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Purpose: To perform vertical bone augmentation on rat parietal bone by coating the inner surface of dense polytetrafluoroethylene (d-PTFE) domes with hydroxyapatite (HA) using Erbium Yttrium Aluminum Garnet (Er:YAG) pulsed laser deposition in a rat model., Methods: The d-PTFE plate surface, α-tricalcium phosphate (α-TCP) coating, and HA coating were measured using scanning electron microscopy and X-ray diffraction to confirm the replacement of α-TCP with HA via high-pressure steam sterilization. The dome was glued to the center of the rat parietal bone and closed with periosteal and epithelial sutures. The rats were euthanized at 4, 8, and 12 weeks postoperatively, and the d-PTFE dome and surrounding tissues were harvested for radiological and histological evaluation., Results: In a group without HA coating (control group), a small amount of layered neosteum was observed in the d-PTFE dome. Conversely, a new balloon-shaped bone was observed in a HA-coated group (experimental group). Bone growth and number of bone beams were higher in the HA-coated group from 4 to 12 weeks postoperatively than those in the control group. However, no significant differences in bone mineral density values or bone bridge widths were observed., Conclusions: The HA coating enhanced bone augmentation without bone replacement. Histological findings indicated that the new bone comprised normal bone tissue with cartilage lumen and matrix. Therefore, our results indicate that bone augmentation with a HA coating may be mediated by endochondral ossification, a process similar to that of ectopic ossification.

In recent years, with the advent of a super-aged society, lifelong dental care has gained increasing emphasis, and implant therapy for patients with an edentulous jaw has become a significant option. However, for implant therapy to be suitable for elderly patients with reduced regenerative and immunological capabilities, higher osteoconductive and antimicrobial properties are required on the implant surfaces. Silicon nitride, a non-oxide ceramic known for its excellent mechanical properties and biocompatibility, has demonstrated high potential for inducing hard tissue differentiation and exhibiting antibacterial properties. In this study, silicon nitride was deposited on pure titanium metal surfaces and evaluated for its biocompatibility and antibacterial properties. The findings indicate that silicon nitride improves the hydrophilicity of the material surface, enhancing the initial adhesion of rat bone marrow cells and promoting hard tissue differentiation. Additionally, the antibacterial properties were assessed using Staphylococcus aureus, revealing that the silicon nitride-coated surfaces exhibited significant antibacterial activity. Importantly, no cytotoxicity was observed, suggesting that silicon nitride-coated titanium could serve as a novel implant material.

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 β4 and laminin 332, were increased. Surface modification to PEEK was suggested to enhance cell activity on PEEK.

We aimed to improve the biocompatibility and osteoinductive potential of Ti implants using a simulated intraoral hydroxyapatite (HAp) coating. We devised a novel surface treatment for aggressive induction of osteoblast adhesion and bone regeneration on the implant surface. A thin α-tricalcium phosphate (α-TCP) film was deposited on the implant surface using a pulsed Er:YAG laser. The coating was converted to HAp through artificial saliva immersion, which was confirmed using scanning electron microscopy (SEM) and X-ray diffraction (XRD). SEM showed needle-like HAp crystals on the Ti disks and sandblasted implant surfaces after immersion in artificial saliva for 96 h. Microcomputed tomography and histological evaluation 4 and 8 weeks after implantation into beagle dog mandibles showed that the HAp-coated implant was biocompatible and exhibited superior osteoinduction compared to that of sandblasted implants. Coating the implant surface with HAp using an Er:YAG laser has potential as a new method of the implant-surface debridement.

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.

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.

This study aimed to develop a novel culture method for rat adipose-derived stem cells (rADSC) and evaluate their osteogenic potential. The rADSC cultured in xeno-free culture medium (XF-rADSCs) or conventional culture medium containing fetal bovine serum (FBS-rADSCs) were combined with micropieces of xeno-free recombinant collagen peptide to form 3-dimensional aggregates (XF-rADSC-CellSaic or FBS-rADSC-CellSaic). Both FBS-rADSC and XF-ADSC in CellSaic exhibited multilineage differentiation potential. Compared to FBS-rADSC-CellSaic, XF-rADSC-CellSaic accelerated and promoted osteogenic differentiation in vitro. When transplanted into rat mandibular congenital bone defects, the osteogenically differentiated XF-rADSC-CellSaic induced regeneration of bone tissue with a highly maturated structure compared to FBS-rADSC-CellSaic. In conclusion, XF-rADSC-CellSaic is a feasible 3-dimensional platform for efficient bone formation.

