Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

Evaluation of the effect of topical and systemic ozone application in periodontitis: an experimental study in rats

Abstract Objective: The goal of the present study was to determine the effect of systemic and topical ozone application on alveolar bone loss (ABL) by evaluating the effect of Hypoxia-inducible factor −1 alpha (HIF-1-α) and receptor activator of NF-kB ligand (RANKL)-positive cells on histopathological and immunohistochemical changes in a rat periodontitis model. Methodology: Thirty male Wistar rats were divided into three groups: 1) Group C (control group); 2) Group SO (systemic ozone group) and 3) Group TO (topical ozone group). Experimental periodontitis was induced with a 3/0 silk suture placed at the mandibular left first molars of rats, and the suture was removed 14 days later. Ozone gas was injected intraperitoneally (0.7 mg/kg) in SO group. Topical ozone application protocol was performed using an ozone generator at 80% concentration (4th grade) 90- degree probe for the duration of 30 s. Both ozone applications were carried out for two weeks at intervals of two days. Histomorphometric and immunohistochemical analysis were performed. Results: ABL was significantly lower in Group SO compared to Group C (p: 0.0052). HIF-1α- positive cells were significantly lower in Group TO than in Group C (p: 0.0043). RANKL-positive cells were significantly lower in Group SO and in Group TO compared to the control group (p: 0.0033, p: 0.0075, respectively). Conclusion: Both ozone applications decreased RANKL-positive cell counts, TO application decreased HIF-1-α positive cells counts, and SO application was found to be more effective in reducing ABL compared to control group.

Avaliação da regeneração óssea guiada por meio histológico, histométrico, imunoistoquímico e perfil inflamatório de membranas reabsorvíveis de colágeno porcino em defeitos críticos em calotas de ratos / Evaluation histological, histometric, immunohistochemistry analysis and inflammatory profile of bone-guided regeneration with membranes of reabsorbable porcine collagen in critical defects in rodent skullcaps

A regeneração óssea guiada (RGO) tornou-se uma prática comum e importante na odontologia, sendo necessário o uso de membranas para sua realização, uma vez que são barreiras que evitam o crescimento de tecido mole nas áreas de defeitos ósseos. Entre as características mais relevantes das membranas absorvíveis estão: o suporte sanguíneo (diretamente relacionado com a porosidade do material) e suporte mecânico ósseo que depende do tempo de reabsorção da membrana. O objetivo desse estudo foi avaliar e comparar duas membranas de colágeno por meio de estudo histológico, histomorfométrico, imunoistoquímico e por contagem de células inflamatórias o processo de regeneração óssea guiada utilizando a membrana de colágeno derivada de pericárdio porcino (Jason®-Instituto Straumann AG, Suíça) em defeitos críticos de 7 mm de diâmetro criados em setenta e duas calvárias de ratos (Rattus Albinus, variedade Wistar). Esses animais foram divididos em 3 grupos: grupo membrana de colágeno porcino (BioGide® - Geistlich Wohlhusen, Suíça), grupo membrana de colágeno de pericárdio porcino (Jason®-Instituto Straumann AG, Suíça) e grupo coágulo, sendo este preenchidos somente com coágulo sem membrana. Esses 3 grupos forma subdivididos em quatro subgrupos de acordo com os tempos avaliados: 7, 15, 30 e 60 dias. Como resultado tivemos na análise histológica e histométrica maior neoformação óssea com o grupo de membrana pericárdio porcino nos períodos de 7 dias (199 pontos) e não foi significante, com 15 dias (494 pontos) estatisticamente significativo, com 30 dias (979 pontos) não significante. Esses valores se modificam 60 dias, mostrando maior superioridade a membrana de colágeno porcino (BioGide®- Geistlich Wohlhusen, Suíça) (1151 pontos) estatisticamente significativo. No analises global a membrana de colágeno porcino (Jason®-Instituto Straumann AG, Suíça) foi superior que a membrana de pericárdio porcino (BioGide®- Geistlich Wohlhusen, Suíça) (p= 0,021) estatisticamente significativo. A análise imunoistoquímica confirmou os achados histométricos, demostrando maior presença da osteocalcina no grupo de pericárdio porcino aos 7 e 15 dias e presença da osteopontina pouco evidente. Já com a membrana de colágeno porcino a osteopontina foi mais imunomarcada aos períodos de 7 e 15 dias, e a presencia de osteocalcina mais evidente aos 30 e 60 dias, corroborando com os resultados iniciais. Na contagem de células inflamatórias para o tempo de 7 dias não houve diferenças estatísticas, já na contagem de vasos sanguíneos houve diferença significativa no período de 15 dias com maior quantidade de vasos para membrana de colágeno porcino, com estes achados concluímos que tanto a membrana de colágeno de pericárdio porcino (Jason® -Instituto Straumann AG, Suíça) quanto a membrana de colágeno porcino (BioGide®-Geistlich Wohlhusen, Suíça) podem ser consideradas como material de escolha apropriada para regeneração óssea guiada, com maior proporção de osso neoformado com a membrana de colágeno porcino(AU)

