AGNETA SIMIONESCU, PhD
Assistant Professor of Bioengineering
Stem Cell Differentiation and Cardiovascular Tissue Engineering in Diabetes
Tissue engineered constructs based on scaffolds and autologous progenitor cells are currently being developed1, but very little information exists regarding the fate of tissue engineered devices in the compromised patient, and more specifically in diabetic environments. Diabetes is a major risk factor for cardiovascular diseases and diabetics have a significantly greater frequency of cardiovascular disorders. As a consequence, diabetics are more prone to undergo surgery for repair or replacement of tissues such as blood vessels. Diabetes is characterized by elevated levels of glucose, which interacts irreversibly with proteins, lipids and nucleic acids via oxidation and crosslinking processes, resulting in formation of advanced glycosylation end products (AGEs). Glycoxidation induces severe cell and matrix alterations that result in endothelial dysfunction, accelerated atherosclerosis, activation of inflammation, fibrosis, impaired healing and ectopic calcifications all of which are not conducive to the desired integration and remodeling of tissue engineered constructs.
Three specific aims will be pursued:
Aim 1: To identify biochemical and mechanical alterations of vascular scaffolds in diabetes.
Hypothesis: Diabetes induces chemical crosslinking and stiffening of the collagen-elastin based biological scaffolds.
Approach: Normal and diabetic Yucatan minipigs will be used for these studies. Small diameter vascular scaffolds will be prepared by complete decellularization of adult porcine femoral arteries obtained from local abattoirs. Vascular scaffolds will be intra-luminally seeded with autologous endothelial progenitor cells (to avoid thrombogenicity) and implanted as femoro-popliteal bypass grafts in normal and diabetic minipigs. Grafts will be monitored for up to 6 months for patency and thrombogenicity, endothelial dysfunction, AGE formation, calcification, and mechanical properties.
Aim 2: To mitigate diabetes-induced alterations of vascular scaffolds by treating them with PGG.
Hypothesis: PGG protects ECM proteins from irreversible cross-link formation, by virtue of its anti-oxidative and matrix-binding properties.
Approach: Small diameter vascular scaffolds prepared by decellularization of adult porcine femoral arteries will be treated with PGG, intra-luminally seeded with autologous endothelial progenitor cells, and implanted as femoro-popliteal bypass grafts in normal and diabetic Yucatan minipigs. Grafts will be monitored for up to 6 months AGE formation, mechanical properties, biocompatibility, and calcification, as well as for patency, thrombogenicity, and endothelial dysfunction.
Aim 3: To maturate the vascular constructs in vitro in order to attain adequate remodeling after implantation.
Hypothesis: In vitro bioreactor conditions, that provide controlled stimuli for cell differentiation and construct maturation, will prevent later adverse remodeling in diabetic animals.
Approach: PGG-treated vascular scaffolds seeded with autologous adipose-derived stem cells within the arterial media and the adventitia and endothelialized with autologous endothelial progenitor cells, will be exposed to controlled biomolecules and physical forces in a pulsatile bioreactor before implantation in normal and diabetic minipigs as femoro-popliteal bypass grafts. Grafts will be monitored for up to 6 months for patency and thrombogenicity, endothelial activation and dysfunction, stem cell differentiation into tunic-specific cells, matrix remodeling, stiffening, calcification, and integration; results will be compared to control grafts implanted without prior bioreactor conditioning.
My long-term goal is to develop constructs adapted to withstand high glycoxidation stress for translational and clinical applications. My working hypothesis is that both scaffolds and cells are susceptible to diabetes-induced complications and that stabilization of scaffolds would improve the outcome of tissue engineering in diabetes. To test this hypothesis we propose to test constructs and autologous stem cells in vitro and in vivo diabetic models and compare their properties to non-diabetic control environments.
There is a major need for tissue engineered vascular grafts with long-term patency and durability to be used in diabetic cardiovascular patients. Tissue engineered constructs based on scaffolds and autologous progenitor cells are currently being developed, but very little information exists regarding their fate in diabetic environments. Diabetes is characterized by elevated levels of blood glucose, which interacts irreversibly with proteins, lipids and nucleic acids via oxidation and crosslinking processes, resulting in formation of advanced glycosylation end products (AGEs). Glycoxidation induces severe cell and matrix alterations that result in endothelial dysfunction, activation of inflammation, fibrosis and impaired healing, all of which are not conducive to the desired integration and remodeling of tissue engineered constructs. My long-term goal is to develop constructs adapted to withstand high glucose and oxidative stress typical of diabetes. My working hypothesis is that both scaffolds and cells are susceptible to diabetes-induced complications and that chemical stabilization of scaffolds would improve the outcome of tissue engineering in diabetes. To test this hypothesis I propose to test constructs in large animal diabetic models and compare their properties to non-diabetic environments.
