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A major limitation in tissue engineering strategies for congenital birth defects is the inability to provide a significant source of oxygen, nutrient, and waste transport in an avascular scaffold. Successful vascularization requires a reliable method to generate vascular cells and a scaffold capable of supporting vessel formation. The broad potential for differentiation, high proliferation rates, and autologous availability for neonatal surgeries make amniotic fluid-derived stem cells (AFSC) well suited for regenerative medicine strategies. AFSC-derived endothelial cells (AFSC-EC) express key proteins and functional phenotypes associated with endothelial cells. Fibrin-based hydrogels were shown to stimulate AFSC-derived network formation in vitro but were limited by rapid degradation. Incorporation of poly(ethylene glycol) (PEG) provided mechanical stability (65%±9% weight retention vs. 0% for fibrin-only at day 14) while retaining key benefits of fibrin-based scaffolds—quick formation (10±3 s), biocompatibility (88%±5% viability), and vasculogenic stimulation. To determine the feasibility of AFSC-derived microvasculature, we compared AFSC-EC as a vascular cell source and AFSC as a perivascular cell source to established sources of these cell types—human umbilical vein endothelial cells (HUVEC) and mesenchymal stem cells (MSC), respectively. Cocultures were seeded at a 4:1 endothelial-to-perivascular cell ratio, and gels were incubated at 37°C for 2 weeks. Mechanical testing was performed using a stress-controlled rheometer (G′=95±10 Pa), and cell-seeded hydrogels were assessed based on morphology. Network formation was analyzed based on key parameters such as vessel thickness, length, and area, as well as the degree of branching. There was no statistical difference between individual cultures of AFSC-EC and HUVEC in regard to these parameters, suggesting the vasculogenic potential of AFSC-EC; however, the development of robust vessels required the presence of both an endothelial and a perivascular cell source and was seen in AFSC cocultures (70%±20% vessel length, 90%±10% vessel area, and 105%±10% vessel thickness compared to HUVEC/MSC). At a fixed seeding density, the coculture of AFSC with AFSC-EC resulted in a synergistic effect on network parameters similar to MSC (150% vessel length, 147% vessel area, 150% vessel thickness, and 155% branching). These results suggest that AFSC-EC and AFSC have significant vasculogenic and perivasculogenic potential, respectively, and are suited for in vivo evaluation.

The major limitation of current engineered myocardial patches for the repair of heart defects is that insulating polymeric scaffold walls hinder the transfer of electrical signals between cardiomyocytes. This loss in signal transduction results in arrhythmias when the scaffolds are implanted. We report that small, subtoxic concentrations of single-walled carbon nanotubes, on the order of tens of parts per million, incorporated in a gelatin–chitosan hydrogel act as electrical nanobridges between cardiomyocytes, resulting in enhanced electrical coupling, synchronous beating, and cardiomyocyte function. These engineered tissues achieve excitation conduction velocities similar to native myocardial tissue (22 ± 9 cm/s) and could function as a full-thickness patch for several cardiovascular defect repair procedures, such as right ventricular outflow track repair for Tetralogy of Fallot, atrial and ventricular septal defect repair, and other cardiac defects, without the risk of inducing cardiac arrhythmias.

A novel cardiac scaffold comprised of decellularized porcine heart matrix was investigated for use as a biodegradable patch with a potential for surgical reconstruction of the right ventricular outflow tract. Powdered heart matrix solution was blended with chitosan and lyophilized to form three-dimensional scaffolds. For this investigation, we examined the influence of different blending ratios of heart matrix to chitosan on porosity and mechanical properties, then gene expression and electrophysiological function of invading neonatal rat ventricular myocytes (NRVM) compared to type-A gelatin/chitosan composite scaffolds. Heart matrix/chitosan-blended hydrogels (1.6 mg/mL heart matrix) had similar porosity (109±34 μm), and elastic modulus (13.2±4.0 kPa) as previously published gelatin/chitosan scaffolds. Heart matrix/chitosan hydrogels maintained80% viability and had higher NRVM retention (∼1000 cells/mm(2)) than gelatin/chitosan scaffolds. There was a significant increase in α-myosin heavy chain and connexin-43 expression in NRVM cultured on heart matrix/chitosan scaffolds after 14 days compared with gelatin/chitosan scaffolds. Further, heart matrix/chitosan scaffolds had significantly higher conduction velocity (12.6±4.9 cm/s) and contractile stress (0.79±0.13 mN/mm(2)) than gelatin/chitosan scaffolds. In summary, NRVM cultured on heart matrix scaffold showed improvements in contractile and electrophysiological function.

