Alveolar ridge preservation in rat tooth extraction model by chitosan-derived epigenetic modulation scaffold.

PURPOSE: Alveolar ridge preservation is a surgical technique used to prevent dimensional changes in the alveolar bone by dressing biomaterials in the extraction socket. Recently, a chitosan biphasic calcium phosphate loaded with trichostatin A (CS/BCP/TSA) scaffold was introduced as an excellent bone-regeneration material. This study aimed to explore the biological properties of released trichostatin A (TSA) and evaluate the potential of the CS/BCP/TSA scaffold in preserving the alveolar ridge in a rat tooth extraction model. METHODS: In vitro biocompatibility, histone deacetylase (HDAC) activity, and osteogenic differentiation of MC3T3-E1 cells were tested. For in vivo studies, the maxillary first molars (M1) of Wistar rats were extracted, and alveolar ridge preservation was performed using a CS/BCP/TSA scaffold or commercial bone graft. Micro-Computed Tomography (micro-CT), polyfluorochrome labeling, and histological analysis were used to evaluate the ridge-preservation ability. RESULTS: The released TSA was cytocompatible. Inhibition of histone deacetylase (HDAC) activity and induction of osteogenic differentiation in MC3T3-E1 cells were confirmed. The socket dressing with the CS/BCP/TSA scaffold showed increased socket bone fill and preserved the buccal and middle aspects of the alveolar ridge compared with the conventional graft. Further analysis of the bone regeneration ability by histomorphometric and histological analyses demonstrated that CS/BCP/TSA showed a significantly higher potential to induce bone formation and complete healing in the extraction socket than the other groups. CONCLUSIONS: The CS/BCP/TSA scaffold is a novel candidate for alveolar ridge preservation.

Effectiveness of iodine for continuous decontamination of dental unit waterline.

OBJECTIVE: Dental Unit Waterlines (DUWLs) are contaminated by various species of microorganisms. DUWLs should be disinfected appropriately to control microbial contamination. This study investigated the effectiveness of devices continuously releasing iodine to control microbial contamination in DUWLs. MATERIALS AND METHODS: Ten dental chair units (DCU) at Chulalongkorn University were randomized into the iodine and control groups. After setting iodine treatment devices, the DCU was allowed to operate normally. 25 ml of water from airotors lines were collected weekly for enumerating bacteria. The viability of biofilms in DUWLs was quantified by ATP testing kit. The amount of iodine released into the procedural water was also quantified. RESULTS: The continuous presence of iodine could significantly control bacterial contamination in the DUWL to be less than 500 CFU/mL, the standard level recommended by the Centre for Disease Control and Prevention (CDC). Iodine treatment can reduce bacterial CFU up to 98-100%. Biofilm viability in the iodine group was slightly lower than that of the control group though not statistically significant. After eleven months, the average iodine release was measured to be 3.6 ppm which is still effective in controlling bacterial contamination. CONCLUSION: Continuously supplying iodine in DUWLs effectively controls microbial contamination.

Effect of Storage Time and Temperature on the Bioactivity of a Chitosan-Derived Epigenetic Modulation Scaffold.

The appropriate storage protocol is one of the main limitations of translating tissue engineering technology to commercialized clinical applications. Recently, the development of a chitosan-derived composite scaffold incorporated with bioactive molecules has been reported as an excellent material to repair a critical size bony defect in mice calvaria. This study aims to determine the storage time and appropriate storage temperature of Chitosan/Biphasic Calcium Phosphate/Trichostatin A composite scaffold (CS/BCP/TSA scaffold) in vitro. The mechanical properties and in vitro bioactivity of trichostatin A (TSA) released from CS/BCP/TSA scaffolds in different storage times and temperatures were evaluated. Different storage times (0, 14, and 28 days) and temperatures (-18, 4, and 25 °C) did not affect the porosity, compressive strength, shape memory, and amount of TSA released. However, scaffolds stored at 25 °C and 4 °C were found to lose their bioactivity after 3- and 7-day storage periods, respectively. Thus, the CS/BCP/TSA scaffold should be stored in freezing conditions to preserve the long-term stability of TSA.

Paradigm shift in infection control practices in dental clinics in response to COVID-19 among dental professionals in Thailand.

