Previous research into the problem of postsurgical cardiac adhesions has produced a hydrogel barrier that can be applied to the surface of the heart, preventing the formation of adhesions in the weeks following trauma during surgery. When developing this type of gel for clinical translation, two issues that must be addressed are the biocompatibility of the material as well as a method of hydrogel delivery to the tissue surface. Previous research yielded a hydrogel made from poly(ethylene glycol) functionalized with aldehyde, aminooxy and dopamine groups. Cell viability is reduced in culture treated with this material due to the inclusion of dopamine. Examination of the hydrogel in culture through fluorescence microscopy and spectrophotometry indicate that this is primarily due to oxidative stress, demonstrated by increased levels of hydrogen peroxide and lipid peroxidation. However, metabolic and morphological assays performed in vitro demonstrate that the biological antioxidant glutathione, a mediator of dopamine effects, can maintain cell viability when added as a culture supplement. Within this study, the effective application of the gel is achieved through air-assisted liquid spray, which allows equal distribution of two separate polymer solutions onto the surface of the heart. This result is predicted computationally through simulation of separate intersecting sprays and verified by spectroscopy and rheological testing of the material produced by a novel device. By investigating these factors, better understanding of the material's biological properties and implementation can be obtained to guide future research.

Myocardial infarction (MI) is a leading cause of death in the world. Many patients survive the MI; however, the ischemic damage remains, and there are currently no treatments available to repair the heart. Decellularized extracellular matrix (ECM) has been widely used for tissue engineering applications and is becoming increasingly versatile as it can take many forms, including patches, powders, and hydrogels. Following additional processing, decellularized ECM can form an inducible hydrogel that can be injected, providing opportunities for minimally invasive delivery. We have developed a new ECM hydrogel therapy, the soluble fraction derived from decellularized, digested ECM, for intravascular infusion. This new form of ECM is capable of gelation in vivo and can be delivered via intravascular infusion following an injury to specifically target the injured tissue, promote cell survival, and improve vascularization. In this dissertation, we show proof-of-concept for the feasibility of ECM infusions using models of acute myocardial infarction and intracoronary infusion for delivery. Following infusion, the ECM material was retained specifically in the injured tissue and colocalized with endothelial cells, coating the leaky microvasculature. Functional improvements, specifically reduced left ventricular volumes, were observed after ECM infusion post-MI. A range of biological activity modulation was implicated, as pathways associated with angiogenesis, cell-substrate/junction, reactive oxygen species and nitric oxide metabolism, and interleukin 6 were differentially expressed. The soluble ECM was also delivered intravascularly using a clinically relevant catheter in a large animal model of acute MI, mitigated negative left ventricular remodeling, and showed improved left ventricle wall motion. This study shows proof-of-concept for a new therapy and intravascular delivery strategy for ECM biomaterials with potential implications for a variety of pathologies to target injured tissues and injured vasculature.

Over the past several decades, coronary heart disease leading to myocardial infarction (MI) and subsequent heart failure (HF) has continued to the leading cause of death in the Western world and worldwide. Sudden death and limited renewal of cardiomyocytes post-myocardial leads to progressive expansion of tissue necrosis, negative left ventricular remodeling, and loss of function eventually causing heart failure. Treatments for end-stage HF, heart transplants and left ventricular assist devices, are hampered by healthy organ availability, limited medical resources, and negative impacts on patients’ quality of life, thus prompting the need for novel therapies. Amongst hydrogel therapies, Injectable extracellular matrix (ECM) hydrogels derived from decellularized porcine left ventricular tissue have rapidly developed into

a leading injectable hydrogel therapy based on shown therapeutic potential post-myocardial infarction demonstrated in both small and large animal models. To continue developing this and general decellularized platforms, improved understanding of the underlying cellular mechanisms contributing to the observed myocardial repair is needed. Based on previous transcriptomic and histological assessments, further examination into the cellular response of cardiomyocyte and immune cell populations is studied to determine their involvement in the observed tissue repair. We show with pre-labeling methods to track events of DNA synthesis and proliferation in in vivo and in vitro models, respectively, that myocardial matrix material properties relevant to supporting proliferative characteristics in cardiomyocytes. Additional examination of immune cell populations has determined that the myocardial matrix supports a dynamic pro-inflammatory to pro-remodeling immune response indicative of induced tissue repair. Finally, we determined the involvement of mast cells in the biomaterial induced tissue repair, highlighting this understudied cell type for the field to consider when developing new biomaterial therapies.

