Agneta Simionescu

Stabilized Collagen Scaffolds for Heart Valve Tissue Engineering

Scaffolds for heart valve tissue engineering must function immediately after implantation but also need to tolerate cell infiltration and gradual remodeling. We hypothesized that moderately cross-linked collagen scaffolds would fulfill these requirements. To test our hypothesis, scaffolds prepared from decellularized porcine pericardium were treated with penta-galloyl glucose (PGG), a collagen-binding polyphenol, and tested for biodegradation, biaxial mechanical properties, and in vivo biocompatibility. For controls, we used un-cross-linked scaffolds and glutaraldehyde-treated scaffolds. Results confirmed complete pericardium decellularization and the ability of scaffolds to encourage fibroblast chemotaxis and to aid in creation of anatomically correct valve-shaped constructs. Glutaraldehyde cross-linking fully stabilized collagen but did not allow for tissue remodeling and calcified when implanted subdermally in rats. PGG-treated collagen was initially resistant to collagenase and then degraded gradually, indicating partial stabilization. Moreover, PGG-treated pericardium exhibited excellent biaxial mechanical properties, did not calcify in vivo, and supported infiltration by host fibroblasts and subsequent matrix remodeling. In conclusion, PGG-treated acellular pericardium is a promising scaffold for heart valve tissue engineering.

Biochemical Pathways of Tissue Degeneration in Bioprosthetic Cardiac Valves; The Role of Matrix Metalloproteinases

Degeneration processes that affect bioprosthetic heart valves made from glutaraldehyde treated bovine pericardium are poorly understood. The present study undertook the identification and characterization of matrix metalloproteinases (MMPs) in extracts obtained from 28 pericardial derived bioprosthetic heart valves explanted at surgery. A lysosomal marker was used to assess the incidence of infiltrating extracellular matrix degrading cells. The major biochemical features that were associated with tissue degeneration and bioprosthetic heart valve failure were increased levels of MMP 9, high levels of beta-glucuronidase, and constant levels of active collagenase and MMP 2. The MMPs extracted from ruptured bioprostheses were inhibited by calcium chelators and zinc binding compounds. These data suggest that tissue failure, in addition to known mechanical and calcification related factors, may be contributed to by the intervention of proteolytic enzymes. A schematic working model was proposed that described the major biochemical pathways underlying tissue degeneration, starting from bioprostheses preparation and ending with clinical failure.

Small noncytotoxic carbon nano-onions: First covalent functionalization with biomolecules

Small carbon nano-onions (CNOs, 6-8 shells) were prepared in high yield and functionalized with carboxylic groups by chemical oxidation. After functionalization these nanostructures were soluble in aqueous solutions. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 tetrazolium (MTS) tests showed excellent cytocompatibility of all CNOs analyzed at 30 and 300 microg mL(-1), so these carbon nanostructures can be safely used for biological applications. The first covalent functionalization of oxidized CNOs (ox-CNOs) with biomolecules, by using biotin-avidin interactions is reported here. Multilayers were prepared on a gold surface by layer-by-layer assembly and the process was monitored by surface plasmon resonance (SPR) spectroscopy and atomic force microscopy (AFM). Covalent binding of molecules to the short amine-terminated organosulfur monolayers was assessed by Fourier transform infrared spectroscopy using total attenuated reflactance mode (FT-IR/HATR).

Inflammation in Cardiovascular Tissue Engineering: The Challenge to a Promise: A Minireview

Tissue engineering employs scaffolds, cells, and stimuli brought together in such a way as to mimic the functional architecture of the target tissue or organ. Exhilarating advances in tissue engineering and regenerative medicine allow us to envision in vitro creation or in vivo regeneration of cardiovascular tissues. Such accomplishments have the potential to revolutionize medicine and greatly improve our standard of life. However, enthusiasm has been hampered in recent years because of abnormal reactions at the implant-host interface, including cell proliferation, fibrosis, calcification and degeneration, as compared to the highly desired healing and remodeling. Animal and clinical studies have highlighted uncontrolled chronic inflammation as the main cause of these processes. In this minireview, we present three case studies highlighting the importance of inflammation in tissue engineering heart valves, vascular grafts, and myocardium and propose to focus on the endothelial barrier, the “final frontier” endowed with the natural potential and ability to regulate inflammatory signals.

Janus magnetic cellular spheroids for vascular tissue engineering.

Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly.

