Myocardial Matrix Hydrogels Mitigate Negative Remodeling and Improve Function in Right Heart Failure Model.

This study evaluates the effectiveness of myocardial matrix (MM) hydrogels in mitigating negative right ventricular (RV) remodeling in a rat model of RV heart failure. The goal was to assess whether a hydrogel derived from either the right or left ventricle could promote cardiac repair. Injured rat right ventricles were injected with either RV-or left ventricular-derived MM hydrogels. Both hydrogels improved RV function and morphology and reduced negative remodeling. This study supports the potential of injectable biomaterial therapies for treating RV heart failure.

Myocardial matrix hydrogel acts as a reactive oxygen species scavenger and supports a proliferative microenvironment for cardiomyocytes.

As the native regenerative potential of adult cardiac tissue is limited post-injury, stimulating endogenous repair mechanisms in the mammalian myocardium is a potential goal of regenerative medicine therapeutics. Injection of myocardial matrix hydrogels into the heart post-myocardial infarction (MI) has demonstrated increased cardiac muscle and promotion of pathways associated with cardiac development, suggesting potential promotion of cardiomyocyte turnover. In this study, the myocardial matrix hydrogel was shown to have native capability as an effective reactive oxygen species scavenger and protect against oxidative stress induced cell cycle inhibition in vitro. Encapsulation of cardiomyocytes demonstrated an enhanced turnover in in vitro studies, and in vivo assessments of myocardial matrix hydrogel treatment post-MI showed increased thymidine analog uptake in cardiomyocyte nuclei compared to saline controls. Overall, this study provides evidence that properties of the myocardial matrix material provide a microenvironment mitigating oxidative damage and supportive of cardiomyocytes undergoing DNA synthesis, toward possible DNA repair or cell cycle activation. STATEMENT OF SIGNIFICANCE: Loss of adult mammalian cardiomyocyte turnover is influenced by shifts in oxidative damage, which represents a potential mechanism for improving restoration of cardiac muscle after myocardial infarction (MI). Injection of a myocardial matrix hydrogel into the heart post-MI previously demonstrated increased cardiac muscle and promotion of pathways associated with cardiac development, suggesting potential in promoting proliferation of cardiomyocytes. In this study, the myocardial matrix hydrogel was shown to protect cells from oxidative stress and increase proliferation in vitro. In a rat MI model, greater presence of tissue free thiol content spared from oxidative damage, lesser mitochondrial superoxide content, and increased thymidine analog uptake in cardiomyocytes was found in matrix injected animals compared to saline controls. Overall, this study provides evidence that properties of the myocardial matrix material provide a microenvironment supportive of cardiomyocytes undergoing DNA synthesis, toward possible DNA repair or cell cycle activation.

Injectable ECM Scaffolds for Cardiac Repair.

Injectable biomaterials have been developed as potential minimally invasive therapies for treating myocardial infarction (MI) and heart failure. Christman et al. first showed that the injection of a biomaterial alone into rat myocardium can improve cardiac function after MI. More recently, hydrogel forms of decellularized extracellular matrix (ECM) materials have shown substantial promise. Here, we present the methods for fabricating an injectable cardiac-specific ECM biomaterial with demonstrated positive outcomes in small and large animal models for cardiac repair as well as initial safety in a Phase I clinical trial. This chapter also covers the methods for the injection of a biomaterial into rat myocardium using a surgical approach through the diaphragm. Although the methods shown here are for injection of an acellular biomaterial, cells or other therapeutics could also be added to the injection for testing other regenerative medicine strategies.

Preventing post-surgical cardiac adhesions with a catechol-functionalized oxime hydrogel.