Congenital heart defects are the leading cause of right ventricular heart failure (RVHF) in pediatric patients. In particular, hypoplastic left heart syndrome is a condition in which infants are born with an underdeveloped left side of the heart and must undergo three corrective open-heart surgeries to make their right ventricle their main systemic pump. Due to the increased afterload on the RV due to systemic circulation, the RV undergoes a cascade of negative remodeling characterized myocardial apoptosis, maladaptive hypertrophy, vessel rarefaction, metabolic shifts from lipid oxidation to glucose metabolism, inflammation, fibrosis, ischemia from mismatched oxygen supply/demand, and oxidative stress that cannot be corrected by standard treatments that only alleviate the underlying symptoms. Regenerative therapies such as c-kit cardiac progenitor cells (CPCs) and decellularized myocardial matrix (MM) hydrogels are promising therapeutics that mitigate negative remodeling in the RV and promote repair and improve overall cardiac function. CPCs, however, are limited due to their low survival and engraftment rates while MM hydrogels have yet to be investigated in models of RVHF. Furthermore, MM hydrogels that have been investigated are derived from the porcine left ventricle (LV) and have only been investigated in models of left ventricular heart failure, neglecting the fact that the LV and RV are two distinct tissues that arise separately during cardiac development. Thus, leading to a need to evaluate RV derived MM hydrogels to treat RVHF. Here we fabricate and characterize a new RV MM hydrogel and compare it to its predecessor, evaluate the effect of both MM on CPC behavior in vitro, compare CPC alone therapy with MM and CPC combined therapy in vivo, and investigate the mechanisms action of LV MM and RV MM and their effects on negative RV remodeling and function in a small animal model of RVHF. We have developed a new RV MM hydrogel, that while it is physically and mechanically similar to LV MM, it is distinctly different based on proteomic makeup. We further showed that both MM enhance the survival of CPCs against common implant hazards such as needle forces and reactive oxygen species as well as enhance their angiogenic paracrine signaling in vitro. Qualitative assessment showed the benefit of delivering CPCs with either MM to improve CPC retention in vivo while echocardiography demonstrated improvements to cardiac function only when comparing the combinatorial therapies to themselves at the baseline indicating a limited repair timeframe. CPCs alone however showed no functional improvement in echo or MRI when compared to the other treatment groups. CPCs also demonstrated detrimental differentially expressed genes when compared to saline and the MM group suggesting the adult cells hold no therapeutic benefit. Finally, we show proof-of-concept of intramyocardial injections of MM in a rat pulmonary artery banded model of RVHF. Animals injected with either LV MM or RV MM demonstrated significant improvements in tricuspid annular plane systolic excursion, RV end-diastolic volume, RV end-systolic volume, and RV free wall thickness. Both MM also affected pathways related to neovascularization, cardiac contractility, and cardiac development at 1 week post injection. RV MM, however, induced overexpression of pro-inflammatory and pro-fibrotic gene expression, suggesting it may induce a prolonged inflammatory response. Despite this, both MM didn’t affect macrophage, capillary, or myofibroblast density at the 1-week timepoint but did induce significant arteriole growth when compared to saline. Both MM further mitigated negative remodeling pathways by reducing hypertrophy, fibrosis and myofibroblast density and by increasing arteriole density when compared to saline. This study shows proof-of-concept of MM hydrogel therapies, tissue specific or otherwise, to not only mitigate pediatric RVHF with implications of being translated into other cases of RVHF.

