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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 AlBr3. The possibility of further chemical modifications using carboxyl groups was examined. The samples were subjected to dehydration–condensation reactions with 1H,1H-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mechanical and resorbable scaffolds are in high demand for stem cell-based regenerative medicine, to treat refractory bone defects in craniofacial abnormalities and injuries. Multipotent progenitor cells, such as dedifferentiated fat (DFAT) cells, are prospective sources for regenerative therapies. Herein, we aimed to demonstrate that a composite gelatin sponge (α-TCP/GS) of alfa-tricalcium phosphate (α-TCP) mixed with gelatin scaffolds (GS), with/without DFATs, induced bone regeneration in a rat calvarial defect model in vivo. α-TCP/GS was prepared by mixing α-TCP and 2% GS using vacuum-heated methods. α-TCP/GS samples with/without DFATs were transplanted into the model. After 4 weeks of implantation, the samples were subjected to micro-computed tomography (μ-CT) and histological analysis. α-TCP/GS possessed adequate mechanical strength; α-TCP did not convert to hydroxyapatite upon contact with water, as determined by X-ray diffraction. Moreover, stable α-TCP/GS was formed by electrostatic interactions, and verified based on the infrared peak shifts. μ-CT analyses showed that bone formation was higher in the α-TCP/GS+ DFAT group than in the α-TCP/GS group. Therefore, the implantation of α-TCP/GS comprising DFAT cells enhanced bone regeneration and vascularization, demonstrating the potential for healing critical-sized bone defects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, using electrolytic reducing ionic water (S-100®), a novel surface treatment method safely and easily modifying the surface properties was evaluated in vitro and in vivo. Ti-6Al-4V disks were washed and the disks were kept standing on a clean bench for one and four weeks for aging. These disks were immersed in S-100® (S-100 group), immersed in ultra-pure water (Control group), or irradiated with ultraviolet light (UV group), and surface analysis, cell experiment, and animal experiment were performed using these disks. The titanium surface became hydrophilic in the S-100 group and the amount of protein adsorption and cell adhesion rate were improved in vitro. In vivo, new bone formation was noted around the disk. These findings suggested that surface treatment with S-100® adds bioactivity to the biologically aged titanium surface. We are planning to further investigate it and accumulate evidence for clinical application. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 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 on day 14, and alkaline phosphatase and periodontal ligament-associated protein 1 expression peaked at 5 g/cm 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. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the difference in the adsorption behavior of different types of bovine salivary proteins on the PMMA and Ti QCM sensors are fabricated by spin-coating and sputtering onto bare QCM sensors by using QCM and X-ray photoelectron spectroscopy (XPS). SPM, XPS, and contact angle investigations were carried out to determine the chemical composition and surface wettability of the QCM surface. We discuss the quality of each sensor and evaluate the potential of the high-frequency QCM sensors by investigating the binding between the QCM sensor and the proteins albumin and mucin (a salivary-related protein). The SPM image showed a relatively homogeneous surface with nano-order roughness. The XPS survey spectra of the thin films coated on the sensors were similar to the binding energy of the characteristic spectra of PMMA and Ti. Additionally, the amount of salivary-related protein on the PMMA QCM sensor was higher than those on the Ti and Au QCM sensors. The difference of protein adsorption is proposed to be related to the wettability of each material. The PMMA and Ti QCM sensors are useful tools to study the adsorption and desorption of albumin and mucin on denture surfaces. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, l-ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering. [ABSTRACT FROM AUTHOR]

Orthodontists use a self-etching adhesive system when attaching brackets, to enamel. The purpose of this study was to evaluate the erosion effects of common clinically used adhesive systems on human enamel surfaces by atomic force microscopy (AFM). Four commercially available adhesive systems (i.e., Kurasper F, Beauty Ortho Bond, Orthophic LC, and Transbond XT) were applied to ground enamel surfaces of extracted human teeth. Enamel surface roughness (ESR), absolute depth profile (ADP), and surface hardness were evaluated by AFM. The ESR and ADP were significantly higher after the pretreatment with the phosphoric acid-etching adhesive system than after the pretreatments with the three self-etching adhesive systems. The surface nanohardness decreased after the pretreatment with the phosphoric acid-etching adhesive system but increased after the pretreatments with the self-etching adhesive systems. These results suggest that the use of a self-etching primer for enamel conditioning might prevent decalcification caused by phosphoric acid etching. [ABSTRACT FROM AUTHOR]