Guided bone regeneration (RGO) has become a common and important practice in dentistry, requiring the use of membranes to perform it, since they are barriers that prevent the growth of soft tissue in areas of bone defects. Among the most relevant characteristics of absorbable membranes are: blood support (directly related to the porosity of the material) and mechanical bone support that depends on the time of membrane resorption. The goal of this study was to evaluate and compare two collagen membranes by means of histological, histomorphometric, immunohistochemical study and by inflammatory cell counting the guided bone regeneration process using the collagen membrane derived from porcine pericardium (Jason®-Instituto Straumann AG, Switzerland) in critical defects of 7 mm in diameter created in seventy-two calvaria of rats (Rattus Albinus, Wistar variety). These animals were divided into 3 groups: porcine collagen membrane group (BioGide® - Geistlich Wohlhusen, Switzerland), porcine pericardium collagen group (Jason®-Instituto Straumann AG, Switzerland) and clot group, which were filled with only a clot without membrane. These 3 groups were subdivided into four subgroups according to the evaluated times: 7, 15, 30 and 60 days. As a result, we had a greater bone neoformation in the histological and histometric analysis with the porcine pericardial membrane group in the periods of 7 days (199 points) and it was not statistically significant with 15 days (494 points), with 30 days (979 points) not significant. These values change 60 days, showing greater superiority to the porcine collagen membrane (BioGide®- Geistlich Wohlhusen, Switzerland) (1151 points) statistically significant. In the global analysis, the porcine collagen membrane (Jason®Instituto Straumann AG, Switzerland) was superior than the porcine pericardium membrane (BioGide®- Geistlich Wohlhusen, Switzerland) (p = 0.021) statistically significant. The immunohistochemical analysis confirmed the histometric findings, showing a greater presence of osteocalcin in the porcine pericardium group at 7 and 15 days and the presence of osteopontin little evident. As for the porcine collagen membrane, osteopontin was more immunostained at 7 and 15 days, and the presence of osteocalcin was more evident at 30 and 60 days, corroborating the initial results. In the inflammatory cell count for the 7-day period, there were no statistical differences, whereas in the blood vessel count, there was a significant difference in the 15-day period with a greater number of vessels for porcine collagen membrane, with these findings we conclude that both the porcine pericardial collagen (Jason® - Straumann AG Institute, Switzerland) and porcine collagen membrane (BioGide®-Geistlich Wohlhusen, Switzerland) can be considered as the material of choice for guided bone regeneration, with a higher proportion of neoformed bone according to porcine collagen membrane(AU)

Difference of brain-derived neurotrophic factor expression and pyramid cell count during mastication of food with varying hardness

Abstract Previous studies suggested that mastication activity can affect learning and memory function. However, most were focused on mastication impaired models by providing long-term soft diet. The effects of chewing food with various hardness, especially during the growth period, remain unknown. Objective: To analyze the difference of hippocampus function and morphology, as characterized by pyramidal cell count and BDNF expression in different mastication activities. Materials and Methods: 28-day old, post-weaned, male-Wistar rats were randomly divided into three groups (n=7); the first (K0) was fed a standard diet using pellets as the control, the second (K1) was fed soft food and the third (K2) was fed hard food. After eight weeks, the rats were decapitated, their brains were removed and placed on histological plates made to count the pyramid cells and quantify BDNF expression in the hippocampus. Data collected were compared using one-way ANOVA. Results: Results confirmed the pyramid cell count (K0=169.14±27.25; K1=130.14±29.32; K2=128.14±39.02) and BDNF expression (K0=85.27±19.78; K1=49.57±20.90; K2=36.86±28.97) of the K0 group to be significantly higher than that of K1 and K2 groups (p<0.05); no significant difference in the pyramidal cell count and BNDF expression was found between K1 and K2 groups (p>0.05). Conclusion: A standard diet leads to the optimum effect on hippocampus morphology. Food consistency must be appropriately suited to each development stage, in this case, hippocampus development in post-weaned period.

A comparison of the sealing abilities between Biodentine and MTA as root-end filling materials and their effects on bone healing in dogs after periradicular surgery

ABSTRACT Objectives: To compare the sealing ability and biocompatibility of Biodentine with mineral trioxide aggregate (MTA) when used as root-end filling materials. Methodology: The Cell Counting Kit-8 (CCK-8) assay was used to compare the cytotoxicity of MTA and Biodentine. Twenty-one extracted teeth with a single canal were immersed in an acidic silver nitrate solution after root-end filling. Then, the volume and depth of silver nitrate that infiltrated the apical portion of the teeth were analyzed using micro-computed tomography (micro-CT). Seventy-two roots from 3 female beagle dogs were randomly distributed into 3 groups and apical surgery was performed. After six months, the volume of the bone defect surrounding these roots was analyzed using micro-CT. Results: Based on the results of the CCK-8 assay, MTA and Biodentine did not show statistically significant differences in cytotoxicity (P>0.05). The volume and the depth of the infiltrated nitrate solution were greater in the MTA group than in the Biodentine group (P<0.05). The volume of the bone defect was larger in the MTA group than in the Biodentine group. However, the difference was not significant (P>0.05). The volumes of the bone defects in the MTA and Biodentine groups were smaller than the group without any filling materials (P<0.05). Conclusions: MTA and Biodentine exhibited comparable cellular biocompatibility. Biodentine showed a superior sealing ability to MTA in root-end filling. Both Biodentine and MTA promoted periradicular bone healing in beagle dog periradicular surgery models.

Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Effects of platelet-rich plasma on human gingival fibroblast proliferation and migration in vitro

Abstract Objective This study evaluated the influence of platelet-rich plasma (PRP) on the behaviour of human gingival fibroblasts (hGFs), including fibroblast proliferation, migration and colony formation. Methods PRP was obtained from the human peripheral blood of a healthy volunteer and then was diluted into platelet concentrations of 1%, 2% and 5%. The proliferation of hGFs was determined by two methods: (1) Cell-number counting with a haemocytometer method at days 1, 3, 5 and 7; (2) Colony-forming unit-fibroblast (CFU-F) assay at 2 weeks. The migration of hGFs was evaluated with scratch assay, then recorded digital images were analysed by Image-Analysis J 1.51j8 software to compare the remaining artificial wound areas between PRP groups at 0, 24 and 48 hours. Results All hGFs that were cultivated in media with 1%, 2% and 5% PRP showed their ability to proliferate and migrate. Cell numbers incubated with 1% PRP increased significantly during the first three days and peaked at day 5, tending to be similar to their proliferation in complete medium. With concentrations of 2% and 5% PRP, hGFs outgrew and peaked at day 3, which was faster than with those in medium with 1% PRP. Especially, hGFs in the group 5% PRP proliferated with higher cell numbers than those in the other remaining groups at day 3. The hGF colony number that was formed in the group 5% PRP was significantly higher than those in the groups 1% and 2% PRP. Scratch assay showed hGFs in the groups 2% and 5% PRP almost filled the artificial wound and migrated more effectively than in the group 1% PRP at 24 hours, which was significant. Conclusion In this study, perhaps the medium with 5% PRP is the dominant option, promoting the abilities of hGFs to heal wounds, because of its fast and effective impact on cell proliferation, colony formation and migration.

