Potencial de reparo cutâneo de células-tronco mesenquimais dermais associadas à Nanomatrizes de celulose bacteriana

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2016.Lesões de espessura total na pele, como queimaduras extensas e úlceras crônicas, resultam em numerosos problemas fisiológicos, funcionais e psicológicos para os pacientes. Apesar de existirem diversos tratamentos para essas lesões, nenhum deles resulta em uma pele totalmente íntegra e funcional. Por isso, buscou-se com este trabalho um melhor método de reparo cutâneo, associando células-tronco mesenquimais dermais (dCTMs) com matrizes de celulose bacteriana (MCBs). Trabalhos têm mostrado que CTMs melhoram o reparo de tecidos lesados, por meio de sua diferenciação nos fenótipos afetados ou da liberação de fatores parácrinos. As MCBs, por sua vez, são polímeros naturais produzidos por bactérias. Essas membranas são atrativas para o reparo tecidual, por apresentarem características como biocompatibilidade, alta capacidade de retenção de água, e boa permeabilidade. Neste trabalho as MCBs produzidas foram caracterizadas fisicamente em seu estado úmido e liofilizado. As MCBs hidratadas apresentaram uma alta capacidade de retenção de água, fibras nanométricas e porosidade superficial de cerca de 30%. Quando submetidas ao processo de liofilização as MCBs não mantiveram sua conformação tridimensional, apresentando porosidade quase nula, fibras achatadas e baixa taxa de reabsorção de água, não sendo capazes de retornar ao seu estado original. Desse modo, a associação de dCTMs foi analisada apenas com as MCBs hidratadas. As dCTMs foram obtidas a partir de fragmentos de pele, de pacientes saudáveis, que se submeteram a cirurgias de lifting facial, e foram cultivadas sobre as MCBs em condições de cultivo padrão. Os experimentos demonstraram que as dCTMs permaneceram viáveis por 21 dias sobre as MCBs, análises com microscopia de varredura e confocal indicaram que as dCTMs se aderem progressivamente sobre as MCBs, são capazes de se proliferar, não migram para o interior das membranas e mantém sua morfologia típica. Nessas condições as dCTMs também mantiveram suas características imunofenotípicas de CTMs como visto por citometria de fluxo. Esses resultados mostram que as MCBs são biocompatíveis às dCTMs. Considerando estas análises, o potencial terapêutico dessa associação foi avaliado in vivo em lesões cutâneas de camundongos. Foram avaliados os seguintes parâmetros: taxa de fechamento das lesões, infiltrado inflamatório, angiogênese, espessura e homogeneidade da epiderme e comprimento das cicatrizes. Os resultados revelaram que, quando comparado com o grupo controle, o grupo tratado com dCTM+MCB apresentou um aumento significativo na angiogênese e no infiltrado de neutrófilos. Além disso, o tratamento influenciou a espessura do tecido de granulação, levou a formação de uma epiderme mais fina e homogênea, e aparentemente diminuiu as cicatrizes. Em conjunto, os resultados sugerem que a associação esteja acelerando a maturação das lesões cutâneas, e favorecendo o processo de reparo cutâneo.Abstract : Full-thickness skin injuries, such as extensive burns and chronic ulcers result in numerous physiological functional and psychological problems for the patients. Even though there are various current treatments for these lesions, none of them lead to a fully reconstituted and functional skin. For this reason, this work search for a better method to improve skin repair, associating dermal mesenchymal stem cells (dMSCs) with bacterial cellulose membranes (BCMs). Studies have shown that MSCs improve tissue repair through differentiation in affected phenotypes or by releasing paracrine factors. The BCMs, in turn, are natural polymers produced by bacteria. These membranes are attractive for tissue repair by presenting biocompatibility, high capacity of water retention, and good permeability. In the present work, the produced BCMs were physically characterized in their wet and freeze-dried states. The wet BCMs showed a high capacity for water retention, nanometric fibers and a surface porosity of about 30%. When subjected to the freeze drying process, BCMs did not keep their three-dimensional conformation, presented almost no porosity, flattened fibers and low water absorption rate, not being able to return to its original state. Thus, dMSCs association was analyzed only with wet BCMs. The dMSCs were obtained from fragments of skin of healthy patients who had undergone facelift surgery. The cells were then cultured on BCMs in standard culture conditions. The experiments showed that dMSCs remained viable for 21 days on BCMs. Scanning electron and confocal microscope analyses revealed that dMSCs progressively adhered on BCMs, were able to proliferate, do not migrate to the interior of the matrices and maintained their typical morphology. In this condition, cells maintained the MSCs immunophenotypic characteristics as assessed by flow cytometry. These results show that the MCBs are biocompatible with the dMSCs. Therefore, the therapeutic potential of the dMSCs associated with BCMs was evaluated in vivo, in mice cutaneous lesions. The following parameters were evaluated: closing rate of lesions, inflammatory infiltration, neovascularization, thickness and homogeneity of the epidermis and length of scars. The results revealed that, when compared to the control group, the treated group (MSC+BCM) showed a significant increase in neovascularization and neutrophil infiltration. Besides, the treatment also influenced the thickness of granulation tissue, and allowed the formation of a thin and uniform epidermis. The lenght of scras int he treated gruoup were relatively smaller. Together, the results suggest that the MSC+BCM treatment is accelerating the maturation of skin lesions, and promoting the skin repair process

