Structural and biomechanical characterizations of porcine myocardial extracellular matrix.

Extracellular matrix (ECM) of myocardium plays an important role to maintain a multilayered helical architecture of cardiomyocytes. In this study, we have characterized the structural and biomechanical properties of porcine myocardial ECM. Fresh myocardium were decellularized in a rotating bioreactor using 0.1 % sodium dodecyl sulfate solution. Masson's trichrome staining and SEM demonstrated the removal of cells and preservation of the interconnected 3D cardiomyocyte lacunae. Movat's pentachrome staining showed the preservation of cardiac elastin ultrastructure and vascular elastin distribution/alignment. DNA assay result confirmed a 98.59 % reduction in DNA content; the acellular myocardial scaffolds were found completely lack of staining for the porcine α-Gal antigen; and the accelerating enzymatic degradation assessment showed a constant degradation rate. Tensile and shear properties of the acellular myocardial scaffolds were also evaluated. Our observations showed that the acellular myocardial ECM possessed important traits of biodegradable scaffolds, indicating the potentials in cardiac regeneration and whole heart tissue engineering.

Assembly and testing of stem cell-seeded layered collagen constructs for heart valve tissue engineering.

Tissue engineering holds great promise for treatment of valvular diseases. Despite excellent progress in the field, current approaches do not fully take into account each patient's valve anatomical uniqueness, the presence of a middle spongiosa cushion that allows shearing of external fibrous layers (fibrosa and ventricularis), and the need for autologous valvular interstitial cells. In this study we propose a novel approach to heart valve tissue engineering based on bioreactor conditioning of mesenchymal stem cell-seeded, valve-shaped constructs assembled from layered collagenous scaffolds. Fibrous scaffolds were prepared by decellularization of porcine pericardium and spongiosa scaffolds by decellularization and elastase treatment of porcine pulmonary arteries. To create anatomically correct constructs, we created silicone molds from native porcine aortic valves, dried two identical fibrous scaffolds onto the molds, and stabilized them with penta-galloyl-glucose a reversible collagen-binding polyphenol that reduces biodegradation. The layers were fused with a protein/aldehyde scaffold bio-adhesive and neutralized to reduce cytotoxicity. Spongiosa scaffolds, seeded with human bone marrow-derived stem cells, were inserted within the valve-shaped layered scaffolds and sutured inside the original aortic root. The final product was mounted in a heart valve bioreactor and cycled in cell culture conditions. Most cells were alive after 8 days, elongated significantly, and stained positive for vimentin, similar to native human valvular interstitial cells, indicating feasibility of our approach.

Lectin and antibody-based histochemical techniques for cardiovascular tissue engineering.

Tissue engineering holds immense potential for treatment of cardiovascular diseases by creating living structures to replace diseased blood vessels, heart valves, and cardiac muscle. In a traditional approach, scaffolds are seeded with stem cells and subjected to stimuli in bioreactors that mimic physiologic conditions or are directly implanted into target sites in animal models. The expected results are significant cell changes, extensive remodeling of the scaffolds and creation of surrogate structures that would be deemed acceptable for tissue regeneration. Histochemical techniques are increasingly becoming essential tools in tissue engineering research. In our studies, we used lectin and antibody-based techniques to characterize novel collagen and elastin scaffolds and to ensure efficient removal of xenoantigens. Scaffolds were implanted in animals and infiltrated host cells were identified using antibodies to activated fibroblasts, macrophages, and lymphocytes. Stem cell-seeded scaffolds were subjected to mechanical strains and tested for differentiation into cardiovascular cells using antibody-based double immunofluorescence methods. Finally, living heart valves were constructed from scaffolds and stem cells, subjected to conditioning in a bioreactor and stem cell differentiation evaluated by immunofluorescence. Overall, these techniques have proven to be outstanding companions to biochemical, molecular biology and cell analysis methods used in tissue engineering research and development.

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.