Effects of mesenchymal stem cell culture on radio sterilized human amnion or radio sterilized pig skin in burn wound healing.

Deep second and third degree burns treatment requires fibroblasts, keratinocytes and other skin cells in order to grow new dermis and epidermis. Cells can proliferate, secrete growth factors and extracellular matrix required to repair the damaged tissue. Radiosterilized human amnion and radiosterilized pig skin have been used as natural origin skin dressings for burned patients. Adipose-derived mesenchymal stem cells can differentiate into fibroblasts and keratinocytes and improve wound-healing progress. These cells can stimulate vascular tissue formation, release growth factors, synthetize new extracellular matrix and immunoregulate other cells. In this study, we developed mesenchymal stem cells-cellularized skin substitutes based from radiosterilized human amnion or pig skin. Third-degree burns were induced in mice animal models to evaluate the effect of cellularized skin substitutes on burn wound healing. Mesenchymal phenotype was immunophenotypically confirmed by flow cytometry and cell viability was close to 100%. Skin recovery was evaluated in burned mice after seven and fourteen days post-coverage with cellularized and non-cellularized sustitutes. Histological techniques and immunofluorescence were used to evaluate re-epithelization and type I collagen deposition. We determined that cellularized-human amnion or cellularized-pig skin in combination with mesenchymal stem cells improve extracellular matrix deposition. Both cellularized constructs increase detection of type I collagen in newly formed mouse skin and can be potentially used as skin coverage for further clinical treatment of burned patients.

Characterization of pediatric microtia cartilage: a reservoir of chondrocytes for auricular reconstruction using tissue engineering strategies.

The external ear is composed of elastic cartilage. Microtia is a congenital malformation of the external ear that involves a small reduction in size or a complete absence. The aim of tissue engineering is to regenerate tissues and organs clinically implantable based on the utilization of cells and biomaterials. Remnants from microtia represent a source of cells for auricular reconstruction using tissue engineering. To examine the macromolecular architecture of microtia cartilage and behavior of chondrocytes, in order to enrich the knowledge of this type of cartilage as a cell reservoir. Auricular cartilage remnants were obtained from pediatric patients with microtia undergoing reconstructive procedures. Extracellular matrix composition was characterized using immunofluorescence and histological staining methods. Chondrocytes were isolated and expanded in vitro using a mechanical-enzymatic protocol. Chondrocyte phenotype was analyzed using qualitative PCR. Microtia cartilage preserves structural organization similar to healthy elastic cartilage. Extracellular matrix is composed of typical cartilage proteins such as type II collagen, elastin and proteoglycans. Chondrocytes displayed morphological features similar to chondrocytes derived from healthy cartilage, expressing SOX9, COL2 and ELN, thus preserving chondral phenotype. Cell viability was 94.6 % during in vitro expansion. Elastic cartilage from microtia has similar characteristics, both architectural and biochemical to healthy cartilage. We confirmed the suitability of microtia remnant as a reservoir of chondrocytes with potential to be expanded in vitro, maintaining phenotypical features and viability. Microtia remnants are an accessible source of autologous cells for auricular reconstruction using tissue engineering strategies.

Chondrocyte differentiation for auricular cartilage reconstruction using a chitosan based hydrogel.

Tissue engineering with the use of biodegradable and biocompatible scaffolds is an interesting option for ear repair. Chitosan-Polyvinyl alcohol-Epichlorohydrine hydrogel (CS-PVA-ECH) is biocompatible and displays appropriate mechanical properties to be used as a scaffold. The present work, studies the potential of CS-PVA-ECH scaffolds seeded with chondrocytes to develop elastic cartilage engineered-neotissues. Chondrocytes isolated from rabbit and swine elastic cartilage were independently cultured onto CS-PVA-ECH scaffolds for 20 days to form the appropriate constructs. Then, in vitro cell viability and morphology were evaluated by calcein AM and EthD-1 assays and Scanning Electron Microscopy (SEM) respectively, and the constructs were implanted in nu/nu mice for four months, in order to evaluate the neotissue formation. Histological analysis of the formed neotissues was performed by Safranin O, Toluidine blue (GAG's), Verhoeff-Van Gieson (elastic fibers), Masson's trichrome (collagen) and Von Kossa (Calcium salts) stains and SEM. Results indicate appropriate cell viability, seeded with rabbit or swine chondrocyte constructs; nevertheless, upon implantation the constructs developed neotissues with different characteristics depending on the animal species from which the seeded chondrocytes came from. Neotissues developed from swine chondrocytes were similar to auricular cartilage, while neotissues from rabbit chondrocytes were similar to hyaline cartilage and eventually they differentiate to bone. This result suggests that neotissue characteristics may be influenced by the animal species source of the chondrocytes isolated.