Comparison of different DNA preservation solutions for oral cytological samples.

OBJECTIVE: The objective of this study was to compare the DNA preservation capacity of buccal mucosa exfoliated cells when stored in different solutions under varying time and temperature conditions. DESIGN: DNA preservation solutions, including Dimethyl sulphoxide disodium-EDTA-saturated NaCl (DESS), Tris-EDTA-NaCl-Tween20 buffer (TENT), Nucleic Acid Preservation Buffer (NAP), and phosphate-buffered saline (PBS), were prepared. Buccal mucosa cells from a single patient were collected, dispensed into these solutions, and stored at room temperature (RT) and 4 °C for 24 h, 72 h, 30 days, 90 days, and 180 days. DNA was extracted using the salting-out method and the QIAamp DNA Mini Kit. DNA concentration and purity were determined using the QuBit device and NanoDrop, while DNA integrity was assessed using the Agilent 4200 TapeStation system. The ability to amplify the IFNA primer was also evaluated by PCR. RESULTS: The salting-out method yielded better concentration and purity results, with PBS, TENT, and DESS buffers demonstrating superior concentration values when stored at 4 °C, resulting in mean values exceeding 10 ng/µL for up to 30 days. DESS consistently exhibited the best integrity values over time for both temperature conditions. Amplification capacity was enhanced when samples were stored at 4 °C. When stored at RT, PBS achieved 100% amplification within 24 h. NAP yielded the poorest results. CONCLUSION: In the context of long-term preservation, the DESS buffer emerges as the most effective solution, maintaining requisite DNA quality and quantity standards for up to 30 days at RT and up to 3 months at 4 °C.

Synthesis of films based on chitosan and protic ionic liquids to be used as wound dressing on the oral mucosa.

Oral mucosal ulcerations expose connective tissue to different pathogens and this can progress to systemic infection. This study aimed to synthesize environmentally-friendly films with chitosan and protic ionic liquids, possessing mucoadhesive properties, activity against opportunistic microorganisms, enhanced malleability and mechanical resistance to be used as a wound dressing on the oral mucosa. Therefore, films with chitosan and 10, 35, and 50 % (wt/wt) of 2-hydroxy diethylammonium lactate, salicylate, and maleate protic ionic liquids were synthesized. Thickness measurements and mechanical properties analysis were performed. In addition, oral mucoadhesion, antimicrobial activity, and cytotoxicity properties were investigated. Results showed that the addition of 35wt% and 50wt% of all kinds of protic ionic liquids tested presented significant improvements in film thickness and mechanical properties. Films based on chitosan and the protic ionic liquid 2-hydroxy diethylammonium salicylate at percentages of 35 and 50wt% exhibited superior mucoadhesive properties, antimicrobial activity on opportunistic microorganisms and an improvement in their flexibility after immersion in synthetic saliva. Cytotoxicity results suggest that all kinds of chitosan/protic ionic liquids films tested are safe for intra-oral use. Therefore, the results of this study indicate that these materials could be good candidates for efficient and environmentally-friendly wound dressing films on the oral mucosa.

TLR4 Expression in Ex-Lichenoid Lesions-Oral Squamous Cell Carcinomas and Its Surrounding Epithelium: The Role of Tumor Inflammatory Microenvironment.

Toll-like receptors (TLRs) regulate innate and adaptive immune responses. Moreover, TLRs can induce a pro-survival and pro-proliferation response in tumor cells. This study aims to investigate the expression of TLR4 in the epithelium surrounding oral squamous cell carcinomas (OSCC) in relation to its inflammatory microenvironment. This study included 150 human samples: 30 normal oral control (NOC), 38 non-lichenoid epithelium surrounding OSCC (NLE-OSCC), 28 lichenoid epithelium surrounding OSCC (LE-OSCC), 30 OSCC ex-non oral lichenoid lesion (OSCC Ex-NOLL), and 24 OSCC ex-oral lichenoid lesion (OSCC Ex-OLL). TLR4 expression was investigated by immunohistochemistry and the percentage of positive cells was quantified. In addition, a semiquantitative analysis of staining intensity was performed. Immunohistochemical analysis revealed that TLR4 is strongly upregulated in LE-OSCC as compared to normal control epithelium and NLE-OSCC. TLR4 expression was associated with the inflammatory environment, since the percentage of positive cells increases from NOC and NLE-OSCC to LE-OSCC, reaching the highest value in OSCC Ex-OLL. TLR4 was detected in the basal third of the epithelium in NLE-OSCC, while in LE-OSCC, TLR4 expression reached the intermediate layer. These results demonstrated that an inflammatory microenvironment can upregulate TLR4, which may boost tumor development.

The role of tumor acidification in aggressiveness, cell dissemination and treatment resistance of oral squamous cell carcinoma.