ARYLSULFATASE-I: A NOVEL SULFATASE REGULATING PROTEOGLYCAN SULFATION DURING ENDOCHONDRAL OSSIFICATION

Endochondral ossification consists of bone formation from a previous cartilaginous template. This process starts during embryonic development when mesenchymal cells condense in presumptive skeletal regions and differentiate into chondrocytes, which undergo a progressive maturation process. Chondrocytes are arranged in morphologically distinct zones within the cartilaginous template, reflecting their maturation states. While mature chondrocytes are in the medial region, immature chondrocytes are in the distal areas of the template. The chondrocytes pronounced cellular changes during maturation are accompanied by prominent extracellular matrix (ECM) remodelling and reorganization. Sulfated proteoglycans (PGs) are one of the main components of cartilage ECM, and their sulfation levels are critical for proper endochondral ossification. PG sulfation is regulated by sulfotransferases and sulfatases - that add and remove, respectively, sulfate esters from PGs. Deficiencies in sulfatases lead to diseases, including mucopolysaccharidosis and multiple sulfatase deficiency. Disease phenotypes include various skeletal defects seemingly related to chondrocyte maturation, including impaired chondrocyte proliferation and hypertrophy, severe growth retardation, and delayed primary and secondary ossification center formation. The mechanisms of how sulfation leads to those phenotypes and how sulfur levels are modulated across endochondral ossification are still to be unveiled. Here the hypothesis that a novel sulfatase - Arylsulfatase I (ARSI) - decreases PG sulfation during cartilage maturation thus promoting endochondral ossification, was tested. Synchrotron X-ray fluorescence (XRF) imaging confirmed that sulfate esters decreased significantly in mature cartilage during endochondral ossification. ARSB and GALNS- the two known chondroitin sulfate PG sulfatases- were not specifically expressed in mature cartilage. Utilizing laser-capture microdissection and RNAseq data previously obtained in the lab, we were able to identify a novel sulfatase - Arsi - to be upregulated in mature cartilage. Increased ARSI protein expression in mature cartilage was further demonstrated in vivo in chick and mouse sections and during the maturation of ATDC5 chondrocytes in vitro. Biochemical assays overexpressing ARSI in HeLa cells confirmed that ARSI is a novel PG sulfatase. Taken together, biochemical and expression results strongly indicate that ARSI controls the decrease of sulfate esters in mature cartilage. To test ARSI function during endochondral ossification, four putative transgenic zebrafish (Tg) lines for this gene were generated by Gateway-Tol2 transgenesis. All generated Gateway constructs had the correct sequence and were integrated into the zebrafish genome, but possibly due to posttranscriptional mechanisms, protein overexpression was not confirmed in putative Tg fish. Our hypothesis was partially addressed, and more studies are needed to investigate the potential ARSI roles in endochondral ossification. In this thesis, we showed for the first time ARSI mRNA expression in chick, ARSI protein expression in chick and mouse tissues, and we presented the first biochemical characterization of ARSI as a PG sulfatase. Our results supplement the scarce literature about ARSI and potentiate future work uncovering its role in skeletal development and disease

Queimaduras: estudo epidemiológico e avaliação da associação de células tronco mesenquimais às matrizes de celulose bacteriana para o reparo cutâneo