Post-surgical cardiac adhesions represent a significant problem during routine cardiothoracic procedures. This fibrous tissue can impair heart function and inhibit surgical access in reoperation procedures. Here, we propose a hydrogel barrier composed of oxime crosslinked poly(ethylene glycol) (PEG) with the inclusion of a catechol (Cat) group to improve retention on the heart for pericardial adhesion prevention. This three component system is comprised of aldehyde (Ald), aminooxy (AO), and Cat functionalized PEG mixed to form the final gel (Ald-AO-Cat). Ald-AO-Cat has favorable mechanical properties, degradation kinetics, and minimal swelling, as well as superior tissue retention compared to an initial Ald-AO gel formulation. We show that the material is cytocompatible, resists cell adhesion, and led to a reduction in the severity of adhesions in an in vivo rat model. We further show feasibility in a pilot porcine study. The Ald-AO-Cat hydrogel barrier may therefore serve as a promising solution for preventing post-surgical cardiac adhesions.

Injectable Myocardial Matrix Hydrogel Mitigates Negative Left Ventricular Remodeling in a Chronic Myocardial Infarction Model.

A first-in-man clinical study on a myocardial-derived decellularized extracellular matrix hydrogel suggested the potential for efficacy in chronic myocardial infarction (MI) patients. However, little is understood about the mechanism of action in chronic MI. In this study, the authors investigated the efficacy and mechanism by which the myocardial matrix hydrogel can mitigate negative left ventricular (LV) remodeling in a rat chronic MI model. Assessment of cardiac function via magnetic resonance imaging demonstrated preservation of LV volumes and apical wall thickening. Differential gene expression analyses showed the matrix is able to prevent further negative LV remodeling in the chronic MI model through modulation of the immune response, down-regulation of pathways involved in heart failure progression and fibrosis, and up-regulation of genes important for cardiac muscle contraction.

Dose optimization of decellularized skeletal muscle extracellular matrix hydrogels for improving perfusion and subsequent validation in an aged hindlimb ischemia model.

Peripheral artery disease (PAD) affects more than 27 million individuals in North America and Europe, and current treatment strategies mainly aim to restore blood perfusion. However, many patients are ineligible for existing procedures, and these therapies are often ineffective. Previous studies have demonstrated success of an injectable decellularized skeletal muscle extracellular matrix (ECM) hydrogel in a young rat hindlimb ischemia model of PAD, but further pre-clinical studies are necessary prior to clinical translation. In this study, varying concentrations of a skeletal muscle ECM hydrogel were investigated for material properties and in vivo effects on restoring blood perfusion. Rheological measurements indicated an increase in viscosity and mechanical strength with the higher concentrations of the ECM hydrogels. When injecting dye-labelled ECM hydrogels into a healthy rat, differences were also observed for the spreading and degradation rate of the various concentrations. The three concentrations for the ECM hydrogel were then further examined in a young rat hindlimb ischemia model. The efficacy of the optimal ECM hydrogel concentration was then further confirmed in an aged mouse hindlimb ischemia model. These results further validate the use of decellularized skeletal muscle ECM hydrogels for improving blood perfusion in small animal models of PAD.

Enzyme-responsive progelator cyclic peptides for minimally invasive delivery to the heart post-myocardial infarction.

Injectable biopolymer hydrogels have gained attention for use as scaffolds to promote cardiac function and prevent negative left ventricular (LV) remodeling post-myocardial infarction (MI). However, most hydrogels tested in preclinical studies are not candidates for minimally invasive catheter delivery due to excess material viscosity, rapid gelation times, and/or concerns regarding hemocompatibility and potential for embolism. We describe a platform technology for progelator materials formulated as sterically constrained cyclic peptides which flow freely for low resistance injection, and rapidly assemble into hydrogels when linearized by disease-associated enzymes. Their utility in vivo is demonstrated by their ability to flow through a syringe and gel at the site of MI in rat models. Additionally, synthetic functionalization enables these materials to flow through a cardiac injection catheter without clogging, without compromising hemocompatibility or cytotoxicity. These studies set the stage for the development of structurally dynamic biomaterials for therapeutic hydrogel delivery to the MI.

Engineering an Injectable Muscle-Specific Microenvironment for Improved Cell Delivery Using a Nanofibrous Extracellular Matrix Hydrogel.

