MicroRNA-200c Release from Gelatin-Coated 3D-Printed PCL Scaffolds Enhances Bone Regeneration.

The fabrication of clinically relevant synthetic bone grafts relies on combining multiple biodegradable biomaterials to create a structure that supports the regeneration of defects while delivering osteogenic biomolecules that enhance regeneration. MicroRNA-200c (miR-200c) functions as a potent osteoinductive biomolecule to enhance osteogenic differentiation and bone formation; however, synthetic tissue-engineered bone grafts that sustain the delivery of miR-200c for bone regeneration have not yet been evaluated. In this study, we created novel, multimaterial, synthetic bone grafts from gelatin-coated 3D-printed polycaprolactone (PCL) scaffolds. We attempted to optimize the release of pDNA encoding miR-200c by varying gelatin types, concentrations, and polymer crosslinking materials to improve its functions for bone regeneration. We revealed that by modulating gelatin type, coating material concentration, and polymer crosslinking, we effectively altered the release rates of pDNA encoding miR-200c, which promoted osteogenic differentiation in vitro and bone regeneration in a critical-sized calvarial bone defect animal model. We also demonstrated that crosslinking the gelatin coatings on the PCL scaffolds with low-concentration glutaraldehyde was biocompatible and increased cell attachment. These results strongly indicate the potential use of gelatin-based systems for pDNA encoding microRNA delivery in gene therapy and further demonstrate the effectiveness of miR-200c for enhancing bone regeneration from synthetic bone grafts.

CaCO 3 Nanoparticles Delivering MicroRNA-200c Suppress Oral Squamous Cell Carcinoma.

MicroRNA (miR)-200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c -based gene therapy to inhibit OSCC growth and metastasis has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA encoding miR-200c delivered via non-viral CaCO 3 -based nanoparticles to inhibit OSCC tumor growth. CaCO 3 -based nanoparticles with various ratios of CaCO 3 and protamine sulfate (PS) were utilized to transfect pDNA encoding miR-200c into OSCC cells and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c were also quantified. It was observed that, while CaCO 3 -based nanoparticles improve transfection efficiencies of pDNA miR-200c , the ratio of CaCO 3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line-derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO 3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO 3 /pDNA miR-200c may potentially be used to reduce oral cancer recurrence and metastasis and improve clinical outcomes in OSCC treatment. (227 words).

A Medium Chain Fatty Acid, 6-hydroxyhexanoic acid (6-HHA), Protects Against Obesity and Insulin Resistance.

Obesity, a worldwide health problem, increases the risk for developing metabolic diseases such as insulin resistance and diabetes. It is well recognized that obesity-associated chronic inflammation plays a key role in the pathogenesis of systemic metabolic dysfunction. Previously, we revealed an anti-inflammatory role for spent culture supernatants isolated from the oral commensal bacterial species Streptococcus gordonii (Sg-SCS). Here, we identified that 6-hydroxyhexanoic acid (6-HHA), a medium chain fatty acid (MCFA), is the one of the key components of Sg-SCS . We found that treatment of 6-HHA in mice fed a high-fat diet (HFD) significantly reduced HFD-mediated weight gain which was largely attributed to a decrease in fat mass. Systemically, 6-HHA improves obesity-associated glucose intolerance and insulin resistance. Furthermore, administration of 6-HHA suppressed obesity-associated systemic inflammation and dyslipidemia. At the cellular level, treatment of 6-HHA ameliorated aberrant inflammatory and metabolic transcriptomic signatures in white adipose tissue of mice with diet-induced obesity (HFD). Mechanistically, we found that 6-HHA suppressed adipocyte-proinflammatory cytokine production and lipolysis, the latter through Gαi-mediated signaling. This work provides direct evidence for the anti-obesity effects of a novel MCFA, which could be a new therapeutic treatment for combating obesity. KEY POINTS: Hydroxyhexanoic medium chain fatty acids (MCFAs) are dietary and bacterial-derived energy sources, however, the outcomes of using MCFAs in treating metabolic disorders are diverse and complex. The MCFA 6-hydroxyhexanoic acid (6-HHA) is a metabolite secreted by the oral bacterial commensal species Streptococcus gordonii; here we investigated its role in modulating high-fat diet (HFD)-induced metabolic dysfunction. In a murine model of obesity, we found 6-HHA-mediated improvement of diet-mediated adiposity, insulin resistance and inflammation were in part due to actions on white adipose tissue (WAT).6-HHA suppressed proinflammatory cytokine production and lipolysis through Gi-mediated signaling in differentiated white adipocytes.

