Objetivo: O presente estudo avaliou, clinica e histomorfometricamente, a matriz dérmica acelular (MDA) como barreira física na regeneração óssea guiada. Materiais e Métodos: Inicialmente foram extraídos, em seis cães, os quatro prémolares mandibulares bilateralmente. Após um período de seis semanas, foram analisadas a quantidade e qualidade de mucosa ceratinizada (Exame Clínico 1). Duas semanas depois, os animais foram submetidos a um segundo procedimento cirúrgico (Confecção do defeito e Regeneração Óssea Guiada ROG), no qual foi criado um defeito ósseo padronizado em cada lado da mandíbula. Sobre o defeito de um hemiarco, escolhido aleatoriamente, foi posicionada e fixada a membrana absorvível de copolímeros de ácido polilático e poliglicólico e carbonato trimetileno (Grupo Controle - GC) e no defeito do hemiarco oposto, a matriz dérmica acelular - MDA (Grupo Teste - GT). Imediatamente após a sutura, foram feitas tomadas radiográficas padronizadas através de raio-X digital, as quais foram novamente realizadas 8 e 16 semanas após a cirurgia de colocação das membranas. Após quatorze semanas de cicatrização, a quantidade e qualidade da mucosa ceratinizada foram novamente avaliadas (Exame Clínico 2). Dezesseis semanas depois da ROG os animais foram sacrificados e foi então realizado o preparo histológico das peças. A análise histomorfométrica, em microscopia óptica, avaliou a quantidade e qualidade de osso neoformado. A neoformação óssea também foi quantificada através das imagens radiográficas. Resultados: Em relação aos parâmetros clínicos, o GT e o GC apresentaram aumento na EMQ e redução na AMQ. Na análise radiográfica, após o período de 16 semanas, as radiografias de subtração apresentaram ganho médio de área radiopaca em relação ao defeito inicial de 83,38 % para o GT e de 83,68% para o GC. Houve um aumento progressivo da densidade radiográfica para ambos os grupos, sem diferenças estatisticamente significantes. Histologicamente, após 16 semanas o tecido ósseo apresentou características de um osso predominantemente maduro. Histomorfometricamente, a ANO correspondeu a 58,99% da ANT no GT e a 61 % da ANT no GC. A altura óssea máxima no GT foi de 7,21 ± 0,80 mm e no GC de 7,70 ± 1,21 mm. A porcentagem de osso cortical na ANO foi de 42,47 % no GT e 38,08 % no GC. Não foram observadas diferenças estatisticamente significantes entre os grupos para nenhum destes parâmetros. Conclusão: A MDA atuou como barreira mecânica na ROG com resultados clínicos, radiográficos e histomorfométricos similares aos obtidos com a membrana absorvível. / Objective: The purpose of this study was to evaluate the acellular dermal matrix (ADM) as membrane for guided bone regeneration (GBR), comparing it to a bioabsorbable membrane. Material and Methods: In six dogs, the mandibular premolars were extracted. After 8 weeks, bone defects were surgically created bilaterally and the GBR was performed. Each side was randomly assigned to the control group (CG: bioabsorbable membrane made of glycolide and lactide copolymer) or test group (TG: ADM as a membrane). Immediately following GBR, standardized digital X-Ray radiographs were taken, and were repeated at 8 and 16 weeks post-operatively. Previously to the GBR and to euthanasia, clinical measurements of the width and thickness of the keratinized tissue (WKT and TKT, respectively) were performed. The dogs were sacrificed 16 weeks following GBR, and histomorphometric analysis was made. Area measurements of new tissue and new bone, and linear measurements of bone height were performed. Results: There were no statistically significant differences between groups for any histomorphometric measurement. Clinically, both groups showed an increase in the TKT and reduction program in the WKT. Radiographically, it could be observed an image suggestive of new bone formation in both groups 8 and 16 weeks following GBR. Conclusion: ADM acted as a mechanical barrier in GBR, with clinical, radiographic and histomorphometric results similar to those obtained with the bioabsorbable membrane.