This study radiologically and histologically compared two bioresorbable bone substitutes with different compositions carbonate apatite (Cytrans ® Granules; CGs) and β-tricalcium phosphate (β-TCP) for vertical bone augmentation on a rat skull using a polytetrafluoroethylene (PTFE) tubes. This PTFE tube was placed at the center of the skull, fixed with Super Bond, and augmented with CGs or β-TCP granules. Specimens with surrounding tissue were harvested at 4, 8, and 12 weeks postoperatively, and radiological and histological evaluations were performed. The bone volume to total volume ratio (BV/TV) of the β-TCP-implanted group was markedly higher than that of the CG-implanted group at 4 and 12 weeks postoperatively. Compared to CGs, β-TCP exhibited the ability to form blood vessels into the graft material for a short period after transplantation, as well as an elevated production of collagen into β-TCP granules during the bone formation process.

Recently, polyetheretherketone (PEEK) has shown promising dental applications. Surface treatment is essential for dental applications owing to its poor surface energy and wettability; however, no consensus on an effective treatment method has been achieved. In this study, we attempted to carboxylate PEEK sample surfaces via Friedel-Crafts acylation using succinic anhydride and AlBr 3 . The possibility of further chemical modifications using carboxyl groups was examined. The samples were subjected to dehydration-condensation reactions with 1 H ,1 H -pentadecafluorooctylamine and N , N '-dicyclohexylcarbodiimide. Furthermore, the sample's surface properties at each reaction stage were evaluated. An absorption band in the 3300-3500 cm -1 wavenumber region was observed. Additionally, peak suggestive of COOH was observed in the sample spectra. Secondary modification diminished the absorption band in 3300-3500 cm -1 and a clear F1s signal was observed. Thus, Friedel-Crafts acylation with succinic anhydride produced carboxyl groups on the PEEK sample surfaces. Further chemical modification of the carboxyl groups by dehydration-condensation reactions is also possible. Thus, a series of reactions can be employed to impart desired chemical structures to PEEK surfaces.

Polyetheretherketone (PEEK) is one of the most promising implant materials for hard tissues due to its similar elastic modulus; however, usage of PEEK is still limited owing to its biological inertness and low osteoconductivity. The objective of the study was to provide PEEK with the ability to sustain the release of growth factors and the osteogenic differentiation of stem cells. The PEEK surface was sandblasted and modified with polydopamine (PDA). Moreover, successful sandblasting and PDA modification of the PEEK surface was confirmed through physicochemical characterization. The gelatin hydrogel was then chemically bound to the PEEK by adding a solution of glutaraldehyde and gelatin to the surface of the PDA-modified PEEK. The binding and degradation of the gelatin hydrogel with PEEK (GPEEK) were confirmed, and the GPEEK mineralization was observed in simulated body fluid. Sustained release of bone morphogenetic protein (BMP)-2 was observed in GPEEK. When cultured on GPEEK with BMP-2, human mesenchymal stem cells (hMSCs) exhibited osteogenic differentiation. We conclude that PEEK with a gelatin hydrogel incorporating BMP-2 is a promising substrate for bone tissue engineering.

Bone regeneration requires cells, growth factors, and scaffolds that should have biocompatibility, porosity, and physical strength. Therefore, coral granules (CG) with diameters of 600-1,000 µm were prepared as a potential graft material from cultured edaphic thermostable corals. X-ray and electron microscopy characterization revealed that CGs were porous and permeable with lumen diameters of approximately 200 µm. Human periodontal ligament fibroblasts showed significantly increased mitochondrial activity in culture seven days after adding CG. After CG filling into an experimentally created one-wall infrabony defect in a beagle dog jawbone, the defect almost completely disappeared within approximately 8 weeks, and bone tissue growth was observed in the replacement area. This could indicate extremely rapid healing of a bone defect previously considered incapable of self-healing. Based on stable supply of cultured coral (Montipora digitata), CG is potentially an ideal replacement material for alveolar and jawbone defects.