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Biológicas. Biologia.A pele é um tecido com alta complexidade e múltiplas funções, grandes traumas neste tecido, como queimaduras, podem ocasionar desequilíbrios osmóticos, cicatrizações hipertróficas, infecções e até mesmo a morte. As queimaduras são lesões cutâneas de alta complexidade que no Brasil afetam mais de 1 milhão de pessoas por ano. Elas possuem uma epidemiologia própria e podem na maioria das vezes ser evitadas. Problemas com a regeneração e cicatrização da pele, após a ocorrência de queimaduras, são frequentes, e podem provocar deformações e perda de função. Assim, novas estratégias para a regeneração da pele, visando à obtenção de uma pele íntegra, morfológica e funcionalmente, são necessárias. Uma das abordagens de estudos para a regeneração cutânea é a associação de células tronco (CTs) à biomateriais, para posterior enxertia. As CT Mesenquimais (CTMs) tem sido descritas como uma fonte atrativa para a engenharia de tecidos, elas podem agir em lesões se diferenciando nos tipos celulares afetados, ou através de efeitos parácrinos, além de serem naturalmente recrutadas pelo organismo para reparar tecidos danificados. Tendo em vista estes aspectos, o presente trabalho foi dividido em duas partes. Primeiramente, com o objetivo de analisar o perfil epidemiológico e obter um maior conhecimento sobre a problemática das queimaduras, foi realizado um estudo retrospectivo no Hospital Infantil Joana de Gusmão (HIJG), onde foram analisados 174 prontuários de crianças queimadas entre 2007-2009, quanto às seguintes variáveis: idade, sexo, agente causador, profundidade, extensão corporal, localização corpórea, ambiente de ocorrência das queimaduras e necessidade de enxerto. A partir deste estudo foi concluído que o perfil epidemiológico das crianças queimadas no HIJG neste período é de um menino, em idade pré-escolar, que se queima em casa, com líquidos aquecidos. 62.6% dos pacientes apresentaram queimaduras de 2º grau; 70% apresentaram até 20% da superfície corporal queimada (SCQ); a maioria dos pacientes apresentou epitelização da SCQ espontaneamente. Crianças menores de 6 anos de idade se queimaram mais com líquidos aquecidos, crianças a cima dessa idade com inflamáveis, especialmente álcool líquido. Esse agente foi o maior responsável por queimaduras compreendendo mais de 20% da SCQ, e por queimaduras de 3º grau, sendo que dentre todas as queimaduras com álcool, 72% necessitaram de enxerto. A segunda parte deste trabalho teve como objetivo avaliar o potencial terapêutico da associação de CTMs derivadas da derme (dCTMs) em matrizes de celulose bacteriana (MCBs), visando uma futura aplicação no reparo de lesões cutâneas. Para isso, foram avaliadas a viabilidade, aderência e morfologia das dCTMs sobre MCBs, além da padronização de um método de aplicação das MCBs em feridas feitas em camundongos. As dCTMs foram isoladas de pele humana, proveniente de lifting facial, e as MCBs foram produzidas por bactérias Gluconacetobacter hansenii cultivadas em meio de cultivo apropriado e estático, após o plaqueamento foram realizadas análises in vitro em dias variados. O teste de viabilidade demonstrou que as dCTMs continuam viáveis após 7 dias de cultivo sobre as MCBs, estas células aderiram nas MCBs progressivamente, porém não migraram para o interior das mesmas, além disso, mantiveram morfologia fibroblastóide típica e características imunofenotípicas de CTMs após 4 dias de cultivo sobre as MCBs. Ao serem implantadas em camundongos, as MCBs foram recobertas com Tegaderm® para não perderem sua umidade característica e suturadas para aumentar seu tempo sobre as feridas. Não foram encontrados outros estudos utilizando dCTMs associadas à MCBs. De acordo com os resultados, concluímos que MCBs parecem ser um bom veículo para inoculação das dCTMs sobre lesões cutâneas, ao mesmo tempo em que são um curativo efetivo para as mesmas

Electrochemotherapy Effectiveness Loss due to Electric Field Indentation between Needle Electrodes: A Numerical Study

