Evaluation of a hydroxyapatite-crosslinked fish gelatin membranes.

Background/purpose: Porcine collagen is widely used in regenerative therapies to generate membranes for bone augmentation. However, porcine or bovine gelatin or collagen is often not appropriate for patients with creed and religious beliefs or for allergic reasons. In this study, we evaluated the potential of fish gelatin to generate membranes. Materials and methods: Fish gelatin and hydroxyapatite (HAp) were used at three different ratios (2:0, 2:1, 2:1.5, and 2:2) to prepare gelatin-hydroxyapatite (G-HAp) membranes via freeze-drying and heat-crosslinking. The surface morphology and cell attachment of G-HAp membranes were observed using scanning electron microscopy and confocal laser microscopy. G-HAp membrane was placed at the bottom of a well plate, and MC3T3-E1 cells were seeded on it. Cell viability and cytotoxicity were tested after 1 and 3 days of culture. Alkaline phosphatase (ALP) and alizarin red staining was performed at 10 and 21 days, respectively. Results: Viability of cells on G-HAp membrane with the gelatin:HAp ratio of 2:1.5 was significantly higher than that on membranes with other gelatin:HAp ratios. ALP and alizarin red staining showed that ALP-positive areas and calcium deposition were the highest on G-HAp membrane with the gelatin:HAp ratio of 2:1. These membranes showed negligible cytotoxicity. Conclusion: Fish-derived G-HAp membranes have the potential to promote osteogenic differentiation of MC3T3-E1 cells with negligible cytotoxicity.

Development of bioinspired damage-tolerant calcium phosphate bulk materials.

Improving the damage tolerance and reliability of ceramic artificial bone materials, such as sintered bodies of hydroxyapatite (HAp), that remain in vivo for long periods of time is of utmost importance. However, the intrinsic brittleness and low damage tolerance of ceramics make this challenging. This paper reports the synthesis of highly damage tolerant calcium phosphate-based materials with a bioinspired design for novel artificial bones. The heat treatment of isophthalate ion-containing octacalcium phosphate compacts in a nitrogen atmosphere at 1000°C for 24 h produced an HAp/ß-tricalcium phosphate/pyrolytic carbon composite with a brick-and-mortar structure (similar to that of the nacreous layer). This composite exhibited excellent damage tolerance, with no brittle fracture upon nailing, likely attributable to the specific mechanical properties derived from its unique microstructure. Its maximum bending stress, maximum bending strain, Young's modulus, and Vickers hardness were 11.7 MPa, 2.8 × 10‒2, 5.3 GPa, and 11.7 kgf/mm2, respectively. The material exhibited a lower Young's modulus and higher fracture strain than that of HAp-sintered bodies and sintered-body samples prepared from pure octacalcium phosphate compacts. Additionally, the apatite-forming ability of the obtained material was confirmed in vitro, using a simulated body fluid. The proposed bioinspired material design could enable the fabrication of highly damage tolerant artificial bones that remain in vivo for long durations of time.

Effect of chemical and electrochemical decontamination protocols on single and multiple-used healing abutments: A comparative analysis of contact surface area, micro-gap, micro-leakage, and surface topography.