Senescence-associated secretory phenotype (SASPs) secreted from senescent cells often cause the deleterious damages to the surrounding tissues. Although dedifferentiated fat (DFAT) cells prepared are considered a promising cell source for regenerative therapies, SASPs from DFAT cells undergoing long-term cell culture, which latently induce replicative senescence, have barely been explored. The present study was designed to investigate senescent behaviors in rat-derived DFAT cells at high passage numbers and to analyze the possible types of SASPs. Our data show that DFAT cells undergo senescence during replicative passaging, as determined by multiple senescent hallmarks including morphological changes in cell shape and nucleus. Moreover, RT 2 PCR array analysis indicated that senescent DFAT cells expressed higher levels of 16 inflammatory cytokines (Ccl11, Ccl12, Ccl21, Ccl5, Csf2, Cxcl1, Cxcl12, Ifna2, IL11, IL12a, IL13, IL1a, IL1rn, IL6, Mif, and Tnf) associated with SASPs than non-senescent cells. This study implicates that rat DFAT cells undergo cellular senescence after long-term cell culture; cautious consideration should be paid to treat SASP secretion when senescent DFAT cells are used in regenerative medicine.

Composite scaffolds obtained by the combination of biodegradable porous scaffolds and hydroxyapatite with bone regeneration potential are feasible materials for bone tissue engineering. However, most composite scaffolds have been fabricated by complicated procedures or under thermally harsh conditions. We have previously demonstrated that hydroxyapatite coating onto various substrates under a thermally mild condition was achieved by erbium-doped yttrium aluminum garnet (Er: YAG) pulsed laser deposition (PLD). The purpose of this study was to prepare a polycaprolactone (PCL) porous scaffold coated with the hydroxyapatite by the Er: YAG-PLD method. Hydroxyapatite coating by the Er: YAG-PLD method was confirmed by morphology, crystallographic analysis, and surface chemical characterization studies. When cultured on PCL porous scaffold coated with hydroxyapatite, rat bone marrow-derived mesenchymal stem cells adhered, spread, and proliferated well. The micro-CT and staining analyses after the implantation of scaffold into the critical-sized calvaria bone defect in rats indicate that PCL porous scaffold coated with hydroxyapatite demonstrates accelerated and widespread bone formation. In conclusion, PCL porous scaffold coated with hydroxyapatite obtained by the Er: YAG-PLD method is a promising material in bone tissue engineering.

Resin blocks and ceramic blocks for CAD/CAM crowns were cut into powders and separated into three particle size groups. Oxidative stress and cell viability were measured in 3T3 and FRSK cells. The results of cytotoxicity tended to be slightly higher for resin than for ceramics. The values also increased as the particle size decreased in the powders. In addition, incorporation into cells was frequently observed under SEM, suggesting that the particle size of easily incorporated dust is different among cell types. Fluorescence-activated cell sorter (FACS) showed an increase in apoptosis and a decrease in cell viability in most of the sample groups compared to the control group. Hematoxylin and eosin staining of the cells showed deep staining of the nuclei in the sample groups. It was found that oxidative stress cell viability and apoptosis appeared differently depending on the size of the particles and the type of cells.

The study was done to compare cell viability between ES-D3 and Balb/c 3T3 cells, and evaluate the difference in cell viability between these cell lines using denture-base lining materials for prosthetic dentistry. To compare the cytotoxicity, three acrylic and three silicone dental materials were used. The cell viability was examined by MTT and lactate dehydrogenase (LDH) methods. The cell viability immediately after malaxation or light irradiation was lower only for the acrylic materials in 3T3 cells, and for both silicone and acrylic materials in ES-D3 cells. However, the cell viability determined 24 h after malaxation or light irradiation by the MTT and LDH methods did not significantly differ between samples. It was observed that ES-D3 cells are more sensitive depending on the type of material. The results suggest that ES-D3 cells can be used as in vitro systems for conducting biosafety assessment to predict embryotoxicity.