A matriz démica acelular, MDA, figura dentre os biomateriais que têm por objetivo restaurar defeitos mucogengivais. A correção de defeitos mucogengivais a partir de constituintes autógenos são os procedimentos mais comumente usados, no entanto, em decorrência da quantidade insuficiente de tecido doador, esses procedimentos se tornam limitados. Diante disso, o objetivo desse estudo foi avaliar, in vitro, diferentes aspectos relacionados ao cultivo prévio de fibroblastos gengivais humanos em MDA. Fibroblastos gengivais humanos foram cultivados pela técnica do explante a partir de amostras de tecido gengival queratinizado removido de três pacientes saudáveis. A MDA foi cultivada com esses fibroblastos por períodos de 14 e 21 dias para posterior análise dos eventos de: adesão celular, proliferação e viabilidade. Os resultados mostraram que em 7 dias, os fibroblastos estavam aderidos, espraiados e dispersos sobre a superfície externa da MDA, em 14 dias formavam monocamada de células de morfologia alongada e quiescentes (Ki-67 negativos) em sua maioria, sendo apenas ocasionalmente observadas no interior da MDA. Em 21 dias a monocamada exibia menor densidade celular. Os resultados sugerem que o cultivo de fibroblastos em MDA em períodos de 14 dias permite boas condições de adesão e espraiamento das células sobre a matriz, porém, a alta densidade de fibras colágenas parece ser um fator limitante à migração celular. / Acellular Dermal Matrix, ADM, is a biomaterial that has been used in periodontal procedures to treat mucogingival defects. Mucogingival defects can be corrected by autogenous grafts that are the most common procedure used in periodontology, however, because of the limited source of donor\'s tissue this procedure became limited. The aim of this investigation was to verify, in vitro, different aspects related to human gingival fibroblasts seeding on to the ADM. Human gingival fibroblasts were established from explant cultures from the connective tissue of keratinized gingiva collected from three healthy patients. ADM was seeded with gingival fibroblasts for 14 and 21 days, and then cell adherence, proliferation and viability were analyzed. Results revealed that, at day 7, fibroblasts were adherent and spreading on the ADM surface, and were unevenly distributed, forming a discontinuous single cell layer, at day 14, a confluent fibroblastic monolayer lining ADM surface was noticed. At day 21, the cell monolayer exhibited a reduction in cell density. The results suggests that fibroblasts seeding on the ADM for 14 days can allow good conditions for cell adhesion and spread on the matrix, however, because of the high collagen fiber bundle density cell, migration inside the matrix was limited.

En cancérologie cervico-faciale, la couverture d’éléments nobles ou de pertes de substance profondes fait souvent appel à l’utilisation de lambeaux locaux, locorégionaux ou libres. La réalisation de lambeaux autologues nécessite de sacrifier une structure indemne au profit de la structure lésée. Lorsque la réalisation d’un lambeau n’est pas envisageable, l’utilisation de substitut dermique peut être une alternative.L’ingénierie tissulaire permet de cultiver presque tous les types cellulaires (cellules différenciées, cellules souches) seuls ou en association (pe peau totale reconstruite) avec des matrices telles que le collagène pour obtenir des cultures tridimensionnelles. Utilisées in vivo en tant que substrat dermique, les matrices de collagène vont servir de guide aux cellules de l’hôte qui vont la coloniser, synthétiser leur propre matrice extra-cellulaire et développer un réseau vasculaire.Les tissus reconstruits se comportent comme des greffes : leur nutrition se fait d’abord par imbibition à partir du site receveur, puis par recolonisation vasculaire à partir du lit de la greffe. L’épaisseur du tissu greffable est donc limitée.Une revue de la littérature sur la peau reconstruite par ingénierie tissulaire et des différentes stratégies de vascularisation d’un tissu reconstruit est présentée. Le but de notre travail était d’évaluer les capacités des substituts dermiques comme vecteur de cellules souches pour la régénération tissulaire in vivo et comme support au développement d’une neovascularisation à partir d’un vaisseau sanguin ouvrant la voie au lambeau microanastomosable reconstruit in vitro. / In head and neck cancer, coverage of noble elements or deep wounds often requires the use of local, locoregional or free flaps. Autologous flaps consist of transferring the patient’s own tissues, but require sacrifice of the healthy structure to replace the damaged structure. When performing a flap is not an option, the use of dermal substitute may be an alternative.Tissue engineering is a rapidly growing discipline comprising multiple fields of research. Almost all cell types can be cultured alone or in combination (e.g. reconstructed full-thickness skin) with matrices such as collagen to obtain three dimensional cultures. Collagen matrices, used in vivo as dermal substrates, are used to guide the growth of host cells (fibroblasts, endothelial cells, etc.) that colonize the matrix, synthesize their own extracellular matrix and develop a vascular network.Reconstructed tissues behave like tissue grafts: nutrition of these tissues is initially based on diffusion from the recipient site and then by vascular recolonization from the bed of the graft. The thickness of graftable tissue is therefore limited.A review of the literature on skin tissue engineering and current strategies to create vascularized tissue is presented. The aim of our study was to evaluate the capacity of dermal substitutes as a carrier of stem cells for tissue regeneration in vivo and as a support to the development of neovascularization from a blood vessel opening the way to flap suitable for microvascular transplantation reconstructed in vitro.