INTRODUCTION: Although the combined use of chemical and electrochemical decontamination protocols can completely remove contaminants from the surfaces of one-time used healing abutments (HAs), their effectiveness in multiple-used HAs remains unknown. We aimed to investigate the effect of reused HAs frequency on the implant-HA contact surface area, micro-gap, microleakage, and surface topography following chemical and combined chemical and electrochemical decontamination protocols. METHODS: Ninety bone level titanium implants were assembled with 90 bone level HAs, in which 80 contaminated HA samples were collected from human participants. The retrieved HAs were randomly divided into two groups according to the cleaning protocol: ultrasonication with 5.25% NaOCl solution for 15 min and steam autoclaving (group I); ultrasonication with 5.25% NaOCl solution for 15 min, followed by electrochemical cleaning and steam autoclaving (group II). The control group (group III) comprised 10 new unused HAs. The cleaning protocol was applied after each insertion as follows: (a) single-use and cleaning, (b) double-use and double cleaning cycles, (c) triple-use and triple cleaning cycles, and (d) more than triple-use and more than triple cleaning cycles. The contact surface area and micro-gap were assessed with micro-computed tomography scanning technique, microleakage test using 2% methylene blue staining, surface morphology with scanning electron microscopy, and surface elemental composition with energy-dispersive X-ray spectroscopy analysis. RESULTS: Group Id exhibited the smallest contact surface area. The values of the micro-gap volumes and microleakage were significantly different (p < 0.001) in the descending order of Id > Ic > Ib > IId > Ia, IIa, and III. Morphological evaluation of Groups IIa, IIb, and IIc revealed that residual biological debris was optimally removed without altering their surface properties. CONCLUSIONS: Chemical and electrochemical decontamination protocols are more effective than NaOCl cleaning methods, particularly for multiple consecutive uses with better decontamination levels, which decreases micro-gap volume and microleakage without surface alterations. Although the use of combined decontamination protocols for the contact surface area at the implant-HA interface showed comparable results with the control, change in the contact surface area was observed following the NaOCl cleaning methods. Therefore, titanium HA reuse can be considered in multiple times, if they are cleaned and sterilized using combined chemical and electrochemical decontamination protocols.

A modal analysis of implant-supported overdentures installed on differently positioned sets of dental implants.

This study evaluated the three vibration characteristics, namely, natural frequency, damping ratio, and natural mode, together with maximum displacement of a two-implant-supported overdenture (IOD) at different locator attachment positions using experimental modal analysis (EMA). Edentulous mandibular models with a gingival thickness of 1 mm or 3 mm were prepared, into which dental implants were placed using a fully guided surgical template designed with simulation software, the locator abutments were fastened, and the IODs were then fabricated. The implant positions were bilaterally marked at the lateral incisor, first premolar, and first molar regions. EMA was performed by hammering the test structures to measure the impulse response and obtain the vibration characteristics (n = 5). The Kruskal-Wallis test was performed for natural frequency and maximum displacement, and the Games-Howell test for damping ratio. The significance level was set at α = 0.05. The study indicated that the gingival thickness had a significant effect on the vibration characteristics. Moreover, the natural frequency and damping ratio results showed that the vibration subsided faster when the attachment was placed on the molar implants in the thick gingival model. Furthermore, according to the effect of lateral force on IODs, the difference in maximum displacement between the anterior and posterior regions of the IOD was smaller when the attachments were designed on the pair of lateral incisors. Thus, within the limits of this experiment, our results suggested that two anterior implant-supported IODs are preferable treatment designs in terms of vibration engineering, especially when the gingiva is thick; the molar attachment design could be considered for thin gingival conditions. The differences in gingival thickness and abutment position affected the vibration characteristics of the IOD. Further in vivo studies would be necessary to validate the implant positions and their IOD designs for the mandibular edentulous shapes and the occlusal relationship.

Dental implant healing abutment decontamination: A systematic review of in vitro studies.

PURPOSE: To investigate various cleaning protocols employed to enable the reuse of healing abutments in the past decade. MATERIALS AND METHODS: The review followed the guidelines set out in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement, with guidance from the Cochrane Collaboration Handbook. Electronic searching and handsearching were performed using the National Library of Medicine (MEDLINE via PubMed) and the Cochrane Central Register of Controlled Trials from January 2010 to July 2022, respectively. Studies published in English were evaluated. Two independent examiners conducted the search and the review process. The risk of bias of the included studies was evaluated. RESULTS: In total, 178 articles were evaluated for review, but only 15 of them were selected for full-text reading. Regarding cleaning efficacy, chemical decontamination using sodium hypochlorite produced better results than laser and mechanical decontamination with airflow. Similar efficacy was found between chemical and electrochemical decontamination. Combined use of chemical and electrochemical decontamination protocols demonstrated the greatest efficacy. Chemical and electrochemical decontamination methods were found to achieve better outcomes in preserving the surface properties of decontaminated healing abutments than laser and mechanical methods. CONCLUSION: The present review found that combined decontamination protocols (chemical, electrochemical processing and autoclave treatment) are favourable for obtaining healing abutments with optimally cleaned surfaces. Moreover, healing abutments located in an area that is difficult to access can be cleaned without affecting the surface properties. This information could benefit researchers and clinicians when multiple-use healing abutments are considered.