Unlike many organs, skeletal muscle possesses the ability to naturally regenerate. However, chronic muscle injuries, including ischemia and chronic unloading, interrupt this regenerative process. We sought to investigate the therapeutic potential of a decellularized skeletal muscle extracellular matrix (ECM) hydrogel in ischemia and chronic unloading muscle injury models with a main focus on translating this material into the clinic. A rat hindlimb ischemia model was utilized for a dose optimization study in which three concentrations (4 mg/mL, 6 mg/mL, and 8 mg/mL) were compared to a saline control and non-tissue specific myocardial ECM hydrogel. The 6 mg/mL concentration produced the largest increase in blood perfusion, and efficacy of the 6 mg/mL skeletal muscle ECM hydrogel was then further confirmed in an aged mouse hindlimb ischemia model to more accurately depict patient pathophysiology. Similar to the dose optimization study, significant increases in blood perfusion were observed after 4 weeks. Since the hindlimb ischemia model is an acute model, the skeletal muscle ECM hydrogel was also probed in a more chronic model, specifically a rabbit model of chronic rotator cuff tears. The timing for the ECM hydrogel injection was investigated, and a delayed injection encouraged more muscle regeneration, as demonstrated by upregulation of key muscle transcription factors and the presence of larger diameter arterioles. Although ECM hydrogels possess regenerative capabilities, efficacy may be limited, and, therefore, the material was also evaluated as a delivery vehicle for microRNAs and exosomes. Incorporation of these therapeutics into the hydrogels yielded prolonged release profiles, and the released molecules remained bioactive in vitro. Lastly, several manufacturing considerations were investigated to ensure efficacy of the final product would be maintained during scale-up. Animal-to-animal variability and a bioburden reducing step did not present issues for manufacturing, but the various harvesting conditions yielded differences with the protein content of the final ECM product. All in all, the skeletal muscle ECM hydrogels demonstrated efficacy in multiple skeletal muscle injury models and as a delivery platform for small therapeutics. In addition, the manufacturing of these materials for clinical translation remains feasible amidst additional processing steps.

With improved management of patients with acute myocardial infarctions (MI), the prevalence in heart failure (HF) post-MI is expected to rise. Currently, the only successful treatments for HF are total heart transplantation and left ventricular (LV) assist devices, but their uses are limited by the availability of donor hearts and invasiveness of the procedure. In the last decade, advancements have been made towards developing injectable hydrogels for the purpose of cardiac repair. Injections of hydrogels alone have been shown to attenuate the decline in cardiac function and LV remodeling typically seen after MI in both large and small animals models. One of these hydrogels was previously developed by our lab and derived from decellularized porcine ventricular myocardium. The goal of this thesis was study to the mechanisms by which injections of the myocardial matrix hydrogel improve cardiac repair post-MI and improve upon its cardioreparative effects. To better understand how this myocardial matrix is able to induce the beneficial effects observed post-MI, a whole transcriptome microarray was performed on infarct tissue collected from matrix or saline injected infarcts. We showed through pathway analysis that the effects of the injection were dividable into several tissue level phenotypes. To better understand these in vivo phenomena, we wanted to recapitulate the observations by cell culture in vitro with the myocardial matrix. Several cell behaviors relevant to the infarct milieu were studied, including cardiac progenitor cell migration, cardiomyocyte apoptosis, cardiac fibroblast metallomatrix proteinase (MMP) production, and macrophage polarization. We demonstrated that the form of the matrix that is presented to the cells have a dramatic effect on the cellular response, whether through the 3D hydrogel or as soluble peptides released during degradation. In addition, different fractions of the degradation products also have different bioactivity. Results from these in vitro experiments suggested that the bioactivity of the myocardial matrix and its degradation products seemed to be essential to its cardioreparative effects post-MI; thus, we investigated whether this could be enhanced by prolonging the degradation rate of the hydrogel. Through these studies, we provided the first steps towards elucidating the mechanism of actions of the myocardial matrix, by defining the tissue level changes that it induces in infarcted myocardium and identifying the bioactivity in both the hydrogel form and degradation products.