Injection of skeletal muscle progenitors has the potential to be a minimally invasive treatment for a number of diseases that negatively affect vasculature and skeletal muscle, including peripheral artery disease. However, success with this approach has been limited because of poor transplant cell survival. This is primarily attributed to cell death due to extensional flow through the needle, the harsh ischemic environment of the host tissue, a deleterious immune cell response, and a lack of biophysical cues supporting exogenous cell viability. We show that engineering a muscle-specific microenvironment, using a nanofibrous decellularized skeletal muscle extracellular matrix hydrogel and skeletal muscle fibroblasts, improves myoblast viability and maturation in vitro. In vivo, this translates to improved cell survival and engraftment and increased perfusion as a result of increased vascularization. Our results indicate that a combinatorial delivery system, which more fully recapitulates the tissue microenvironment, can improve cell delivery to skeletal muscle.

Modulating In Vivo Degradation Rate of Injectable Extracellular Matrix Hydrogels.

Extracellular matrix (ECM) derived hydrogels are increasingly used as scaffolds to stimulate endogenous repair. However, few studies have examined how altering the degradation rates of these materials affect cellular interaction in vivo. This study sought to examine how crosslinking or matrix metalloproteinase (MMP) inhibition by doxycycline could be employed to modulate the degradation rate of an injectable hydrogel derived from decellularized porcine ventricular myocardium. While both approaches were effective in reducing degradation in vitro, only doxycycline significantly prolonged hydrogel degradation in vivo without affecting material biocompatibility. In addition, unlike crosslinking, incorporation of doxycycline into the hydrogel did not affect mechanical properties. Lastly, the results of this study highlighted the need for development of novel crosslinkers for in situ modification of injectable ECM-derived hydrogels, as none of the crosslinking agents investigated in this study were both biocompatible and effective.

Extracellular Matrix Hydrogel Promotes Tissue Remodeling, Arteriogenesis, and Perfusion in a Rat Hindlimb Ischemia Model.

OBJECTIVE: This study aimed to examine acellular extracellular matrix based hydrogels as potential therapies for treating peripheral artery disease (PAD). We tested the efficacy of using a tissue specific injectable hydrogel, derived from decellularized porcine skeletal muscle (SKM), compared to a new human umbilical cord derived matrix (hUC) hydrogel, which could have greater potential for tissue regeneration because of its young tissue source age. BACKGROUND: The prevalence of PAD is increasing and can lead to critical limb ischemia (CLI) with potential limb amputation. Currently there are no therapies for PAD that effectively treat all of the underlying pathologies, including reduced tissue perfusion and muscle atrophy. METHODS: In a rodent hindlimb ischemia model both hydrogels were injected 1-week post-surgery and perfusion was regularly monitored with laser speckle contrast analysis (LASCA) to 35 days post-injection. Histology and immunohistochemistry were used to assess neovascularization and muscle health. Whole transcriptome analysis was further conducted on SKM injected animals on 3 and 10 days post-injection. RESULTS: Significant improvements in hindlimb tissue perfusion and perfusion kinetics were observed with both biomaterials. End point histology indicated this was a result of arteriogenesis, rather than angiogenesis, and that the materials were biocompatible. Skeletal muscle fiber morphology analysis indicated that the muscle treated with the tissue specific, SKM hydrogel more closely matched healthy tissue morphology. Short term histology also indicated arteriogenesis rather than angiogenesis, as well as improved recruitment of skeletal muscle progenitors. Whole transcriptome analysis indicated that the SKM hydrogel caused a shift in the inflammatory response, decreased cell death, and increased blood vessel and muscle development. CONCLUSION: These results show the efficacy of an injectable ECM hydrogel alone as a potential therapy for treating patients with PAD. Our results indicate that the SKM hydrogel improved functional outcomes through stimulation of arteriogenesis and muscle progenitor cell recruitment.

Evidence for Mechanisms Underlying the Functional Benefits of a Myocardial Matrix Hydrogel for Post-MI Treatment.

