Involvement of ferroptosis in Porphyromonas gingivalis lipopolysaccharide-stimulated periodontitis in vitro and in vivo.

OBJECTIVES: Ferroptosis is associated with multiple inflammatory diseases. Periodontitis is an inflammatory disease mainly caused by oral opportunistic pathogens. However, the ferroptosis-periodontitis relationship has not been thoroughly described. We here analyzed whether ferroptosis is involved in periodontitis. MATERIALS AND METHODS: Human gingival fibroblasts (HGFs) were stimulated with P. gingivalis-LPS and ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and changes in mitochondrial morphology, ferroptosis-related factors, and inflammation levels were detected. After the rat experimental periodontitis model was established, changes in ferroptosis-related factors and inflammation levels were re-evaluated in the same manner. RESULTS: Porphyromonas gingivalis-LPS-induced mitochondrial shrinkage, an increase in mitochondrial membrane density, and upregulation of reactive oxygen species in HGFs. The expression of prostaglandin-endoperoxide synthase 2, transferrin receptor 1, and malondialdehyde and inflammation levels were upregulated, whereas the expression of solute carrier family seven member 11, glutathione peroxidase 4, superoxide dismutase, and glutathione were downregulated. Fer-1 attenuated these aforementioned changes and inflammation levels induced by P. gingivalis-LPS. The in vivo experiment results were consistent with the in vitro experiment results. CONCLUSIONS: Ferroptosis is involved in inflammatory processes in HGFs upon P. gingivalis-LPS stimulation. Ferroptosis is observed in the gingival tissue of periodontitis rats.

Docosahexaenoic Acid-Loaded Nanostructured Lipid Carriers for the Treatment of Peri-Implantitis in Rats.

Being the most common cause of implant failure, peri-implantitis is defined as a pathological condition associated with the occurrence of peri-implant plaque, characterized by peri-implant mucosal inflammation and progressive loss of the supporting bone tissue attributed to the persistence of pro-inflammatory cytokines. Docosahexaenoic acid (DHA), which is a type of omega-3 polyunsaturated fatty acid, is generally used for the treatment of many inflammatory diseases. However, a suitable form for dosing and its therapeutic effect on peri-implantitis remain unclear. In this study, a novel nanostructured lipid carrier (NLC) loaded with squalene and DHA was fabricated (DHA-loaded NLC). The encapsulation efficiency and drug loading efficiency values of the DHA-loaded NLC were 78.13% ± 1.85% and 28.09% ± 0.48%, respectively. The release of DHA was gradual and steady until 144 h. In addition, the free-radical-scavenging rate of DHA-loaded NLC (0.57 ± 0.03) was much higher than that of sole DHA (0.17 ± 0.003). By inhibiting nuclear factor-κB p65 nuclear translocation, DHA-loaded NLC prevented the activation of nuclear factor-κB downstream inflammatory pathways and exerted anti-inflammatory effects on macrophages. Moreover, DHA-loaded NLC showed better effects on preventing alveolar bone resorption of rat peri-implantitis model than sole DHA. Hence, DHA-loaded NLC enhanced the anti-inflammatory bioavailability of DHA, offering a novel approach for the treatment of peri-implantitis.

D-aspartic acid protects against gingival fibroblasts inflammation by suppressing pyroptosis.

BACKGROUND: Peri-implantitis is the main cause of dental implant failure, which is associated with pyroptosis. The roles of D-aspartic acid (D-Asp) on pyroptosis and the mechanism of the protective effect of D-Asp on human gingival fibroblasts (HGFs) remain unknown. This study investigated the effects of D-Asp on the pyroptosis of HGFs induced by high mobility group box 1 protein (HMGB1). METHODS: The cytotoxic effects of D-Asp on HGFs was detected by Cell Counting Kit-8 assay, the membrane permeability was investigated by propidium iodide/ Hoechst 33,342 double staining, flow cytometry analysis, and lactate dehydrogenase releasing, The gene and protein expression levels were detected by real-time quantitative PCR, enzyme-linked immunosorbent assay, and Western blot, respectively. RESULTS: Cell viability analysis showed that D-Asp ≤ 30 mM had no cytotoxicity to HGFs. HMGB1 drastically raised the membrane permeability of HGFs, while 1/10/30 mM D-Asp suppressed the permeability and remained the integrity of the membrane. HMGB1 promoted the mRNA expression of NLRP3, caspase-1, GSDMD, IL-1ß, and IL-18, and the protein expression of IL-1ß, IL-18, caspase-1, GSDMD, and NLRP3. CONCLUSIONS: With the pretreatment of HGFs with D-Asp of 1/10/30 mM for 24 h, the cell membrane permeability was reduced and the expression of NLRP3, caspase-1, GSDMD, IL-1ß, and IL-18 was significantly decreased compared with the HMGB1 group, indicating the competitive antagonism of D-Asp against HMGB1 on the binding with toll-like receptors. Hence, this study may provide a novel insight into preventing pyroptosis and propose a new strategy for the treatment of peri-implantitis.

