UC San Diego

Ischemic disease, which involves tissue death and dysfunction due to vessel blockage, is one of the largest causes of morbidity and mortality across the world. Ischemia that targets the brain can lead to a stroke, which causes functional impairment. Blockage of the coronary artery can lead to a myocardial infarction (MI) which can eventually lead to heart failure. Ischemia of the vessels in the skeletal muscle causes peripheral artery disease, which can lead to tissue damage that may necessitate amputation of the affected limb. The severity of the downstream effects of ischemia indicates the need for some sort of treatment to repair the tissue after ischemic attack. Yet, there are few clinical treatments available for patients, creating a need for novel therapies for treating this disease. The use of biomaterials in tissue engineering strategies have recently been studied to alleviate these conditions, however this approach has been met with limited success as many of these therapies require an invasive surgery for delivery to the affected site. Injectable biomaterials offer the advantage of minimally invasive delivery to improve patient outcomes, which would be attractive to reduce patient recovery time. The materials that have been studied often do not mimic the microenvironment of the tissue that it is trying to repair. This is similar to how cells are often cultured on a substrate that do not mimic the in vivo environment, which may be important for assessing cellular function. Thus, the objective of this dissertation was to generate biomaterials derived from decellularized tissue from the brain, skeletal muscle, and cardiac tissue, and test whether they could be used as cell culture platforms that would provide biomimetic substrates and be used as scaffolds for tissue engineering. In this work, I have developed a method to decellularize each tissue leaving behind only the extracellular matrix. The matrix material was then characterized using gel electrophoresis, mass spectrometry, glycosaminoglycan quantification and DNA quantification, indicating that the cellular remnants have been removed, but that the biocomplexity has been retained. These tissue specific biomaterials were tested as a cell culture coating platform in vitro and as a potential therapy for ischemia in vivo. The material was enzymatically digested and used as a cell culture coating and compared to conventionally used substrates. It was found that progenitor cells cultured on the tissue-matched coatings display a more mature morphology on the decellularized extracellular matrix (ECM) coatings. For instance, skeletal muscle progenitors differentiate into larger, thicker myotubes, cardiomyocytes derived from human embryonic stem cells localize their intracellular junctions into a more mature organization, and neurons from induced pluripotent stem cells display a clear axon and increased dendritic branching. The maturation of these cell types on the coatings demonstrate a more in vivo like phenotype which could be useful for studying cellular behavior and to translate in vitro findings into an in vivo setting. This material was able to self-assemble upon injection in vivo, forming a nanofibrous and porous scaffold that could be used as an injectable biomaterial for ischemic repair. Additionally, in vitro assays measuring proliferation and migration show that some of the matrix materials act as a chemoattractant and as a mitogenic agent on cells in culture. The skeletal muscle matrix has been used in a rat hindlimb ischemia model and compared to a collagen scaffold. It was shown that the skeletal muscle matrix stimulates increased neovascularization, which is important for bringing blood flow to an ischemic region, as well as recruits endogenous muscle progenitor cells into the scaffold. The cardiac matrix is able to gel in situ upon injection and has been explored by others in our lab. The brain matrix was also able to self-assemble and form a gel after subcutaneous injection into a mouse, demonstrating proof-of-concept for its use as a tissue engineering scaffold. To investigate whether the material might be derived from an allogeneic source instead of from porcine origin, the decellularization process was also performed on human cardiac tissue. It was found that additional steps were needed to fully decellularize the material and render it into a usable form. However, this could provide a potentially allogeneic source for this material. This work demonstrates that decellularized extracellular matrices derived from various tissues provide a biomimetic platform for cell culture that increases maturation of progenitor and stem cells cultured upon the surface. The maturation of these cells could be important for understanding and regulating cellular processes. The same material can be used as an injectable scaffold that could be delivered through minimally invasive means to treat ischemic damage in the brain, heart and skeletal muscle. When applied in a hindlimb ischemia model, the skeletal muscle matrix is able to increase neovascularization, recruit more muscle progenitor cells, and recruit more proliferating muscle cells when compared to a collagen control. This work shows that decellularized matrices hold great potential for both in vitro and in vivo applications