stromal cells (hMSCs) and a 10-µm thick biological apatite (BAp) membrane fabricated with an ArF pulsed laser ablation for periodontal regeneration. X-ray diffraction showed that crystalline hydroxyapatite (HAp) was present in BAp and HAp membranes after post-annealing. Energy dispersive analysis of the BAp membrane revealed peaks of Na and Mg in addition to Ca and P. Approximately 3×10(4) hMSCs were cultured on BAp and HAP membranes for 7 and 14 days. From in vitro assays, hMSCs grew faster and had higher osteoblast differentiation when cultured on the BAp membrane than did the cell culture on the HAp membrane. Stem cell sheets combined with a BAp membrane may have potential applications in guided bone regeneration and osteoconductive scaffolds.

Bone regeneration with a collagen model polypeptides/α-tricalcium phosphate sponge in a canine tibia defect model.

INTRODUCTION: We evaluated the effects of synthesized collagen model polypeptides consisting of a proline-hydroxyproline-glycine (poly(PHG)) sequence combined with porous alpha-tricalcium phosphate (α-TCP) particles on bone formation in a canine tibia defect model. MATERIALS AND METHODS: The porous α-TCP particles were mixed with a poly(PHG) solution, and the obtained sponge was then cross-linked and characterized by x-ray diffraction and scanning electron microscopy. Tibia defects were analyzed in 12 healthy beagles using microcomputed tomography and histological evaluation. RESULTS: At 2 and 4 weeks, the volume density of new bone was higher in the poly(PHG)/α-TCP group than in poly(PHG) alone group (P < 0.05); however, there was no difference at 8 weeks (P > 0.05). Histological evaluation at 4 weeks after implantation revealed that the poly(PHG) had degraded, and newly formed bone was present on the surface of the α-TCP particles. At 8 weeks, continuous cortical bone formation with a Haversian structure covered the top of the bone defects in both groups. CONCLUSION: This study demonstrates that the composite created using porous α-TCP particles and poly(PHG) is sufficiently adaptable for treating bone defects.