Correction: Lu et al. Periodontal Pathogen Adhesion, Cytotoxicity, and Surface Free Energy of Different Materials for an Implant Prosthesis Screw Access Hole. Medicina 2022, 58, 329.

In the original publication [...].

Efficacy of combined chemical and electrochemical decontamination treatments on contaminated healing abutments and their effect on surface topography: An in vitro study.

OBJECTIVE: To evaluate the efficacy of four decontamination protocols on contaminated healing abutments (HAs) and their effects on surface topography. METHODS: Eighty contaminated single-use HA samples collected from human participants were stained with phloxine B and examined microscopically. The retrieved HAs were randomly divided into four test groups: (1) Autoclaving only (AU), (2) 5.25% sodium hypochlorite (NaOCl) + AU, (3) Electrochemical treatment (EC) + AU, (4) NaOCl + EC + AU, and positive control (contaminated without any treatment). Four new unused HAs served as negative controls (NC). The surface features were analyzed using stereo microscopy (SM), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and optical profilometry. RESULTS: The lowest decontamination efficacy was observed for the AU group. The NaOCl + AU and EC + AU groups effectively removed residual contamination, whereas EC + AU showed better decontamination results than NaOCl + AU. SM, SEM, and EDS analyses revealed the best decontamination efficacy in the combined NaOCl + EC + AU group compared to the other groups. Surface roughness (Sa), developed surface area ratio (Sdr), and texture-aspect ratio (Str) in AU, NaOCl + AU, EC + AU, and NaOCl + EC + AU groups were not statistically significant compared to the NC group. CONCLUSIONS: The combination of NaOCl with subsequent EC can remove soft and hard deposits from the surface of HAs compared to NaOCl alone and EC alone, without altering the surface topography of HAs.

An In Vitro Evaluation of Selenium Nanoparticles on Osteoblastic Differentiation and Antimicrobial Properties against Porphyromonas gingivalis.

Despite numerous treatment methods, there is no gold standard for the treatment of peri-implantitis-an infectious peri-implant disease. Here, we examined selenium nanoparticles (SeNPs) at a wide range of concentrations to investigate their cytotoxicity, regulation of osteoblastic differentiation, and assessed the antibacterial effect against Porphyromonas gingivalis. SeNPs (mean size: 70 nm; shape: near-spherical; concentration: 0-2048 ppm) were tested against the MC3T3-E1 osteoblast precursor cell line and P. gingivalis red complex pathogen. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis was used to evaluate the bone morphogenetic protein 2 (BMP-2) signaling pathway. SeNPs at concentrations of 2-16 ppm showed no obvious cytotoxicity and promoted good mineralization and calcification. SeNPs at concentrations 64 ppm and below influenced gene expression promoting osteoblastic differentiation, whereas at high concentrations inhibited the expression of Runt-related transcription factor 2 (Runx2). The growth of P. gingivalis was significantly inhibited at SeNP concentrations of more than 4 ppm. SeNPs at low concentrations promoted osteoblastic differentiation while strongly inhibiting peri-implantitis pathogen growth. This study represents one of the few in vitro assessments of SeNPs against a red complex pathogen and the regulatory effect on osteoblastic differentiation. The findings demonstrate SeNPs could potentially be used for future application on implant coating.

Microbiological Comparison of Different Sealing Materials for the Access Holes of Implant Restorations.