Efeito de campos magnéticos estáticos e compensados na proliferação celular in vitro / Proteomics of the effect of compensated and static magnetic fields on cell proliferation in vitro

Inserido no paradigma da transdisciplinaridade, o presente trabalho foi desenvolvido em etapas, com os seguintes objetivos: a) Construir um dispositivo com base de metal não magnético para ímãs permanentes, visando à geração de um Campo Magnético Estático (CME) ou de um Campo Magnético Compensado (CMC); b) Expor culturas de células mesenquimais a um CME e a um CMC, ou a nenhum campo (controle); c) Analisar a influência destes campos na viabilidade e proliferação celular e nos casos em que houve alteração em pelo menos um destes parâmetros, utilizar a análise proteômica como ferramenta para a compreensão dos mecanismos envolvidos. O dispositivo foi construído utilizando aço inoxidável, capaz de gerar dois tipos de Campos Magnéticos: Compensado (CMC) com intensidade de aproximadamente 0 mT e Estático (CME) com intensidade média de 165 mT. Estes campos foram aplicados a culturas de células mesenquimais de medula óssea de camundongos AJ (MSC/AJ), nos períodos de 0, 24, 48, 72 e 96 h (CMC) e 24 h (CME). Os efeitos sobre a proliferação e a viabilidade foram avaliados por método de contagem manual de células com marcação por azul de tripan. A análise proteômica foi realizada para os experimentos com CMC, com o objetivo de descrever as proteínas envolvidas nas alterações encontradas. A exposição ao CMC tendeu a reduzir a proliferação das células de medula óssea MSC/AJ em relação ao controle em 96 h, porém sem diferença significativa, o que poderia estar relacionado a proteínas que inibem a transcrição, como a Forkhead box protein P2 Foxp2. Este mesmo campo aumentou a viabilidade celular em relação ao baseline para todos os tempos experimentais, o que poderia estar relacionado a proteínas relacionadas à ligação ao Ca+2. Esses mecanismos, entretanto, precisam ser estudados mais profundamente para que possam ser comprovados ou não. Já a exposição ao CME levou a uma tendência à diminuição da proliferação e viabilidade celular em relação ao grupo controle, embora sem diferenças significativas, provavelmente por conta do tamanho amostral e tempo de avaliação (24 h).(AU)

Inserted in the transdisciplinarity paradigm, the present work was developed by steps with the following aims: a) To build a device of non-magnetic metal to hold permanent magnets for the generation of a Static Magnetic Field (SMF) or a Compensated Magnetic Field (CMF); b) To expose mesenchimal cells to the SMF and to CMF or to none of the fields (control); c) To analyze the influence of these fields on cell viability and cell proliferation and in the case where it occurred alteration in at least one of these parameters, to use proteomics as a tool for the comprehension of the involved mechanisms. The device was built in stainless steel, able to generate two kinds of Magnetic Fields: Compesated (CMF) with an intensity of nearly zero mT and Static (SMF) with a mean intensity of 165 mT. These fields were applied to bone marrow mesenchimal cell cultures from AJ mice (MSC/AJ), for 0, 24, 48, 72 and 96 h (CMF) and 24 h (SMF) periods. The effects on the proliferation and viability were assessed by tripan blue dying and manual counting of the cells. Proteomics was done for the experiments with CMF, aiming to describe the involved proteins on found alterations. The exposition to CMF tends to reduce the bone marrow cell proliferation of MSC/AJ in relation to control in 96 h, but with no significant difference, which may be related to proteins that inhibit the transcription, like Forkhead box protein P2 Foxp2. This very field raised the cell viability in relation to the baseline for all the experimental times that could be related to proteins connected to Ca2+ binding. However, these mechanisms need more experiments, so they can be confirmed or not. The exposition to the SMF tends to decrease both cell proliferation and viability in relation to the control group, although with no significant difference, probably because of the sample number and the exposition time (24h).(AU)

Efeitos do PDGF-BB na taxa de proliferação e na adesão de células derivadas da granulação óssea a fragmentos radiculares / Effects of PDGF-BB on the rate of proliferation and on the adhesion of human cells derived from bone granulation tissue to root fragments