The surface of titanium (Ti) dental implants must be modified to improve their applicability, owing to the biological inertness of Ti. This study aims to use sandblasting as a pretreatment method and prepare a hydroxyapatite (HA) coating on Ti to improve its biocompatibility and induce bone bonding and osteogenesis. In this paper, sandblasted Ti discs were coated with α-tricalcium phosphate (α-TCP) via Er:YAG pulsed laser deposition (Er:YAG-PLD). An HA coating was then obtained via the hydrothermal treatment of the discs at 90 °C for 10 h. The surface characteristics of the samples were evaluated by SEM, SPM, XPS, XRD, FTIR, and tensile tests. Rat bone marrow mesenchymal stem cells were seeded on the HA-coated discs to determine cellular responses in vitro. The surface characterization results indicated the successful transformation of the HA coating with a nanorod-like morphology, and its surface roughness increased. In vitro experiments revealed increased cell attachment on the HA-coated discs, as did the cell morphology of fluorescence staining and SEM analysis; in contrast, there was no increase in cell proliferation. This study confirms that Er:YAG-PLD could be used as an implant surface-modification technique to prepare HA coatings with a nanorod-like morphology on Ti discs.

Standard zirconia implants used in restoration still present problems related to inertness and long-term stability. Various physicochemical approaches have been used to modify the implant surfaces to improve early and late bone-to-implant integration; however, no ideal surface modification has been reported. This study used pulsed laser deposition to deposit a fluorinated hydroxyapatite (FHA) film on a zirconia implant to create a biologically active surface. The film prepared was uniform, dense, and crack-free, and exhibited granular surface droplets; it also presented excellent mechanical strength and favorable biological behavior. The FHA-coated implant was implanted on the femur of Sprague-Dawley rats, and various tests and analyses were performed. Results show that the in vitro initial cell activity on the FHA-coated samples was enhanced. In addition, higher alkaline phosphatase activity and cell mineralization were detected in cells cultured on the FHA-coated groups. Further, the newly formed bone volume of the FHA-coated group was higher than that of the bare micro-adjusted composite nano-zirconia (NANOZR) group. Therefore, the FHA film facilitated osseointegration and may improve the long-term survival rates of dental implants, and could become part of a new treatment technology for implant surfaces, promoting further optimization of NANOZR implant materials.

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.

There are treatments available for enamel demineralization or acid erosion, but they have limitations. We aimed to manufacture a device that could directly form a hydroxyapatite (HAp) film coating on the enamel with a chairside erbium-doped yttrium aluminum garnet (Er:YAG) laser using the pulsed laser deposition (PLD) method for repairing enamel defects. We used decalcified bovine enamel specimens and compacted α-tricalcium phosphate (α-TCP) as targets of Er:YAG-PLD. With irradiation, an α-TCP coating layer was immediately deposited on the specimen surface. The morphological, mechanical, and chemical characteristics of the coatings were evaluated using scanning electron microscopy (SEM), scanning probe microscopy (SPM), X-ray diffractometry (XRD), and a micro-Vickers hardness tester. Wear resistance, cell attachment of the HAp coatings, and temperature changes during the Er:YAG-PLD procedure were also observed. SEM demonstrated that the α-TCP powder turned into microparticles by irradiation. XRD peaks revealed that the coatings were almost hydrolyzed into HAp within 2 days. Micro-Vickers hardness indicated that the hardness lost by decalcification was almost recovered by the coatings. The results suggest that the Er:YAG-PLD technique is useful for repairing enamel defects and has great potential for future clinical applications.

Various stresses latently induce cellular senescence that occasionally deteriorates the functioning of surrounding tissues. Nevertheless, little is known about the appearance and function of senescent cells, caused by the implantation of beta-tricalcium phosphate (β-TCP)-used widely in dentistry and orthopedics for treating bone diseases. In this study, two varying sizes of β-TCP granules (<300 μm and 300-500 μm) were implanted, and using histological and immunofluorescent staining, appearances of senescent-like cells in critical-sized bone defects in the calvaria of Sprague Dawley rats were evaluated. Parallelly, bone formation in defects was investigated with or without the oral administration of senolytics (a cocktail of dasatinib and quercetin). A week after the implantation, the number of senescence-associated beta-galactosidase, p21-, p19-, and tartrate-resistant acid phosphatase-positive cells increased and then decreased upon administrating senolytics. This administration of senolytics also attenuated 4-hydroxy-2-nonenal staining, representing reactive oxygen species. Combining senolytic administration with β-TCP implantation significantly enhanced the bone formation in defects as revealed by micro-computed tomography analysis and hematoxylin-eosin staining. This study demonstrates that β-TCP granules latently induce senescent-like cells, and senolytic administration may improve the bone-forming ability of β-TCP by inhibiting senescence-associated mechanisms.