PURPOSE: To evaluate the performance of sealing materials used in the screw-access holes of screw-retained implant final superstructures in vivo and in vitro. MATERIALS AND METHODS: Twenty-one screw-access holes in the final superstructures were randomly divided into three groups (each group, n = 7). Following disinfection and isolation, all access holes were initially filled with sterilised cotton pellets of the same weight. Depending on the group, the access holes were finally sealed with either provisional composite restorations (group A), self-curing resin for provisional sealing (group B), or acrylic resin (group C). After one month of the functional period, the inner cotton pellets were collected as bacterial reservoirs. RESULTS: Total aerobic bacteria and total gram-negative anaerobic bacteria were measured after bacterial culture for 48 h and 72 h, respectively. In vitro evaluation of porosity using scanning electron microscopy (SEM) was also performed. Samples from superstructures sealed with provisional composite restorations showed fewer bacteria and less porosity than samples from superstructures sealed with self-curing resin for provisional sealing and acrylic resin. In this study, provisional composite restorations showed the best sealing properties. Provisional composite restorations may prevent bacterial invasion of the access holes of the final superstructures. CONCLUSION: In this study, provisional composite restorations showed the best sealing properties. Provisional composite restorations may prevent bacterial invasion of the access holes of the final superstructures.

Periodontal Pathogen Adhesion, Cytotoxicity, and Surface Free Energy of Different Materials for an Implant Prosthesis Screw Access Hole.

Background and Objectives: Oral implant restorations are an excellent treatment option for edentulous patients; however, periodontopathogenic bacteria have been found in the microgaps between implant-abutment junctions. Implant designs to limit the microgaps have been extensively studied. However, studies have shown microgaps continue to exist, allowing for the leakage of bacteria into the implant system. Screw access hole materials are used to fill the access hole void. The use of materials with beneficial properties could provide bacterial leakage prevention. The aim of this study was to examine the surface free energy, cytotoxicity, and bacterial adhesion of selected screw access hole materials such as cotton, polytetrafluoroethylene (PTFE) tape, paraffin wax-polyolefin thermoplastic (PF), paraffin wax (Wax), gutta-percha (GP), and caviton EX (CE). Materials and Methods: A sessile drop test was performed to observe the contact angle and calculate the surface free energy of each material in order to determine the level of hydrophobicity. Cytotoxicity was examined in a mouse gingival epithelial cell line for day 1 and day 3. Bacterial adhesion was tested with Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. Results: PTFE, PF, and wax presented low surface free energies of 19.34, 23.041, and 24.883 mN.m-1, respectively. No cytotoxicity was observed, except for GP and CE. Concurrently, the bacterial adhesion was also the lowest in PTFE and PF. Conclusions: Within the limits of this study, PTFE and PF showed an excellent biocompatibility with few bacterial adhesions. These materials could be potential screw access hole materials in clinical settings.

Fluctuation of salivary alpha-amylase activity levels and vital signs during dental implant surgery.

BACKGROUND: Salivary alpha-amylase (sAA) activity level is thought to be an indicator of mental stress. However, the relationship between sAA activity levels and mental stress in patients during dental implant treatment has not been studied. In the present study, we aimed to examine the correlation between sAA activity levels and changes in patients' vital signs during dental implant surgery. RESULTS: Levels of sAA activity were higher after surgery when compared to before-surgery measurements. A significant positive correlation was found between sAA activity and heart rate (HR) (rs=0.434, p=0.007) as well as a positive correlation with oxygen level (rs=0.392, p=0.016). CONCLUSION: Levels of sAA activity tended to increase after the surgical procedures, as did patients' stress levels. SpO2 and sAA activity levels were inversely correlated. There was a positive significant correlation between HR and sAA activity, though there was no correlation between blood pressure and sAA activity levels. Salivary alpha-amylase may be a valuable indicator of stress and anxiety in dental patients undergoing dental implant surgery.

A novel hydroxyapatite fiber material for the regeneration of critical-sized rabbit calvaria defects.

Hydroxyapatite (HA) [Ca10 (PO4)6 (OH)2] has a high degree of chemical similarity with the mineral composition of animal bone. Hydroxyapatite fiber scaffold (HAF) is a biological material with a highly interconnected porous structure. We aimed to study the physical and biological characteristics of HAF and compare the osteogenic effects of HAF, natural osteogenic materials (NOM), and carbonate apatite (CO3Ap-DP) in the parietal defects of a rabbit's skull. X-ray analysis and histological assessment showed that HAF followed a trend of early initial osteogenesis and bone trabecular structure formation, especially at the cortical bone portion.Compared to the other two materials, HAF was more absorptive. Results indicated that HAF had the same osteoconductive and new bone formation properties as NOM and CO3Ap-DP. These findings will provide options for future material development and novel protocols for use in surgeries, ultimately leading to better patient outcomes.