Synergistic effects of D-arginine, D-methionine and D-histidine against Porphyromonas gingivalis biofilms.

Porphyromonas gingivalis biofilms are implicated in the pathology of peri-implantitis and periodontitis. In this study, D-arginine (R), D-methionine (M), D-histidine (H), and a mixture of these D-amino acids (D-AAs) were investigated as an effective therapeutic strategy against P. gingivalis biofilms. The bacterial growth activity and minimum inhibitory concentrations were determined for each D-AA, along with the effects of the D-AAs mixture on biofilm development, morphology, structure, extracellular polysaccharides (EPS), cytotoxicity towards commensals, and bacterial structure. The D-AA mixture delayed the proliferation of P. gingivalis, changed its membrane structure, and decreased biofilm thickness and integrity, as compared with individual D-AAs. The EPS content increased with the concentration of D-AAs. The present study shows that a 4 mM RMH, triple D-AA mixture, enhanced deleterious effects on P. gingivalis biofilms without any cytotoxicity compared with individual D-AAs, thus providing a new strategy for the treatment of peri-implantitis and periodontitis.

The Assessment of Volumetric Changes for Alveolar Ridge Preservation or Reconstruction by Three-dimensional Analysis at Posterior Extraction Sites with Severe Bone Defects Using DBBM-C Collagen Membrane and PRF: A Prospective and Randomized Clinical Trial.

STATEMENT OF PROBLEM: Volumetric resorption of the alveolar ridge often occurs following tooth extraction in both horizontal and vertical directions. There is a specific lack of evidence for alveolar ridge reconstruction at molar and premolar sites with severe bone resorption. PURPOSE: This randomized and controlled trial aimed to use three dimensional and linear analyses to evaluate volumetric changes of the alveolar bone following alveolar ridge reconstruction (ARR) at molar and premolar sites with severe bone resorption as compared with non-assisted socket healing be implant placement. MATERIAL AND METHODS: A total of 31 patients (15 males and 16 females) with more than 50% of hard tissue loss in one or more socket walls were recruited and randomized into either a test group (ARR after extraction using deproteinized bovine bone mineral with 10% collagen (DBBM-C) and platelet-rich fibrin (PRF) with a resorbable collagen membrane) or a control group (natural healing after extraction). Then, the clinical, linear, volumetric implant-related and patient-reported outcomes were analyzed after a 4-month healing process. RESULTS: Linear bone assessments revealed significantly greater gains of ridge width in the test group (25% in the mesial, mid-facial and distal aspects) and less reduction of vertical bone ridge than in the control group (P＜0.05). Furthermore, volumetric bone remodeling was significantly higher in the test group (ARR=35.1±34.9%, control=14.2±12.8%, P＜0.05). Patient-reported discomfort and keratinized mucosal changes were comparable between groups. CONCLUSIONS: Alveolar ridge reconstruction with a combination of DBBM-C, PRF, and a resorbable membrane at posterior sites with severe socket wall deficiency (＞ 50% bone loss) is a safe and more capable therapeutic method when compared with natural healing and non-assisted sockets. CLINICAL IMPLICATIONS: Collectively, our analyses demonstrated that alveolar ridge reconstruction represents an efficient method to maintain and augment crestal bone at posterior extraction sites with severe bone defects when assessed after four months of healing.

Advances and Prospects in Antibacterial-Osteogenic Multifunctional Dental Implant Surface.

