Periodontal ligament cells mobilized by transforming growth factor-beta 1 and migrated without stimuli showed enhanced osteogenic differentiation.

OBJECTIVE: This study aimed to analyze the ability of G-CSF and TGF-ß1 to mobilize periodontal ligament stem cells to obtain populations with better potential for proliferation and osteogenic differentiation. DESIGN: Primary cultures were established from the periodontal ligament of Wistar rats. After a cell migration assay, four experimental groups were obtained: PDLSC, composed of the primary culture, non-mobilized cells; MPDLSC, the spontaneously migrated cells; MPDLSC-GCSF, the cells mobilized with G-CSF; and MPDLSC-TGF-ß1, the cells mobilized with TGF-ß1. The expression of mesenchymal stem cell markers was assessed by flow cytometry. Clonogenicity, viability, proliferative potential, and osteogenic differentiation capacity were also analyzed. RESULTS: All the study groups expressed well-known mesenchymal stem cell markers and exhibited clonogenic capacity. The higher proliferation potential was seen in the PDLSC and MPDLSC groups, while the MPDLSC and MPDLSC-TGFß1 groups showed a higher number of mineralized deposits in vitro and higher ALP activity after osteogenic differentiation induction. Cells of all the groups also expressed mRNA of genes associated with osteogenic differentiation without previous induction. CONCLUSIONS: Both agents were able to mobilize stem cells from the periodontal ligament, but G-CSF did not show an advantage, whereas TGF-ß1 appears to direct the cells towards a state of increased osteogenic differentiation. Furthermore, spontaneous cell migration through a membrane was sufficient to enrich the cell population.

Effect of chronic ethanol ingestion on dendritic cell population during oral mucosal repair: An experimental study.

The aim of this study was to analyze the effect of chronic ethanol ingestion on dendritic cell repopulation during the repair process of rat oral mucosa and in the rat spleen by analyzing the immunohistochemical expression of dendritic cell markers. Wistar rats ingested 20% ethanol solution for 28 days; a surgical wound was performed on the rat tongue after this period. The repair process and the number of CD1a+, CD11c+, and CD207+ cells in the regions adjacent to the wound were determined at day 1, 3, and 7 following the wound as well as in the rat spleen. The wound-only group (no ethanol exposure) had complete reepithelization after 7 days, but this did not occur in the ethanol + wound group at this time point. The inflammatory infiltrate was significantly reduced in animals exposed to ethanol, which also showed significantly lower counts of CD1a+, CD11c+, and CD207+ cells than the wound-only group at all experimental time points. In addition, ethanol exposure also resulted in lower densities of CD11c+ and CD207+ cells in the rat spleen. In conclusion, chronic ethanol intake had a negative impact on dendritic cell numbers, a fact that may contribute to delay in oral mucosa repair.

Temporo-spatial distribution of stem cell markers CD146 and p75NTR during odontogenesis in mice.

Mesenchymal and epithelial stem cells were identified in dental tissues; however, knowledge about the odontogenic stem cells is limited, and there are some questions regarding their temporo-spatial dynamics in tooth development. OBJECTIVE: Our study aimed to analyze the expression of the stem cell markers CD146 and p75NTR during the different stages of odontogenesis. METHODOLOGY: The groups consisted of 13.5, 15.5, 17.5 days old embryos, and 14 days postnatal BALB/c mice. The expression of CD146 and p75NTR was evaluated by immunohistochemistry. RESULTS: Our results showed that positive cells for both markers were present in all stages of tooth development, and the number of positive cells increased with the progression of this process. Cells of epithelial and ectomesenchymal origin were positive for CD146, and the expression of p75NTR was mainly detected in the dental papilla and dental follicle. In the postnatal group, dental pulp cells were positive for CD146, and the reduced enamel epithelium and the oral mucosa epithelium showed immunostaining for p75NTR. CONCLUSIONS: These results suggest that the staining pattern of CD146 and p75NTR underwent temporal and spatial changes during odontogenesis and both markers were expressed by epithelial and mesenchymal cell types, which is relevant due to the significance of the epithelial-ectomesenchymal interactions in tooth development.