Evaluation of a Cetylpyridinium Chloride-, Dipotassium Glycyrrhizinate-, and Tranexamic Acid-based Mouthwash after Implant Placement: A Double-blind Randomised Clinical Trial.

PURPOSE: To evaluate the positive effects of a CPC-, GK2-, and TXA-based (CPC/GK2/TXA) mouthwash after implant placement. MATERIALS AND METHODS: Twenty patients (n = 20) who underwent posterior implant-placement surgery were randomly and evenly allocated to the study or the placebo group. After the mouthwash was used 3x/day for 7 to 10 days postoperatively, sutures were analysed by counting the colony-forming units (CFU) for total aerobes, total G [-] anaerobes, total enterobacteria and total H. influenzae, followed by Real-Time PCR of bacterial-specific DNAs of A. actinomycetemcomitans, P. gingivalis, T. forsythia, T. denticola, P. intermedia, P. micra, F. nucleatum, C. rectus, and E. corrodens. In vitro resistance of P. gingivalis, S. aureus, and P. aeruginosa was analysed. The compatibility of the mouthwash with Straumann SLA implant surfaces was evaluated by scanning electron microscopy (SEM). RESULTS: Sixteen patients (n = 16) completed the trial. A statistically significantly greater number of CFU was found in the placebo group for almost all species, especially for total G [-] anaerobes. No statistically significant in vitro resistance was found for P. gingivalis, S. aureus, and P. aeruginosa. SEM revealed no surface alteration after exposure to the mouthwash. CONCLUSION: The use of a CPC/GK2/TXA mouthwash inhibited propagation of the bacteria extracted from the post-surgical sutures after implant placement.

Low-Intensity Pulsed Ultrasound Stimulates Osteogenic Differentiation of Periosteal Cells In Vitro.

Adequate bone volume is required for osseointegrated implants to restore lost teeth and oral function. Several studies have demonstrated potential advantage of stem cells in regenerative medicine using osteoblasts. The periosteum is composed of osteoblasts, fibroblasts, and osteoprogenitor cells. It may be an alternative source for bone tissue engineering because of easy isolation and rapid proliferation in vivo and in vitro. Low-intensity pulsed ultrasound (LIPUS) has proved successful in recoveries from nonunions, delayed unions, and fracture of the bone in both animal experiments and clinical treatments. The study was to investigate the influence of LIPUS on the osteogenic differentiation in murine periosteum-derived cells (PDCs) and the underlying mechanism of LIPUS. PDCs were treated daily with LIPUS for 20 min up to 21 days with 3 MHz frequency, 30 mW/cm2 intensity, and pulse repetition frequency of 1 kHz. The effects of LIPUS on cell proliferation and viability were investigated. Osteogenic differentiation was analyzed by alkaline phosphatase (ALP)-positive cell staining, ALP activity assay, mineralized nodule formation, real-time reverse transcription-polymerase chain reaction, as well as western blotting. The results indicated that ultrasound stimulation did not significantly affect the proliferation of PDCs. But LIPUS significantly increased ALP activity on day 7 and markedly promoted formation of mineralized nodules on day 21. mRNA expression of ALP and osteocalcin was significantly upregulated by stimulation with LIPUS. LIPUS enhanced gene expression of both bone morphogenetic protein-2 (BMP-2) and osterix only in the presence of osteogenic medium. LIPUS stimulation did not affect Smad 1 and Smad 5 protein expression, but significantly upregulated protein levels of BMP-2 and phosphor-Smad 1/5/9 in PDCs. Thus, LIPUS stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of BMP-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition. Therefore, LIPUS might have potential to promote osteogenesis in PDCs. Impact statement There are few studies on periosteum-derived cells (PDCs) because conventional methods of their isolation are relatively difficult to procure abundant cells for cell culture and the total cell numbers are limited. In this study, a modified isolation technique of murine calvarial PDCs using gelatin is described. PDCs were initiated to emerge as early as day 3 and showed increased proliferation, which can be used for further studies. Low-intensity pulsed ultrasound stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of bone morphogenic protein-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition.