In recent years, dental implantation has become the preferred protocol for restoring dentition defects. Being the direct contact between implant and bone interface, osseointegration is the basis for implant exerting physiological functions. Nevertheless, biological complications such as insufficient bone volume, poor osseointegration, and postoperative infection can lead to implant failure. Emerging antibacterial-osteogenic multifunctional implant surfaces were designed to make up for these shortcomings both during the stage of forming osseointegration and in the long term of supporting the superstructure. In this mini-review, we summarized the recent antibacterial-osteogenic modifications of the dental implant surface. The effects of these modifications on biological performance like soft tissue integration, bone osteogenesis, and immune response were discussed. In addition, the clinical findings and prospects of emerging antibacterial-osteogenic implant materials were also discussed.

D-arginine Enhances the Effect of Alpha-Amylase on Disassembling Actinomyces viscosus Biofilm.

Peri-implantitis is the leading cause of dental implant failure, initially raised by biofilm accumulation on the implant surface. During the development of biofilm, Actinomyces viscosus (A. viscosus) plays a pivotal role in initial attachment as well as the bacterial coaggregation of multispecies pathogens. Hence, eliminating the A. viscosus-associated biofilm is fundamental for the regeneration of the lost bone around implants. Whereas clinical evidence indicated that antimicrobials and debridement did not show significant effects on the decontamination of biofilm on the implant surface. In this study, alpha-amylase was investigated for its effects on disassembling A. viscosus biofilm. Then, in order to substantially disperse biofilm under biosafety concentration, D-arginine was employed to appraise its enhancing effects on alpha-amylase. In addition, molecular dynamics simulations and molecular docking were conducted to elucidate the mechanism of D-arginine enhancing alpha-amylase. 0.1-0.5% alpha-amylase showed significant effects on disassembling A. viscosus biofilm, with definite cytotoxicity toward MC3T3-E1 cells meanwhile. Intriguingly, 8 mM D-arginine drastically enhanced the eradication of A. viscosus biofilm biomass by 0.01% alpha-amylase with biosafety in 30 min. The exopolysaccharides of biofilm were also thoroughly hydrolyzed by 0.01% alpha-amylase with 8 mM D-arginine. The biofilm thickness and integrity were disrupted, and the exopolysaccharides among the extracellular matrix were elusive. Molecular dynamics simulations showed that with the hydrogen bonding of D-arginine to the catalytic triad and calcium-binding regions of alpha-amylase, the atom fluctuation of the structure was attenuated. The distances between catalytic triad were shortened, and the calcium-binding regions became more stable. Molecular docking scores revealed that D-arginine facilitated the maltotetraose binding process of alpha-amylase. In conclusion, these results demonstrate that D-arginine enhances the disassembly effects of alpha-amylase on A. viscosus biofilm through potentiating the catalytic triad and stabilizing the calcium-binding regions, thus providing a novel strategy for the decontamination of biofilm contaminated implant surface.

Construction of a BMP-2 gene delivery system for polyetheretherketone bone implant material and its effect on bone formation in vitro.

Polyetheretherketone (PEEK) has been widely investigated for improving its biological inert to enable it to achieve stronger osteogenic capability and to be a promising material in implant fields. The most important mechanism that makes a successful implantation is osteointegration. Surface modification is an appropriate method to maintain the excellent mechanical properties of PEEK and simultaneously endow PEEK certain biological characters. In this work, we attempted to shape the nano-topography of PEEK surface by nitrogen low-temperature plasma and polydopamine coating on the surface as a secondary reaction platform to bond the aminated poly (lactic-co-glycolic acid) (PLGA) microspheres encapsulating the BMP-2 gene for enhancing the biological activity. Scanning electron microscope, atomic force microscopy, X-ray photoelectron spectroscopy and water contact angle (CA) measurements were applied to characterize the surface of modified or untreated PEEK. Surface characterization showed that the modification was successfully performed on PEEK including a rougher and more hydrophilic surface with nanotopographic features. The influence on cell adhesion, proliferation and differentiation was evaluated by culturing of rat bone marrow mesenchymal stem cells on different modified PEEK substrates in vitro. The biological results indicated that the low-temperature plasma treatment and PDA-coating on PEEK significantly promoted cell adhesion and proliferation. And the osteogenic differentiation was effectively improved by BMP-2 gene releasing from PLGA-NH2 microspheres. The results showed that this novel biological surface modification endowed PEEK with outstanding bioactivity and osteogenic ability, providing a theoretical basis for application in the field of implantation.