Infection control (IC) practice routines depend mainly on knowledge, perception, and awareness of a disease among dental professionals. However, there has been no report on the perception, awareness, and adaptability to the new practice guidelines of Thai dental professionals (dentists, dental nurses, dental assistants, and dental technicians) to the COVID-19 pandemic. This study aims to investigate how dental professionals in Thailand perceive and are aware of COVID-19, and how they have changed their IC practices in response to the pandemic. Online cross-sectional surveys using convenience sampling during September 2021 were sent to Thai dental professionals. The data were analyzed using descriptive statistics and the Chi-square test. Statistical analysis was performed using the Statistical Package for Social Sciences, version 22.0. The tests were two-tailed, with a significance level of p < 0.05 and 95% confidence intervals (CIs). The 1,177 dental professionals who completed the questionnaire were from the public and private sectors. Most respondents obtained their knowledge about COVID-19 from social media (91.8%). 86.7% had adapted to the new IC practice guidelines. The respondents reported that they had modified their work practices in several aspects; changes in administrative control, 1,039 (88.3%); enhancing local source control of dental aerosols, 1,031 (87.6%); heightening sterilization and disinfection procedures, 1,032 (87.7%); and improving the ventilation system, 994 (84.5%). As of October 2021, 1,162 (98.7%) respondents were vaccinated, and 47 (3.99%) had tested positive for COVID-19 compared with 2.30% in the general population. Among infected individuals, 10 (21.3%) were suspected of being infected while working in the dental setting. In conclusion, with an average worry score well over 4.10 out of 5, more than 96% of Thai dental professionals reported seeking updated knowledge and agreed that escalation of IC measures was needed. However, only 86.7% improved their COVID-19 infection prevention practices in 4 aspects and appropriate PPE use. The infection rate in dental professionals was 3.99%, with the highest infection rate in dental assistants. Despite statistical insignificance of infection rate between changed and unchanged group, it cannot be concluded that stricter IC measures are negligible as ones might contract disease from setting other than work.

Novel Epigenetic Modulation Chitosan-Based Scaffold as a Promising Bone Regenerative Material.

Bone tissue engineering is a complicated field requiring concerted participation of cells, scaffolds, and osteoactive molecules to replace damaged bone. This study synthesized a chitosan-based (CS) scaffold incorporated with trichostatin A (TSA), an epigenetic modifier molecule, to achieve promising bone regeneration potential. The scaffolds with various biphasic calcium phosphate (BCP) proportions: 0%, 10%, 20%, and 40% were fabricated. The addition of BCP improved the scaffolds' mechanical properties and delayed the degradation rate, whereas 20% BCP scaffold matched the appropriate scaffold requirements. The proper concentration of TSA was also validated. Our developed scaffold released TSA and sustained them for up to three days. The scaffold with 800 nM of TSA showed excellent biocompatibility and induced robust osteoblast-related gene expression in the primary human periodontal ligament cells (hPDLCs). To evaluate in vivo bone regeneration potential, the scaffolds were implanted in the mice calvarial defect model. The excellent bone regeneration ability was further demonstrated in the micro-CT and histology sections compared to both negative control and commercial bone graft product. New bone formed in the CS/BCP/TSA group revealed a trabeculae-liked characteristic of the mature bone as early as six weeks. The CS/BCP/TSA scaffold is an up-and-coming candidate for the bone tissue engineering scaffold.

Chitosan-Based Scaffold for Mineralized Tissues Regeneration.

Conventional bone grafting procedures used to treat bone defects have several limitations. An important aspect of bone tissue engineering is developing novel bone substitute biomaterials for bone grafts to repair orthopedic defects. Considerable attention has been given to chitosan, a natural biopolymer primarily extracted from crustacean shells, which offers desirable characteristics, such as being biocompatible, biodegradable, and osteoconductive. This review presents an overview of the chitosan-based biomaterials for bone tissue engineering (BTE). It covers the basic knowledge of chitosan in terms of biomaterials, the traditional and novel strategies of the chitosan scaffold fabrication process, and their advantages and disadvantages. Furthermore, this paper integrates the relevant contributions in giving a brief insight into the recent research development of chitosan-based scaffolds and their limitations in BTE. The last part of the review discusses the next-generation smart chitosan-based scaffold and current applications in regenerative dentistry and future directions in the field of mineralized tissue regeneration.