Surface modification of titanium has been extensively investigated in implant science and technology in an effort to improve its osteoconductivity. The rate of protein adsorption on titanium surfaces is known to vary depending on the chemistry, structure, morphology, and titanium-specific biological aging of the surface. It is thus desirable to modify smooth titanium surfaces of mini-implants used as orthodontic anchors immediately prior to use. In this study, we have developed a simple surface modification of titanium alloy that improves its biofunctional activity. The surface of a Ti-6A1-4V disk was modified by applying 3% H2O2 hydrothermal treatment using an autoclave. A nanostructured porous network TiO2 was observed on the treated surface. Treated surfaces exhibited higher hydrophilicity, protein adsorption, and cell proliferation than untreated surfaces. 3% H2O2 hydrothermal treatment is thought to provide biofunctional activity for aged titanium surface. [ABSTRACT FROM AUTHOR]

We evaluated the effects on bone formation of combining synthesized collagen model polypeptides consisting of a Pro-Hyp-Gly [poly(PHG)] sequence and alpha-tricalcium phosphate (α-TCP) particles with various median sizes (large: 580.8 μm; small: 136.2 μm; or large and small mixed: 499.3 μm) in a skull defect model in mini-pigs. Quantitative image analyses for the volume density (VD) of new bone revealed that the VD in each α-TCP group was significantly higher than that in the poly(PHG) control group, with the mixed group showing the highest VD among all the groups at 4 weeks after implantation. Histological assessments revealed that the small α-TCP particles were almost completely degraded at 8 weeks. At 12 weeks, all sizes of α-TCP particles were completely degraded and remodeling of the lamellar bone was observed. The present findings suggest that particle size may influence the success of bone formation in defects. [ABSTRACT FROM AUTHOR]

To date, there has been no study on the development of novel regimens based on the following tissue engineering principles: seeding and culturing mesenchymal stem cells (MSCs) on a scaffold before surgery or injecting cultured MSCs into a scaffold during surgery. The purpose of this study was to assess the in viuo osteogenic ability of scaffold/MSCs implanted beneath the periosteum of the cranial bone of rats in three different sample groups: one in which MSCs were pre-seeded and cultured on a scaffold to produce the 3-D woven fabric scaffold/MSC composite using osteo-lineage induction medium, one in which cultured MSCs produced by osteo- lineage induction in cell cultivation flasks were injected into a scaffold during surgery and a control group, in which only the 3-D woven fabric scaffold was implanted. The results indicate that pre-seeding MSCs on a scaffold leads to a higher osteogenic ability than injecting cultured MSCs into a scaffold during surgery. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of β-tricalcium phosphate (β-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the β-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from β-TCP beads with diameters ranging from 300 to 500 μm (small beads), 500–700 μm (medium beads) and 700–850 μm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In the current study, we examined the cytocompatibility of eight vinyl esters as candidate plasticizers for producing phthalate- and ethanol-free tissue conditioners. We measured the estrogenic activity and cytotoxicity of vinyl esters in human fibroblasts and keratinocytes using an E-screen assay and a mitochondrial dye conversion assay, respectively. We also assessed the cytotoxicity of three prototype materials and commercially available tissue conditioners on human fibroblasts grown in collagen gels. Finally, we measured the effects of these materials on the expression of cytokines in three-dimensional cultures by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. None of the tested vinyl esters had estrogenic activity. Vinyl octanoate and vinyl pivalate were the least cytotoxic of the eight tested vinyl esters. In the same vein, a prototype tissue conditioner containing vinyl octanoate had equivalent or weaker cytotoxicity and induction of cytokine expression than conventional materials. [ABSTRACT FROM AUTHOR]