Recently, the frequency of use of bone substitute materials for the purpose of bone augmentation has increased in implant treatment, but bone formation with bone substitute materials alone is limited. Calcification of bone in the body progresses as Ca 2+ , H 2 PO 4- , and HPO 4 2- in the body form hydroxyapatite (HA) crystals. In this study, therefore, we prepared a biphasic bone substitute with biological activity to promote bone formation by inducing precipitation and growth of HA crystals on the surface of a bone substitute and evaluated it. Biphasic bone substitute granules were prepared by immersing HA granules in a supersaturated calcium phosphate solution prepared by mixing five medical infusion solutions, the precipitate was analyzed, and the biological activities of biphasic HA granules were evaluated in vitro and in vivo. As a result, the precipitated calcium phosphate crystals were identified as low crystalline HA. On the surface of the HA granules, low-crystalline HA grew markedly as needle-shaped crystals and significantly promoted cell proliferation and bone differentiation. In animal experiments, biphasic HA granules had a significantly higher bone mineral density, new bone volume ratio, and new bone area ratio. Therefore, it suggests that biphasic hydroxyapatite is a useful bone substitute for bone augmentation in dental implant treatment.

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.

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.

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis ( P. gingivalis ) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis , a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA ( r FimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal r FimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal r FimA plus FL/CpG resulted in increased levels of r FimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given r FimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by r FimA-stimulated CD4 + T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with r FimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal r FimA plus FL/CpG were challenged with nasal P. gingivalis , the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated r FimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with r FimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial and financial relationships that could be construed as a potential conflict of interest. The reviewer SM declared a shared affiliation with one of the authors, KF, to the handling editor at the time of review. The reviewer ZM declared a shared affiliation with one of the authors, KF, to the handling editor at the time of review., (Copyright © 2021 Kataoka, Kawabata, Koyanagi, Hashimoto, Miyake and Fujihashi.)

Bone regeneration using mesenchymal stem cells has several limitations. We investigated adipose-derived dedifferentiated fat (DFAT) cells as an alternative, and evaluated their cell proliferation rate, osteoblast differentiation, and bone regeneration ability in combination with activated platelet-rich plasma (aPRP). Rat DFATs and aPRP were isolated using ceiling culture and centrifugation, respectively. The cell proliferation rate was measured, and the cells were cultured in an osteoblast differentiation medium under varying concentrations of aPRP for 21 days and stained with Alizarin red. Gene expression was evaluated using real time polymerase chain reaction. Critical defects were implanted with DFAT seeded gelatin sponges under aPRP, and four weeks later, the bone regeneration ability was evaluated using micro-computed tomography and hematoxylin-eosin staining. The cell proliferation rate was significantly increased by the addition of aPRP. Alizarin red staining was positive 21 days after the start of induction, with significantly higher Runt-related transcription factor 2 ( Runx2) and osteocalcin ( OCN) expression levels than those in the controls. A 9 mm critical defect was largely closed (60.6%) after four weeks of gelatin sponge implantation with DFAT and aPRP. Therefore, materials combining DFAT cells and aPRP may be an effective approach for bone regeneration. Further research is needed to explore the long-term effects of these materials.

The Bio-C Sealer is a recently developed high-plasticity, calcium-silicate-based, ready-to-use material. In the present study, chemical elements of the materials were characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The biocompatibility of the Bio-C Sealer was investigated using cytotoxicity tests and histological responses in the roots of dogs' teeth. XRD, SEM, and FTIR produced hydrated calcium silicate in the presence of water molecules. In addition, FTIR showed the formation of calcium hydroxide and polyethylene glycol, a dispersing agent. The 1:4 dilutions of Bio-C Sealer presented weaker cytotoxicity than the Calcipex II in an in vitro system using the V-79 cell line. After 90 d, the periradicular tissue response of beagle dog roots was histologically evaluated. Absence of periradicular inflammation was reported in 17 of the 18 roots assessed with the Bio-C Sealer, whereas mature vertical periodontal ligament fibers were observed in the apical root ends filled with the Bio-C Sealer. Based on these results and previous investigations, the Bio-C Sealer is recommended as an effective root-end filling material. These results are relevant for clinicians considering the use of Bio-C Sealer for treating their patients.