I have shown that experimental diabetes in rats significantly affects extracellular matrix-derived scaffolds and that treatment with a matrix-binding polyphenolic antioxidant stabilizes the scaffolds and renders them “diabetes-resistant”, without impeding on stem cell seeding, infiltration and matrix remodeling after subdermal implantation in diabetic rats.
Scaffolds will be prepared from decellularized vascular tissues, treated with polyphenol stabilizing agents, and implanted in normal and diabetic Yucatan mini-pigs. Adipose tissue derived stem cells isolated from mini-pig fat will be seeded on scaffolds and the constructs will be mounted in bioreactors, with normal and high glucose concentration.
Tissue-Engineered Myocardium for Replacement of Myocardial Infarct Scar Tissue
Myocardial infarction affects nearly 600,000 new individuals each year. Damage from myocardial infarction initiates a pathophysiological progression towards congestive heart failure in over a quarter of these patients. In order to forestall congestive heart failure, therapies must target the cause of the maladaptive cardiac remodeling which precedes it—the akinetic infarct scar—and replace it with functional cardiac muscle. Tissue engineering holds promise for halt or reverse post-myocardial infarct cardiac remodeling, but early efforts to develop and implant thick (> 200 µm), biomimetic, and functional grafts have been hindered by insufficient vascularization. Therefore, our long-term goal is to fulfill the need for thick, fully vascularized, and functional tissue-engineered myocardial grafts to replace infarct scar tissue.
In preliminary studies, we generated a myocardial flap scaffold by removing the cells from porcine left-ventricular myocardium and its associated coronary arteries and veins. The scaffold displayed a fully intact and patent vasculature to the level of capillaries, was devoid of cellular components, retained collagen, elastin, and basal lamina structures; in addition, the scaffold exhibited excellent mechanical properties, was compatible towards seeded cells, and degraded within 4-6 weeks when implanted sub dermally in rats. Encouraged by these results, we are now proposing studies focused upon stabilizing the scaffolds to reduce degradation rate, seeding them with stem cells, and in vitro conditioning the resultant constructs into mature, functional grafts, before testing their potential in replacing infarct scar tissue in a rabbit infarct model.
Aim 1: To adjust in vivo degradation rate of myocardial scaffolds.
Hypothesis: Scaffold treatment with penta-galloyl glucose (PGG), a plant-derived, antioxidative, and matrix-binding polyphenol, will stabilize extracellular matrix components, allowing 50% of the original mass to be retained at 8 weeks post-implantation.
Approach: Acellular myocardial scaffolds will be treated with different concentrations of PGG and implanted subdermally in rats. At 4 and 8 weeks post-implantation, scaffolds will be explanted, weighed, and examined histologically to characterize the host response, as well as the rate and extent of degradation; results will be compared to non PGG-treated scaffolds.
Aim 2: To condition stem cell-seeded myocardial constructs in vitro into functional grafts.
Approach: Scaffolds will be seeded first with fibroblasts to generate “living scaffolds”; rabbit adipose-derived stem cells and endothelial progenitor cell seeding will follow. Then constructs will be accommodated in a bioreactor and exposed to controlled biophysical and biochemical conditions (pressures, electrical stimulation, and media compositions) for 4 weeks to induce stem cell differentiation into electrically integrated and synchronously contracting cardiac myocytes. We will analyze the conditioned grafts through histology, gene and protein expression, and by measuring characteristics of excitation-contraction coupling, such as excitation thresholds, fractional shortening, and force generation; results will be compared to no stimulated control constructs.
Aim 3: To demonstrate functional benefits of infarct scar replacement with functional grafts.
Hypothesis: Replacement of the infarct scar tissue with a functional myocardial graft will halt or reverse ventricular remodeling.
Approach: Coronary ligation in rabbits will be used to establish an infarct model. At 4 weeks post-injury, the infarct scar will be resected and replaced with the pre-conditioned functional myocardial graft). The major vessels will be anastomosed to the host vasculature, and the flap-like portion of the graft will be affixed adjacent to the exposed viable myocardium with fibrin glue and sutures. Using echocardiography, animals will be monitored up to 1 year for changes in cardiac function metrics, such as ejection fraction, left ventricular diameter, and stroke volume. During this period, several grafts will be explanted at 4 week intervals and examined histologically, biochemically, and mechanically for evidence of integration with host tissue. Results will be compared to untreated and sham-operated control groups of animals.
Expectation and Impact: This research harnesses the potential of recent discoveries in stem cell biology, cell-extracellular matrix biochemistry and biology, and tissue engineering in an effort to develop a meaningful therapy and advance it toward practical application in the clinic. By completion of this project, we expect to determine the feasibility of a translational approach which could have far-reaching effects upon efforts to treat a growing population of patients suffering from the devastating consequences of coronary heart disease. Our approach holds promise for enabling clinicians to prevent or reverse the progression towards congestive heart failure, not only saving lives, but also improving the quality of life for recipients of this therapy.