Synthetic biomaterials have been developed and used for bone grafting. Here, we developed a biodegradable sponge composite for bone tissue engineering by combining β-tricalcium phosphate (β-TCP) and collagen. In addition, we sought to determine the optimal β-TCP granules/collagen ratio by evaluating and bone formation in vivo. Porous β-TCP granules were mixed with atelocollagen hydrochloride solution at various ratios -- 0.02, 0.05, 0.1, and 0.2 g/mL. The resultant mixtures were freeze-dried and subjected to dehydrothermal treatment in vacuo. The final composites obtained were designated β-TCP/collagen sponge composites (β-TCP/CS). Through compression testing, it was found that the stress values for β-TCP/CS (0.2 g/mL) were higher than those of the other three composites over the whole strain range. Histological evaluation at four weeks after implantation revealed that the collagen sponge had degraded and newly formed bone was present on the surface of the β-TCP granules. At 12 weeks, the β-TCP granules were completely degraded and remodeling of the lamellar bone was observed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Ca2+, H2PO4-, and HPO42- 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The present study showed differences in morphology between allergic, irritant and negative patch test reactions. Examination of the surface of the skin site of allergic reactions showed an increase in crista, deepening of sulci and papules on the gypsum cast. The use of replicas in the dermatology clinic has previously been studied. Their use has, however, been limited to observation of the reaction of the skin surface to irritants or other factors, not to evaluate patch test results; nor have gypsum casts been used. The present system can store morphological changes during skin reactions on a gypsum cast like a photograph, allowing comparison of patch test results and examination of features of the positive reaction.

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. [ABSTRACT FROM AUTHOR]

Background: The biological aging of titanium (Ti) surface is a time‐dependent degradation of the osteoconductivity, lead to a decrease in the biomechanical strength of the bone–Ti integration. To overcome the aging, ultraviolet light (UV) has been shown to changing the physicochemical properties of Ti surface from hydrophobic to hydrophilic for removal of the surface hydrocarbons without altering the surface topography. However, expensive special equipment was required for UV photo‐functionalization. Aim/Hypothesis: In this study, we focused on electrolyzed deoxidized and ionized water (S – 100) which can easily remove contamination such as carbon without special equipment. The aim of this study is to investigate the influence S‐100 treatment on the recovery of the biological activity of the aged Ti surface. Material and Methods: We prepared mirror polished Ti‐6Al‐4V discs. All the discs cleaned using an ultrasonic cleaner in acetone, ethanol, and double distilled water for 10 minutes each. And then the prepared Ti discs were aged by storing in the clean‐bench at a room temperature for 1 and 4 weeks. After the aging, we divided the Ti disks into two groups. Half of the discs immersed in S‐100 for 3 minutes and the remaining half of the discs immersed in saline water for 3 minutes as a control group. The contact angle on the disc surface were measured by digital contact angle measuring device. The chemical composition of the surface analyzed by X‐ray photoelectron spectroscopy (XPS). As a protein adsorption assay, the discs were immersed into 500 μL of bovine serum albumin (1 mg/mL). After incubating for 24 hours, the amount of protein quantified using a micro plate reader at 595 nm. MC3T3‐E1 cell were cultured on the control and S‐100 treated disks for 24 hours, the cell growth were observed with a fluorescence microscope. Results: Fig. 1 shows the contact angle. The dropping 0.5 μL DDW were formed hemispheric droplets with a contact angle of approximately 65.2 degree on the 4‐week aged control surfaces, indicating that the surfaces were hydrophobic (Fig. 2). In contrast, water droplets on the surface treated with S‐100 were spread immediately, and the contact angle was approximately 18.7 degree. Even on the 1‐week aged surface, the contact angle on the S‐100 treatment significantly decreased and changed to a hydrophilic surface. In XPS analysis, a decrease in C1s was observed on the surface treated with S‐100 in all periods. On the other hand, O1s and Ti increased by S‐100 treatment. The results of protein adsorption on the Ti surface, there were no significant difference between the S‐100 treated surface and the control surface. However, the proliferation of osteoblastic cells on the S‐100 treated surface were higher than on the control surface in 4‐week of culture. Conclusions and Clinical Implications: In this study, we compared the biological activity of untreated and S‐100 treated Ti alloys surface. Aged Ti disks were immersed for only 3 minutes could easily improve the reduction of the surface energy due to carbon attached to the Ti surface. As a result, the hydrophobic Ti surface were changed to hydrophilic, and then the proliferation of osteogenic cells were accelerated. S‐100 treatment suggested that the new bone formation on the Ti surface promotes at early stage of osseointegration. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]