Leslie Sierad

Stabilized Collagen and Elastin-Based Scaffolds for Mitral Valve Tissue Engineering.

There is a significant clinical need for new approaches to treatment of mitral valve disease. The aim of this study was to develop a tissue-engineered mitral valve scaffold possessing appropriate composition and structure to ensure ideal characteristics of mitral valves, such as large orifice, rapid opening and closure, maintenance of mitral annulus-papillary muscle continuity, in vivo biocompatibility and extended durability. An extracellular matrix-based scaffold was generated, based on the native porcine mitral valve as starting material and a technique for porcine cell removal without causing damage to the matrix components. To stabilize these structures and slow down their degradation, acellular scaffolds were treated with penta-galloyl glucose (PGG), a well-characterized polyphenol with high affinity for collagen and elastin. Biaxial mechanical testing presented similar characteristics for the PGG-treated scaffolds compared to fresh tissues. The extracellular matrix components, crucial for maintaining the valve shape and function, were well preserved in leaflets, and in chordae, as shown by their resistance to collagenase and elastin. When extracted with strong detergents, the PGG-treated scaffolds released a reduced amount of soluble matrix peptides, compared to untreated scaffolds; this correlated with diminished activation of fibroblasts seeded on scaffolds treated with PGG. Cell-seeded scaffolds conditioned for 5 weeks in a valve bioreactor showed good cell viability. Finally, rat subdermal implantation studies showed that PGG-treated mitral valve scaffolds were biocompatible, nonimmunogenic, noninflammatory, and noncalcifying. In conclusion, a biocompatible mitral valve scaffold was developed, which preserved the biochemical composition and structural integrity of the valve, essential for its highly dynamic mechanical demands, and its biologic durability.

Bioreactor conditioning of valve scaffolds seeded internally with adult stem cells

The goal of this study was to test the hypothesis that stem cells, as a response to valve-specific extracellular matrix “niches” and mechanical stimuli, would differentiate into valvular interstitial cells (VICs). Porcine aortic root scaffolds were prepared by decellularization. After verifying that roots exhibited adequate hemodynamics in vitro, we seeded human adipose-derived stem cells (hADSCs) within the interstitium of the cusps and subjected the valves to in vitro pulsatile bioreactor testing in pulmonary pressures and flow conditions. As controls we incubated cell-seeded valves in a rotator device which allowed fluid to flow through the valves ensuring gas and nutrient exchange without subjecting the cusps to significant stress. After 24 days of conditioning, valves were analyzed for cell phenotype using immunohistochemistry for vimentin, alpha-smooth muscle cell actin (SMA) and prolyl-hydroxylase (PHA). Fresh native valves were used as immunohistochemistry controls. Analysis of bioreactor-conditioned valves showed that almost all seeded cells had died and large islands of cell debris were found within each cusp. Remnants of cells were positive for vimentin. Cell seeded controls, which were only rotated slowly to ensure gas and nutrient exchange, maintained about 50% of cells alive; these cells were positive for vimentin and negative for alpha-SMA and PHA, similar to native VICs. These results highlight for the first time the extreme vulnerability of hADSCs to valve-specific mechanical forces and also suggest that careful, progressive mechanical adaptation to valve-specific forces might encourage stem cell differentiation towards the VIC phenotype.

In Vivo Testing of Xenogeneic Acellular Aortic Valves Seeded with Stem Cells

The main treatment for valvular disease consists in surgical replacement with mechanical or biological artificial devices; these are excellent mid-term replacements which greatly improve the patient’s quality of life. However, long-term use of these devices (beyond 10-15 years) is associated with bleeding risks in the case of mechanical valves and degeneration, for biological valves (1). Heart valve tissue engineering, the science of combining scaffolds and cells has the potential to revolutionize the field of valve surgery by providing functional, durable, and viable valves. To date, several approaches have been tested using synthetic and natural scaffolds and a variety of cells and bioreactors, but overall most approaches have not been successful on the long term when tested in animals or human patients as pulmonary or aortic implants (1-4). We hypothesized that one potential approach would be based on well characterized xenogeneic acellular aortic valve root scaffolds seeded with autologous adipose-tissue derived stem cells (ASCs). To test this hypothesis, we prepared porcine acellular aortic roots, injected them with autologous ASCs and implanted them in the right ventricular outflow tract (RVOT) of juvenile sheep. Porcine valve roots were decellularized with detergents and enzymes using a pressurized perfusion system and then stabilized with polyphenols as described before (5). Male juvenile sheep weighing about 35 kg were used for this study. After acclimation, we collected inter-scapular adipose tissue from each animal, isolated ASCs using a published collagenase-based procedure (6) and propagated them in culture for 3-4 passages. We then mounted each acellular aortic root within a decellularized bovine pericardial tube and seeded each cusp with 4 million autologous ASCs in 200 ul PBS by injection into the base of each cusp. ASC-seeded valved conduits were then implanted under general anesthesia in the right ventricle outflow tract (RVOT) with full ligation of the native pulmonary artery. Six animals (n=6) underwent implantation with autologous ASC-seeded valves and one with a non-cell seeded valve as control. Postoperatively antalgics, antibiotics, and diuretics were administered for 5 days. Anticoagulant (Fraxiparine 2 x 0.3 ml) therapy was maintained for the first 30 days and anti-aggregant treatment (Aspenter 75 mg/day orally) for the entire length of the study with a target follow-up of 4 months. Monthly transthoracic echography was performed under mild sedation, monitoring the valve hemodynamics, leaflet mobility and thickness and evidence for right ventricular remodeling. All animal procedures were performed in accordance to the “Guide for the care and use of laboratory animals”, published by the NIH (NIH Publication No. 85-23, revised 1996) under an animal use protocol approved by the University of Medicine and Pharmacy Targu Mures Ethical Committee (AUP #8/04.02.2012).

Pulmonary heart valve replacement using stabilized acellular xenogeneic scaffolds; effects of seeding with autologous stem cells

Abstract Background: We hypothesized that an ideal heart valve replacement would be acellular valve root scaffolds seeded with autologous stem cells. To test this hypothesis, we prepared porcine acellular pulmonary valves, seeded them with autologous adipose derived stem cells (ADSCs) and implanted them in sheep and compared them to acellular valves. Methods: Fresh porcine pulmonary valve roots were decellularized with detergents and enzymes. ADSCs were isolated from subdermal fat and injected within the acellular cusps. Valves were then implanted in an extra-anatomic pulmonary position as RV to PA shunts: Group A (n=6) consisted of acellular valves and Group B (n=6) of autologous stem cell-seeded acellular xenografts. Sheep were followed up for 6 months by echocardiography and histologic analysis was performed on explanted valves. Results: Early evolution was favorable for both groups. All Group A animals had physiologic growth without any signs of heart failure and leaflets were found with preserved structure and mobility, lacking signs of thrombi, inflammation or calcification. Group B sheep however expressed signs of right ventricle failure starting at one month, accompanied by progressive regurgitation and right ventricle dilatation, and the leaflets were found covered with host tissue. No cells were found in any Group A or B explants. Conclusions: Acellular stabilized xenogeneic pulmonary valves are reliable, stable, non-immunogenic, non-thrombogenic and non-calcifying scaffolds with excellent hemodynamics. Seeding these scaffolds with autologous ADSCs was not conducive to tissue regeneration. Studies aimed at understanding these novel observations and further harnessing the potential of stem cells are ongoing.