Avaliação do potencial terapêutico de células tronco mesenquimais derivadas da pele no reparo de lesões cutâneas

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e do Desenvolvimento, Florianópolis, 2013.Novas estratégias para a regeneração da pele são necessárias a fim de proporcionar um tratamento eficaz para feridas cutâneas e doenças. Dentre estas possíveis estratégias estão o desenvolvimento de novos biomateriais, a realização de terapia celular e a identificação e aplicação de fatores envolvidos no reparo de tecidos. Avanços na área de engenharia tecidual têm possibilitado o desenvolvimento de biomateriais que se assemelham a arquitetura tecidual da pele, como os substitutos dérmicos, que permitem o recobrimento da lesão e facilitam a recolonização celular, auxiliando assim a regeneração do tecido dérmico. Além disso, as células-tronco mesenquimais (CTMs) têm sido descritas como uma fonte atrativa de células para a engenharia de tecidos, em razão da sua multipotencialidade e capacidade de liberação de moléculas ativas importantes para o reparo tecidual. Tendo em vista estes aspectos, o presente trabalho busca estabelecer e avaliar um novo método de tratamento de lesões de pele, tendo como base a associação das CTMs derivadas da pele humana com substitutos dérmicos comerciais, utilizados atualmente em abordagens clínicas. Para isso, primeiramente, CTMs da derme humana (dCTMs) foram isoladas e sua potencialidade e características morfológicas, fenotípicas e migratórias analisadas. Os resultados obtidos mostram que as dCTMs possuem características semelhantes às CTMs derivadas da medula óssea, como: morfologia fibroblastóide, alta capacidade proliferativa, perfil de expressão de marcadores de superfície e potencial de diferenciação para fenótipos mesodermais. Além disso, as células expressam marcadores de pluripotencialidade e de linhagens neurais e mesenquimais, sem indução específica, e possuem potencial de diferenciação para a linhagem epidermal. Ademais, as dCTMs mostram alta capacidade migratória in vitro. Após esta caracterização das dCTMs, foi avaliado o crescimento e a manutenção destas células em um sistema de cultura tridimensional com os substitutos dérmicos Integra® e Pelnac®. Os resultados mostram que ambos os substitutos dérmicos suportam igualmente a adesão, proliferação e migração das dCTMs, mantendo suas características fenotípicas, tais como o perfil de expressão de marcadores de superfície, de pluripotencialidade e de linhagens neurais e mesenquimais. Tendo em vista que a associação das dCTMs com os substitutos dérmicos mostrou-se eficiente, foi avaliado a seguir o potencial terapêutico destas células associadas ao substituto dérmico (SD) Integra® no reparo de lesões de pele em camundongos. Para isso, foi avaliada a inflamação, vascularização, depósito de matriz extracelular e a re-epitelização em lesões tratadas com dCTMs associadas ao SD e em lesões mantidas apenas com o SD (controle). Os resultados demonstram que o tratamento com dCTMs + SDs aumenta o tecido de granulação, o recrutamento de células inflamatórias (neutrófilos e macrófagos), a vascularização, o depósito de colágeno e a re-epitelização, acelerando assim o reparo tecidual. Além disso, o tratamento com dCTMs + SDs modula a expressão de diferentes genes relacionados com o reparo tecidual na área da lesão. Em conclusão, o presente estudo isolou uma população de CTMs da pele humana, denominada de CTMs dermais (dCTM), e mostrou uma eficiente associação destas células com os SDs, representando assim uma nova ferramenta terapêutica para a engenharia tecidual. Esta associação, quando avaliada no tratamento de lesões cutâneas de camundongos, mostrou-se promissora, resultando em um reparo tecidual mais eficiente.Abstract : New strategies for skin regeneration are needed in order to provide effective treatment for cutaneous wounds and diseases. Among these possible strategies are the development of new biomaterials, cell therapy and the identification and application of factors involved in tissue repair. Advances in tissue engineering have provided the development of biomaterials similar to the skin such as dermal substitute (DS), for covering the lesion and to facilitate cell recolonization thereby supporting dermal regeneration. In addition, mesenchymal stem cells (MSCs) have been suggested as an attractive source of cells for tissue engineering because of their multipotentiality and ability to release active molecules for tissue repair. Therefore, the present study established and evaluated a new method for treatment of skin lesions, based on the association of MSCs derived from human skin with commercial dermal substitutes currently used in clinical procedures. Thus, MSCs from human dermis (dCTMs) were isolated and their morphologic, phenotypic migratory characteristics and potentiality analyzed. The results showed that dCTMs have characteristics similar to MSCs derived from bone marrow, such as: fibroblast-like morphology, high proliferative capacity, profile of surface markers and differentiation potential for mesodermal phenotypes. In addition, these cells express pluripotent and mesenchymal and neural lineages markers, without specific induction. Moreover, the dMSCs display the potential for epidermal lineage and show high migratory capacity in vitro. It was also evaluated the growth and maintenance of these cells in three-dimensional (3D) culture system with the dermal substitutes Pelnac® and Integra® . The results showed that both dermal substitutes equally support the adhesion, spread and growth of human dMSCs in 3D-culture, maintaining MSC phenotype, expression of the pluripotent and neural and mesnchymal lineages markers. In view of these results, the therapeutic potential of these cells associated with the Integra DS in the repair of skin lesions in mice was evaluated in vivo. Inflammation, vascularization, deposition of matrix extracellular molecules and reepithelialization in skin lesions were analyzed. The results demonstrated that the treatment with dCTMs + DSs increases the granulation tissue, recruitment of inflammatory cells (neutrophils and macrophages), vascularization, collagen deposition and reepithelization, thus accelerating tissue repair. In addition, the treatment with dCTMs + DSs modulates the expression of different genes related to tissue repair. In conclusion, the present study isolated a population of MSCs from human skin, named dermal MSCs (dMSCs), and showed an efficient association of these cells with DSs, representing a new therapeutic tool for tissue engineering. This combination, when evaluated in the treatment of cutaneous lesions in mice, has shown to be promising, resulting in a more efficient tissue repair

