Ingeniería tisularaplicación de células madre en reparación de defectos epidérmicos

Abstract

Wound healing is a key step in the process whereby a patient’s health is fully recovered. Wounds represent the loss of a physical barrier that protects tissues from invasion by microorganisms and allows the loss of fluids, which can be drastic in patients with burns. Further, several medical or surgical complications can be attributed to deficient wound healing. In the intrahospital setting, these may be a significant cause of morbidity and, indirectly, of mortality. In regenerative medicine several areas of biomedical research merge to give rise to tissue engineering, whose basic tools are cells, scaffolds or matrices, and signals. Interest in tissue engineering has been recently rekindled by new knowledge emerging on the biology of stem cells and their possible therapeutic applications. Previous attempts at obtaining epidermal adult stem cells capable of slow epithelialisation and stabilising the healing processes in large burns, prompted us to consider the possibility of using musclederived stem cells (MDSC) to aid the wound repair process. These cells have been used in the treatment of other diseases but never for skin repair purposes. Our working hypothesis was whether MDSC could be an efficient option for the repair of incisional cutaneous defects. To test this hypothesis, we established two main objectives: 1) To isolate and characterise MDSC and select the best time for grafting. 2) To apply MDSC as cell therapy in an experimental incisional cutaneous wound model. For the first objective, MDSC were harvested from a biopsy specimen taken from the dorsal muscle of New Zealand White rabbits, and then characterised by immunohistochemical techniques and Western blotting. These in vitro studies were completed with a cell proliferation assay based on 3Hthymidine incorporation. Light microscopy and transmission and scanning electron microscopy studies were also performed. Fourteen days of culture rendered a cell population comprised mainly of precursor cells capable of proliferation, adhesion and migration, thus reproducing their biological properties for their application in the skin repair model designed as the second objective of our study. MDSC were applied to a 2 cmdiameter incisional skin defect created on the ear of the experimental animal. The wounds were made on the back part of both ears using a cutting cylindrical instrument avoiding the perichondrial tissue of the ear cartilage. The animals were sacrificed after 14 days and two study groups established according to whether the wounds were left untreated (Control group, left ear) (n=10) or treated with grafted autologous MDSC (Treatment group, right ear) (n=10). The cultured cells used were labelled with PKH26 so that they could be traced inside the wound. The wounds were covered with a custom made polyethylene dressing. The rabbit was fitted with a neck collar to prevent it touching the wound. The wounds were evaluated visually in terms of wound contraction and reepithelialisation established by histomorphometry, including image analysis. Data were compared using the MannWhitney Utest. Light and electron microscopy techniques were used for the histological studies. Our morphometric data revealed significantly greater wound contraction in the control than treatment group (1.83 cm2 vs 2.72 cm2 ; *p<0.05). When expressed as a percentage, contraction was 50.83± 12.84% in the control group versus 22.25± 9.56 % for the cell therapy group. A significantly larger reepithelialised area was recorded for the treatment group (2.50 cm2 vs 1.29 cm2; *p<0.05). Percentage reepithelialised areas were 96.33± 3.09% vs 70.91±6.94% respectively. Light microscopy revealed that the repair process in the control wounds commences at the healthy edges of the defect. In surface areas, white blood cells, cells with epithelial characteristics and vascular buds were often found. Sirius red showed the presence of type III collagen in control wounds and type I, or mature collagen, in the seeded wounds. The PKH26 marker allowed us to monitor the stem cells in the treated wounds. In the presence of the seeded substrate, a continuous surface appeared between the defect’s edges. The substrate promotes the proliferation and migration of the cells from the healthy margins inwards. Keratinocytes proliferate in a centripetal manner towards the center of the defect. The immunohistochemical study performed on control and treated specimens in both cases revealed a lack of vimentin and desmin expression, while labelling for αactin appeared on native blood vessels in both study groups. The expression of myosin in control specimens was greater than in the treated wounds and possibly corresponded to zones with more myofibroblasts and therefore to areas of wound contraction. Our findings allowed us to draw the following conclusions: 1. The cell population isolated from skeletal muscle shows several properties inherent to an undifferentiated population of cells and self renewal, as indicated by the results compared to those observed for the fibroblasts used as the differentiated cell control. 2. After 14 days of culture, the MDSC are undifferentiated, proliferative and very adherent. These are ideal properties for their application in the repair of incisional skin defects. 3. Wounds subjected to cell therapy for two weeks experienced significant less contraction of the defect’s area as well as a significantly faster reepithelialisation than the control untreated wounds. . Thus, isolated undifferentiated MDSC cultured for 14 days fulfill all the requirements for their use as cell therapy, both in terms of numbers and adhesion, proliferation and differentiation properties. In an experimental model, the repair of an incisional wound was significantly improved.