Marcus Cristian Muniz Conde

DENTAL PULP TISSUE ENGINEERING

Dental pulp is a highly specialized mesenchymal tissue that has a limited regeneration capacity due to anatomical arrangement and post-mitotic nature of odontoblastic cells. Entire pulp amputation followed by pulp space disinfection and filling with an artificial material cause loss of a significant amount of dentin leaving as life-lasting sequelae a non-vital and weakened tooth. However, regenerative endodontics is an emerging field of modern tissue engineering that has demonstrated promising results using stem cells associated with scaffolds and responsive molecules. Thereby, this article reviews the most recent endeavors to regenerate pulp tissue based on tissue engineering principles and provides insightful information to readers about the different aspects involved in tissue engineering. Here, we speculate that the search for the ideal combination of cells, scaffolds, and morphogenic factors for dental pulp tissue engineering may be extended over future years and result in significant advances in other areas of dental and craniofacial research. The findings collected in this literature review show that we are now at a stage in which engineering a complex tissue, such as the dental pulp, is no longer an unachievable goal and the next decade will certainly be an exciting time for dental and craniofacial research.

Nanofiller loading level: Influence on selected properties of an adhesive resin.

OBJECTIVES To evaluate the effect of the filler content in the cohesive strength (sigma), Weibull modulus (m) and degree of conversion (DC) of an experimental adhesive system. METHODS A HEMA/Bis-GMA/TEGDMA-based adhesive was formulated and filled with silica nanofillers in the following weight percentages (wt%): R0=0%; R1=1%; R3=3%; R5=5% and R10=10%. The adhesive of Adper Scotchbond Multi-Purpose (SBMP) system was used as a commercial reference. Twenty dumbbell-shaped specimens with cross-sectional area of 0.5mm(2) were made per group and tensile tested with a crosshead speed of 0.5mm/min until fracture. The cohesive strength was calculated in MPa. DC was obtained through FTIR after light curing for 25s. Data were submitted to one-way ANOVA and Tukeys test (alpha=0.05) and to Weibull analysis. RESULTS Mean sigma results were: R0=65.4+/-8.4; R1=73.2+/-8.8; R3=72.0+/-8.4; R5=73.1+/-9.7; R10=85.5+/-13.1 and SBMP=79.0+/-11.0MPa. R10 presented the highest sigma, while R0 showed the lowest. R5 and SBMP did not differ significantly (p<0.05). Weibull analysis revealed no significant difference in structural reliability between groups. The experimental adhesives presented similar results of DC, which, in turn, were significantly higher than the SBMP. CONCLUSIONS The addition of 10% filler in weight improves the cohesive strength of the adhesive, not interfering in the structural reliability or the degree of conversion.

Novel experimental cements for use on the dentin-pulp complex

This aim of this study was to evaluate the physicochemical and biological properties of novel experimental cements (Hybrid, Paste and Resin) based on synergistic combinations of existing materials, including pH, diametral tensile strength (DTS) and cytotoxicity comparing them with mineral trioxide aggregate (MTA - Angelus®) and a glass ionomer cement (GIC) developed at our laboratory. For the physicochemical and biological tests, specimens with standard dimensions were produced. pH measurements were performed with digital pH meter at the following time intervals: 3, 24, 48 and 72 h. For the DTS test, cylindrical specimens were subjected to compressive load until fracture. The MTT assay was performed for cytotoxicity evaluation. Data were analyzed by ANOVA and Tukeys test (α=0.05). Paste group showed pH values similar to MTA, and Hybrid group presented pH values similar to GIC (p>0.05). The tested materials showed pH values ranging from alkaline to near neutrality at the evaluated times. MTA and GIC showed similar DTS values. The lowest and highest DTS values were seen in the Paste and Resin groups, respectively (p<0.05). Cell viability for MTA and experimental Hybrid, Paste and Resin groups was 49%, 93%, 90% and 86%, respectively, when compared with the control group. The photo-cured experimental resin cement showed similar or superior performance compared with the current commercial or other tested experimental materials.