O objetivo deste estudo foi investigar o papel do fator de crescimento derivado de plaquetas-BB (PDGF-BB) na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea humana a fragmentos radiculares periodontalmente comprometidos. Na primeira etapa do estudo, foi estabelecida cultura primária de células da granulação óssea de dois pacientes adultos, sistemicamente saudáveis, não fumantes. Após a expansão celular, as células foram caracterizadas para determinação do fenótipo por meio de ensaios de viabilidade celular, MTT, ensaio de atividade de fosfatase alcalina, ensaio de mineralização e caracterização imunohistoquímica por meio de citometria de fluxo (segunda etapa). Na terceira etapa do estudo, os efeitos da adição de PDGF-BB recombinante humano na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea a superfícies radiculares periodontalmente comprometidas foram investigados. A taxa de proliferação celular estimulada pelo PDGF-BB (grupo teste) ou pelo meio de cultura (grupo controle) foi investigada por meio de contagem de células viáveis nos frascos de cultura após 1, 3, 5 e 7 dias do cultivo celular. Foram obtidos 30 fragmentos dentários a partir de dentes extraídos por razões periodontais. Os fragmentos foram raspados com curetas Gracey e condicionados com solução em gel de EDTA a 24% durante 3 minutos, lavados com solução de soro fisiológico, secos e posicionados em placas de 24 poços. Foram incubadas sobre os fragmentos tratados 1x104 células GO por 24 horas, seguido por fixação e preparo para análise por microscopia eletrônica de varredura (MEV). O número de células aderidas sobre os fragmentos foi analisado nas fotomicrografias. O padrão de crescimento das células GO foi compatível com células ósseas, com modificação do padrão do crescimento com o aumento do número de passagens. Houve atividade de fosfatase alcalina em meio osteogênico e convencional, com pico máximo aos 7 dias e atividade de mineralização estimulada ou não por meio osteogênico, com pico máximo aos 21 dias. A análise por meio de citometria de fluxo demonstrou que as células GO não expressaram CD105 e CD166 na 14a passagem, indicando sua diferenciação celular avançada nesse período. A adição de rhPDGF-BB resultou em mudança na taxa de proliferação celular, observando-se pico máximo de crescimento aos 7 dias, com diferenças estatisticamente significantes (p < 0.005; ANOVA post hoc Tukey) em relação aos períodos de 1, 3 e 5 dias. O ensaio de MTT demonstrou maior viabilidade celular no período de 48 hs, comparativamente aos períodos de 24 e 72 horas, quando a densidade óptica celular diminuiu de forma significativa (p< 0.05; Friedmann pósteste Dunn). No ensaio de adesão celular, pode-se observar que a adição de rhPDGFBB aumentou significativamente o número de células aderidas aos fragmentos dentários (p< 0.05; teste t não pareado com correção Welch), com alteração da morfologia celular. Esses resultados sugerem que as células GO tem características compatíveis com linhagem de células osteoblásticas, de fenótipo mais diferenciado após a 12a passagem. A adição de rhPDGF-BB (300ng/ml) resulta em aumento da taxa de proliferação das células GO e do número de células aderidas a fragmentos radiculares, indicando que, nesta concentração, o fator de crescimento é citocompatível, favorecendo a proliferação e adesão celular.(AU)

The goal of this study was to investigate the effects of recombinant human platelet derived growth factor (rhPDGF-BB) at the concentration of 300ng/ml in the proliferation and adhesion of human bone granulation cells to periodontally diseased root fragments. At the first stage of the study, the granulation tissue existent in healing sockets (21 days after its creation) was collected from two systemically healthy nonsmoking adults to the establishment of primary culture. The in vitro properties of bone granulation (BG) cell lineage were characterized by cell viability, MTT, alkaline phosphatase activity and mineralization assays. The effects of culture medium (control) and rhPGDF-BB 300ng/ml (test) in the proliferation and adhesion of BG cells were investigated. The rate of BG cells proliferation was investigated by the number of viable cells present at 1, 3, 5 and 7 days after platting. Thirty root fragments were obtained from teeth extracted for periodontal reasons. Root fragments were scaled and root planed, conditioned with EDTA 24% for 3 minutes, rinsed in saline solution, air-dryed and positioned in 24-well plates. Each fragment was seeded with 104 BG cells, fixated after 24 hours and prepared for analysis in SEM. The number of cells adhered to the fragments was analysed in photomicrographies. BG cells growth pattern was compatible with osteogenic cell lineage, showing modification with the increasing number of cell passage. GO cells expressed alkaline phosphatase activity in conventional and osteogenic culture medium, with maximum peak at 7 days, as well as mineralization activity stimulated or not by osteogenic or non-osteogenic culture medium, with maximum peak at 21 days. The analysis by flow cytometer showed that BG cells have not expressed CD105 and CD106 at the 14th passage, indicating its advanced cell differentiation. The addition of rhPDGF-BB resulted in modification of proliferation rate, with maximum peak observed at 7 days, significantly different from 1-, 3- and 5-day periods (p< 0.005; ANOVA post hoc Tukey). MTT assay showed greater cell viability after 48 hours than after 24 and 72 hours, when optical density has significantly diminished (p< 0.05; Friedmann post hoc Dunn). At cell adhesion assay, it could be observed that the adhesion of rhPDGF-BB has significantly increased the number of cells adhered to root fragments (p< 0.05; unpaired t test with Welchs correction), and alterations in cell morphology. These results suggest that BG cells present in vitro characteristics compatible with osteoblastic cell lineages, with a more differentiated phenotype after the 12th passage. The addition of rhPDGF-BB (300 ng/ml) results in increase of the rate of BG cell proliferation and in the number of cells adhered to root fragments, indicating that, at this concentration, the growth factor is compatible with BG cells and favors cells proliferation and adhesion.(AU)

Inflammatory response of human dental pulp to at-home and in-office tooth bleaching

ABSTRACT Tooth bleaching is a technique of choice to obtain a harmonious smile, but bleaching agents may damage the dental pulp. Objective: This study evaluated the inflammatory responses of human dental pulp after the use of two bleaching techniques. Material and Methods: Pulp samples were collected from human third molars extracted for orthodontic reasons and divided into three groups: control - no tooth bleaching (CG) (n=7); at-home bleaching with 15% carbamide peroxide (AH) (n = 10), and in-office bleaching with 38% hydrogen peroxide (IO) (n=12). Pulps were removed and stained with hematoxylin-eosin for microscopic analysis of inflammation intensity, collagen degradation, and pulp tissue organization. Immunohistochemistry was used to detect mast cells (tryptase+), blood vessels (CD31+), and macrophages (CD68+). Chi-square, Kruskal-Wallis, and Mann Whitney tests were used for statistical analysis. The level of significance was set at p<.05. Results: The inflammation intensity and the number of macrophages were significantly greater in IO than in AH and CG (p<0.05). The results of CD31+ (blood vessels per mm2) were similar in CG (61.39±20.03), AH (52.29±27.62), and IO (57.43±8.69) groups (p>0.05). No mast cells were found in the pulp samples analyzed. Conclusion: In-office bleaching with 38% hydrogen peroxide resulted in more intense inflammation, higher macrophages migration, and greater pulp damage then at-home bleaching with 15% carbamide peroxide, however, these bleaching techniques did not induce migration of mast cells and increased the number of blood vessels.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity

ABSTRACT Objective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals. Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification. Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001). Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

The effects of supplemental melatonin administration on the healing of bone defects in streptozotocin-induced diabetic rats

ABSTRACT Diabetes mellitus (DM) causes an increased production of free radicals that can impair bone healing. Melatonin is a hormone secreted mainly by the pineal gland, which participates in the neutralization process of free radicals. Objective The aim of this study was to investigate histologic and biochemical effects of supplemental melatonin administration on bone healing and antioxidant defense mechanism in diabetic rats. Material and Methods Eighty-six Sprague-Dawley male rats were used in this study. Diabetes mellitus was induced by intraperitoneal (i.p.) administration of 65 mg/kg streptozotocin (STZ). Surgical bone defects were prepared in the tibia of each animal. Diabetic animals and those in control groups were treated either with daily melatonin (250 μg/animal/day/i.p.) diluted in ethanol, only ethanol, or sterile saline solution. Rats were humanely killed at the 10th and 30th postoperative days. Plasma levels of Advanced Oxidation Protein Products (AOPP), Malondialdehyde (MDA), and Superoxide Dismutase (SOD) were measured. The number of osteoblasts, blood vessels and the area of new mineralized tissue formation were calculated in histologic sections. Results At the 10th day, DM+MEL (rats receiving both STZ and melatonin) group had significantly higher number of osteoblasts and blood vessels as well as larger new mineralized tissue surfaces (p<0.05 for each) when compared with DM group. At the 30th day, DM group treated with melatonin had significantly lower levels of AOPP and MDA than those of DM group (p<0.05). Conclusion Melatonin administration in STZ induced diabetic rats reduced oxidative stress related biomarkers and showed beneficial effects on bone healing at short term.

Evaluation of an experimental rat model for comparative studies of bleaching agents

ABSTRACT Dental materials, in general, are tested in different animal models prior to their clinical use in humans, except for bleaching agents. Objectives To evaluate an experimental rat model for comparative studies of bleaching agents by investigating the influence of different concentrations and application times of H2O2 gel in the pulp tissue during in-office bleaching of rats’ vital teeth. Material and methods The right and left maxillary molars of 50 Wistar rats were bleached with 20% and 35% H2O2 gels, respectively, for 5, 10, 15, 30, or 45 min (n=10 rats/group). Ten animals (control) were untreated. The rats were killed after 2 or 30 days, and the maxillae were examined by light microscopy. Inflammation was evaluated by histomorphometric analysis with inflammatory cell counting in the coronal and radicular thirds of the pulp. The counting of fibroblasts was also performed. Scores were attributed to the odontoblastic layer and to vascular changes. The tertiary dentin area and the pulp chamber central area were histomorphometrically measured. Data were compared by the analysis of variance and the Kruskal-Wallis test (p<0.05). Results After 2 days, the amount of inflammatory cells increased in the occlusal third of the coronal pulp until the time of 15 min for both concentrations of bleaching gels. In 30 and 45 min groups of each concentration, the number of inflammatory cells decreased along with the appearance of necrotic areas. After 30 days, a reduction in the pulp chamber central area and an enlargement of tertiary dentin area were observed without the detection of inflammation areas. Conclusion The rat model of extra coronal bleaching showed to be adequate for bleaching protocols studies, as it was possible to observe alterations in the pulp tissues and in the tooth structure caused by different concentrations and periods of application of bleaching agents.

Influência de diferentes densidades de energia do laser de baixa intensidade em fibroblastos derivados da polpa de dentes decíduos / Influence of low-level laser with different energy densities on pulp fibroblasts from human primary teeth