Porphyromonas gingivalis lipopolysaccharide affects oral epithelial connections via pyroptosis.

BACKGROUND/PURPOSE: Pyroptosis is a form of programmed cell death dependent on the activation of caspase-1. Porphyromonas gingivalis (P. gingivalis) is a major pathogenic bacterium in periodontitis and its lipopolysaccharide (LPS) can trigger inflammation. However, whether P. gingivalis-LPS affects epithelial connections or triggers pyroptosis in the gingival epithelium is unknown. MATERIALS AND METHODS: Gingival samples from human donors were collected and the expression levels of E-cadherin, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1/4/5, interleukin (IL)-18, and IL-1ß were examined. P. gingivalis-LPS was injected into rat gingival sulcus to establish gingivitis models, and the expression levels of E-cadherin, NLRP3, caspase-1/11, IL-18, and IL-1ß were compared via immunohistochemistry. The mRNA levels of E-cadherin, caspase-1, IL-18, and IL-1ß were evaluated in oral mucosa epithelial cells (OMECs) and rat gingival tissues. RESULTS: In the present study, NLRP3 (p < 0.01), caspase-1 (p < 0.01), caspase-4 (p = 0.044), and IL-18 (p = 0.036) expression was greater in the human inflammatory gingival samples, whereas E-cadherin (p = 0.045) had the opposite presentation. Gingivitis models were successfully established in rats with the injection of P. gingivalis-LPS. NLRP3 (p = 0.015), caspase-1 (p < 0.01), caspase-11 (p < 0.01), and IL-18 (p = 0.041) were upregulated, whereas E-cadherin (p = 0.038) expression was decreased. Furthermore, E-cadherin mRNA was decreased while caspase-1, IL-18, and IL-1ß mRNA levels were increased. The addition of a caspase-1 inhibitor reversed the expression changes. CONCLUSION: P. gingivalis-LPS may effectively destroy the epithelial connection by triggering pyroptosis.

The Effect of Porphyromonas gingivalis Lipopolysaccharide on the Pyroptosis of Gingival Fibroblasts.

Periodontitis is a chronic inflammatory disease induced by Porphyromonas gingivalis (P. gingivalis) and other pathogens. P. gingivalis release various virulence factors including lipopolysaccharide (LPS). However, whether P. gingivalis-LPS inducing pyroptosis in human gingival fibroblasts (HGFs) remains unknown. In present study, P. gingivalis-LPS decreased the membrane integrity of HGFs, and pyroptosis-associated cytokines were upregulated at the mRNA level. In addition, pyroptosis proteins were highly expressed in gingival tissues of periodontitis. P. gingivalis-LPS induced gingivitis in the rat model, and the expression level of pyroptosis-associated proteins increased. Together, P. gingivalis-LPS can activate the pyroptosis reaction, which may be a pro-pyroptosis status in a relative low concentration.

X-ray-responsive polypeptide nanogel for concurrent chemoradiotherapy.

Concurrent chemoradiotherapy (CCRT) is a standard treatment regimen for medically inoperable stage III non-small-cell lung carcinoma (NSCLC) owing to its superior prognostics compared with the sequential modality. Nevertheless, the current pattern of CCRT still fails to provide satisfactory survival outcome. Furthermore, CCRT is always accompanied by a higher risk of severe side effects, limiting the dose escalation. Herein, an X-ray-responsive polypeptide nanogel (PNG) was developed for on-demand delivery of chemotherapeutic agent triggered by radiotherapy to synergistically improve the efficacy of CCRT with reduced side effects. The smart PNG was formed by crosslinking methoxy poly(ethylene glycol)-block-poly(L-glutamic acid-co-γ-2-chloroethyl-L-glutamate) (mPEG-b-P(LG-co-CELG)) with a diselenide (Se-Se) bond. The doxorubicin (DOX)-loaded polypeptide nanogel (PNG/DOX) exhibited accelerated drug release when exposed to X-ray irradiation as a result of Se-Se bond degradation. With prolonged circulation and enhanced intratumoral accumulation in vivo, PNG/DOX combined with X-ray irradiation exhibited better synergistic antitumor efficacy and fewer side effects toward human A549 lung carcinoma-bearing nude mice. The smart X-ray-responsive nanogel provides a promising bridge between chemotherapy and radiotherapy and enhances the potential application of CCRT in clinic.