Electrochemotherapy is an anticancer treatment based on applying electric field pulses that reduce cell membrane selectivity, allowing chemotherapy drugs to enter the cells. In parallel to electrochemotherapy clinical tests, in silico experiments have helped scientists and clinicians to understand the electric field distribution through anatomically complex regions of the body. In particular, these in silico experiments allow clinicians to predict problems that may arise in treatment effectiveness. The current work presents a metastatic case of a mast cell tumor in a dog. In this specific treatment planning study, we show that using needle electrodes has a possible pitfall. The macroscopic consequence of the electroporation was assessed through a mathematical model of tissue electrical conductivity. Considering the electrical and geometrical characteristics of the case under study, we modeled an ellipsoidal tumor. Initial simulations were based on the European Standard Operating Procedures for electrochemotherapy suggestions, and then different electrodes’ arrangements were evaluated. To avoid blind spots, multiple applications are usually required for large tumors, demanding electrode repositioning. An effective treatment electroporates all the tumor cells. Partially and slightly overlapping the areas increases the session’s duration but also likely increases the treatment’s effectiveness. It is worth noting that for a single application, the needles should not be placed close to the tumor’s borders because effectiveness is highly likely to be lost

Dermal Substitutes Support the Growth of Human Skin-Derived Mesenchymal Stromal Cells: Potential Tool for Skin Regeneration

<div><p>New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and disease. Mesenchymal stem cells (MSCs) are an attractive source of cells for tissue engineering because of their prolonged self-renewal capacity, multipotentiality, and ability to release active molecules important for tissue repair. In this paper, we show that human skin-derived mesenchymal stromal cells (SD-MSCs) display similar characteristics to the multipotent MSCs. We also evaluate their growth in a three-dimensional (3D) culture system with dermal substitutes (Integra and Pelnac). When cultured in monolayers, SD-MSCs expressed mesenchymal markers, such as CD105, Fibronectin, and α-SMA; and neural markers, such as Nestin and βIII-Tubulin; at transcriptional and/or protein level. Integra and Pelnac equally supported the adhesion, spread and growth of human SD-MSCs in 3D culture, maintaining the MSC characteristics and the expression of multilineage markers. Therefore, dermal substitutes support the growth of mesenchymal stromal cells from human skin, promising an effective tool for tissue engineering and regenerative technology.</p></div

Immunophenotypic profile of human SD-MSCs cultured in Integra and Pelnac.

<p>Flow cytometry analysis of SD-MSCs (CD90, CD73, CD105) and hematopoietic (CD45) markers in SD-MSCs after 3 days of culture in Integra (upper panel) and Pelnac (lower panel). Curves in black show the specific markers, and gray curves correspond to controls.</p

Multilineage potential of SD-MSCs.

<p>(A) Gene expression profile by RT-PCR of SD-MSCs cultivated in standard medium. (B–E) Immunofluorescence staining of (B) CD105 and (C) Fibronectin. (D–F) Double-staining of α-SMA and βIII-Tubulin, (G–I) α-SMA and Nestin and (J–L) βIII-Tubulin and Nestin co-expression. (F, I and L): Merged pictures of D–E, G–H, J–K, respectively. Cell nuclei were stained with DAPI (blue). Arrows: Nestin nuclear staining. Scale bar: 50 µm.</p

MSC phenotypic characterization of human skin-derived cells.

<p>(A) Morphological analysis of skin-derived cells by phase contrast microscopy. (B) MTS cell proliferation/viability assay. (C) Osteogenic and (D) adipogenic differentiation. (C) Cells cultured in inductive medium formed Alizarin Red S-stained mineralized nodules and (D) Oil red O-stained lipid clusters. (E) Flow cytometry analysis of hematopoietic (CD34, CD45) and MSC (CD90, CD73, CD105) markers. Specific markers are shown by black curves and controls by gray curves. ***p<0.001. Scale bar: (C–F): 50 µm. Other pictures: 200 µm.</p

3D cultures of human SD-MSCs in (A–D) Integra and (E–H) Pelnac.

<p>Confocal microscopy of SD-MSCs cultured in (A–C) Integra and (D–F) Pelnac. (A and E) DAPI nuclear staining of SD-MSCs. (B and F) Integra and (F) Pelnac dermal substitutes, respectively (green autofluorescence). (C and G) merged images of (A and B) and (E and F), respectively. (D and H) MTS cell viability assay of SD-MSCs cultivated in Integra and Pelnac, respectively. ***p<0.001, **p<0.01. Scale bar: 100 um.</p