O objetivo deste trabalho foi comparar os efeitos de diferentes densidades de energia e irradiâncias do Laser de Baixa Intensidade (LBI), variando em função do tempo de irradiação e potência, na viabilidade e proliferação de fibroblastos derivados da polpa de dentes decíduos humanos (HPF). HPF foram cultivados em DMEM e usados entre a 4ª e 8ª passagem. Os grupos foram divididos de acordo com diferentes densidades de energia, variando: Tempo de irradiação - Grupo I Ia (1,2 J/cm2 - 5 mW - 10 s), Ib (2,5 J/cm2 - 5 mW - 20 s), Ic (3,7 J/cm2 - 5 mW - 30 s), Id (5,0 J/cm2 - 5 mW - 40 s), e Ie (6,2 J/cm2 - 5 mW - 50 s); ou potência - Grupo II IIa (1,2 J/cm2 - 5 mW - 10 s), IIb (2,5 J/cm2 - 10 mW - 10 s), IIc (3,7 J/cm2 - 15 mW - 10 s), IId (5,0 J/cm2 - 20 mW - 10 s), e IIe (6,2 J/cm2 - 25 mW - 10 s). Células não irradiadas - cultivadas em condições nutricionais regulares - 10% Soro Fetal Bovino (SFB) (If e IIf) e células não irradiadas - cultivadas em déficit nutricional - 1% SFB (Ig e IIg), foram consideradas controles positivos e negativos, respectivamente. A viabilidade e proliferação celular foram avaliadas, repesctivamente, pelas técnicas MTT e Cristal violeta (CV), nos períodos de 24, 48 e 72 horas após a irradiação. Os dados obtidos foram submetidos à análise estatística por ANOVA 2 critérios, seguido pelo teste de Tukey (P<0,05). No ensaio MTT, os controles negativos, Ig e IIg, apresentaram significativamente menor viabilidade em relação aos correspondentes grupos experimentais: IIa e IIb, 24 horas após a irradiação; Ia, Ib, Ie, If e IIf no período de 48 horas; e Ib-If, assim como, IIa-IIf após 72 horas. Nos diferentes períodos de avaliação do ensaio CV, todos os grupos, exceto Ie, IIe e If, exibiram significativamente maior proliferação em comparação aos respectivos controles negativos. Dentro de um mesmo grupo nos diferentes períodos, os grupos If e IIe apresentaram menor viabilidade durante o período de 24 horas em comparação ao período de 72 horas pelo ensaio MTT. Na avaliação intragrupos, o ensaio CV revelou menor proliferação no período de 24 horas em comparação aos períodos de 48 e 72 horas, independente do grupo avaliado. Os diferentes protocolos de irradiação, grupos I e II, não apresentaram diferença estatisticamente significativa na viabilidade e proliferação celular entre densidades de energia iguais com irradiâncias diferentes durante os períodos avaliados. De acordo com os resultados obtidos, as diferentes densidades de energia e irradiâncias propostas não prejudicaram a viabilidade e proliferação de fibroblastos pulpares de dentes decíduos humanos. A variação do protocolo de irradiação LBI, em função do tempo ou da potência, não interferiram nas respostas celulares após a aplicação da mesma densidade de energia com irradiâncias diferentes.(AU)

The aim of this study was to compare the effects of Low-level laser (LLL) with different energy densities and irradiances, varying according to the irradiation time and power, on cell viability and proliferation of pulp fibloblasts from human primary teeth (HPF). HPF were culture in DMEM and used between 4th and 8th passages. Groups were divided according to different energy densities, varying: Time of irradiation Ia (1.2 J/cm2 - 5 mW - 10 s), Ib (2.5 J/cm2 - 5 mW - 20 s), Ic (3.7 J/cm2 - 5 mW - 30 s), Id (5.0 J/cm2 - 5 mW - 40 s), and Ie (6.2 J/cm2 - 5 mW - 50 s); or output power - Grupo II IIa (1.2 J/cm2 - 5 mW - 10 s), IIb (2.5 J/cm2 - 10 mW - 10 s), IIc (3.7 J/cm2 - 15 mW - 10 s), IId (5.0 J/cm2 - 20 mW - 10 s), e IIe (6.2 J/cm2 - 25 mW - 10 s). Non-irradiated cells - grown in regular nutritional conditions - 10% Fetal Bovine Serum (FSB) (If and IIf) and non-irradiated cells - grown in nutritional deficit - 1% FBS (Ig and IIg) were considered positive and negative controls, respectively. Cell viability and proliferation were respectively assessed through MTT and Crystal violet (CV) assays at 24, 48 and 72h after irradiation. Data were submitted to statistical analysis by ANOVA 2 criteria, followed by Tukey test (P<0.05). In the MTT assay, the negative controls, Ig and IIg, showed significantly lower viability in relation to the corresponding groups: IIa and IIb 24 hours after irradiation; Ia, Ib, Ie, If and IIf at 48 hours period; and Ib-If, as IIa-IIf, after 72 hours. At different periods of evaluation of CV assay, all groups, except Ie, IIe and If, exhibited significantly higher proliferation compared to the respective negative controls. Within the same group at different periods, groups If and IIe showed lower viability during 24 hours compared to 72 hours period by MTT assay. In the intragroup evaluation, CV assay revealed lower proliferation at 24 hours compared to 48 and 72 hours periods, regardless of the evaluated group. Different irradiation protocols, groups I and II, showed no statistically significant differences on cell viability and proliferation among equals energy densities with different irradiances at the evaluated periods. According to these findings, different LLL energy densities and irradiances proposed did not impair viability and proliferation of pulp fibloblasts from human primary teeth. The variation of the LLL irradiation protocol, by the time or power, did not interfere in cellular responses after the application of the same energy density with different irradiances.(AU)

Cytocompatibility and biocompatibility of nanostructured carbonated hydroxyapatite spheres for bone repair

ABSTRACT Objective The aim of this study was to investigate the in vitro and in vivo biological responses to nanostructured carbonated hydroxyapatite/calcium alginate (CHA) microspheres used for alveolar bone repair, compared to sintered hydroxyapatite (HA). Material and Methods The maxillary central incisors of 45 Wistar rats were extracted, and the dental sockets were filled with HA, CHA, and blood clot (control group) (n=5/period/group). After 7, 21 and 42 days, the samples of bone with the biomaterials were obtained for histological and histomorphometric analysis, and the plasma levels of RANKL and OPG were determined via immunoassay. Statistical analysis was performed by Two-Way ANOVA with post-hoc Tukey test at 95% level of significance. Results The CHA and HA microspheres were cytocompatible with both human and murine cells on an in vitro assay. Histological analysis showed the time-dependent increase of newly formed bone in control group characterized by an intense osteoblast activity. In HA and CHA groups, the presence of a slight granulation reaction around the spheres was observed after seven days, which was reduced by the 42nd day. A considerable amount of newly formed bone was observed surrounding the CHA spheres and the biomaterials particles at 42-day time point compared with HA. Histomorphometric analysis showed a significant increase of newly formed bone in CHA group compared with HA after 21 and 42 days from surgery, moreover, CHA showed almost 2-fold greater biosorption than HA at 42 days (two-way ANOVA, p<0.05) indicating greater biosorption. An increase in the RANKL/OPG ratio was observed in the CHA group on the 7th day. Conclusion CHA spheres were osteoconductive and presented earlier biosorption, inducing early increases in the levels of proteins involved in resorption.

