Use of collagen and auricular cartilage in bioengineering: scaffolds for tissue regeneration.

The aim of this study was the development of collagen and collagen/auricular cartilage scaffolds for application in dermal regeneration. Collagen was obtained from bovine tendon by a 72 h-long treatment, while bovine auricular cartilage was treated for 24 h and divided into two parts, external (perichondrium, E) and internal (elastic cartilage, I). The scaffolds were prepared by mixing collagen (C) with the internal part (CI) or the external part (CE) in a 3:1 ratio. Differential scanning calorimetry, scanning electron microscopy (SEM) analysis, microcomputed tomography imaging (micro-CT) and swelling degree were used to characterize the scaffolds. Cytotoxicity, cell adhesion, and cell proliferation assays were performed using the cell line NIH/3T3. All samples presented a similar denaturation temperature (Td) around 48 °C, while CE presented a second Td at 51.2 °C. SEM micrographs showed superficial pores in all scaffolds and micro-CT exhibited interconnected pore spaces with porosity above 60% (sizes between 47 and 149 µm). The order of swelling was CE < CI < C and the scaffolds did not present cytotoxicity, showing attachment rates above 75%-all samples showed a similar pattern of proliferation until 168 h, whereas CI tended to decrease after this time. The scaffolds were easily obtained, biocompatible and had adequate morphology for cell growth. All samples showed high adhesion, whereas collagen-only and collagen/external part scaffolds presented a better cell proliferation rate and would be indicated for possible use in dermal regeneration.

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study.

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

Development of collagen/nanohydroxyapatite scaffolds containing plant extract intended for bone regeneration.

In this study scaffolds of nanohydroxyapatite (nHA) and anionic collagen (C) combined with plant extracts intended for bone tissue repair were developed. Grape seed (P), pomegranate peel (R) and jabuticaba peel (J) extracts were used as collagen crosslinker agents in order to improve the materials properties. All crude extracts were effective against Staphylococcus aureus, but only for CR scaffold inhibition zone was noticed. The extracts acted as crosslinking agents, increasing enzymatic resistance and thermal stability of collagen. The extracts showed cytotoxicity at the concentrations tested, while nHA increased cell viability. The scaffolds presented porosity and pore size appropriate for bone growth. CR, CnHAP, CnHAR and CnHAJ increased the cell viability after 24 h. The combination of collagen, nHA and plant extracts offers a promising strategy to design novel biomaterials for bone tissue regeneration.

Candida albicans-Cell Interactions Activate Innate Immune Defense in Human Palate Epithelial Primary Cells via Nitric Oxide (NO) and ß-Defensin 2 (hBD-2).

The presence of Candida albicans in the biofilm underlying the dental prosthesis is related to denture stomatitis (DS), an inflammatory reaction of the oral mucosa. The oral epithelium, a component of the innate immune response, has the ability to react to fungal invasion. In this study, we evaluated the in vitro effect of viable C. albicans on the apoptosis, nitric oxide (NO) production, and ß-defensin 2 (hBD-2) expression and production of human palate epithelial cells (HPECs). We further determined whether or not these effects were correlated with fungal invasion of epithelial cells. Interaction between HPEC primary culture and C. albicans was obtained through either direct or indirect cell-cell contact with a supernatant from a hyphal fungus. We found that the hyphae supernatants were sufficient to induce slight HPEC apoptosis, which occurred prior to the activation of the specific mechanisms of epithelial defense. The epithelial defense responses were found to occur via NO and antimicrobial peptide hBD-2 production only during direct contact between C. albicans and HPECs and coincided with the fungus's intraepithelial invasion. However, although the hBD-2 levels remained constant in the HPEC supernatants over time, the NO release and hBD-2 gene expression were reduced at a later time (10 h), indicating that the epithelial defense capacity against the fungal invasion was not maintained in later phases. This aspect of the immune response was associated with increased epithelial invasion and apoptosis maintenance.

c-Jun, pc-Jun, and p27 are differently expressed in oral leukoplakias in smokers and never-smokers.