Pelvic floor disorders, which include pelvic organ prolapse, and urinary and fecal incontinence, affect millions of women globally and represent a major public health concern. Pelvic floor muscle (PFM) dysfunction has been identified as one of the leading risk factors for the development of these morbid conditions. Even though childbirth, specifically vaginal delivery, has been long recognized as the most important potentially modifiable risk factor for PFM injury, the precise mechanisms of PFM dysfunction following childbirth remain elusive. In addition, treatments for PFM dysfunction are urgently needed as the current therapeutic approaches are delayed and mainly compensatory. Using a pre-clinical rat model, we first demonstrated that PFM undergoes atrophy and fibrosis following birth injury. The transcriptional signature of PFM post-injury indicated a sustained inflammatory response, impairment in muscle anabolism, and persistent expression of extracellular matrix (ECM) remodeling genes. We then evaluated the administration of an injectable skeletal muscle ECM hydrogel to prevent and mitigate the pathological alterations following simulated birth injury. Treatment of PFMs with the biomaterial either at the time of birth injury or 4 weeks post-injury reduced muscle atrophy and mitigated fibrotic degeneration. Through gene expression analyses, we show that improvement in PFM phenotype is potentially associated with the hydrogel-induced modulation of the immune response and intramuscular fibrosis and the enhancement of endogenous myogenesis. Lastly, we investigated the PFM response following repeated simulated birth injuries (SBIs), as multiparity further exacerbates the risk for pelvic floor disorders. We demonstrated that repeated SBIs increase pathological alterations and muscle dysfunction compared to a single injury. Gene expression analyses indicated a prolonged inflammatory response compared to a single SBI. Delayed administration of the acellular biomaterial following repeated injuries improved PFM histological changes and modulated the immune response. Overall, this thesis contributes to the understanding of the mechanisms behind PFM birth injury and demonstrates proof-of-concept for utilizing the pro-regenerative biomaterial for treating injured PFMs.

Skeletal muscle injury is a leading cause of diminished quality of life and morbidity in adults, with limited therapeutic interventions. Synthetic and naturally-derived biomaterials provide a potential avenue for novel treatment modalities that address unmet clinical needs. These materials can improve delivery of biological therapeutics or serve as acellular scaffolds for stimulating endogenous tissue repair processes. The goals of this dissertation were to investigate the efficacy of a decellularized skeletal muscle matrix hydrogel in tissue specific

regeneration and to design tunable enzyme-targeted nanoparticles for minimally invasive delivery to ischemic muscle.

An injectable decellularized skeletal muscle matrix hydrogel has previously been developed by our lab as a therapy to improve perfusion to ischemic skeletal muscle in a preclinical peripheral artery disease model. To better understand how the skeletal muscle matrix induces beneficial effects, transcriptomic analysis of matrix or saline-injected muscle was performed in both ischemic and acute muscle regeneration models. We showed that the skeletal muscle matrix increased cell survival, neovascularization, and muscle regeneration and confirmed these pathways through histological analysis at short, medium, and long time points. In acute muscle regeneration, the skeletal muscle matrix increased the density of skeletal muscle progenitors over non-specific controls (lung matrix or saline). This suggests the tissue specific source of the decellularized matrix can have an effect on regenerative pathways.

Enzyme-targeted nanoparticles are an alternative biomaterial approach for more minimally invasive therapeutic delivery as they can be injected intravenously and will target ischemic tissue due to overexpression of matrix metalloproteinases. We were the first group to show minimally invasive targeting of peptide-polymer nanoparticles to ischemic skeletal muscle. We also showed tunable nanoparticle targeting through altering the packing density of enzyme cleavable peptide and the surface charge of the nanoparticles.