BACKGROUND: There is increasing need for better therapies to prevent the development of heart failure after myocardial infarction (MI). An injectable hydrogel derived from decellularized porcine ventricular myocardium has been shown to halt the post-infarction progression of negative left ventricular remodeling and decline in cardiac function in both small and large animal models. OBJECTIVES: This study sought to elucidate the tissue-level mechanisms underlying the therapeutic benefits of myocardial matrix injection. METHODS: Myocardial matrix or saline was injected into infarcted myocardium 1 week after ischemia-reperfusion in Sprague-Dawley rats. Cardiac function was evaluated by magnetic resonance imaging and hemodynamic measurements at 5 weeks after injection. Whole transcriptome microarrays were performed on RNA isolated from the infarct at 3 days and 1 week after injection. Quantitative polymerase chain reaction and histologic quantification confirmed expression of key genes and their activation in altered pathways. RESULTS: Principal component analysis of the transcriptomes showed that samples collected from myocardial matrix-injected infarcts are distinct and cluster separately from saline-injected control subjects. Pathway analysis indicated that these differences are due to changes in several tissue processes that may contribute to improved cardiac healing after MI. Matrix-injected infarcted myocardium exhibits an altered inflammatory response, reduced cardiomyocyte apoptosis, enhanced infarct neovascularization, diminished cardiac hypertrophy and fibrosis, altered metabolic enzyme expression, increased cardiac transcription factor expression, and progenitor cell recruitment, along with improvements in global cardiac function and hemodynamics. CONCLUSIONS: These results indicate that the myocardial matrix alters several key pathways after MI creating a pro-regenerative environment, further demonstrating its promise as a potential post-MI therapy.

Cardiac-Derived Extracellular Matrix Enhances Cardiogenic Properties of Human Cardiac Progenitor Cells.

The use of biomaterials has been demonstrated as a viable strategy to promote cell survival and cardiac repair. However, limitations on combinational cell-biomaterial therapies exist, as cellular behavior is influenced by the microenvironment and physical characteristics of the material. Among the different scaffolds employed for cardiac tissue engineering, a myocardial matrix hydrogel has been shown to promote cardiogenesis in murine cardiac progenitor cells (mCPCs) in vitro. In this study, we investigated the influence of the hydrogel on Sca-1-like human fetal and adult CPCs (fCPCs and aCPCs) when encapsulated in three-dimensional (3D) material in vitro. fCPCs encapsulated in the myocardial matrix showed an increase in the gene expression level of cardiac markers GATA-4 and MLC2v and the vascular marker vascular endothelial growth factor receptor 2 (VEGFR2) after 4 days in culture, and a significant increase in GATA-4 up to 1 week. Increased gene expression levels of Nkx2.5, MEF2c, VEGFR2, and CD31 were also observed when aCPCs were cultured in the matrix compared to collagen. Cell survival was sustained in both hydrogels up to 1 week in culture with the myocardial matrix capable of enhancing the expression of the proliferation marker Ki-67 after 4 days in culture. When encapsulated CPCs were treated with H2O2, an improved survival of the cells cultured in the myocardial matrix was observed. Finally, we evaluated the use of the myocardial matrix as hydrogel for in vivo cell transplantation and demonstrated that the gelation properties of the hydrogel are not influenced by the cells. In summary, we showed that the myocardial matrix hydrogel promotes human CPC cardiogenic potential, proliferation, and survival and is a favorable hydrogel for 3D in vitro culture. Furthermore, we demonstrated the in vivo applicability of the matrix as a potential vehicle for cell transplantation.

Degradable acetalated dextran microparticles for tunable release of an engineered hepatocyte growth factor fragment.