Therapeutic potential of periodontal ligament stem cells.

Inflammatory periodontal disease known as periodontitis is one of the most common conditions that affect human teeth and often leads to tooth loss. Due to the complexity of the periodontium, which is composed of several tissues, its regeneration and subsequent return to a homeostatic state is challenging with the therapies currently available. Cellular therapy is increasingly becoming an alternative in regenerative medicine/dentistry, especially therapies using mesenchymal stem cells, as they can be isolated from a myriad of tissues. Periodontal ligament stem cells (PDLSCs) are probably the most adequate to be used as a cell source with the aim of regenerating the periodontium. Biological insights have also highlighted PDLSCs as promising immunomodulator agents. In this review, we explore the state of knowledge regarding the properties of PDLSCs, as well as their therapeutic potential, describing current and future clinical applications based on tissue engineering techniques.

Effects of serum-free culture media on human apical papilla cells properties.

OBJECTIVE: Aiming at more effective and safer cell therapies, the objective of this study was to evaluate the biological properties of human apical papilla cells cultured in the absence of serum supplementation in comparison to cells cultured with fetal bovine serum (FBS). DESIGN: Two apical papilla cell populations were isolated from third molars with incomplete rhizogenesis, and cultured in four different media: minimum essential Eagle medium - alpha modification (alpha-MEM); alpha-MEM supplemented with FBS (alpha-MEM + FBS); Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12); and DMEM/F12 supplemented with FBS (DMEM/F12 + FBS). We evaluated their proliferation, clonogenicity, and in vitro osteogenic and chondrogenic differentiation potential. RESULTS: Apical papilla cells cultured in DMEM/F12 + FBS and alpha-MEM + FBS were more proliferative than those grown in serum-free media, and also exhibited greater efficiency in colony cell formation. Despite this, all study groups showed immunostaining for the marker of mitosis anti-PHH3. Also, alpha-MEM + FBS, alpha-MEM, and DMEM/F12 + FBS exhibited higher amount of mineralized deposits in vitro than DMEM/F12, while only cells cultured with FBS were able to form spheres in chondrogenic differentiation assay. CONCLUSIONS: Our results showed that, although the cultivation of apical papilla cells in a serum-free medium has reduced the properties of cell proliferation and differentiation, these cells are still capable of maintaining their desirable characteristics.

Temporo-spatial distribution of stem cell markers CD146 and p75NTR during odontogenesis in mice

Abstract Mesenchymal and epithelial stem cells were identified in dental tissues; however, knowledge about the odontogenic stem cells is limited, and there are some questions regarding their temporo-spatial dynamics in tooth development. Objective Our study aimed to analyze the expression of the stem cell markers CD146 and p75NTR during the different stages of odontogenesis. Methodology The groups consisted of 13.5, 15.5, 17.5 days old embryos, and 14 days postnatal BALB/c mice. The expression of CD146 and p75NTR was evaluated by immunohistochemistry. Results Our results showed that positive cells for both markers were present in all stages of tooth development, and the number of positive cells increased with the progression of this process. Cells of epithelial and ectomesenchymal origin were positive for CD146, and the expression of p75NTR was mainly detected in the dental papilla and dental follicle. In the postnatal group, dental pulp cells were positive for CD146, and the reduced enamel epithelium and the oral mucosa epithelium showed immunostaining for p75NTR. Conclusions These results suggest that the staining pattern of CD146 and p75NTR underwent temporal and spatial changes during odontogenesis and both markers were expressed by epithelial and mesenchymal cell types, which is relevant due to the significance of the epithelial-ectomesenchymal interactions in tooth development.