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties.

Plasma Sterilization Effectively Reduces Bacterial Contamination in Dental Unit Waterlines.

OBJECTIVE: To investigate the effectiveness of plasma sterilization in reducing bacterial contamination and controlling biofilms in dental unit waterlines. MATERIALS AND METHODS: Ten identical dental chair units (DCUs) were used. Five DCUs were installed with an automated plasma sterilization system (PSS) and the other five were kept as nontreated controls (CTL). Water flushed from the airotor line served as the output water of the dental unit waterlines (DUWLs). Water samples were collected at the beginning and on a weekly basis for 4 months. Water was analyzed for bacterial contamination (CFU/mL). Scanning electron microscopy (SEM) was used to investigate the amount of biofilm in the waterlines. Biofilm viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. All statistical analyses were performed using the Mann-Whitney U test. A value of p < 0.05 was considered significant. RESULTS: The DCU output water was found to be heavily contaminated with bacteria. Plasma sterilization effectively reduced bacterial contamination from an average of 212 CFU/mL to 8 CFU/mL. During the entire period of 4 months, the level remained below 500 CFU/mL, the standard level recommended by the Centers for Disease Control and Prevention (CDC) of the USA. The reduction in the bacterial count was significant compared with the CTL group (p < 0.05). Plasma sterilization could not eradicate the existing biofilms in the waterlines, and it did reduce biofilm mass and viability. Moreover, treatment with plasma sterilization did not induce a change in the composition of microorganisms, as analyzed by Gram staining. CONCLUSION: Plasma sterilization, which is part of electrochemically activated water, effectively reduces bacterial contamination and reduces biofilms in dental unit waterlines.

Influence of Adjuncts to Irrigation in the Disinfection of Large Root Canals.

INTRODUCTION: The aims of this study were to evaluate the effectiveness of disinfection methods and determine the most promising irrigation protocol for regenerative endodontics in teeth with large root canals. METHODS: Sterilized root samples with 0.8-mm wide apical foramen (n = 94) were prepared from human mandibular premolars. Ninety-two samples were infected with Enterococcus faecalis for 21 days and biofilm formation was verified using scanning electron microscopy (n = 2). The 90 infected samples were randomly assigned into 9 groups: no intervention (initial), 1.5% NaOCl irrigation (1.5 N), 2.5% NaOCl irrigation (2.5 N), 1.5 N + intermittent passive ultrasonic irrigation (PUI), 1.5 N + intermittent canal brushing with Navitip FX (NFX), 1.5 N + intermittent XP-endo Finisher (XPF), 1.5 N + circumferential filing (CF), 1.5 N + 1-min Self-adjusting File (SAF), and 1.5 N + mechanical instrumentation using #90‒110 files (MI). Subsequently, the root canal walls were shaved for microbial analysis. The mean colony-forming units (CFUs) were determined and analyzed using 1-way analysis of variance. RESULTS: The mean CFU count was lowest in the MI group (63.5 CFU/mL), followed by the NFX, XPF, SAF, 2.5 N, CF, PUI, 1.5 N, and initial groups. The remaining bacteria in the 1.5 N group was 3.6-fold higher than that of the PUI group; 4- to 5-fold higher than that of the 2.5 N, CF, and SAF groups; and 22-fold and 36-fold higher than that of the XPF and NFX groups, respectively. The 2.5 N and 1.5 N groups with adjunctive treatments, excluding the PUI group, had significantly fewer remaining bacteria compared with the 1.5 N group (P < .05). CONCLUSIONS: Performing various procedures supplemental to 1.5 N improved large root canal disinfection. Adjunctive NFX most effectively reduced the number of bacteria without dentin removal.

Application of D-Amino Acids as Biofilm Dispersing Agent in Dental Unit Waterlines.