Injectable biomaterials are promising as new therapies to treat myocardial infarction (MI). One useful property of biomaterials is the ability to protect and sustain release of therapeutic payloads. In order to create a platform for optimizing the release rate of cardioprotective molecules we utilized the tunable degradation of acetalated dextran (AcDex). We created microparticles with three distinct degradation profiles and showed that the consequent protein release profiles could be modulated within the infarcted heart. This enabled us to determine how delivery rate impacted the efficacy of a model therapeutic, an engineered hepatocyte growth factor fragment (HGF-f). Our results showed that the cardioprotective efficacy of HGF-f was optimal when delivered over three days post-intramyocardial injection, yielding the largest arterioles, fewest apoptotic cardiomyocytes bordering the infarct and the smallest infarcts compared to empty particle treatment four weeks after injection. This work demonstrates the potential of using AcDex particles as a delivery platform to optimize the time frame for delivering therapeutic proteins to the heart.

Intramyocardial injection of hydrogel with high interstitial spread does not impact action potential propagation.

Injectable biomaterials have been evaluated as potential new therapies for myocardial infarction (MI) and heart failure. These materials have improved left ventricular (LV) geometry and ejection fraction, yet there remain concerns that biomaterial injection may create a substrate for arrhythmia. Since studies of this risk are lacking, we utilized optical mapping to assess the effects of biomaterial injection and interstitial spread on cardiac electrophysiology. Healthy and infarcted rat hearts were injected with a model poly(ethylene glycol) hydrogel with varying degrees of interstitial spread. Activation maps demonstrated delayed propagation of action potentials across the LV epicardium in the hydrogel-injected group when compared to saline and no-injection groups. However, the degree of the electrophysiological changes depended on the spread characteristics of the hydrogel, such that hearts injected with highly spread hydrogels showed no conduction abnormalities. Conversely, the results of this study indicate that injection of a hydrogel exhibiting minimal interstitial spread may create a substrate for arrhythmia shortly after injection by causing LV activation delays and reducing gap junction density at the site of injection. Thus, this work establishes site of delivery and interstitial spread characteristics as important factors in the future design and use of biomaterial therapies for MI treatment. STATEMENT OF SIGNIFICANCE: Biomaterials for treating myocardial infarction have become an increasingly popular area of research. Within the past few years, this work has transitioned to some large animals models, and Phase I & II clinical trials. While these materials have preserved/improved cardiac function the effect of these materials on arrhythmogenesis, which is of considerable concern when injecting anything into the heart, has yet to be understood. Our manuscript is therefore a first of its kind in that it directly examines the potential of an injectable material to create a substrate for arrhythmias. This work suggests that site of delivery and distribution in the tissue are important criteria in the design and development of future biomaterial therapies for myocardial infarction treatment.

Enzyme-Responsive Nanoparticles for Targeted Accumulation and Prolonged Retention in Heart Tissue after Myocardial Infarction.

A method for targeting to and retaining intravenously injected nanoparticles at the site of acute myocardial infarction in a rat model is described. Enzyme-responsive peptide-polymer amphiphiles are assembled as spherical micellar nanoparticles, and undergo a morphological transition from spherical-shaped, discrete materials to network-like assemblies when acted upon by matrix metalloproteinases (MMP-2 and MMP-9), which are up-regulated in heart tissue post-myocardial infarction.

Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction.

Hepatocyte growth factor (HGF) has been shown to have anti-fibrotic, pro-angiogenic, and cardioprotective effects; however, it is highly unstable and expensive to manufacture, hindering its clinical translation. Recently, a HGF fragment (HGF-f), an alternative c-MET agonist, was engineered to possess increased stability and recombinant expression yields. In this study, we assessed the potential of HGF-f, delivered in an extracellular matrix (ECM)-derived hydrogel, as a potential treatment for myocardial infarction (MI). HGF-f protected cardiomyocytes from serum-starvation and induced down-regulation of fibrotic markers in whole cardiac cell isolate compared to the untreated control. The ECM hydrogel prolonged release of HGF-f compared to collagen gels, and in vivo delivery of HGF-f from ECM hydrogels mitigated negative left ventricular (LV) remodeling, improved fractional area change (FAC), and increased arteriole density in a rat myocardial infarction model. These results indicate that HGF-f may be a viable alternative to using recombinant HGF, and that an ECM hydrogel can be employed to increase growth factor retention and efficacy.