Efeito dos fatores FGF-2, EGF E beta-catenina no potencial de diferenciação das células tronco mesenquimais de placenta humana

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 20092012-10-24T12:03:06

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

Comparative Experimental Study of Wound Healing in Mice: Pelnac versus Integra

<div><p>Strategies for skin regeneration have been developed to provide effective treatment for cutaneous wounds and disease. Dermal substitutes have been used to cover the lesion to facilitate cell colonization, thereby promoting dermal regeneration. However, very little is known about Pelnac matrix especially at histological level. Therefore, the present work carried out an experimental <i>in vivo</i> comparative analysis between Pelnac and Integra, the most used dermal templates, in a mouse model of full-thickness skin wounds. Histological sections performed at the 3^rd, 6^th and 9^th days after surgery were analyzed with regard to inflammatory response and vascularization. Both templates were completely incorporated in all animals at the end of the analyzed period. Pelnac-treated animals displayed reduced granulation tissue during the first 6 days of treatment compared to the animals treated with Integra at the same time period. The number of inflammatory cells (neutrophils) was similar in both groups during the period, significantly reducing at the end of inflammatory phase (9^th day of treatment) consistent with the progression of healing process. In addition, the density of blood vessels was also statistically similar in both matrices. Therefore, the two dermal templates displayed comparable biological behavior in tissue repair. It is noteworthy that this is the first experimental study comparing Pelnac and Integra dermal templates with focus on full-thickness skin wounds.</p></div

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

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

Scanning electron microscopy (SEM) images of SD-MSCs cultured in (A–D) Integra and (E–H) Pelnac.

<p>(A) Cross-sectional view of Integra dermal substitute alone showing the silicone (*) and the inner layer. (B–D) 3D culture of SD-MSCs in Integra 48 hours after seeding. (E) Surface and (F) cross-sectional views of Pelnac showing the collagen layer. (G–H) 3D culture of SD-MSCs in Pelnac 48 hours after seeding. Insets in B: different magnifications of a SD-MSC.</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

Histological aspects of wounds treated with dermal substitutes.

<p>HE-stained transversal sections of a full-thickness skin wound treated with (A-C) Integra or (D-F) Pelnac collected at (A, D) 3, (B, E) 6 and (C, F) 9 days after the procedure show a clear distinction between the dermal substitute (DM) and the wound bed (WB). Original magnification 100X.</p