Odontoblast RNA stability in different temperature-based protocols for tooth storage

AIM To evaluate the effect of four tooth storage temperature-based methods on quality of RNA obtained from cells retrieved from human dental pulps and human pre-dentine. METHODOLOGY RNA was isolated from dental pulp tissue and from cells retrieved by scraping the pre-dentine of freshly extracted human third molars (n = 15) using TRIzol(®) reagent. Teeth were randomly assigned to the following temperature conditions: immediate RNA isolation after tooth extraction, liquid nitrogen (24 h), -80 °C (24 h), 20 °C (24 h) and 4 °C (6 h). RNA integrity was checked by the density of 28S and 18S ribosomal RNA. RT-PCR was used to analyse the expression of odontoblast makers (DSPP, DMP1 and MEPE) and the housekeeping gene GAPDH. RESULTS All experimental conditions evaluated preserved RNA integrity. The three odontoblastic markers were amplified from the pulp tissue and from the cells associated with pre-dentine. CONCLUSION The four storage options allowed RNA isolation for RT-PCR analysis. These findings may facilitate the use of clinically derived human dental pulp and odontoblasts for endodontic research.

Comparison between DNA obtained from buccal cells of the upper and lower gutter area

This study compared quantitatively and qualitatively the DNA extracted from buccal cells collected from the upper or lower gutter areas. Buccal cells were collected from the upper (n=15) and lower gutter (n=15) region from 15 volunteers using a special cytobrush (Gentra), totaling 2 collections from each individual. DNA was extracted from the samples according to the manufacturers instructions. The DNA obtained was qualitatively and quantitatively evaluated by 2 calibrated blind examiners using spectrophotometry and analysis of DNA bands (0.8% agarose gel electrophoresis). Data was statistically analyzed by one-way ANOVA (alpha=0.05). Means and standard derivation (SD) for total DNA yield from the upper and lower gutter area were 12.2 microg (12.0) and 9.4 microg (8.5), respectively (p=0.821). There was higher (p<0.05) DNA purity for the upper gutter (1.79; 0.05) when compared to lower gutter area (1.66; 0.10). Regarding to the DNA quality, no differences were observed between the 2 location sites, but all samples showed similar degree of degradation. In conclusion, it would be recommendable that buccal cells for DNA extraction be collected from the upper gutter area in the attempt to increase DNA purity.

Does Cryopreservation Affect the Biological Properties of Stem Cells from Dental Tissues? A Systematic Review

This systematic review evaluated if different cryopreservation protocols could affect biological properties (Cell survival rate (CSR), proliferation, differentiation, maintenance of stem cell markers) of stem cells obtained from dental tissues (DSC) post-thaw. An electronic search was carried out within PubMed and ISI Web Science by using specific keyword. Two independent reviewers read the titles and abstracts of all reports respecting predetermined inclusion/exclusion criteria. Data were extracted considering the biological properties of previously cryopreserved DSCs and previously cryopreserved dental tissues. DSCs cryopreserved as soon as possible after their isolation presents a CSR quite similar to the non-cryopreserved DSC. Dimethyl sulfoxide (DMSO) [10%] showed good results related to cell recovery post-thaw to cryopreserve cells and tissues for periods of up to 2 years. The cryopreservation of DSC in a mechanical freezer (-80°C) allows the recovery of stem cells post-thaw. The facilities producing magnetic field (MF), demand a lower concentration of cryoprotectant, but their use is not dispensable. It is possible to isolate and cryopreserve dental pulp stem cell (DPSC) from healthy and diseased vital teeth. Cryopreservation of dental tissues for late DSC isolation, combined with MF dispensability, could be valuable to reduce costs and improve the logistics to develop teeth banks.

Platelet-Poor Plasma as a Supplement for Fibroblasts Cultured in Platelet-Rich Fibrin

The aim of this study was to evaluate the proliferation and adhesion of mesenchymal cells (3T3/NIH) in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with Platelet-Poor Plasma (PPP) in aPlatelet-Rich Fibrin (PRF) scaffold. Human blood was obtained and processed in a centrifuge considering the equation G=1.12xRx(RPM/1000)2 to obtain PRF and PPP.Cell adhesion and maintenance analyses were performed by MTTassays in a 96 well plate withsupplemented DMEM: PPP (90:10) for 24 hours. Besides, the PRF was deposited in a 48 well plate and 10x104 cells were seeded above each PRF (n=3) with 800µl of DMEM: PPP (90:10) and cultured for 7 days. Histological analysis and the immunohistochemical staining for Vimentin were performed. Results were analyzed by one-way ANOVA in Stata12®. A significant decrease (p<0.05) of cells adhesion in relationship to FBSwas observed. However, a similar ability of cell-maintenance for PPP 10% was observed (P>0.05). Fibroblasts culture for 7 days in PRF supplemented with PPP 10% was possible, showing positive staining for Vimentin. Therefore, PPP cell supplementation decreased the initial adhesion of cells but was able to maintain the proliferation of adhered cells and able to support their viability in PRF.It seems that this method has many clinical advantagessince it provides an autologous and natural scaffold with their respective supplement for cell culture by only one process, without using xenogeneic compounds. This could improve the potential of clinical translational therapies based on the use of PRF cultured cells, promoting the regenerative potential for future use in medicine and dentistry.