OBJECTIVE: Oral cancer may be preceded by potentially malignant lesions, and smoking is a risk factor. Oral leukoplakia (OL), which is the most common among these lesions, is defined by the World Health Organization as "a white plaque of questionable risk having excluded known diseases or disorders that carry no increased risk for cancer." Thus, OL is a clinical diagnosis used to designate oral white lesions, which are histologically represented by hyperkeratosis associated or not associated with epithelial dysplasia. It is known that c-Jun and pc-Jun have a role in cell proliferation and that p27 is decreased during carcinogenesis; thus, the aim of this study was to investigate whether these proteins are differently expressed in OL in smokers and never-smokers. STUDY DESIGN: Seventy-three cases diagnosed as OL were selected and divided into four groups according to the presence or absence of dysplasia and patients' smoking status (smokers: 39 cases, 24 dysplastic; never-smokers: 34 cases, 20 dysplastic). The immunoexpressions of c-Jun, pc-Jun, and p27 were evaluated. RESULTS: A significant correlation between smoking condition and the percentages of c-Jun (P = .0356) and pc-Jun (P = .0216) was found and was more intense in cases that underwent malignant transformation (6/47). CONCLUSIONS: Smoking habits may be linked to the expression of proteins directly associated with cell cycle progression.

Evaluation of microRNA expression in head and neck squamous cell carcinoma cell lines and in primary culture of oral keratinocytes.

OBJECTIVE: Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. METHODS: The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. RESULTS: Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. CONCLUSIONS: Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.

Avaliação da expressão de microRNAs em linhagens celulares de carcinoma epidermoide de cabeça e pescoço e em cultura primária de queratinócitos orais / Evaluation of microRNA expression in head and neck squamous cell carcinoma cell lines and in primary culture of oral keratinocytes

Objective: Functional in vitro studies are fundamental to understand the role of microRNAs, small non coding RNA molecules that function as post-transcriptional regulators, in cancer. The objective of this study was to determine the applicability of head and neck squamous cell carcinoma cell lines and human oral keratinocytes as models for functional studies on microRNAs previously identified as deregulated in head and neck squamous cell carcinomas. Methods: The expression level of four microRNAs was assessed in cell lines and in primary cultures of oral keratinocytes using specific real-time polymerase chain reactions. The identity of oral squamous cell carcinoma cell lines was confirmed by means of STR (short tandem repeats) profiling. The possible impact of feeder-layer gene expression in global microRNA expression results from keratinocyte primary culture was also evaluated. Results: Significant differences in microRNA gene expression were observed among squamous cell carcinoma cell lines, particularly among cells lines from distinct subsites, as well as between primary culture of human keratinocytes and immortalized keratinocyte cell lines. Conclusions: Primary cultures of human keratinocytes and diverse tumor cell lines are relatively easy to obtain. However, each cell model possesses a characteristic phenotype; whereas one may be useful for a specific study, it may be inappropriate for another. Therefore, it is imperative that suitable cell lines are cautiously selected for functional studies in cancer.

Objetivo: Estudos funcionais in vitro são essenciais para a compreensão do papel de microRNAs, pequenas moléculas de RNA que desempenham papel importante na regulação gênica, no câncer. Neste estudo, analisamos a viabilidade de linhagens celulares derivadas de carcinoma epidermoide de cabeça e pescoço, queratinócitos orais provenientes de culturas primárias e queratinócitos imortalizados, como modelos para estudos funcionais de microRNAs previamente identificados como desregulados nesse tipo de carcinoma. Métodos: Avaliamos a expressão de quatro microRNAs em linhagens celulares e em cultura primária de queratinócitos orais por meio de reações em cadeia da polimerase em tempo real específica. As linhagens celulares de carcinoma epidermoide de boca foram previamente caracterizadas quanto ao seu perfil de sequências de DNA do tipo STR (do inglês short tandem repeats ou repetições curtas em sequência) com o objetivo de confirmar a identidade da linhagem. Avaliamos ainda a possível influência da expressão gênica detectada na camada de sustentação usada no cultivo de queratinócitos no resultado global obtido. Resultados: Nossos resultados apontam diferenças significativas na expressão dos microRNAs entre linhagens celulares passíveis de serem utilizadas como modelos para estudos funcionais em carcinoma epidermoide de cabeça e pescoço. Ressaltam-se diferenças entre linhagens de carcinoma de língua e de faringe, bem como diferenças expressivas entre a linhagem de queratinócitos orais imortalizados e queratinócitos orais normais provenientes de culturas primárias. Conclusão: Culturas primárias de queratinócitos orais bem como linhagens tumorais são obtidas de forma relativamente simples. Entretanto, cada modelo celular possui características particulares que os tornam mais ou menos adequados para um determinado estudo. Conclui-se que a seleção cuidadosa das linhagens é fundamental para estudos funcionais sobre câncer.