Keratinized mucosa augmentation guided by double xenogeneic collagen matrix membranes around implants in the posterior mandible: A case report.

RATIONALE: Traditional free gingival graft (FGG) technique is usually used for patients with insufficient peri-implant keratinized mucosa. However, this technique often requires a second surgical area which increases the pain as well as the risk of infection in patients. Xenogeneic collagen matrix (XCM) membrane technique can obtain good results for keratinized mucosa increment. PATIENT CONCERNS: The patient was a 66-year-old healthy female with loss of left mandibular first molar and second molar (FDI #36, #37) for 5 years. Two implants were placed submucosally for 3 months with no interference, while a stage II surgery was needed. DIAGNOSIS: Probing depth measurements suggested that the mesial, medial, and distal widths of buccal keratinized mucosa within the edentulous area were 0.5, 0.5, and 1 mm, respectively, which were insufficient to maintain the health of peri-implant tissues. INTERVENTIONS: Keratinized mucosa augmentation guided by XCM membranes was performed to increase the inadequate buccal keratinized mucosa. OUTCOMES: After 2 months of healing, the widths of mesial, medial, and distal buccal keratinized mucosa were 4, 3, and 3 mm, respectively, and the thickness of the augmented mucosa was 4 mm. Then a stage II surgery was followed. The patient was satisfied with the outcomes of keratinized mucosa augmentation. LESSONS: Keratinized mucosa augmentation guided by double XCM membrane technique can be applied to cases with keratinized mucosa width within 2 mm around implants.

A Fast Online Replanning Algorithm Based on Intensity Field Projection for Adaptive Radiotherapy.

Purpose: The purpose of this work was to propose an online replanning algorithm, named intensity field projection (IFP), that directly adjusts intensity distributions for each beam based on the deformation of structures. IFP can be implemented within a reasonably acceptable time frame. Methods and Materials: The online replanning method is based on the gradient-based free form deformation (GFFD) algorithm, which we have previously proposed. The method involves the following steps: The planning computed tomography (CT) and cone-beam CT image are registered to generate a three-dimensional (3-D) deformation field. According to the 3-D deformation field, the registered image and a new delineation are generated. The two-dimensional (2-D) deformation field of ray intensity in each beam direction is determined based on the 3-D deformation field in the region of interest. The 2-D ray intensity distribution in the corresponding beam direction is deformed to generate a new 2-D ray intensity distribution. According to the new 2-D ray intensity distribution, corresponding multi-leaf collimator (MLC), and jaw motion data are generated. The feasibility and advantages of our method have been demonstrated in 20 lung cancer intensity modulated radiation therapy (IMRT) cases. Results: Substantial underdosing in the CTV is seen in the original and the repositioning plans. The average prescription dose coverage (V100%) and D95 for CTV were 100% and 60.3 Gy for the IFP plans compared to 82.6% (P < 0.01) and 44.0 Gy (P < 0.01) for original plans, 86.7% (P < 0.01), and 58.5 Gy (P < 0.01) for repositioning plans. On average, the mean total lung doses were 12.2 Gy for the IFP plan compared to the 12.4 Gy (P < 0.01) and 12.6 Gy (P < 0.01) for the original and the repositioning plans. The entire process of IFP can be completed within 3 min. Conclusions: We proposed an online replanning strategy for automatically correcting interfractional anatomy variations. The preliminary results indicate that the IFP method substantially increased planning speed for online adaptive replanning.

Effects of D-arginine on Porphyromonas gingivalis biofilm.

