Intrathoracic organs and tissues biological scaffolds creation in rat and nonhuman primate models
Tissue engineering as a part of the regenerative medicine is an interdisciplinary field, which applies principles of cell transplantation, materials science and engineering to develop biological substitutes that establish or restore physiological function. The one of three main principles of tissue engineering is use of a scaffold, i.e. a three-dimensional structure for cells to adhere to and grow on which can be either natural or artificially derived. Perfect biomaterial scaffolds for intrathoracic organs and tissue grafts should be non-toxic, resistant to infection, durable, elastic, biodegradable and supporting in vitro adhesion, growth and function of several cell types. In vivo acellular matrices should act as a template allowing the ingrowth of host cells and remodeling into a living tissue. Biomaterials should not: provide inflammatory reaction or rejection, allergy or sensitization, shrink in the healing process, be carcinogenic or initiate local complications. For biologically derived scaffold creation we used decellularization of donor organs. Decellularization involves use of physical or chemical means to eliminate immunogenic cells from an organ or tissue while preserving a native ultrastructure and composition of the extracellular matrix (ECM), which maintains biomechanical properties of the organ. An optimal decellularization method varies depending on the tissue/organ. For intrathoracic organs and tissues (heart, lungs, diaphragm, esophagus) decellularization in rat and nonhuman primate models a detergent-enzymatic method (DEM) was used. We applied a modified protocol with reduced detergent and enzymes exposure time and sequence: [Aqua MilliQ, Deoxycholate 4% (Sigma, Sweden), Triton-X 100 (Sigma, Sweden), PBS, DNAse I, 2000ku in 200ml PBS (Invitrogen, Sweden), EDTA, 800 um in 200 MilliQ (Sigma, Sweden)]. Absence of nuclei and other cell elements was demonstrated in decellularized organs. Moreover, the reciprocal orientation of fibers in scaffold walls resembled that of the control (i. e. native organ), and there also were no signs of collagen and elastin degradation. Architectonics of intrafibrous connective tissue remained intact and preserved adventitia of small vessels. Nuclear structures fluoresced intensively in native organs, while in decellularized organs no fluorescence was observed. In vitro studies revealed that 24 hours was necessary for complete removal of cellular part of tissue, including elimination of specific tissue protein tropomyosin and MHC class I, MHC class II, von Willebrand factor. We determined that these protocols have a minimal negative impact on extracellular matrix composition, ultrastructure and biocompatibility properties. Our suggestion was confirmed by undamaged histo-architecture and ultrastructure (SEM) of extracellular matrix without fragmentation or structure loss. Moreover, we observed presence of extracellular proteins such as collagen I, collagen IV, laminin, fibronectin, elastin. Lack of immunogenicity was also confirmed by DNA quantification of decellularized matrices. Moreover, decellularized matrices preserved important biomechanical properties. Measuring of mechanical properties demonstrated that both native and decellularized organs possess almost the same properties. Our data obtained in vitro from MTT assay, Live/Dead assay suggested that the seeded graft contained attached cells that were viable and proliferating on surfaces. It could be demonstrated that matrix degradation products did not exert any toxic effect on cell viability.