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.

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-β-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-β-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.

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., Competing Interests: The authors declare no conflict of interest.

We have previously fabricated hydroxyapatite (HAP) nanoparticle-assembled powder (nano-HAP) plates and granules by assembling low-crystallinity HAP nanoparticles without template/binder molecules or high-temperature/pressure treatments. In this study, we combined the nano-HAP with fibroblast growth factor (FGF) 2, which promotes odontoblast differentiation, and used this as a pulpcapping agent for dentin defects created in rat molars. The tissue response was then radiologically and histologically assessed at 1 and 2 weeks after capping, to assess the biocompatibility and ability of this material to promote hard tissue formation. The application of nano-HAP/FGF2 induced the invasion of dental pulp cells and vessels, and was consistently found to stimulate formation of a dentinal bridge containing numerous dentinal tubules. We thus succeeded in treating the pulp exposure by using a physiological approach to promote tissue regeneration. Further investigations should be performed to explain exactly how the nano-HAP/FGF2 combination contributes to calcified tissue formation.

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.

Tying shape memory wires to crowded teeth causes the wires to deform according to the dental arch. This deformation results in a resilient force that is delivered to the tooth. The appropriate amount of force can activate the osteogenetic and osteoclastic ability of the periodontal ligament (PDL) and the tooth can be moved. This is the biological basis of orthodontic treatment. To achieve further insight into the mechanisms underlying orthodontic treatment, we examined whether accelerated construction of an in vitro human PDL fibroblast (HPdLF) stretching model can be achieved by combining fibronectin coating and vacuum plasma treatment with polydimethylsiloxane (PDMS) cell-culture chambers. Each chamber was randomly assigned to a no-surface modification (NN), fibronectin coating (FN), vacuum plasma treatment (PN), or vacuum plasma treatment followed by a fibronectin coating (PF) treatment protocol. The physical and chemical features and ability to promote cellular proliferation of the PDMS chamber surfaces were evaluated. Cellular adhesion of four materials were evaluated and two best-proliferated groups were considered as better model-constructing surfaces and used in subsequent experiments and used in subsequent experiments. HPdLFs were cultured on these two kinds of chambers without stretching for 3 days, then with stretching for 7 days. Time-course gene expression cellular morphology were evaluated. Chambers in the PN group had high wettability and surface component changes. The FN and PF chambers had high cellular proliferation ability. They were selected into subsequent experiments. After 3 days of culturing HPdLFs on the PF and PN chambers, the cells in the PF chambers had significantly higher levels of runt-related transcription factor 2 ( Runx-2 ) and osteocalcin ( OCN ) gene expression compared with the cells in the PN chambers. After cyclic stretch application to the cells in the PN and PF chambers, expression of the type-3 collagen ( COL-3 ) gene in PF group continued to increase for 7 days and was significantly higher than that in the PN group from day 5 onwards. The HPdLFs in the PF group showed parallel alignment from days 3 to 7 after imposition of cyclic stretch, while those in the PN group aligned in parallel from day 5 on. Our results suggested that applying a fibronectin coating to a PDMS chamber after plasma treatment can accelerate establishment of an in vitro PDL stretching model., Competing Interests: The authors declare there are no competing interests.

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/cm 2 on an area 1×1 mm 2 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.

Chemical modification of gelatin using epigallocatechin gallate (EGCG) promotes bone formation in vivo. However, further improvements are required to increase the mechanical strength and bone-forming ability of fabricated EGCG-modified gelatin sponges (EGCG-GS) for practical applications in regenerative therapy. In the present study, we investigated whether vacuum heating-induced dehydrothermal cross-linking of EGCG-GS enhances bone formation in critical-sized rat calvarial defects. The bone-forming ability of vacuum-heated EGCG-GS (vhEGCG-GS) and other sponges was evaluated by micro-computed tomography and histological staining. The degradation of sponges was assessed using protein assays, and cell morphology and proliferation were verified by scanning electron microscopy and immunostaining using osteoblastic UMR106 cells in vitro. Four weeks after the implantation of sponges, greater bone formation was detected for vhEGCG-GS than for EGCG-GS or vacuum-heated gelatin sponges (dehydrothermal cross-linked sponges without EGCG). In vitro experiments revealed that the relatively low degradability of vhEGCG-GS supports cell attachment, proliferation, and cell-cell communication on the matrix. These findings suggest that vacuum heating enhanced the bone forming ability of EGCG-GS, possibly via the dehydrothermal cross-linking of EGCG-GS, which provides a scaffold for cells, and by maintaining the pharmacological effect of EGCG., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: The authors declare no conflict of interest.