Demographic and Clinicopathologic Features of Actinic Cheilitis and Lip Squamous Cell Carcinoma: a Brazilian Multicentre Study.

Data on the occurrence and clinicopathological characteristics of actinic cheilitis (AC) and lip squamous cell carcinoma (LSCC) are well studied; however, they are based on studies limited to a single centre. Herein, we described the frequency of AC and LSCC submitted to microscopic examination from representative geographic regions of Brazil. A retrospective multicentre study was performed on biopsies obtained from 1953 to 2018 at 10 Brazilian oral and maxillofacial pathology centres. A total of 198,709 biopsy specimens were surveyed. Sociodemographic data and clinicopathologic characteristics were analysed. A total of 2017 cases of ACs (1.0%) and 850 cases of LSCCs (0.4%) were recorded. A strong fair-skinned (> 87%) male (> 70%) predilection was observed in both conditions. The mean age was 54.8 ± 18.7 for individuals with AC and 57.8 ± 19.0 for individuals with LSCC. The most commonly affected site was the lower lip (> 90%). This is a large multicentre study of AC and LSCC from Brazil. The frequency and clinicopathological features of AC and LSCC were similar to those described worldwide. This study provides robust and representative epidemiological data of these conditions for the scientific community.

Photodynamic Therapy Mediated by 5-aminolevulinic Acid Promotes the Upregulation and Modifies the Intracellular Expression of Surveillance Proteins in Oral Squamous Cell Carcinoma.

Expression of proteins related to cell surveillance has been described in tumors presenting resistance to photodynamic therapy (PDT). The aim of this study was to verify whether there was upregulation of proteins related to resistance in oral squamous cell carcinoma (OSCC) after PDT. OSCC was chemically induced in rats and treated after one cycle of PDT mediated by 5-aminolevulinic acid (5-ALA-PDT). Immunolabeling of p-NFκB, Bcl-2, survivin, iNOS, p-Akt, p-mTOR and cyclin D1 was performed after the treatment. There was increased expression of Bcl-2 (P = 0.008), iNOS (P = 0.020), p-Akt (P = 0.020) and p-mTOR (P = 0.010) by surviving neoplastic cells after PDT when compared to the control. In conclusion, after one cycle of 5-ALA-mediated PDT, Bcl-2, p-Akt, p-mTOR and iNOS were upregulated in neoplastic cells of OSCC, suggesting an activation of antiapoptosis and cell proliferation pathways. This fact must be considered in the establishment of PDT protocols for OSCC treatment, mainly those in which PDT will be combined with chemotherapy drugs targeted at the studied proteins.

Apical Papilla Cells Are Capable of Forming a Pulplike Tissue with Odontoblastlike Cells without the Use of Exogenous Growth Factors.

INTRODUCTION: Dental pulp is a complex tissue with highly differentiated cells, which makes its reconstruction a challenging task. The apical papilla is an undifferentiated tissue considered as the remnant of the dental papilla that forms the dentin-pulp complex. Aiming to analyze morphologic features of the tissue formed in an in vivo pulp model, we used human apical papilla as a cell source without the use of exogenous growth factors. METHODS: A construct was built using newborn mice molar crowns treated with TrypLE (Fisher Scientific, Loughborough, UK) and EDTA. The crowns were filled with PuraMatrix (Corning Inc, Corning, NY) and a pool population of human apical papilla cells. As a control, we used crowns filled only with PuraMatrix and empty crowns. The constructs were transplanted under severe combined immunodeficient mice kidney capsules. Immunohistochemistry for lamin A, dentin sialophosphoprotein, and dentin matrix protein 1 was performed. RESULTS: Morphologic analysis of all transplanted crowns showed the formation of a loose connective tissue of variable cellularity with the presence of well-formed functional vessels. In the study group, lamin A-positive cells represented the majority of cells within the pulp chamber and a few cells in the vessel lining. We also found positivity for dentin sialophosphoprotein and dentin matrix protein 1, an indicator of odontoblast differentiation. CONCLUSIONS: In our study model, human transplanted apical papilla cells mixed with the host cells and formed a vascularized viable tissue, and these cells were able to differentiate into odontoblastlike cells without the use of exogenous growth factors.