Phenotype and cell proliferation activity of duct-like structures in human sublingual glands: a histological and immunohistochemical study

There are several age-related microscopic changes in the salivary glands, including the increase in the number of duct-like structures (DLS). However, the true origin and the phenotype of the DLS are not known. Objective To evaluate the phenotype and the cell proliferation index of the DLS of human sublingual glands. Material and Methods Sixty sublingual glands obtained from human cadavers were divided into two groups - 0-30 and 61-90 years old. The phenotype was estimated by immunostaining for cytokeratin 19 (CK 19) and the S-100 protein as well as by the presence of mucin and glycogen. The cell proliferation index was determined by the Ki-67 antibody. The histochemical techniques used periodic acid-Schiff (PAS) and Alcian Blue. In each captured microscopic field, the DLS were counted to establish a percentage for the staining profile. The statistical analysis was accomplished using Student's t-test, the Mann-Whitney test and Pearson's correlation coefficient (p<0.05). Results Comparing both groups, only CK 19 showed a statistically significant difference (p=0.033), with the strongest expression in the elderly group. There was no significant difference between PAS and Alcian Blue (p=0.270). In both groups, the immunostaining for CK 19 was stronger than that for S-100 (p=0.004;p<0.001), but there was no correlation between the two immunomarkers (ρ=-0.163; p=0.315). There was no immunostaining for Ki-67. Conclusions DLS demonstrate a ductal phenotypic profile and do not present cell proliferation activity. DLS may represent a regressive process arising from acini or represent the result of metaplasia. .

Effect of sumac extract on serum oxidative status, RANKL/OPG system and alveolar bone loss in experimental periodontitis in rats

Objectives Sumac (Rhus coriaria L.) is widely used spice which has several properties such as antioxidant, anti-inflammatory and antimicrobial. The purpose of this animal study was to evaluate the effects of sumac extract on levels of receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG) expression, serum oxidative status, and alveolar bone loss in experimental periodontitis. Material and Methods Twenty-four Wistar rats were separated into three groups: non-ligated (NL, n=8), ligature only (LO, n=8), and ligature and treated with sumac extract (S, n=8) (20 mg/kg per day for 11 days). A 4/0 silk suture was placed around the mandibular right first molars subgingivally; after 11 days, the rats were sacrificed, and alveolar bone loss was histometrically measured. The detection of RANKL and OPG were immunohistochemically performed. Levels of serum total antioxidant status (TAS)/total oxidant status (TOS), and oxidative stress index (OSI) were also analyzed. Results Alveolar bone loss was significantly greater in the LO group compared to the S and NL groups (p<0.05). The number of inflammatory cell infiltrate (ICI) and osteoclasts in the LO group was significantly higher than that of the NL and S groups (p<0.05). The number of osteoblasts in the LO and S groups was significantly higher than that of the NL group (p<0.05). There were significantly more RANKL-positive cells in the LO group than in the S and NL groups (p<0.05). OPG-positive cells were higher in S group than in LO and NL groups (p<0.05). TOS and OSI levels were significantly reduced in S group compared to LO group (P<0.05) and TAS levels were similar in S and NL group (p>0.05). Conclusions The present study showed that systemic administration of sumac extract may reduce alveolar bone loss by affecting RANKL/OPG balance, TOS and OSI levels in periodontal disease in rats. .

Avaliação do efeito do flúor na osteoclastogênese in vitro / Fluoride affects in vitro osteoclastogenesis

O tecido ósseo possui alto grau de rigidez e resistência à pressão, característica relacionada às suas funções de proteção, sustentação e locomoção. Patologias como periodontite, artrite reumatóide e osteoporose decorrem do desequilíbrio da dinâmica óssea, que ocorre quando os osteoclastos estão em maior atividade em relação aos osteoblastos. O fluoreto (F-) é incorporado nos tecidos mineralizados após ingestão e a sua administração aumenta a formação óssea in vivo, de maneira dependente da dose e da linhagem dos animais. As linhagens A/J e 129P3/J possibilitam a análise dos fenômenos moleculares envolvidos na suscetibilidade e resistência de células ósseas aos efeitos do F- no osso. Neste estudo, avaliou-se a administração in vivo de F- na osteoclastogênese, através da análise da atividade da enzima fosfatase ácida resistente ao tartarato (TRAP), contagem de células TRAP- positivas e quantificação da atividade das metaloproteinases MMP-2 e -9. Para tal, células hematopoiéticas provenientes da medula óssea de camundongos das linhagens 129P3/J e A/J foram cultivadas com M-CSF e RANKL em associação ou não com F-, nas concentrações de 10- 9, 10-7, 10-5, 5x10-5, 5x10-5, 10-4 e 10-3 M. Os dados mostraram que a atividade da TRAP foi semelhante entre as células das duas linhagens de animais, independente do F-. Concomitante à diferenciação osteoclástica, o tratamento das células com F-, independente da concentração, aumentou significativamente a atividade da TRAP em células da linhagem A/J. O aumento da atividade de TRAP foi associada com o aumento do número de osteoclastos formados, na concentração de 10-3 M F-. Já em células dos animais 129P3/J, o F- não alterou a atividade da TRAP bem como a osteoclastogênese. A atividade de MMP-9 foi aumentada em relação à da MMP-2, porém ambas não foram moduladas pelo F-, independente da linhagem. Assim, concluiu-se que as células de ambas as linhagens não diferem com relação à diferenciação de osteoclastos,