We evaluated the effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on periodontal regeneration in a canine model of 2-wall periodontal defects. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), and the remaining defects were filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed at 2, 4, and 8 weeks post-implantation. The bone mineral content of the bFGF group was higher than that of the control group at 2 and 4 weeks (p < 0.05). Histological evaluation at 2 weeks post-implantation revealed degradation of the porous α-TCP, and bone had formed on the surface of α-TCP particles in the bFGF group. Some of these collagen fibers connected the newly formed cementum with the alveolar bone, revealing the formation of new periodontal ligaments with Sharpey's fibers. At 8 weeks, continuous cortical bone with a Haversian structure covered the top of the bone defects in the bFGF group. These findings indicate that porous α-TCP with immobilized bFGF could promote periodontal regeneration at the early regeneration phase in a canine model of 2-wall periodontal defects.

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.

The effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on bone regeneration was evaluated in a canine mandibular bone defect model. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), whereas the other was filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed two, four and eight weeks after implantation. The bone mineral density of the bFGF group was higher than that of the control group at each time point ( p < 0.05), and the bone mineral content of the bFGF group was higher than that of the control group at four and eight weeks ( p < 0.05). Histological evaluation two weeks after implantation revealed that the porous α-TCP had degraded and bone had formed on the surface of α-TCP particles in the bFGF group. At eight weeks, continuous cortical bone with a Haversian structure covered the top of bone defects in the bFGF group. These findings demonstrate that porous α-TCP with immobilized bFGF can promote bone regeneration.

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., (Copyright © 2016 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.)

This study investigated cell survival and gene expression under various compressive stress conditions mimicking orthodontic force by using a newly developed in vitro model of human periodontal ligament-like tissue (HPdLLT). The HPdLLT was developed by three-dimensional culturing of human periodontal ligament fibroblasts in a porous poly-L-lactide matrix with threefold increased culture media permeability due to hydrophilic modification. In vitro HPdLLTs in experimental groups were subjected to 5, 15, 25 and 35 g/cm(2) compressive stress for 1, 3, 7 or 14 days; controls were cultured over the same periods without compressive stress. Cell morphology and cell apoptosis in the experimental and control groups were investigated using scanning electron microscopy and caspase-3/7 detection. Real-time polymerase chain reaction was performed for seven osteogenic and osteoclastic genes. Similar extracellular matrix and spindle-shaped cells were observed inside or on the surface of in vitro HPdLLTs, with no relation to compressive stress duration or intensity. Similar caspase-3/7 activity indicating comparable apoptosis levels was observed in all samples. Receptor activator of nuclear factor kappa-B ligand and bone morphogenetic protein 2 genes showed characteristic "double-peak" expression at 15 and 35 g/cm(2) on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm(2) on day 14; other genes also showed time-dependent and load-dependent expression patterns. The in vitro HPdLLT model system effectively mimicked the reaction and gene expression of the human periodontal ligament in response to orthodontic force. This work provides new information on the effects of compressive stress on human periodontal ligament tissue.

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.

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.

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.

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.