Expression of cancer stem cell markers CD44, ALDH1 and p75NTR in actinic cheilitis and lip cancer.

PURPOSE: The aim of this work was to evaluate the expression of the cancer stem cell (CSC) markers CD44, ALDH1 and p75NTR in the ultraviolet-induced lesions actinic cheilitis (AC) and lip squamous cell carcinoma (LSCC), and to correlate it with p53 expression. METHODS: Immunohistochemistry was performed in 4 cases of normal lip (NL), 43 of AC and 20 of LSCC. RESULTS: All cases were positive for CD44, showing a membranous staining without differences between the groups. ALDH1 showed cytoplasmic staining and it was invariable amongst the grades of epithelial dysplasia and between AC and LSCC. p75NTR presented membranous/cytoplasmic staining in the basal and parabasal layer of NL and AC, while LSCC presented cytoplasmic staining in the peripheral layers of the tumor islands. p75NTR showed different expression amongst the dysplasia grades (p < 0.001) but no differences between AC and LSCC. p53 expression was similar amongst the dysplasia grades and between AC and LSCC. CD44, ALDH1 and p75NTR were unrelated amongst themselves and to p53 expression. CONCLUSIONS: CSC markers are expressed in potentially malignant and malignant lesions of the lip. Their expressions were invariable between AC and LSCC and unrelated to p53. p75NTR expression increased with the worsening of epithelial dysplasia grade.

Resistance of oral cancer cells to 5-ALA-mediated photodynamic therapy.

Photodynamic therapy (PDT) has been indicated for oral squamous cell carcinoma (OSCC) at early stages. Chemo and radio-resistance are frequently observed in OSCC, but it is unknown whether this tumor can develop resistance to PDT. It was investigated the process of acquiring resistance to multiple cycles of PDT by using OSCC cells. We also analyzed the expression of anti-apoptotic proteins and those related to Akt/mTOR pathway. Sub-lethal doses of PDT were applied, consisting of a constant concentration of 5-aminolevulinic acid (5-ALA) (1 mM, 4-h incubation) and increasing irradiation dose with LED (from 5.86 to 10.54 J/cm2 ). Cell viability, migration capacity, intracellular expression of protoporphyrin IX (PpIX), mitochondrial density, and pro-survival proteins were investigated in PDT-resistant cells. Six OSCC cell generations resistant to PDT were isolated. The resistant cells exhibited a survival phenotype characterized by a reduction in the expression of endogenous PpIX, increase in mitochondrial density, increase in migration capacity, and up-regulation of p-NFκB, p-survivin, iNOS, p-Akt Ser473 , cyclin D1, p-mTOR Ser2481 , and p-mTOR Ser2448 . OSCC cells are able to survive doses of 5-ALA-PDT by reducing PpIX synthesis and activating signaling pathways related to the inhibition of apoptosis and improvement of cell proliferation. Further studies are necessary to confirm whether this PDT-resistance phenotype can be clinically present, mainly in OSCC showing clinical recurrences after exposure to different PDT protocols.

Photobiomodulation of mesenchymal stem cells encapsulated in an injectable rhBMP4-loaded hydrogel directs hard tissue bioengineering.