Câncer e doença periodontal / Cancer and periodontal disease

Atualmente, tem se discutido a influência e a repercussão da doença periodontal sobre o organismo humano. O termo “medicina periodontal” foi introduzido em 1996, focando na validação desta relação. As evidências já foram constatadas de que a saúde periodontal deficiente está associada à deficiência da saúde sistêmica. Seu envolvimento está cada vez mais comprovado nas doenças cardiovasculares, diabetes, complicações na gestação e parto, bebês de baixo peso, acidentes vasculares cerebrais, artrite reumatoide e vários tipos de câncer. Segundo a literatura existente nessa área, o fator causal está na inflamação crônica provocada pela doença periodontal e a resposta do hospedeiro frente à inflamação. Com base nessas informações, o objetivo deste trabalho foi buscar na literatura atual informações que sustentam e comprovam tais hipóteses. Como conclusão, temos a confirmação do envolvimento da saúde periodontal na saúde sistêmica e a comprovação de que esta associação está relacionada não somente ao papel das bactérias, mas principalmente à inflamação crônica provocada pelas mesmas. Concluiu-se também que, tanto a Medicina como a Odontologia, devem estar cada vez mais integradas, a fim de proporcionar aos pacientes melhores condições de saúde, destacando o papel do cirurgião-dentista na prevenção da doença periodontal, assegurando que a mesma não contribua com a doença sistêmica.

Ação do Viscum album em cultivo celular de carcinoma epidermoide de cabeça e pescoço / Viscum album effect in head and neck squamous cell carcinoma cell culture

O carcinoma epidermoide de cabeça e pescoço, hoje considerada mundialmente uma das neoplasias mais frequentes desta região, tornou-se um problema de saúde pública, necessitando urgente de medidas a serem tomadas, a fim de melhorar a qualidade de vida dos pacientes acometidos, consequentemente aumentando a sobrevida, avaliada em cinco anos. O carcinoma epidermoide de cabeça e pescoço é uma doença complexa, e inclui vários fatores etiológicos, além de alterações moleculares, capazes de desencadear e dar continuidade a alguns eventos. No geral os carcinomas orais são tratados primeiramente com cirurgia excisional ou radioterapia individualmente, ou em combinação para os estágios mais avançados. As preparações dos extratos fermentados de Viscum album (VA), uma planta semiparasita da família das Lorantáceas, vêm sendo utilizada, principalmente em países da Europa, com resultados promissores no âmbito das terapias coadjuvantes, especialmente a medicina antroposófica. Em conjunto com as terapias convencionais, tem demonstrado uma melhora na qualidade de vida dos pacientes portadores de neoplasias malígnas. Estudos in vitro, realizados em células cancerígenas, têm demonstrado que vários tipos de VA podem apresentar citotoxicidade em células de carcinoma, sendo capazes de ativar a cascata apoptótica ou levando as células à necrose. Este estudo teve por objetivo verificar a ação de três tipos de extratos de VA (Iscador Qu Spezial, Iscador P e Iscador M), em linhagens celulares de carcinoma epidermoide de língua (SCC9 e SCC25). Para isso foi verificada a ação citotóxica do fármaco Iscador Qu Spezial, por meio do teste de viabilidade celular, para a obtenção da IC50...

Head and neck squamous cell carcinoma, one of the most common malignancies worldwide in this area, became a public health problem that requires urgent attitudes to be taken in order to improve the quality of life of the affected patients, increasing survival that today valued at five years. Head and neck squamous cell carcinoma is a complex disease which includes several etiologic factors and different molecular changes that may trigger and get on with some events. In general, oral carcinomas are treated primarily with surgical excision or radiotherapy alone or in combination for cases in more advanced stages. Preparations of fermented extracts from fermented Viscum album (VA), a plant from the Loranthaceae family have been used, mainly in European countries, with promising results as adjuvant therapies, especially in the Anthroposophy Medicine. In combination with conventional therapies, VA has lead to improvement in quality of life of patients with cancer. in vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells being able to activate the apoptotic cascade or leading cells to necrosis. The present study aimed to verify the effect of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue (SCC9 and SCC25). The cytotoxic action of Iscador Qu Spezial was verified by cell viability test obtaining the IC50. The three drugs were tested as follow: Tunnel to evaluate apoptosis, Annexin V and FITC/propidium iodide were evaluated by flow cytometry to quantify the apoptosis rate induced by the drugs...