Development of a completely biological tissue engineered heart valve.

Abstract

In the United States alone, over 100,000 heart valve replacement procedures are performed each year, with approximately 45% of patients below age 65. While current mechanical and bioprosthetic heart valves are viable options, they have several limitations. The most significant limitation is for pediatric patients, since neither of these valve types grow and remodel with the patient. Tissue engineering provides a methodology to create functional heart valves that can grow and remodel similar to native tissue once implanted. Several tissue engineering approaches have been proposed using decellularized native scaffolds, synthetic biopolymers, and biological polymers seeded with cells. Fibrin provides a scaffold to create tissue-engineered heart valves (TEHV) that are completely biological with an environment permissive for extracellular matrix (ECM) deposition. Previous research in our lab has demonstrated the feasibility of creating a fibrin-based TEHV with neonatal human dermal fibroblast (nHDF) that yields valve leaflets with structural and mechanical anisotropy similar to native leaflets. However, the TEHV had sub-optimal tensile mechanical properties and was thus unable to withstand physiological forces. The development of tissue can be accelerated by both chemical and mechanical stimulus. Previously, fibrin based TEHV were cultured with chemical stimulus in the form of growth factors supplemented in the culture medium resulting in improved ECM deposition by the cells; however, no mechanical stimulation was applied. Prior research in both our lab and by other researchers has shown cyclic stretching with constant strain amplitude is a method to stimulate remodeling of biological scaffolds seeded with cells. Initial experiments were conducted to evaluate the effect of cyclic stretching on fibrin-based tubular constructs seeded with porcine valve interstitial cells (PVIC) and nHDF. Cyclic stretching with 10% constant strain amplitude applied for 3 weeks led to modest improvement in tensile properties of the tubular constructs. We hypothesized that long-term cyclic stretching, as was used in this study, could induce cellular adaptation, minimizing the benefits of cyclic stretching. This hypothesis was tested in subsequent experiments using tubular constructs cultured with incremental strain amplitude cyclic stretching, with an average strain of 10% for 3 weeks. Both PVIC and nHDF seeded constructs exhibited a 2-fold improvement in ultimate tensile strength (UTS) and collagen density over samples conditioned with constant strain amplitude strteching. To verify that this was the result of a cellular response, phosphorylation of extracellular signal-regulated kinase (ERK) was measured by western blot. At 5 weeks, the phosphorylated ERK was 255% higher in incremental cyclic strained samples compared to constant strain samples. nHDF-seeded tubular constructs were also used to optimize the use of transforming growth factor beta (TGF-β). Studies showed that under cyclic stretching conditions, TGF-β has detrimental effects on total collagen deposition and collagen maturation. Western blot analysis showed a decrease in p-ERK signaling in TGF-β treated samples. However, TGF-β use demonstrated a benefit by increasing the elastin content of the tissue constructs. In subsequent experiments, a sequence of cyclic stretching and TGF-β supplementation was used to optimize tensile mechanical properties and elastin content of the engineered tissue. Based on the results with tubular constructs, a novel bioreactor was designed to apply controlled cyclic stretching to the fibrin-based TEHV. Briefly, the valve was mounted on two plastic end-pieces with elastic latex tube placed around TEHV. Using a reciprocating syringe pump, culture medium was cyclically pumped into the bioreactor. The root distension, which was determined by the stiffer latex was used as a control parameter, and in turn stretched the leaflets. A separate flowloop (connected to the bioreactor end-pieces) was used to control nutrient transport to the TEHV. Using an incremental strain amplitude stretching regime, fibrin-based TEHV were conditioned in the bioreactor for 3 weeks. Cyclically stretched valves (CS valve) had improved tensile properties and collagen deposition compared to statically-cultured valves. The mechanical stiffness (modulus) and anisotropy (measured as ratio of leaflet modulus in circumferential to radial directions) in the leaflets was comparable to native sheep pulmonary valve leaflets. Collagen organization/ maturation also improved in CS valves over statically-cultured valves as observed by picrosirius red staining of tissue crosssections. In addition, the CS valve root could withstand pressures of up to 150 mmHg and its compliance was comparable to that of the sheep pulmonary artery at physiological pressures. To assess in vivo remodeling TEHV were implanted in the pulmonary artery of two sheep for 4.5 weeks with the pulmonary valve either left intact or rendered incompetent by leaflet excision. Echocardiography immediately after implantation showed functional coapting leaflets, with normal right heart function. It was also performed just prior to explantation, revealing functional leaflets although with moderate regurgitation in both cases and a partial detachment of one leaflet from the root in one case. The explanted leaflets had thickness and tensile properties comparable the implanted leaflets. There was endothelialization on the lumenal surface of the TEHV root. These preliminary results are unprecedented for a TEHV developed from a biological scaffold; however, many issues remain to be surmounted. In further development of the TEHV with a fibrin scaffold, photo-cross linking of the fibrin gel was utilized as a method to stiffen the matrix, thereby inhibiting excessive cell-induced compaction. Preliminary studies with tubular constructs demonstrated reduced compaction of cross-linked fibrin gel during cyclic stretching with no effect on nHDF proliferation or deposited collagen. In addition, a preliminary investigation using blood outgrowth endothelial cells (BOEC) has been conducted to assess their adhesion to the remodeled TEHV surface. Studies showed BOEC adhesion and proliferation on remodeled fibrin surface creating a confluent layer after 4 days of culture. Successful seeding of sheep BOEC on the TEHV surface prior to implantation would reduce the risk of clotting. Overall, the studies presented in this dissertation advance the development of a completely biological tissue-engineered heart valve. These studies improve our understanding of the role of cyclic stretching in tissue remodeling and have furthered the science of mechnotransduction and tissue remodeling.