Comparison of splinted and non-splinted superstructures of three implants placed in a mandibular distal extension model with missing teeth using modal analysis.

This study evaluated the effects of two types of superstructures (splinted crown and non-splinted crown) on four vibration characteristics (natural frequency, damping ratio (DAR), vectors in antiphase, maximum displacement (MDP)) by using modal analysis. These structures were fabricated on three implants placed in the mandibular free-end defect model. After placing three implants on a mandibular distal extension model with missing teeth, the three-unit splinted and non-splinted crowns were designed on the CAD software. Subsequently, the zirconia disc was milled using a milling machine to produce the superstructures (n = 5). After establishing four measurement points on each crown of the prostheses, a vibration was applied to the mandible model with an impact hammer, and the transfer function of each measurement point was identified. Using the modal analysis software, the natural frequency and damping ratios were calculated from the transfer function, modal shapes at each natural frequency were observed, and maximum displacement that takes into account the lateral force during mastication was simulated. The t-test was performed for each of the averaged values of natural frequency, maximum displacement, and damping ratio (p < 0.05). Mann-Whitney U test was performed on the average of the number of vectors in antiphase (p < 0.05). The mean natural frequency was significantly higher in the splinted crown (758.2 ± 2.8 Hz) than that in the non-splinted crown (752.8 ± 3.7 Hz) (P = 0.047). The mean damping ratio was not significantly different for the splinted (3.3 ± 0.2%) and non-splinted crowns (3.2 ± 0.3%) (P = 0.535). The average number of vectors in antiphase was significantly smaller in the non-splinted crown (4.4 ± 0.9) than in the splinted crown (10 ± 2.5) (P = 0.008). The mean value of the maximum displacement was significantly smaller in the splinted crown (6.7 ± 1.1 µm) than that in the non-splinted crown (7.3 ± 0.6 µm) (P = 0.048). Within the limitations of this study, the vibration characteristics of the superstructures differed between designs with splinted and non-splinted crowns. Crown splinting increased the rigidity and natural frequency and decreased the MDP. However, the higher number of vectors in antiphase indicated more twists in the superstructures during vibration. The non-splinted crowns showed a lower natural frequency and a greater maximum displacement; however, they comprised fewer vectors in antiphase, indicating that the twist during vibration was less than that observed with the splinted crowns. Thus, our results suggest that crown splinting reduces the deformation of the superstructure, implants, and the surrounding tissues in comparison to the deformation observed when no splinting is performed.

Evaluation of residual contamination on healing abutments after cleaning with a protein-denaturing agent and detergent.

OBJECTIVE: This study evaluated the cleaning potential of a protein-denaturing agent with or without anionic detergent by monitoring the residual contamination on healing abutments used for dental implant treatment. METHOD AND MATERIALS: Forty contaminated healing abutments removed from patients were randomized and immediately treated with differing cleaning methods; either Method A (presoaking in 1% sodium dodecyl sulfate followed by ultrasonication with 4 mol/L guanidine hydrochloride), or Method B (soaking in distilled water followed by ultrasonication with 4 mol/L guanidine hydrochloride) was used. Samples were stained with phloxine B and photographed using a light microscope. The proportion of stained and contaminated areas on each healing abutment was then calculated using Image J. The surface was examined with a scanning electron microscope and energy-dispersive x-ray spectroscopy. RESULTS: The percentages of contaminated surfaces of the screwdriver engagement region, upper body, and lower body for methods A and B were 50% and 38%, 10% and 80%, and 38% and 18%, respectively. There was a statistically significant difference (engagement region [P < .001], upper body [P = .043], and lower body [P = .017]; Mann-Whitney) regarding the residually stained areas between the two cleaning methods. No surface alterations were seen by scanning electron microscopy. Energy-dispersive x-ray spectroscopy confirmed that the cleaned surfaces of the healing abutments revealed no signs of organic contamination. CONCLUSION: Although the combination of a strong denaturing agent and detergent effectively cleaned contaminated healing abutments, perfect cleaning was not always possible, indicating that the reuse of healing abutments in different patients is not recommended.