Photobiomodulation (PBM) therapy displays relevant properties for tissue healing and regeneration, which may be of interest for the tissue engineering field. Here, we show that PBM is able to improve cell survival and to interact with recombinant human Bone Morphogenetic Protein 4 (rhBMP4) to direct and accelerate odonto/osteogenic differentiation of dental derived mesenchymal stem cells (MSCs). MSCs were encapsulated in an injectable and thermo-responsive cell carrier (Pluronic® F-127) loaded with rhBMP4 and then photoactivated. PBM improved MSCs self-renewal and survival upon encapsulation in the Pluronic® F-127. In the presence of rhBMP4, cell odonto/osteogenic differentiation was premature and markedly improved in the photoactivated MSCs. An in vivo calvarial critical sized defect model demonstrated significant increase in bone formation after PBM treatment. Finally, a balance in the reactive oxygen species levels may be related to the favorable results of PBM and rhBMP4 association. PBM may act in synergism with rhBMP4 and is a promise candidate to direct and accelerate hard tissue bioengineering.

Disposição dos Cirurgiões-Dentistas para identificar condições médicas em consultório odontológico / Dentists' willingness to identify medical conditions in a dental setting

Objetivo: As doenças crônicas não transmissíveis bem como a infecção pelo HIV e as hepatites virais representam hoje problemas de saúde importantes liderando as causas de mortes e causando grande gasto de dinheiro público. O objetivo desta pesquisa foi avaliar as atitudes e vontade de Cirurgiões- Dentistas brasileiros em relação a detecção de condições médicas em consultório odontológico, e comparar os resultados com pesquisa semelhante conduzida com Cirurgiões-Dentistas americanos. Material e métodos: Entrevistamos 323 Cirurgiões-Dentistas de São Paulo. O questionário anônimo avaliou a opinião e disposição dos Cirurgiões-Dentistas em realizar procedimentos médicos no consultório odontológico. A escala de respostas de 5 pontos incluía 1 (muito importante/ muito disposto) até 5 (muito pouco importante/ muito pouco disposto). Resultados: Igualmente aos Cirurgiões-Dentistas americanos, a maioria dos entrevistados acredita ser muito importante detectar doença cardiovascular (80.8%), hipertensão (83.3%), diabetes (83.3%), infecção pelo HIV (86%), e hepatite (86.7%). A maioria está disposta a realizar exame com resultado imediato (54% dos Cirurgiões-Dentistas), mas apenas 47% estão dispostos a discutir o resultado de exames com o paciente imediatamente após realizá-lo. Menos da metade dos entrevistados (47%) consideram seguro saúde como "muito importante" na decisão de incorporar ou não os exames médicos no consultório. Tempo, custo, responsabilidade legal e a vontade do paciente foram considerados "muito importantes" pela maioria dos entrevistados. Conclusão: A maioria dos Cirurgiões-Dentistas brasileiros entrevistados acredita que a triagem para condições médicas em cadeira odontológica seja muito importante e estão dispostos a conduzir um rastreio para condições médicas específicas em consultório odontológico..

Objective: Non-communicable diseases as well as HIV infection and viral hepatitis represents today a major health problem leading causes of death and great expenditures for society. The objective of this survey was to assess Brazilians dentists' attitudes and willingness regarding chairside medical screening in the dental office, and compare the results with a similar survey conducted with American dentists. Material and methods: We interviewed 323 dentists from São Paulo city. The anonymous questionnaire aimed assessing the opinions of dentists in relation to medical procedures in the dental office. The 5-point response scale enclose 1 (very important/very willing) to 5 (very unimportant/very unwilling). Results: In the same way as the American dentists, the majority of the respondents believed it is very important for dentists to conduct screening for cardiovascular disease (80.8%), hypertension (83.3%), diabetes (83.3%), human immunodeficiency virus (86%), and hepatitis (86.7%). Most are willing to conduct screening that generated immediate results (54%), but only 47% are disposed to discuss results immediately with the patient during the dental visit. Less than half of interviewed dentists (47%) considered that having insurance coverage was "very important" in deciding whether or not incorporating medical screening into dental practice. Time, cost, liability and patient´s willing were considered more important among majority of the respondents. Conclusion: Most respondents believed that chair side screening for medical conditions was very important and were disposed to conduct screening for specified medical conditions in a dental setting.