Spent culture supernatant of Streptococcus gordonii mitigates inflammation of human periodontal cells and inhibits proliferation of pathogenic oral microbes.

BACKGROUND: The oral commensal bacterial species Streptococcus gordonii has been reported to regulate the inflammation of oral epithelial cells stimulated by the periodontal pathogen Porphyromonas gingivalis. This study investigated the activities of S. gordonii metabolites in S. gordonii spent culture supernatants (Sg-SCS) on periodontal-related bacterial growth and periodontitis-associated inflammatory cytokines. METHODS: Sg-SCS was collected from S. gordonii cultures grown in Dulbecco Modified Eagle Medium and added to the growth media of representative health- and disease-related oral species: S. gordonii, Streptococcus sanguinis, Streptococcus mitis, Streptococcus oralis, P. gingivalis, Tannerella forsythia, and Treponema denticola. The Sg-SCS was also tested for its ability to regulate the expression of proinflammatory cytokines by human macrophages, epithelial cells, and gingival fibroblasts upon stimulation with P. gingivalis-derived lipopolysaccharide (Pg-LPS). RESULTS: Sg-SCS significantly reduced transcript and protein levels of interleukin (IL)-1ß, 6, and 8 induced by Pg-LPS stimulation in multiple types of periodontal cells. mRNA sequencing and bioinformatics analyses indicated that Sg-SCS significantly affects 10 inflammatory pathways. Additionally, Sg-SCS exhibited suppression of the growth of periodontal disease-related bacteria, including T. denticola and P. gingivalis, along with the primary plaque-colonizing species S. oralis. At a low concentration, Sg-SCS also inhibits P. gingivalis adhesion. CONCLUSIONS: These results strongly suggest that S. gordonii-derived SCS contains metabolites that have anti-inflammatory properties and an ability to inhibit periodontitis-associated pathogenic bacteria. Further investigation will be needed to identify the individual metabolites within the Sg-SCS to develop a novel metabolite-based approach to treating and preventing periodontitis.

Evaluation of the Bond Strength of Epoxy Resin-based Sealer Following Different Calcium Hydroxide Paste Removal Methods in Oval-shaped Root Canal.

AIM: The study aimed to investigate the bond strength of epoxy resin-based sealer following five different calcium hydroxide paste removal methods in oval-shaped root canals. MATERIALS AND METHODS: Sixty single-rooted human mandibular premolars having an oval-shaped root canal as evidenced by radiographs were decoronated and instrumented to size 40/0.04, medicated with calcium hydroxide paste for 7 days, before being randomly assigned to the conventional needle irrigation (CNI), manual dynamic agitation (MDA), sonic irrigation (SI), passive ultrasonic irrigation (PUI), and XP-endo Finisher (XP) groups to be irrigated using different irrigation systems. All specimens were then obturated using an epoxy resin-based root canal sealer and warm vertical compaction obturation technique. After 7 days, each specimen was sectioned into 1 mm root slices at the coronal, middle, and apical third of the root canal and tested for the push-out bond strength using a universal testing machine. RESULTS: The XP group was the only group that had comparable bond strength to the control group at every level of the root canal following removal of calcium hydroxide medicament (p >0.05). The control group had higher bond strength than the CNI group at every level of the root canal (p < 0.05). CONCLUSION: Calcium hydroxide interfered with the bonding of epoxy resin-based sealer to root canal walls. Irrigation with the XP increased the bonding sealer at every level of the root canal. CLINICAL SIGNIFICANCE: The XP was efficacious as a final rinse agitation technique to promote the bonding of the epoxy resin-based sealer at every level of the root canal following calcium hydroxide medication.

Effect of a heat-based root canal obturation technique on push-out bond strength of the classical bioceramic and new HiFlow sealer.