Porphyromonas gingivalis (P. gingivalis) is one of the major pathogenic bacteria of periodontitis or peri-implantitis. P. gingivalis tends to attach to the implant's neck with the formation of biofilm, leading to peri-implantitis. d-arginine has been shown to have a potential antimicrobial role. In this study, P. gingivalis was cultured in Brain Heart Infusion broth together with d-arginine. After 3 days (inhibition) or 6 days (dissociation), these were characterized using crystal violet (CV) staining for the biofilm, extracellular polysaccharide (EPS) production from the biofilm, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for biofilm activation. Furthermore, the P. gingivalis biofilm was observed by scanning electron microscopy (SEM). d-arginine effectively reduced biomass accumulation and promoted dissociation at concentrations of ≥50 mM and 100 mM, respectively. Through CV staining, d-arginine concentrations of EPS production from the biofilm for inhibition and dissociation effects was ≥50 mM and 100 mM, respectively. In addition, d-arginine affected biofilm activation for the corresponding concentrations: ≥60 mM for inhibition and ≥90 mM for dispersal. Under SEM observation, d-arginine changed the P. gingivalis biofilm structure in relatively high concentrations for inhibition or dissociation, respectively. The authors concluded that d-arginine could inhibit the formation of P. gingivalis biofilm and promote the dissociation of P. gingivalis biofilm.

Effects of d-valine on periodontal or peri-implant pathogens: Porphyromonas gingivalis biofilm.

BACKGROUND: When presented with a surface or an interface, bacteria often grow as biofilms in which cells are held together by an extracellular matrix. Biofilm formation on implants is an initiating factor for their failure. Porphyromonas gingivalis is the primary etiologic bacteria of initiation and progression of periodontal disease. This microorganism is also the risk factor of many systemic diseases, such as cardiovascular disease, diabetes, and pulmonary infection. To date, no medication that can remove such biofilm has been accepted for clinical use. D-valine (D-val) can reportedly inhibit the formation of biofilm and/or trigger the scattering of mature biofilm. Accordingly, this study investigated the effects of d-val on single-species P. gingivalis biofilms in vitro. METHODS: P. gingivalis grown in brain heart infusion culture with or without d-val was inoculated in 24- or 96-well plates. After incubation for 72 hours, biomass via crystal violet staining, extracellular polysaccharide production by biofilms, and scanning electron microscopy (SEM) were used to determine the d-val concentration that can effectively prevent P. gingivalis biofilm formation. RESULTS: Experimental results showed that d-val effectively inhibited biofilm formation at concentrations ≥50 mM (mMol/L), and that d-val inhibition increased with increased concentration. Moreover, at high concentrations, the bacterial form changed from the normal baseball form into a rodlike shape. d-val also notably affected extracellular polysaccharide production by P. gingivalis. CONCLUSIONS: d-val can inhibit P. gingivalis biofilm formation, and high concentrations can affect bacterial morphology.

Water-soluble fluorescent unimolecular micelles: ultra-small size, tunable fluorescence emission from the visible to NIR region and enhanced biocompatibility for in vitro and in vivo bioimaging.

Fluorescent unimolecular micelles (FUMs) with multicolor emission acting as fluorescent nanoagents for optical fluorescence imaging have, for the first time, been reported. The FUMs show good water-solubility, ultra-small size, and enhanced biocompatibility, which endow the FUMs with versatile applications including organelle labeling, multicolor markers and high tumor accumulation, revealing that our design can serve as a rational strategy for the development of UM-based fluorescent nanoagents for bioprocess monitoring.

Effects of the micro-nano surface topography of titanium alloy on the biological responses of osteoblast.

In clinical applications, osseointegration is essential for the long-term stability of dental implants. Inspired by the hierarchical structure of natural bone, we applied the electrochemical etching (EC) technique to form a micro-nano structure on a titanium alloy (Ti6Al4V) substrate, called EC surface. Sand blasting and acid etching (SLA) and machined (M) methods were employed to generate micro and smooth textures, respectively, as the control groups. The surface topographies of the three substrates were characterized using scanning electron microscopy (SEM). Then, human osteoblast-like cells (MG63) were cultured on substrates, and adhesion, proliferation, morphology, alkaline phosphatase activity (ALP), and gene expression levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and type I collagen (COLIA 1) were analyzed. MG63 cells cultured on the EC Ti alloy substrates displayed better cell adhesion, significant proliferation, and a higher production level of ALP, gene expressions of RUNX2, OCN, OPN and COLIA 1 (p < 0.01 or p < 0.05) compared with those of SLA and M substrates. These results indicate that the micro-nano structure fabricated by electrochemical etching method is beneficial for the biological functions of MG63 cells and may be a promising candidate in dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 757-769, 2017.