AIMS: To investigate the role of tumor acidification in cell behavior, migration, and treatment resistance of oral squamous cell carcinoma (OSCC). MAIN METHODS: The SCC4 and SCC25 cell lines were exposed to acidified (pH 6.8) cell culture medium for 7 days. Alternatively, a long-term acidosis was induced for 21 days. In addition, to mimic dynamic pH fluctuation of the tumor microenvironment, cells were reconditioned to neutral pH after experimental acidosis. This study assessed cell proliferation and viability by sulforhodamine B and flow cytometry. Individual and collective cell migration was analyzed by wound healing, time lapse, and transwell assays. Modifications of cell phenotype, EMT induction and stemness potential were investigated by qRT-PCR, western blot, and immunofluorescence. Finally, resistance to chemo- and radiotherapy of OSCC when exposed to acidified environmental conditions (pH 6.8) was determined. KEY FINDINGS: The exposure to an acidic microenvironment caused an initial reduction of OSCC cells viability, followed by an adaptation process. Acidic adapted cells acquired a mesenchymal-like phenotype along with increased migration and motility indexes. Moreover, tumoral extracellular acidity was capable to induce cellular stemness and to increase chemo- and radioresistance of oral cancer cells. SIGNIFICANCE: In summary, the results showed that the acidic microenvironment leads to a more aggressive and treatment resistant OSCC cell population.

Evaluation of the biological and physicochemical properties of calcium silicate-based and epoxy resin-based root canal sealers.

Biocompatibility, dimensional stability, radiopacity, flow, and low solubility are the characteristics of an ideal endodontic sealer. This study evaluated and compared in vivo and in vitro biological and physicochemical properties of calcium silicate-based sealers: Sealer Plus BC (BC), MTA Fillapex (MF); and resin-based sealers: AH Plus (AHP) and Sealer Plus (SP). Apical papilla cells were exposed to sealer extracts and subjected to MTT, SRB, scratch, alkaline phosphatase enzyme activity (ALP) and Alizarin red staining (ALZ) assays. Sealers were histologically evaluated in connective tissue of Wistar rats in different periods. Radiopacity, film thickness, flow, setting time, pH and element analyses were investigated. BC had better results compared to AHP and MF at hour 72 for MTT assay (p < .05), and the highest cell viability under SRB (p < .05). All sealers presented ALP activity. BC presented the highest mineralized deposition under ALZ (p < .05). BC and MF promoted wound healing. All sealers induced an initial inflammation reaction that decreased over time. Eosinophils were observed at day 7 in MF (p < .05). Despite MF did not present final setting time, the sealers properties were in accordance to ISO 6876/2012 and ASTM C266-08. All sealers presented cell viability and biocompatibility. BC presented higher pH values and bioactivity. The materials tested showed physico-chemical properties in accordance with standards, except for MF setting time.

Synthesis of sol-gel derived calcium silicate particles and development of a bioactive endodontic cement.

OBJECTIVE: The aim of this study is to produce sol-gel derived calcium silicate particles (CS) and evaluate the influence of different concentration of calcium tungstate in the physical, chemical, mechanical and biological properties of developed cements. METHODS: Sol-gel route were used to synthesize calcium silicate particles that were characterized with x-ray difraction, Fourier transformed infrared spectroscopy, scanning electron microscopy, laser diffraction and nitrogen absorption. Cements were formulated with the addition of different concentrations of calcium tungstate (CaWO4), resulting in four experimental groups according to the CS:CaWO4 ratio: CS100 (100:0), CS90 (90:10), CS80 (80:20), CS70 (70:30). The setting time, radiopacity, compressive strength, pH, calcium release, cell proliferation and cell differentiation were used to characterize the cements. RESULTS: CS particles were succesfully sinthesized. The addition of CaWO4 increased the radiopacity and did not influenced the setting time and the mechanical properties of cements. The pH of distilled water was increased for all groups and the CS100 and CS90 groups presented incresed calcium release. Reduced cell viability was found for CS70 while CS100 and CS90 presented higher ALP activity and % of mineralized nodules after 21 days. SIGNIFICANCE: Sol-gel derived CS particles were sucssfully developed with potential to applied for the production of bioactive ceramic cements. The addition of 10% of CaWO4 resulted in cements with adequate properties and bioactivity being an alternative for regenerative endodontic treatments.

Effects of extracellular acidity on resistance to chemotherapy treatment: a systematic review.

Metabolic alterations in the tumor microenvironment have a complex effect on cancer progression. Extracellular acidity is a consequence of metabolic switch in cancer and results in cell phenotypes with higher resistance to chemotherapeutics. However, mechanisms underlying the relationship between the extracellular acidity and chemoresistance are not clearly understood. This systematic review was carried out by searching the databases PubMed and EMBASE using the keywords "cancer" and "acidosis" or "acidic" and "chemoresistance" or "drug resistance." In vitro and in vivo studies that evaluated the effects of acidification of the tumor microenvironment on chemotherapeutic treatments were included. Literature reviews, letters to the editor, and articles that were not published in English were excluded. The search resulted in a total of 352 articles. After discarding 75 duplicate references, 277 articles were analyzed by sequentially reading through their titles, abstracts, and finally full-text. A total of 14 articles was selected. Acidification of the tumor microenvironment can trigger resistance through different mechanisms, such as increase in drug efflux transporters, inhibition of proton pumps, induction of the unfolded protein response (UPR), and cellular autophagy.