This study aimed to investigate the effect of the heat-based obturation technique on the push-out bond strength of the classical bioceramic and new HiFlow sealer. Fifty single-rooted human mandibular premolars were obturated using one of the following techniques: (1) BC-SC; (2) BC-W; (3) HiFlow-SC; (4) HiFlow-W; and (5) AH Plus-W. Following obturation, specimens were sectioned into 1-mm-thick root slices 4 and 8 mm from the working length and were subjected to a push-out bond test using a universal testing machine and examined for their modes of failure under an inverted phase-contrast microscope. At both distances tested, there was no difference in the push-out bond strength among the four bioceramic groups (P > 0.05). The AH Plus-W group had lower bond strength than every other group at both distances from the working length. The most common failure mode was mixed failure.

Optimal antimicrobial concentration of mixed antibiotic pastes in eliminating Enterococcus faecalis from root dentin.

This study aims to identify the optimal concentrations of triple antibiotic paste (TAP), modified triple antibiotic paste (mTAP) and double antibiotic paste (DAP) that could completely eradicate Enterococcus faecalis in dentinal tubules. One hundred and ninety extracted human premolars inoculated with E. faecalis were medicated with calcium hydroxide and mixed antibiotic pastes (TAP, mTAP and DAP at 0.1, 1.25, 5, 10 and 20 mg mL-1 of each drug) for seven days before being frozen, pulverised and cultured. Specimens treated with the lowest concentration of the mixed antibiotics capable of eradicating E. faecalis were examined using scanning electron and confocal laser scanning microscopy to confirm the eradication of the microbial. TAP at 10 mg mL-1 of each drug was able to completely eliminate bacteria inside dentinal tubules, whereas mTAP and DAP required 20 mg mL-1 of each drug. Calcium hydroxide was not effective in eradicating E. faecalis inside dentinal tubules.

Smear Layer Removal from Root Canal Dentin and Antimicrobial Effect of Citric Acid-modified Chlorhexidine.

OBJECTIVE: To study the effectiveness of various concentrations of citric acid (CA) added to 2% chlorhexidine (CHX) on smear layer removal from the root canal wall and antimicrobial efficacy against Enterococcus faecalis (E. faecalis) and Candida albicans (C. albicans). METHODS: Fifty-three single-rooted mandibular premolars were decoronate and the root canals underwent mechanical instrumentation using MTwo rotary files to size 40/0.06. The samples were then randomly divided into 5 groups according to the root canal irrigants to be used: 17% ethylenediaminetetraacetic acid (EDTA), 2% CHX, 1%, 6%, and 10% citric acid-modified 2% chlorhexidine (CAmCHX). Three teeth irrigated with phosphate-buffered saline (PBS) were used as a negative control. The smear layer removal effectiveness was evaluated under scanning electron microscopy (SEM). Images were randomly taken at the apical, middle, and coronal third level. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney U tests. Disc diffusion and direct exposure tests were performed along with three additional control groups consisting of 1%, 6%, and 10% CA groups to assess and compare the antimicrobial efficacy of irrigants against E. faecalis and C. albicans. Statistical analysis was conducted using one-way ANOVA and Dunnett's T3 tests. RESULTS: Smear layer removal effectiveness in 17% EDTA group and 6% and 10% CAmCHX groups were not significantly different in the coronal and apical third of the root canal (P>0.05), however at the middle third of the root canal, the 10% CAmCHX group had significantly less remaining smear layer than all of the other experimental groups (P<0.05). There was significantly more smear layer remnant in the CHX group (P<0.01). For antimicrobial efficacy, the largest growth inhibition zone against E. faecalis was recorded in the 10% CAmCHX group (P<0.05). For planktonic E. faecalis, 1%, 6%, and 10% CAmCHX demonstrated an insignificant difference in antimicrobial efficacy compared to CHX (P>0.05). CA demonstrated no antifungal effect against C. albicans. Whereas, 6% and 10% CAmCHX resulted in the largest growth inhibition zone. Also, adding CA to CHX resulted in an insignificant difference in antifungal effect against planktonic C. albicans compared to CHX (P>0.05). CONCLUSION: When CA was added into CHX, the mixed irrigant demonstrated smear layer removal ability. Additionally, its antimicrobial effect remained the same.