Dedifferentiated fat cells (DFAT cells) isolated from adipose tissue have been demonstrated to differentiate into chondrogenic cells in vitro. Nevertheless, an efficient method to facilitate its chondrogenic differentiation is still unexplored, hampering the extensive application of these cells in cartilage regeneration therapies. Here we provide the evidence that supplementation of strontium ions (Sr) in a chondrogenic medium (CM) significantly promotes early chondrogenic differentiation of DFAT cells. Human DFAT cells and the mesenchymal stem cell line (RCB2153) were subjected to the CM supplemented with/without Sr. After 14 days, alcian blue staining intensity significantly increased in DFAT cells, but not in RCB2153, subjected to CM with Sr. mRNA expression analysis revealed that the CM with 1.5 mM Sr increased the expression of chondrogenic marker, collagen type 2 alpha 1, whereas there was no significant change in osteogenic markers, collagen type 1 alpha 1, runt-related transcription factor 2, and osteocalcin, and hypertrophic chondrogenic marker, collagen type 10 alpha 1. Inhibitors for extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and calcium-sensing receptor (CaSR) pathways significantly diminished the alcian blue staining intensity, providing the first evidence that these signal pathways are associated with chondrogenic differentiation of DFAT cells. CaSR and ERK1/2 pathways independently induced Sr-mediated early chondrogenic differentiation. These results suggest that Sr supplementation into the CM may provide a powerful platform for preparing chondrogenically differentiated DFAT cells for cartilage regeneration.

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.

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.

We compared the osteoblastic differentiation abilities of dedifferentiated fat cells (DFATs) and human bone marrow mesenchymal stem cells (hMSCs) as a cell source for bone regeneration therapies. In addition, the utility of DFATs in bone tissue engineering in vitro was assessed by an alpha-tricalcium phosphate (α-TCP)/collagen sponge (CS). Human DFATs were isolated from the submandibular of a patient by ceiling culture. DFATs and hMSCs at passage 3 were cultured in control medium or osteogenic medium (OM) for 14 days. Runx2 gene expression, alkaline phosphatase (ALP) activity, as well as osteocalcin (OCN) and calcium contents were analyzed to evaluate the osteoblastic differentiation ability of both cell types. DFATs seeded in a α-TCP/CS and cultured in OM for 14 days were analyzed by scanning electron microscopy (SEM) and histologically. Compared with hMSCs, DFATs cultured in OM generally underwent superior osteoblastogenesis by higher Runx2 gene expression at all days tested, as well as higher ALP activity at day 3 and 7, OCN expression at day 14, and calcium content at day 7. In SEM analyses, DFATs seeded in a α-TCP/CS were well spread and covered the α-TCP/CS by day 7. In addition, numerous spherical deposits were found to almost completely cover the α-TCP/CS on day 14. Von Kossa staining showed that DFATs differentiated into osteoblasts in the α-TCP/CS and formed cultured bone by deposition of a mineralized extracellular matrix. The combined use of DFATs and an α-TCP/CS may be an attractive option for bone tissue engineering.

Objectives: The purpose of this study was to evaluate and compare the osteoblastic differentiation ability of dedifferentiated fat (DFAT) cells and adipose stem cells (ASCs) from the buccal fat pad (BFP)., Materials and Methods: We isolated human DFAT cells and ASCs from the BFP of a patient who underwent oral and maxillofacial surgery and then analyzed their cell surface antigens by flow cytometry. Then, the cells were cultured in osteogenic medium for 14 days. Measurement of bone-specific alkaline phosphatase (BAP), osteocalcin (OCN), and calcium deposition and alizarin red staining were performed to evaluate the osteoblastic differentiation ability of both cell types., Results: ASCs and DFAT cells were positive for CD90 and CD105 and negative for CD11b, CD34, and CD45. BAP (days 3 and 7), OCN (day 14), and calcium deposition (days 7 and 14) within DFAT cell cultures were significantly higher than those in ASC cultures. The alizarin red-stained area in DFAT cell cultures, which indicates mineralized matrix deposition, was stained more strongly than that in ASC cultures., Conclusions: The cell surface antigens of ASCs and DFAT cells tend to be similar. Furthermore, the osteoblastic differentiation ability of human DFAT cells is higher than that of ASCs from the BFP., Clinical Relevance: Isolation of DFAT cells from the BFP has an esthetic advantage because the BFP can be obtained via the oral cavity without injury to the external body surface. Therefore, we consider that DFAT cells from the BFP are an ideal cell source for bone tissue engineering.

The objective of this study was to investigate the feasibility of biodegradable gelatin-β-tricalcium phosphate (β-TCP) composites as a cell scaffold and controlled-release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and β-TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical-sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin-β-TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and β-TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF-free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin-β-TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF., (Copyright © 2012 John Wiley & Sons, Ltd.)