Injectable ECM scaffolds for cardiac repair.

Injectable biomaterials have been developed as potential minimally invasive therapies for treating myocardial infarction (MI) and heart failure. Christman et al. first showed that the injection of a biomaterial alone into rat myocardium can improve cardiac function after MI (Christman et al. Tissue Eng 10:403-409, 2004). More recently, hydrogel forms of decellularized extracellular matrix (ECM) materials have shown substantial promise. Here we present the methods for fabricating an injectable cardiac specific ECM biomaterial shown to already have positive outcomes in small and large animal models for cardiac repair (Singelyn et al. Biomaterials 30:5409-5416, 2009; Singelyn et al. J Am Coll Cardiol 59:751-763, 2012; Seif-Naraghi et al. Sci Transl Med 5:173ra25, 2013). Also covered are the methods for the injection of a biomaterial into rat myocardium using a surgical approach through the diaphragm. Although the methods shown here are for injection of an acellular biomaterial, cells or other therapeutics could also be added to the injection for testing other regenerative medicine strategies.

Tunable protein release from acetalated dextran microparticles: a platform for delivery of protein therapeutics to the heart post-MI.

The leading cause of death in the United States is cardiovascular disease. The majority of these cases result from heart failure post-myocardial infarction (MI). We present data providing evidence for use of acetalated dextran (AcDex) microparticles as a delivery vehicle for therapeutics to the heart post-MI. We harnessed the tunable degradation and acid-sensitivity of AcDex in the design of microparticles for intramyocardial injection. The particles released a model protein, myoglobin, and a sensitive growth factor, basic fibroblast growth factor (bFGF), over a wide range of time frames (from days to weeks) based on the percentage of cyclic acetals in the AcDex, which was easily controlled with acetalation reaction time. The release was shown in low pH environments, similar to what is found in an infarcted heart. bFGF maintained activity after release from the microparticles. Finally, biocompatibility of the microparticles was assessed.

In vivo response to dynamic hyaluronic acid hydrogels.

Tissue-specific elasticity arises in part from developmental changes in extracellular matrix over time, e.g. ~10-fold myocardial stiffening in the chicken embryo. When this time-dependent stiffening has been mimicked in vitro with thiolated hyaluronic acid (HA-SH) hydrogels, improved cardiomyocyte maturation has been observed. However, host interactions, matrix polymerization, and the stiffening kinetics remain uncertain in vivo, and each plays a critical role in therapeutic applications using HA-SH. Hematological and histological analysis of subcutaneously injected HA-SH hydrogels showed minimal systemic immune response and host cell infiltration. Most importantly, subcutaneously injected HA-SH hydrogels exhibited time-dependent porosity and stiffness changes at a rate similar to hydrogels polymerized in vitro. When injected intramyocardially host cells begin to actively degrade HA-SH hydrogels within 1week post-injection, continuing this process while producing matrix to nearly replace the hydrogel within 1month post-injection. While non-thiolated HA did not degrade after injection into the myocardium, it also did not elicit an immune response, unlike HA-SH, where visible granulomas and macrophage infiltration were present 1month post-injection, likely due to reactive thiol groups. Altogether these data suggest that the HA-SH hydrogel responds appropriately in a less vascularized niche and stiffens as had been demonstrated in vitro, but in more vascularized tissues, in vivo applicability appears limited.

Oxime cross-linked injectable hydrogels for catheter delivery.

Catheter delivery of therapeutic materials is important for developing minimally invasive treatment approaches. However, the majority of injectable materials gel rapidly upon mixing and/or heating to body temperature. The application of an oxime cross-linked hydrogel system is demonstrated. The system has a broad range of tunable gelation rates, is capable of injection through a catheter, and exhibits rapid gelation upon injection into tissue.