Recapitulation of the embryonic cardiovascular progenitor cell niche.

Stem or progenitor cell populations are often established in unique niche microenvironments that regulate cell fate decisions. Although niches have been shown to be critical for the normal development of several tissues, their role in the cardiovascular system is poorly understood. In this study, we characterized the cardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signaling pathways and extracellular matrix (ECM) proteins that are crucial for CPC maintenance and expansion. We demonstrate that collagen IV (ColIV) and ß-catenin-dependent signaling are essential for maintaining and expanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated the impact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning technologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimensionality of these structures increased mouse embryonic stem cell differentiation into CPCs when compared to 2D control cultures, which was further enhanced by incorporation of ColIV into the substrates. Inhibiting p300-dependent ß-catenin signals with the small molecule IQ1 facilitated further expansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that can serve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology.

The use of three-dimensional nanostructures to instruct cells to produce extracellular matrix for regenerative medicine strategies.

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(epsilon-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.

Cardiomyopathy is associated with structural remodelling of heart valve extracellular matrix.

AIMS: To increase the supply, many countries harvest allograft valves from explanted hearts of transplant recipients with ischaemic (ICM) or dilated cardiomyopathy (DCM). This study determines the structural integrity of valves from cardiomyopathic hearts. METHODS AND RESULTS: Extracellular matrix (ECM) was examined in human valves obtained from normal, ICM, and DCM hearts. To confirm if ECM changes were directly related to the cardiomyopathy, we developed a porcine model of chronic ICM. Histology and immunohistostaining, as well as non-invasive multiphoton and second harmonic generation (SHG) imaging revealed marked disruption of ECM structures in human valves from ICM and DCM hearts. The ECM was unaffected in valves from normal and acute ICM pigs, whereas chronic ICM specimens showed ECM alterations similar to those seen in ICM and DCM patients. Proteins and proteinases implicated in ECM remodelling, including Tenascin C, TGFbeta1, Cathepsin B, MMP2, were upregulated in human ICM and DCM, and porcine chronic ICM specimens. CONCLUSION: Valves from cardiomyopathic hearts showed significant ECM deterioration with a disrupted collagen and elastic fibre network. It will be important to determine the impact of this ECM damage on valve durability and calcification in vivo if allografts are to be used from these donors.

Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering.

Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(epsilon-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.

Hypoxic cell death is reduced by pH buffering in a model of engineered heart tissue.

INTRODUCTION: In this report, we tested the ability of HEPES-buffered culture medium to reduce acidotic cell death in hypoxic monolayer cell cultures and in a diffusion-limited model of engineered heart tissue (EHT). METHODS: Neonatal rat cardiomyocytes were either plated (monolayers) or suspended in a hydrogel disc containing HEPES. RESULTS: Monolayers cultured in 0% oxygen exhibited a pH drop to 5.46 +/- 0.27 after 4 days with no HEPES, or 7.11 +/- 0.09 with 50 mM HEPES. The lowest observed pH in EHTs was estimated as 6.2 with no HEPES and 7.1 with 50 mM HEPES, which were endpoints of noticeably different pH gradients across the EHTs. Addition of HEPES to hypoxic monolayers corresponded with fewer propidium iodide-positive cells and TUNEL-positive cells; addition of HEPES to EHTs resulted in greater calcein staining and less LDH release. CONCLUSION: Effective pH buffering reduces cell death by attenuating the acidosis that accompanies anaerobic metabolism.

Analysis of pH gradients resulting from mass transport limitations in engineered heart tissue.

Transport limitations of critical nutrients are a major obstacle in the construction of engineered heart tissues (EHTs), and the importance of oxygen in this regard is well-documented throughout the literature. An indirect effect of cellular hypoxia is the shunt to the less-efficient glycolytic metabolism, which is accompanied by a reduction in extracellular pH. Image analysis of phenol red coloration in an experimental model of EHTs demonstrated pH gradients towards the center of the construct, which were dependent on experimental variables. Based on these observations, a four-species, 2-D diffusion-reaction mathematical model was developed to predict pH in a radial-diffusion model. The mathematical model predicted lethal values of pH (<6.5) in EHTs comprised of a nominal cell density of 10(6) cells/cm(3). pH predictions were moderately dependent on O(2) concentration, and strongly dependent on cell density, CO(2) concentration, and diffusion path length. It can be concluded from this study that hypoxia-induced acidosis is an important element in the mass transport problem, and future experiments measuring pH with more sensitive methods is expected to further elucidate the extent of this effect.

Modulation of gene expression in neonatal rat cardiomyocytes by surface modification of polylactide-co-glycolide substrates.

Myocardial tissue engineering presents a potential treatment option for heart disease. Cardiomyocytes isolated at various stages of development retain the ability to form contractile networks in vitro, which suggests that it should be possible to reconstitute viable myocardium given the appropriate architecture, stimuli, and cardiomyogenic cell source. This study investigates the effects of modifying substrate surface energy (by plasma etching) and protein coating (by fibronectin adsorption) on neonatal rat ventricular myocyte (NRVM) function. Primary NRVMs were cultured for 96 h on modified and control films of a common degradable polymer, polylactide-co-glycolide. Cultures were analyzed for cell spreading, protein content, and mRNA expression of atrial natriuretic factor and beta-myosin heavy chain. The results demonstrate that NRVMs cultured on etched films significantly increased in spreading, myofibril development, protein content, and gene expression of atrial natriuretic factor and beta-myosin heavy chain compared with unetched films, and that this surface energy effect is overwhelmed by the addition of fibronectin. Conclusions from this study are that surface energy and protein adsorption influence the gene expression of adherent NRVMs, and may be important for modulating the function of engineered myocardium.