AIM AND PURPOSE: Biofilms in dental unit waterlines (DUWLs) are extremely difficult to eliminate. Aim of this study is to evaluate the efficacy of a mixture of four D-amino acids on biofilm dispersion in DUWLs. MATERIALS AND METHODS: A mixture of four D-amino acids (D-methionine, D-tryptophan, D-leucine, and D-tyrosine, 10 mM each), distilled water (control), and 0.1 M hydrochloric acid (HCl) was used in the experiment. In laboratory, pieces of DUWLs covered with biofilms were submerged in different solutions for 5 days, flushed, and measured OD600 of the dispersed biofilms. Remnants of biofilms on the DUWLs were evaluated by SEM. In clinic, fifteen DCUs were incubated with test and control solutions, flushed, and measured OD600 of the dispersed biofilms. Microbial count of DUWL output water was enumerated twice a week for four weeks. RESULTS: There was a slight, but not significant, increase in OD600 of flushing water in D-amino acids group. D-amino acids effectively reduced bacterial plaque as demonstrated by SEM. Incubation with D-amino acids significantly reduced biofilms especially after the first day of flushing. Bacterial count in DUWL output water was significantly reduced after treatment with D-amino acids. CONCLUSION: D-amino acids are applicable as biofilm dispersing agents in DUWLs.

Histone deacetylases and their roles in mineralized tissue regeneration.

Histone acetylation is an important epigenetic mechanism that controls expression of certain genes. It includes non-sequence-based changes of chromosomal regional structure that can alter the expression of genes. Acetylation of histones is controlled by the activity of two groups of enzymes: the histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs remove acetyl groups from the histone tail, which alters its charge and thus promotes compaction of DNA in the nucleosome. HDACs render the chromatin structure into a more compact form of heterochromatin, which makes the genes inaccessible for transcription. By altering the transcriptional activity of bone-associated genes, HDACs control both osteogenesis and osteoclastogenesis. This review presents an overview of the function of HDACs in the modulation of bone formation. Special attention is paid to the use of HDAC inhibitors in mineralized tissue regeneration from cells of dental origin.

Histone deacetylase inhibition enhances in-vivo bone regeneration induced by human periodontal ligament cells.

Periodontal ligament cells have the potential to differentiate into bone forming osteoblasts and thus represent a good cellular candidate for bone regeneration. This study aimed to investigate the effect of inhibition of histone deacetylases, using the inhibitor Trichostatin A (TSA), on bone regeneration by human periodontal ligament cells (hPDLCs) in a mouse calvaria bone defect. METHODS: RUNX2 protein and its acetylation was analyzed by immunoprecipitation and western blotting. The effect of TSA on osteogenic differentiation of hPDLCs was investigated using in vitro 3D cultures. hPDLCs were pre-incubated with and without TSA and implanted in mouse calvaria defects with polycaprolactone/polyethylene glycol (PCL/PEG) co-polymer scaffold. Micro-CT scanning and bone histomorphometric analysis were used to quantify the amount of bone. Survival of hPDLCs as xenogenic grafts was verified by immunohistochemistry with anti-human ß1-integrin. The immunological response of mice against hPDLCs xenografts was evaluated by measuring total IgG and hPDLCs-specific IgG. RESULTS: Beside affecting histone protein, TSA also induced hyper-acetylation of RUNX2 which might be a crucial mechanism for enhancing osteogenesis by hPDLCs. TSA enhanced mineral deposition by hPDLCs in in vitro 3D cultures and had no effect on cell viability. In vivo bone regeneration of mouse calvaria defects was significantly enhanced by TSA pre-treated hPDLCs. By using anti-human ß1 integrin hPDLCs were shown to differentiate into osteocyte-like cells that were present in newly formed bone. hPDLCs, as a xenograft, slightly but not significantly induced an immunological response in recipient mice as demonstrated by the level of total IgG and hPDLCs-specific IgG. CONCLUSION: Inhibition of histone deacetylases by TSA enhanced in vivo bone regeneration by hPDLCs. The data strongly suggest a novel approach to regenerate bone tissue.

Rinsing with Saline Promotes Human Gingival Fibroblast Wound Healing In Vitro.