Electrochemical Cathodic Polarization, a Simplified Method That Can Modified and Increase the Biological Activity of Titanium Surfaces: A Systematic Review.

Background The cathodic polarization seems to be an electrochemical method capable of modifying and coat biomolecules on titanium surfaces, improving the surface activity and promoting better biological responses. Objective The aim of the systematic review is to assess the scientific literature to evaluate the cellular response produced by treatment of titanium surfaces by applying the cathodic polarization technique. Data, Sources, and Selection The literature search was performed in several databases including PubMed, Web of Science, Scopus, Science Direct, Scielo and EBSCO Host, until June 2016, with no limits used. Eligibility criteria were used and quality assessment was performed following slightly modified ARRIVE and SYRCLE guidelines for cellular studies and animal research. Results Thirteen studies accomplished the inclusion criteria and were considered in the review. The quality of reporting studies in animal models was low and for the in vitro studies it was high. The in vitro and in vivo results reported that the use of cathodic polarization promoted hydride surfaces, effective deposition, and adhesion of the coated biomolecules. In the experimental groups that used the electrochemical method, cellular viability, proliferation, adhesion, differentiation, or bone growth were better or comparable with the control groups. Conclusions The use of the cathodic polarization method to modify titanium surfaces seems to be an interesting method that could produce active layers and consequently enhance cellular response, in vitro and in vivo animal model studies.

Immunohistochemical Expression of TGF-β1 and Osteonectin in engineered and Ca(OH)2-repaired human pulp tissues

The aim of the present study was to evaluate the expression of transforming growth factor-β1 (TGF-β1) and osteonectin (ON) in pulp-like tissues developed by tissue engineering and to compare it with the expression of these proteins in pulps treated with Ca(OH)2 therapy. Tooth slices were obtained from non-carious human third molars under sterile procedures. The residual periodontal and pulp soft tissues were removed. Empty pulp spaces of the tooth slice were filled with sodium chloride particles (250-425 µm). PLLA solubilized in 5% chloroform was applied over the salt particles. The tooth slice/scaffold (TS/S) set was stored overnight and then rinsed thoroughly to wash out the salt. Scaffolds were previously sterilized with ethanol (100-70°) and washed with phosphate-buffered saline (PBS). TS/S was treated with 10% EDTA and seeded with dental pulp stem cells (DPSC). Then, TS/S was implanted into the dorsum of immunodeficient mice for 28 days. Human third molars previously treated with Ca(OH)2 for 90 days were also evaluated. Samples were prepared and submitted to histological and immunohistochemical (with anti-TGF-β1, 1:100 and anti-ON, 1:350) analyses. After 28 days, TS/S showed morphological characteristics similar to those observed in dental pulp treated with Ca(OH)2. Ca(OH)2-treated pulps showed the usual repaired pulp characteristics. In TS/S, newly formed tissues and pre-dentin was colored, which elucidated the expression of TGF-β1 and ON. Immunohistochemistry staining of Ca(OH)2-treated pulps showed the same expression patterns. The extracellular matrix displayed a fibrillar pattern under both conditions. Regenerative events in the pulp seem to follow a similar pattern of TGF-β1 and ON expression as the repair processes.

Produção especializada no SUS em capitais brasileiras com centros de especialidades odontológicas: uma análise descritiva

Dental Specialty Centers (Centros de Especialidades Odontológicas – CEOs) are specialized health facilities that should perform a minimum number of procedures. Objectives: this study aimed to describe the specialized dental production and report the achievement of goals in Brazilian capitals with CEOs. Materials and method: a retrospective longitudinal study was performed with a search for the CEOs listed in the National Registry of Health Establishments. The dental production was searched in the Outpatient Information System of the Brazilian Unified Health System for the period from May 2015 to April 2016. Results: fifty-nine CEOs were found and considered eligible for the present study. They were located in 19 Brazilian capitals and in the Federal District, whereas 48% were CEOs Type II. Approximately 730 thousand specialized procedures were performed during the 12 months evaluated. A rate of 86% of goals was met and surgery presented the highest achievement (92%), followed by periodontics (89%) and endodontics (76%). One of the capitals achieved only 33% of the goals. Three capitals did not achieve any of the goals set for endodontic procedures. Conclusion: there was a great variation in the achievement of goals among capitals with CEOs. While some capitals showed high achievement of goals, others presented concerning data, especially for endodontic procedures.