The objective of this study was to investigate a bioreactor suitable for human bladder regeneration. Simulations were performed using the computational fluid dynamic tools. The thickness of the bladder scaffold was 3 mm, similar to the human bladder, and overall hold-up volume within the spherical shape scaffold was 755 ml. All simulations were performed using (i) Brinkman equation on porous regions using the properties of 1% chitosan-1% gelatin structures, (ii) Michaelis-Menten type rate law nutrient consumption for smooth muscle cells (SMCs) and (iii) Mackie-Meares relationship for determining effective diffusivities. Steady state simulations were performed using flow rates from 0.5 to 5 ml/min. Two different inlet shapes: (i) straight entry at the centre (Design 1) and (ii) entry with an expansion (Design 2) were simulated to evaluate shear stress distribution. Also, mimicking bladder shape of two inlets (Design 3) was tested. Design 2 provided the uniform shear stress at the inlet and nutrient distribution, which was further investigated for the effect of scaffold locations within the reactor: (i) attached with a 3-mm open channel (Design 2-A), (ii) flow through with no open channel (Design 2-B) and (iii) porous structure suspended in the middle with 1.5-mm open channel on either side (Design 2-C). In Design 2-A and 2-C, fluid flow occurred by diffusion dominant mechanisms. Furthermore, the designed bioreactor is suitable for increased cell density of SMCs. These results showed that increasing the flow rate is necessary due to the decreased permeability at cell densities similar to the human bladder.

The stiffness of myocardial tissue changes significantly at birth and during neonatal development, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. However, effects of substrate stiffness on the electrophysiology and ion currents in cardiomyocytes have not been fully characterized. In this study, neonatal rat ventricular myocytes were cultured on the surface of flat polyacrylamide hydrogels with elastic moduli ranging from 1 to 25 kPa. Using whole-cell patch clamping, action potentials and L-type calcium currents were recorded. Cardiomyocytes cultured on hydrogels with a 9 kPa elastic modulus, similar to that of native myocardium, had the longest action potential duration. Additionally, the voltage at maximum calcium flux significantly decreased in cardiomyocytes on hydrogels with an elastic modulus higher than 9 kPa, and the mean inactivation voltage decreased with increasing stiffness. Interestingly, the expression of the L-type calcium channel subunit α gene and channel localization did not change with stiffness. Substrate stiffness significantly affects action potential length and calcium flux in cultured neonatal rat cardiomyocytes in a manner that may be unrelated to calcium channel expression. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

This article reviews current applications and novel candidate biomaterials for use as tissue-engineered scaffolds in pediatric cardiac tissue engineering. This overview of different types of biomaterials includes naturally derived and synthetic polymers and their biological, physical, and biomechanical properties for the use as a patch or baffle for surgical reconstruction of congenital heart defects. Applications and characteristics of composite biomaterials are highlighted with their respective feasibilities in use as cardiac scaffolds. Currently, a wide range of biomaterials has been introduced for cardiovascular reconstruction for complex congenital cardiac defects. However, there are still many remaining challenges for engineered tissue implantations.