Rinsing the mouth with sodium chloride (NaCl) solution is believed to promote healthy gum and improve oral ulcer healing. Scientific evidence to support this assumption is, however, lacking. This study aims to investigate the effect and clarify underlying mechanisms of short-term rinsing with NaCl on human gingival fibroblast (hGFs) wound healing. Isolated primary hGFs and human normal oral keratinocytes (hNOKs) were rinsed with 0-7.2% NaCl for 2 min, 3 times a day. Scratch-tests, trans-well migration assays and MTT activity were performed. mRNA expression was assessed of type-I collagen, fibronectin and FAK. Changes in FAK and F-actin were detected by immunofluorescence. KCl, NaH2PO4, KH2PO4 were used to clarify the molecules involved. Rinsing with 0.9-1.8% NaCl significantly promoted hGFs cell migration but not proliferation. However, it had no effect on hNOKs. Rinsing with 1.8% NaCl significantly up-regulated the expression of type-I collagen and fibronectin. FAK and F-actin, molecules responsible for cytoskeleton re-organization and cell migration, were also up-regulated. Cl- seemed to be essential since rinsing with KCl resulted in a similar effect as noted with NaCl. In conclusion, short-term rinsing with NaCl promoted hGFs migration, and increased the expression of extracellular matrix as well as cytoskeletal proteins. These data strongly support the long held belief in the benefits of using NaCl mouth-rinse.

Inhibition of Histone Deacetylases Enhances the Osteogenic Differentiation of Human Periodontal Ligament Cells.

One of the characteristics of periodontal ligament (PDL) cells is their plasticity. Yet, the underlying mechanisms responsible for this phenomenon are unknown. One possible mechanism might be related to epigenetics, since histone deacetylases (HDACs) have been shown to play a role in osteoblast differentiation. This study was aimed to investigate the role of HDACs in osteogenic differentiation of human PDL (hPDL) cells. HDAC inhibitor trichostatin A (TSA) had no effect on cell viability as was assessed by MTT assay. Osteogenic and adipogenic differentiation was analyzed by gene expression, ALP activity and mineral deposition. Western blotting was used to investigate the effect of TSA on histone acetylation and protein expression. In the presence of the HDAC inhibitor osteogenic differentiation was induced; osteoblast-related gene expression was increased significantly. ALP activity and mineral nodule formation were also enhanced. Inhibition of HDACs did not induce differentiation into the adipocyte lineage. hPDL highly expressed HDACs of both class I (HDAC 1, 2, 3) and class II (HDAC 4, 6). During osteogenic differentiation HDAC 3 expression gradually decreased. This was apparent in the absence and presence of the inhibitor. The level of acetylated Histone H3 was increased during osteogenic differentiation. Inhibition of HDAC activity induced hyperacetylation of Histone H3, therefore, demonstrating Histone H3 as a candidate target molecule for HDAC inhibition. In conclusion, hPDL cells express a distinguished series of HDACs and these enzymes appear to be involved in osteogenic differentiation. This finding suggests a potential application of TSA for bone regeneration therapy by hPDL cells.

Effectiveness of Different Disinfection Protocols on the Reduction of Bacteria in Enterococcus faecalis Biofilm in Teeth with Large Root Canals.

INTRODUCTION: This study compared the effectiveness of different disinfection protocols in reducing bacteria in an Enterococcus faecalis biofilm in teeth with large root canals. METHODS: Fifty-five roots were prepared from human mandibular premolars with large single root canals, and 50 roots were infected with E. faecalis for 21 days. Four roots were observed using scanning electron microscopy to verify biofilm formation. The remaining specimens were assigned into 5 experimental groups and a sterile control group: mechanical instrumentation using files size 60-90 and 2.5% sodium hypochlorite (NaOCl) (MI-N), irrigation with 2.5% NaOCl (IR-N), irrigation with 2.5% NaOCl using intermittent passive ultrasonic irrigation (PUI-N), irrigation with normal saline, and no intervention (initial). After root canal disinfection, dentin specimens were collected for microbial analysis. Mean colony forming units were determined and compared between groups using 1-way analysis of variance. RESULTS: The lowest number of intracanal bacteria (24.5 colony-forming units/mL) was recovered from the MI-N group followed by the PUI-N and IR-N groups. The irrigation with normal saline group did not show a significant reduction compared with the initial group. However, there were significant differences between groups (P < .01). The remaining bacteria in the PUI-N group was 4.5-fold lower compared with the IR-N group; however, it was 1862-fold higher compared with the MI-N group. CONCLUSIONS: MI-N was the most effective method to disinfect large root canals. Although ultrasonic activation enhanced the efficacy of root canal irrigation, it could not substitute for mechanical instrumentation even in large root canals with unlimited irrigant access to the apical portion.