Comparison between placental and skeletal muscle ECM: in vivo implantation.

PURPOSE OF THE STUDY: Several tissues have been decellularized and their extracellular matrices used as allogeneic or xenogeneic scaffolds, either in orthotopic or heterotopic implantations, for tissue engineering purposes. Placentas have abundant matrix, extensive microvascular structure, immunomodulatory properties, growth factors and are discarded after birth, representing an interesting source of extracellular matrix. This study aimed at comparing decellularized canine placentas and murine skeletal muscles to regenerate skeletal muscles in a rat model. MATERIALS AND METHODS: Muscle pockets were created at the posterior limbs of male Wistar rats, where the muscle- and placenta-derived extracellular matrices were implanted. Macroscopic, histological, and immunohistochemical analyses were performed after 3, 15, and 45 days of surgeries. RESULTS: On the third day, intense inflammatory reaction, with macrophages (CD163+) and proliferative cells (PCNA+) being observed in control group and adjacent to the decellularized matrices. The percentage of proliferative cells was higher in placenta than in muscle matrices. Macrophages CD163+ high were higher in muscles than in placentas, whereas CD163+ low were higher in placentas than in muscle ECM, at days 3 and 15. Placental matrices were not completely degraded at day 15, as opposed to the muscular ones. After 45 days, both matrices were resorbed and morphologically normal myofibers, with reduction of cell infiltration, were observed. CONCLUSIONS: These results demonstrated that xenogeneic placental ECM, implanted heterotopically (representing a biologically critical and challenging microenvironment), induced local inflammatory reactions similar to the allogeneic muscle ECM, implanted orthotopically. Thus, placenta-derived extracellular matrix must be further explored in regenerative medicine.

Effects of chemical and physical methods on decellularization of murine skeletal muscles.

Volumetric muscle loss causes functional weakness and is often treated with muscle grafts or implant of biomaterials. Extracellular matrices, obtained through tissue decellularization, have been widely used as biological biomaterials in tissue engineering. Optimal decellularization method varies among tissues and have significant impact on the quality of the matrix. This study aimed at comparing the efficacy of four protocols, that varied according to the temperature of tissue storage and the sequence of chemical reagents, to decellularize murine skeletal muscles. Tibialis anterior muscles were harvested from rats and were frozen at -20°C or stored at room temperature, followed by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100, applied in different sequences. Samples were analyzed for macroscopic aspects, cell removal, decrease of DNA content, preservation of proteins and three-dimensional structure of the matrices. Processing protocols that started with incubation in SDS solution optimized removal of cells and DNA content and preserved the matrix ultrastructure and composition, compared to those that were initiated with EDTA + Tris. Freezing the samples before decellularization favored cell removal, regardless of the sequence of chemical reagents. Thus, to freeze skeletal muscles and to start decellularization with 1% SDS solution showed the best results.

Effects of chemical and physical methods on decellularization of murine skeletal muscles

Volumetric muscle loss causes functional weakness and is often treated with muscle grafts or implant of biomaterials. Extracellular matrices, obtained through tissue decellularization, have been widely used as biological biomaterials in tissue engineering. Optimal decellularization method varies among tissues and have significant impact on the quality of the matrix. This study aimed at comparing the efficacy of four protocols, that varied according to the temperature of tissue storage and the sequence of chemical reagents, to decellularize murine skeletal muscles. Tibialis anterior muscles were harvested from rats and were frozen at -20°C or stored at room temperature, followed by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100, applied in different sequences. Samples were analyzed for macroscopic aspects, cell removal, decrease of DNA content, preservation of proteins and three-dimensional structure of the matrices. Processing protocols that started with incubation in SDS solution optimized removal of cells and DNA content and preserved the matrix ultrastructure and composition, compared to those that were initiated with EDTA + Tris. Freezing the samples before decellularization favored cell removal, regardless of the sequence of chemical reagents. Thus, to freeze skeletal muscles and to start decellularization with 1% SDS solution showed the best results.

Comparison between placental and skeletal muscle ECM: in vivo implantation

Purpose of the study: Several tissues have been decellularized and their extracellular matrices used as allogeneic or xenogeneic scaffolds, either in orthotopic or heterotopic implantations, for tissue engineering purposes. Placentas have abundant matrix, extensive microvascular structure, immunomodulatory properties, growth factors and are discarded after birth, representing an interesting source of extracellular matrix. This study aimed at comparing decellularized canine placentas and murine skeletal muscles to regenerate skeletal muscles in a rat model. Materials and Methods: Muscle pockets were created at the posterior limbs of male Wistar rats, where the muscle- and placenta-derived extracellular matrices were implanted. Macroscopic, histological, and immunohistochemical analyses were performed after 3, 15, and 45 days of surgeries. Results: On the third day, intense inflammatory reaction, with macrophages (CD163+) and proliferative cells (PCNA+) being observed in control group and adjacent to the decellularized matrices. The percentage of proliferative cells was higher in placenta than in muscle matrices. Macrophages CD163+ high were higher in muscles than in placentas, whereas CD163+ low were higher in placentas than in muscle ECM, at days 3 and 15. Placental matrices were not completely degraded at day 15, as opposed to the muscular ones. After 45 days, both matrices were resorbed and morphologically normal myofibers, with reduction of cell infiltration, were observed. Conclusions: These results demonstrated that xenogeneic placental ECM, implanted heterotopically (representing a biologically critical and challenging microenvironment), induced local inflammatory reactions similar to the allogeneic muscle ECM, implanted orthotopically. Thus, placenta-derived extracellular matrix must be further explored in regenerative medicine.