The performance of cross-linked acellular arterial scaffolds as vascular grafts; pre-clinical testing in direct and isolation loop circulatory models.

There is a significant need for small diameter vascular grafts to be used in peripheral vascular surgery; however autologous grafts are not always available, synthetic grafts perform poorly and allografts and xenografts degenerate, dilate and calcify after implantation. We hypothesized that chemical stabilization of acellular xenogenic arteries would generate off-the-shelf grafts resistant to thrombosis, dilatation and calcification. To test this hypothesis, we decellularized porcine renal arteries, stabilized elastin with penta-galloyl glucose and collagen with carbodiimide/activated heparin and implanted them as transposition grafts in the abdominal aorta of rats as direct implants and separately as indirect, isolation-loop implants. All implants resulted in high patency and animal survival rates, ubiquitous encapsulation within a vascularized collagenous capsule, and exhibited lack of lumen thrombogenicity and no graft wall calcification. Peri-anastomotic neo-intimal tissue overgrowth was a normal occurrence in direct implants; however this reaction was circumvented in indirect implants. Notably, implantation of non-treated control scaffolds exhibited marked graft dilatation and elastin degeneration; however PGG significantly reduced elastin degradation and prevented aneurismal dilatation of vascular grafts. Overall these results point to the outstanding potential of crosslinked arterial scaffolds as small diameter vascular grafts.

Matrix metalloproteinases in the pathology of natural and bioprosthetic cardiac valves

Abstract Degenerative dysfunction of cardiac valves may be accounted for by uncontrolled extracellular matrix degradation processes in which matrix metalloproteinases could play a major role. In this study, 24 pathologic human valves and 26 pericardial-derived bioprostheses were analysed for metalloproteinases by gelatin zymography. Compared to controls, human stenotic valves and bioprostheses explanted because of either calcifying or noncalcifying degeneration revealed three notable biochemical aspects: (1) an amplification in the levels of metalloproteinase 9 (gelatinase B), suggestive of its active role in valvular pathology; (2) minimal modifications in the gelatinolytic levels of metalloproteinase 2 (gelatinase A), indicative of a constitutive secretion; and (3) activation products derived from both gelatinase A and B. All gelatinolytic activities identified in pathologic specimens were inhibited in vitro by zinc and calcium chelators (captopril, doxycycline, dithiothreitol, and ethylenediaminotetraacetic acid), suggesting potential therapeutic approaches. High levels of beta-glucuronidase (a lysosomal marker enzyme for phagocytic cells) were found in human calcified stenotic valves and in ruptured and calcified pericardial-derived bioprostheses. Mononuclear recruitment was minimal to moderate in pathologic human valves, and in noncalcified ruptured bioprostheses infiltrating mononuclear cells were concentrated in large numbers at the cuspal free edge. These findings suggest the involvement of infiltrating phagocytic cells and putative common mechanisms in the degeneration of both the natural and the bioprosthetic valvular extracellular matrix (ECM).

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.

Immunomodulatory effects of adipose tissue-derived stem cells on elastin scaffold remodeling in diabetes

Diabetes is a major risk factor for the progression of vascular disease, contributing to elevated levels of glycoxidation, chronic inflammation and calcification. Tissue engineering emerges as a potential solution for the treatment of vascular diseases however there is a considerable gap in the understanding of how scaffolds and stem cells will perform in patients with diabetes. We hypothesized that adipose tissue-derived stem cells (ASCs) by virtue of their immunosuppressive potential would moderate the diabetes-intensified inflammatory reactions and induce positive construct remodeling. To test this hypothesis, we prepared arterial elastin scaffolds seeded with autologous ASCs and implanted them subdermally in diabetic rats and compared inflammatory markers, macrophage polarization, matrix remodeling, calcification and bone protein expression to control scaffolds implanted with and without cells in nondiabetic rats. ASC-seeded scaffolds exhibited lower levels of CD8+ T-cells and CD68+ pan-macrophages and higher numbers of M2 macrophages, smooth muscle cell-like and fibroblast-like cells. Calcification and osteogenic markers were reduced in ASCseeded scaffolds implanted in non-diabetic rats but remained unchanged in diabetes, unless the scaffolds were first pre-treated with penta-galloyl glucose (PGG), a known anti-oxidative elastin-binding polyphenol. In conclusion, autologous ASC seeding in elastin scaffolds is effective in combating diabetes-related complications. To prevent calcification, the oxidative milieu needs to be reduced by elastin-binding antioxidants such as PGG.

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.