Os modelos de reconstrução do microambiente são úteis para investigar as propriedades biológicas dos melanócitos humanos com a matriz e como plataforma para testes de novos fármacos. Existe uma demanda crescente para a utilização de pele e derme reconstruídas em laboratório, em ensaios in vitro de citotoxicidade, viabilidade celular, crescimento celular, irritabilidade e avaliação dos constituintes da matriz extracelular. Caracterizamos, em equivalente dérmico, alguns mecanismos de viabilidade e invasão de melanoma metastático humano quando na presença da matriz, a fim de ampliar o conhecimento a respeito de novas terapias contra o melanoma e entender os mecanismos que favorecem seu potencial invasivo, mimetizando com mais fidelidade o que ocorre in vivo. Através da validação deste modelo, constatamos que ele é essencial para resgatar a fisiopatologia do tumor, pois o melanoma, na presença de equivalente dérmico, torna-se capaz de evadir mecanismos de morte e aumentar a secreção de metaproteinases e citocinas que favorecerão a evolução tumoral. Avaliamos também as propriedades antineoplásicas do ácido clorogênico, que já foram relatadas em um grande número de tumores, porém os mecanismos que levam à sua ação antitumoral ainda não foram bem elucidados. Diante dos trabalhos enfatizando o efeito quimioprotetor dos polifenóis, referentes à sua ação antioxidante em células neoplásicas e com potencial metastático, não há na literatura estudos que comprovem a eficácia do ácido clorogênico sobre células de melanoma metastático humano. Portanto, este estudo visou recriar a estrutura dérmica in vitro e, a partir disso, comparar a resposta de fármacos em células de melanoma cultivada em equivalente dérmico, a fim de avaliar se há modulação diferencial nesse substrato, sugerindo, portanto, um protocolo mais eficaz para a prospecção de fármacos antimelanoma. / The microenvironment reconstruction models are useful for investigating the biological properties of human melanocytes onto the matrix and as platform for testing new drugs. There is a growing demand for the use of reconstructed skin and dermis in the laboratory for in vitro assays of cytotoxicity, viability, cell growth, irritability, and evaluation of the extracellular matrix. We characterize in dermal equivalent some mechanisms for cell viability and invasion of human metastatic melanoma in the presence of matrix, the order to increase knowledge about new therapies against melanoma and to understand the mechanisms that favor its invasive potential, to more closely mimic what occurs in vivo. By means of this model, we find that it is essential to rescue the tumor physiopathology as melanoma, in the presence of dermal equivalent, it is able to evade death mechanisms and increase the expression of metalloproteinases and cytokines which favor the tumor evolution. We also evaluated the antineoplastic properties of chlorogenic acid, that have been reported in a large number of tumors, but the mechanisms that lead to its antitumor action has not been well elucidated. Before the work emphasizing the chemoprotective effect of polyphenols related to its antioxidant action in neoplastic cells and with metastatic potential, there is no literature studies confirming the effectiveness of chlorogenic acid on human metastatic melanoma cells. Therefore, this study aims to rebuild the dermal structure in vitro, and from this, to compare the response to drugs in melanoma cells dermal equivalent cultured in order to evaluate whether modulation differential on the substrate, thus suggesting a protocol more effective for prospecting antimelanoma drugs.