Structural cardiac defects, such as Tetralogy of Fallot, often requires surgical placement of a patch to expand the right ventricular outflow tract (RVOT) in an area normally consisting of contractile myocardial tissue. Current cardiac patch materials are biologically inert and will not grow with a pediatric patient, often requiring reoperations. In this study, novel multi-layered scaffolds with a polycaprolactone core, a chitosan-based scaffold, and either gelatin or decellularized porcine heart matrix were implanted into a full thickness rat right ventricle defect for up to 8 weeks. The results show that engineered scaffolds were biodegradable and promoted tissue remodeling. Histological analysis of control fixed pericardium patches showed little to no cellular infiltration, while engineered scaffolds had significant muscular and vascular cell remodeling. Quantitative MRI revealed that left ventricular ejection fractions were stabilized in all patched hearts after 8 weeks, and the right ventricular ejection fraction in hearts with engineered patches was significantly greater than hearts with control pericardium patches. In addition, patches with heart matrix promoted a denser vascular network and a higher M2/M1 inflammatory macrophage ratio when compared to patches containing only gelatin. Collectively, these results show that these multi-layered patches are capable of full thickness defect repair and regeneration.

A three-dimensional scaffold composed of self-assembled polycaprolactone (PCL) sandwiched in a gelatin-chitosan hydrogel was developed for use as a biodegradable patch with a potential for surgical reconstruction of congenital heart defects. The PCL core provides surgical handling, suturability and high initial tensile strength, while the gelatin-chitosan scaffold allows for cell attachment, with pore size and mechanical properties conducive to cardiomyocyte migration and function. The ultimate tensile stress of the PCL core, made from blends of 10, 46 and 80kDa (Mn) PCL, was controllable in the range of 2-4MPa, with lower average molecular weight PCL blends correlating with lower tensile stress. Blends with lower molecular weight PCL also had faster degradation (controllable from 0% to 7% weight loss in saline over 30 days) and larger pores. PCL scaffolds supporting a gelatin-chitosan emulsion gel showed no significant alteration in tensile stress, strain or tensile modulus. However, the compressive modulus of the composite tissue was similar to that of native tissue (∼15kPa for 50% gelatin and 50% chitosan). Electron microscopy revealed that the gelatin-chitosan gel had a three-dimensional porous structure, with a mean pore diameter of ∼80μm, showed migration of neonatal rat ventricular myocytes (NRVM), maintained NRVM viability for over 7 days, and resulted in spontaneously beating scaffolds. This multi-layered scaffold has sufficient tensile strength and surgical handling for use as a cardiac patch, while allowing migration or pre-loading of cardiac cells in a biomimetic environment to allow for eventual degradation of the patch and incorporation into native tissue.

In this study, a novel process of dissolving polycaprolactone (PCL) matrices in glacial acetic acid was explored in which matrices spontaneously formed upon contact with water. Scanning electron microscopy analysis showed rough architecture and holes on the self-assembled matrix relative to matrices formed after dissolving in chloroform. Immersion in the gelatin solution reduced its roughness and number of micropores. Atomic force microscopy (AFM) analysis confirmed the increased roughness of the self-assembled matrices. The roughness of the matrices decreased after incubation in 1N NaOH for 10 min. AFM analysis also revealed that the self-assembled matrix had a net positive surface charge, whereas chloroform-cast matrix had a negative surface charge. The surface charge of self-assembled matrix after immersion in gelatin changed to negative. However, incubation in NaOH did not affect the surface charge. The tensile properties were tested in both the dry state (25 degrees Celsius) and the wet state (37 degrees Celsius) by immersion in phosphate-buffered saline. Self-assembled matrix had lower elastic modulus, break stress and break strain than chloroform-cast matrix in both states. The elastic modulus in the wet condition was reduced by half in self-assembled matrix but tensile strain increased. Samples were further analyzed by ramp-hold test for assessing stress relaxation behavior. Both self-assembled and chloroform-cast matrices had similar trends in stress relaxation behavior. However, stress accumulation in self-assembled matrix was half that of chloroform-cast matrix. In vitro cell cultures were conducted using human foreskin fibroblast (HFF-1) in serum-free medium. Cytoskeletal actin staining showed cell adhesion and spreading on all matrices. Cell retention was significantly increased in self-assembled matrix compared to chloroform-cast matrix. Addition of gelatin improved the retention of seeded cells on the surface. In summary, PCL matrices generated using this novel technique show significant promise in biomedical applications.