Biocompatibility of dental implants coated with hydroxyapatite using pulsed Er:YAG laser deposition.

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.

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

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

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

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

Evaluation of Osteogenic Potential for Rat Adipose-Derived Stem Cells under Xeno-Free Environment.

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.

Novel Carboxylation Method for Polyetheretherketone (PEEK) Surface Modification Using Friedel-Crafts Acylation.

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.

Carbonate apatite versus ß-tricalcium phosphate for rat vertical bone augmentation: A comparison of bioresorbable bone substitutes using polytetrafluoroethylene tubes.

This study radiologically and histologically compared two bioresorbable bone substitutes with different compositions carbonate apatite (Cytrans® Granules; CGs) and ß-tricalcium phosphate (ß-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 ß-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 ß-TCP-implanted group was markedly higher than that of the CG-implanted group at 4 and 12 weeks postoperatively. Compared to CGs, ß-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 ß-TCP granules during the bone formation process.

Bioactive Polyetheretherketone with Gelatin Hydrogel Leads to Sustained Release of Bone Morphogenetic Protein-2 and Promotes Osteogenic Differentiation.

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 augmentation with a prototype coral exoskeleton-derived bone replacement material applied to experimental one-wall infrabony defects created in alveolar bone.

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 phenotypes in rat-derived dedifferentiated fat cells with replicative senescence.

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, RT2 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.

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

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

Effect of Hydroxyapatite Coating by Er: YAG Pulsed Laser Deposition on the Bone Formation Efficacy by Polycaprolactone Porous Scaffold.

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.

Structural Characterization and Osseointegrative Properties of Pulsed Laser-Deposited Fluorinated Hydroxyapatite Films on Nano-Zirconia for Implant Applications.

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.

Characterization of Hydroxyapatite Film Obtained by Er:YAG Pulsed Laser Deposition on Sandblasted Titanium: An In Vitro Study.

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.

Cell viability of fine powders in hybrid resins and ceramic materials for CAD/CAM.

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.

Comparison of cell viability between mouse-derived ES-D3 cells and Balb/c 3T3 cells using denture-base lining materials.

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.

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

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

Hydroxyapatite Film Coating by Er:YAG Pulsed Laser Deposition Method for the Repair of Enamel Defects.

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.

Enhancement of Bone-Forming Ability on Beta-Tricalcium Phosphate by Modulating Cellular Senescence Mechanisms Using Senolytics.

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 (ß-TCP)-used widely in dentistry and orthopedics for treating bone diseases. In this study, two varying sizes of ß-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 ß-TCP implantation significantly enhanced the bone formation in defects as revealed by micro-computed tomography analysis and hematoxylin-eosin staining. This study demonstrates that ß-TCP granules latently induce senescent-like cells, and senolytic administration may improve the bone-forming ability of ß-TCP by inhibiting senescence-associated mechanisms.

Bioactivity Evaluation of Biphasic Hydroxyapatite Bone Substitutes Immersed and Grown with Supersaturated Calcium Phosphate Solution.

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.

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

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