Comparison of gingiva-derived and bone marrow mesenchymal stem cells for osteogenesis.

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva-derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK-8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT-qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT-qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90-positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.

Modal analysis of two different types of fixed implant-supported prostheses embedded in edentulous maxillae.

PURPOSE: Modal analysis has been used to monitor and quantify the vibration of each component of a prosthesis at its natural frequency. An understanding of the vibrational characteristics of a prosthesis can guide selection of the appropriate prosthesis configuration to prevent excessive stress. In this study, modal analysis of fixed implant-supported prostheses with 4 or 6 abutments in edentulous maxillae was performed. METHODS: Sixteen patients underwent maxillary dental implant treatment and received screw-fixed implant-supported prostheses (9 patients received 4-abutment prostheses and 7 patients received 6-abutment prostheses) in edentulous maxillae. An impact hammer was used for excitation of the prostheses, and vibration was detected on every crown of the prostheses. The modal parameters were determined in each subject. Furthermore, the modal shapes were compared at each natural frequency. RESULTS: The median natural frequencies of subjects with 4- and 6-abutment prostheses were 697 and 781Hz, respectively. The mean damping ratios did not differ significantly between 4- (8.2±1.7%) and 6-abutment (6.6±1.7%) prostheses (p=0.125). The mean maximum displacements of fixed prostheses were significantly lower for 6-abutment (10.3±3.3µm) than for 4- (20.3±9.1µm) prostheses (p=0.004). The median number of vectors in antiphase was significantly lower for 6- (4) than for 4-abutment prostheses (14) (p=0.001). CONCLUSIONS: The current study demonstrated less adverse vibration in the 6-abutment prostheses than in the 4-abutment prostheses, suggesting that modal analysis can contribute to novel future developments in the designs of dental implant prostheses.

Indirect osteoblast differentiation by liposomal clodronate.

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow-derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real-time RT-PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow-derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre-treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate-injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK-containing exosomes in response to clodronates.

Osteogenic Potential of Adipose-Derived Macrospheroids Cocultured with CD11b+ Monocytes.

PURPOSE: Among potential cell-based therapies, adipose-derived stem cells (ASCs) have been proposed as a promising source of stem cells for tissue regeneration. Although many recent clinical trials have investigated the use of adipose tissue or ASCs in transplantation, analysis of the microstructures of outgrowing macrosized spheroids (macrospheroids) or three-dimensional coculture of ASC spheroids and monocyte/macrophage lineages has not been performed. The aim of this study was to analyze the microstructures of murine-derived ASC macrospheroids and the growth and osteogenic potential of these macrospheroids in a three-dimensional environment and after calcification induction by coculture with monocytes. MATERIALS AND METHODS: The histologic structures of murine-derived ASC macrospheroids and the expression of marker genes for multipotency within these macrospheroids were analyzed by hematoxylin and eosin staining and in situ hybridization. ASC macrospheroid microstructures were observed by transmission electron microscopy, and cell proliferation in the spheroids was analyzed. Additionally, the growth and osteogenic potential of these macrospheroids were assessed in two-dimensional and three-dimensional environments and after calcification induction by coculture with monocytes. RESULTS: The expression of Oct3/4, Nanog, and Sox2 was detected even in the deep zone of spheroids, although higher expression was observed at the surface. Cell proliferation was detected within the spheroid centers. Observation of spheroid microstructure revealed extracellular matrix production within the spheroid architecture. Transplantation of a spheroid on the hydroxyapatite disc resulted in three-dimensional cell growth, filling the disc. Coculture of the spheroids with monocytes led to the formation of many osteoclast-like, multinucleated cells, and calcification was observed after 3 weeks of coculture. CONCLUSION: ASC spheroids exhibited high capacity for dynamic three-dimensional growth and osteogenic differentiation. Furthermore, ASC spheroids promoted monocyte differentiation into osteoclast-like cells, which may enhance the osteogenic potential of ASC spheroids.