The bone tissue has a high degree of rigidity and pressure resistance, characteristic related to its protection, support and locomotion functions. Diseases such as periodontitis, osteoporosis and rheumatoid arthritis arise from dynamic bone imbalance that occurs when osteoclasts present increased activity compared to osteoblasts. Fluoride (F-) is incorporated in mineralized tissues after ingestion and its administration increases in vivo bone formation in dose and strain-dependent manner. The strains A/J and 129P3/J make possible the analysis of molecular phenomena involved in susceptibility and resistance of bone cells to the effect of F- in bone. In this study, we evaluated the effect of F- on the in vitro osteoclastogenesis, by analyzing the fosfatase tartrate-resistant acid (TRAP) activity, TRAP positive cell counts and the activity of MMP-2 and -9 measurements in A/J and 129P3/J differentiated hematopoietic stem cells. Hematopoietic cells were differentiated with M-CSF and RANKL and treated or not with F- in the concentrations of 10-9, 10-7, 10-5, 5x10-5, 5x10-5, 10-4 and 10-3 M.The data show that TRAP activity, an osteoclast marker, was similar between cells from both strains of mice, regardless of F-. In addition, the treatment of A/J cells with F- significantly increased TRAP activity, in a dose-independent manner. This was accompained by the increased TRAP+-osteoclast counts, at 10-3 M F- dose. However, while the MMP-9 activity was aumented when compared to MMP-2, it was not altered by F-, regardless of the strain. Thus, we concluded that cells from both strains do not differ regarding the osteoclast differentiation potential, but they respond differently to F-, whereas A/J and 129P3/J cells are sensitive and resistant to F--induced osteoclastogenesis, respectively.

Estudo microscópico morfométrico e genotípico de pacientes portadores de lesão central de células gigantes / Microscopic morphometric and genotypic assessment of patients with central giant cell lesions

A lesão central de células gigantes (LCCG) é uma afecção benigna dos maxilares, de comportamento biológico incerto, variando de discreta tumefação assintomática e de crescimento lento à uma forma agressiva, associada a dor, reabsorção radicular e óssea, com destruição cortical. Sua etiologia permanece desconhecida, havendo controvérsias entre processo reacional, neoplásico ou genético. Mutações no gene SH3BP2 foram identificadas em pacientes com querubismo, condição que compartilha várias características clínicas, radiográficas e histopatológicas com a LCCG. Para testar a hipótese de que tais mutações seriam responsáveis por, ou estariam associadas a LCCG e na tentativa de melhor entender a diferenciação microscópica/morfométrica das lesões agressivas e não agressivas, vinte e cinco pacientes portadores de LCCG foram selecionados para o estudo. O DNA foi obtido através do sangue e de espécimes em blocos de parafina, oriundos de biópsias e tratamento cirúrgico. Um estudo microscópico morfométrico foi paralelamente realizado, para avaliar o número de células gigantes e densidade de volume das mesmas nas lesões agressivas e não agressivas. O sequenciamento genético dos treze exons do gene SH3BP2 nos vinte e cinco pacientes estudados evidenciou uma alteração no códon do exon 4 em 10 pacientes. A densidade de volume de células gigantes foi maior nas lesões agressivas quando comparadas às não agressivas (p=0,013). Não houve diferença significante quanto ao número de células gigantes/mm2 em lesões agressivas e não agressivas (p =0,245).

Central giant cell lesion (CGCL) is a benign disease of the jaws, with uncertain behavior, ranging from mild asymptomatic slow-growing swelling to an aggressive form, with pain, radicular and bone resorption and cortical destruction. Its aetiology is still unknown and there is discussion whether it is a reactive, neoplastic or genetic disease. Mutations on gene SH3BP3 were identified in patients with cherubism, which shares several clinical, radiographic and histopathological features with CGCL. In order to test the hypothesis that such mutations would be responsible for or would be related to CGCL and also in order to better understand microscopic morphometric differentiation of the aggressive and non-aggressive lesions, 25 patients with CGCL were selected to this study. DNA was extracted from blood samples and from tissue samples, obtained by biopsy or surgical treatment. Microscopic morphometric assessment was also performed, in order to evaluate the number and the volume density of the giant cells in aggressive and in non-aggressive lesions. Gene sequencing of all 13 exons in gene SH3BP3, performed on each of the 25 patients, showed an alteration in one codon from exon 4, in ten patients. Volume density of giant cells was greater in aggressive lesions than in non-aggressive ones (p=0,013). There was no significant difference on the number of giant cells per mm2 when comparing aggressive and non-aggressive lesions.

A comparative study of p53 expression in hyperplastic, dysplastic epithelium and oral squamous cell carcinoma

Aim: To study oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma to determine (1) the prevalence of p53 protein immunoreactivity, (2) number of p53 positive cells, and (3) the area of localization of p53 protein immunoreactivity. Methods: Two contiguous sections from 30 tissue specimens (10 each from oral hyperplastic epithelium, dysplastic epithelium and squamous cell carcinoma) were subjected to hematoxylin and eosin (H/E) staining forhistopathological diagnosis and immunohistochemical (IHC) staining for demonstration of p53. p53 positivity was looked for in each IHC stained slide and the number of positive cells amongst 1,000 epithelial cells were recorded. The localization of these p53 positive cells within the strata (i.e.basal/suprabasal, spinous and superficial layers) of epithelium between 3 groups, and also with ineach group according to histological grades was recorded. Results: Higher p53 positive cell counts were demonstrated in oral squamous cell carcinoma compared to hyperplastic and dysplastic tissues. The expression of p53 in epithelial hyperkeratosis was mainly localized to basal epithelialcells whereas in epithelial dysplasia, it was predominantly localized to spinous epithelial cells. Conclusions: Qualitatively p53 is not a specific marker for malignancy of oral epithelium. However the quantitative analysis of p53 positive cells and their localization in oral epithelium is of importance as a marker for oral squamous cell carcinoma.