Orientador: Rosemary Adriana Chiérici Marcantonio / Banca: Joni Augusto Cirelli / Banca: Elizabeth Pimentel Rosetti / Banca: Washington Rodrigues Camargo / Banca: Giuseppe Alexandre Romito / Resumo: O propósito deste estudo foi avaliar histológica e histometricamente o resultado da utilização das matrizes de colágeno de Pericárdio Bovino (MPB) e Dérmica Acelular (MAD) no tratamento de deiscências ósseas criadas cirurgicamente em cães. Foram criados defeitos periodontais do tipo deiscência óssea na superfície vestibular dos 2os, 3os e 4os pré-molares inferiores de seis cães. Estes dentes foram divididos em 3 grupos: grupo I: MAD; grupo II: controle e grupo III: MPB, ambos com reposição coronária do retalho. Após 70 dias os animais foram sacrificados, os dentes retirados em bloco e processados histologicamente. Realizou-se análise histológica descritiva e histométrica, medindo-se a extensão do tecido epitelial, neoformação óssea e cementária e nível gengival para os grupos I, II e III. Os resultados demonstraram valores estatisticamente iguais para as medianas (em mm) dos valores de epitélio: 1,49, 1,91 e 1,82; cemento: 3,18, 3,09 e 3,31 respectivamente para os grupos I, II e III. Foi encontrada diferença estatisticamente significante em relação à neoformação óssea, com medianas (em mm) de 1,23, 1,82 e 1,62, respectivamente para os grupos I, II e III. Foi observado escasso infiltrado inflamatório. Com isso, pudemos concluir que as matrizes de MPB e MAD são materiais que permitem a reparação dos tecidos periodontais. / Abstract: The goal of this investigation was to compare, histologically and histometrically, the healing process of dehiscence-type defects treated by acellular dermal matrix (AD) and bovine pericardium matrix (PB). Six mongrel dogs were used. Buccal osseous deiscences were surgically created on mesial roots of the mandibular second, third and fourth premolars. The defects were randomly assigned to one of the treatments: AD, control and PB. After 70 days of healing, the dogs were sacrificed and the blocks were processed. The histometric parameters evaluated included: gingival level, epithelial length, new cementum and new bone. No statistically significant differences were found between PB, AD and control groups for epithelial length and new cementum parameters (p<0,05). Statistically significant differences were found between PB, AD and control groups for new bone (minor for AD). The presence of a slight inflammation was observed. Withim the limits of this study, it can be concluded that PB and AD showed good biocompatibility, but did not bring a complete periodontal tissue regeneration. / Doutor

The sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2a) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2a with an ablated C1P interaction site (KI) to examine the cPLA2a/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was dramatically enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (e.g., PGE2) and increased levels of specific HETE species (e.g., 5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2a was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key in vivo biological mechanisms by a defined protein:lipid interaction and provide new insights into cPLA2a function.

acase@tulane.edu

acase@tulane.edu

Background: Respiratory conditions are prevalent and cause an enormous burden on society. In recent decades, there has been a global increase in asthma in children and adults, yet the diagnosis of asthma must be made on clinical grounds as the diagnostic use of pharmacological reversibility of airway obstruction remains controversial. It is possible however that tools exist from different medical paradigms that may assist in the clinical diagnosis of asthma. Tools such as Traditional Chinese Medicine (TCM) enquiry, Breath Holding Time (BHT) or Electro-Dermal Screening Test (EDST) may provide useful clinical information, yet their use has not been widely explored or validated. Integrative medicine may be considered to represent a new frontier in medicine where each therapy and diagnostic method is seen to have its own advantages and limitations and where an integration of both diagnostic and therapeutic techniques from conventional and complementary medicine is seen to produce the best results. However, while there is a high community use of complementary therapies for conditions such as asthma, there is also a need to maintain accepted standards of medical and scientific principles and foster high quality research into complementary therapies. Objectives: The current study sought to determine: • If there is a correlation between conventional measures of lung function such as Forced Expiratory Volume in one second (FEV1) and less conventional measures such as airways expired NO level (eNO), electrodermal measures, TCM enquiry and BHT. • If any of the lung function measures are able to distinguish between asthmatic and healthy subjects. • If the use of a rye grass extract is better than placebo in improving requirements for bronchodilator medication, peak flow, forced expiratory volume in one second or quality of life in mild to moderate childhood asthma. Rational for carrying out two separate studies as one study; The author was interested on both the integration of diagnostic techniques as well as the effect of the herbal extract on asthma so it was decided to use the opportunity and put both in one study. Method: A range of conventional and non-conventional measures were conducted in healthy and asthmatic children including demographic details, quality of life data, spirometry measures, airway nitric oxide levels, electro-dermal measures, TCM history enquiries, breath holding time, and skin prick tests. The data were analysed to determine any significant correlations between these measures. A double blind randomized controlled pilot clinical trial was also performed to assess the effect of using rye grass extract in asthmatic children aged 8 to 16 years. Results: There were significant correlations between forced expiratory volume in one second and active quality of life, TCM spleen score and fraction of exhaled nitric oxide measurements. There were also statistically significant differences between asthmatic and healthy subjects in TCM history, breath holding time and exhaled nitric oxide. The use of rye grass extract did not produce any significant improvement over placebo in any of the asthma outcome measures. Conclusions: The use of inexpensive measures such as BHT and TCM enquiry may provide useful clinical information when assessing respiratory conditions such as asthma. However, further research is required in larger populations to confirm their use. The use of the rye grass extract at the dose given in this study did not provide any clinical benefit for the asthmatic children in this study.

Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors. One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin. The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications. Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV). In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.