Loraine L.Y. Chiu

Scaffolds with covalently immobilized VEGF and Angiopoietin-1 for vascularization of engineered tissues.

The aim of this study was to engineer a biomaterial capable of supporting vascularization in vitro and in vivo. We covalently immobilized vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang1) onto three-dimensional porous collagen scaffolds using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) chemistry. Over both 3 and 7 days in vitro, seeded endothelial cells (ECs) had increased proliferation on scaffolds with immobilized VEGF and/or Ang1 compared to unmodified scaffolds and soluble growth factor controls. Notably, the group with co-immobilized VEGF and Ang1 showed significantly higher cell number (P=0.0079), higher overall lactate production rate (P=0.0044) and higher overall glucose consumption rate (P=0.0034) at Day 3, compared to its corresponding soluble control for which growth factors were added to culture medium. By Day 7, hematoxylin and eosin, live/dead, CD31, and von Willebrand factor staining all showed improved tube formation by ECs when cultivated on scaffolds with co-immobilized growth factors. Interestingly, scaffolds with co-immobilized VEGF and Ang1 showed increased EC infiltration in the chorioallantoic membrane (CAM) assay, compared to scaffolds with independently immobilized VEGF/Ang1. This study presents an alternative method for promoting the formation of vascular structures, via covalent immobilization of angiogenic growth factors that are more stable than soluble ones and have a localized effect.

Biodegradable collagen patch with covalently immobilized VEGF for myocardial repair.

Vascularization of engineered tissues in vitro and in vivo remains a key problem in translation of engineered tissues to clinical practice. Growth factor signalling can be prolonged by covalent tethering, thus we hypothesized that covalent immobilization of vascular endothelial growth factor (VEGF-165) to a porous collagen scaffold will enable rapid vascularization in vivo. Covalent immobilization may be preferred over controlled release or cell transfection if the effects are desired within the biomaterial rather than the surrounding tissue. Scaffolds were prepared with 14.5 ± 1.4 ng (Low) or 97.2 ± 8.0 ng (High) immobilized VEGF, or left untreated (control), and used to replace a full right ventricular free wall defect in rat hearts. In addition to rapid vascularization, an effective cardiac patch should exhibit neither thinning nor dilatation upon implantation. In vitro, VEGF enhanced the growth of endothelial and bone marrow cells seeded onto scaffolds. In vivo, High VEGF patches had greater blood vessel density (p < 0.01) than control at Day 7 and 28 due to increased cell recruitment and proliferation (p < 0.05 vs. control). At Day 28, VEGF-treated patches were significantly thicker (p < 0.05) than control, and thickness correlated positively with neovascularization (r = 0.67, p = 0.023). Importantly, angiogenesis in VEGF scaffolds contributed to improved cell survival and tissue formation.

Perfusable branching microvessel bed for vascularization of engineered tissues

Vascularization is critical for the survival of engineered tissues in vitro and in vivo. In vivo, angiogenesis involves endothelial cell proliferation and sprouting followed by connection of extended cellular processes and subsequent lumen propagation through vacuole fusion. We mimicked this process in engineering an organized capillary network anchored by an artery and a vein. The network was generated by inducing directed capillary sprouting from vascular explants on micropatterned substrates containing thymosin β4-hydrogel. The capillary outgrowths connected between the parent explants by day 21, a process that was accelerated to 14 d by application of soluble VEGF and hepatocyte growth factor. Confocal microscopy and transmission electron microscopy indicated the presence of tubules with lumens formed by endothelial cells expressing CD31, VE-cadherin, and von Willebrand factor. Cardiac tissues engineered around the resulting vasculature exhibited improved functional properties, cell striations, and cell–cell junctions compared with tissues without prevascularization. This approach uniquely allows easy removal of the vasculature from the microfabricated substrate and easy seeding of the tissue specific cell types in the parenchymal space.

Microfabricated poly(ethylene glycol) templates enable rapid screening of triculture conditions for cardiac tissue engineering

The purpose of this study was to design a simple system for cultivation of micro-scale cardiac organoids and investigate the effects of cellular composition on the organoid function. We hypothesized that cultivation of cardiomyocytes (CM) on preformed networks of fibroblasts (FB) and endothelial cells (EC) would enhance the structural and functional properties of the organoids, compared to simultaneously seeding the three cell types or cultivating enriched CM alone. Microchannels for cell seeding were created by photopolymerization of poly(ethylene glycol) diacrylate. In the preculture group the channels were seeded with a mixture of NIH 3T3 FB and D4T EC, following by addition of neonatal rat CM after 2 days of FB/EC preculture. The control microchannels were seeded simultaneously with FB/EC/CM (simultaneous triculture) or with enriched CM alone (enriched CM). Preculture resulted in cylindrical, contractile, and compact cardiac organoids that contained elongated CM expressing connexin-43 and cardiac troponin I. In contrast, simultaneous triculture resulted in noncontractile organoids with clusters of CM growing separately from elongated FBs and ECs. The staining for Connexin-43 was absent in the simultaneous triculture group. When fixed or frozen FB/EC were utilized as a preculture substrate for CM, noncontractile organoids were obtained; while preculture on a single cell type (either FB or EC) resulted in contractile organoids but with inferior properties compared to preculture with both FB/EC. These results emphasize the importance of living cells, presence of both nonmyocyte cell types as well as sequential seeding approach for cultivation of functional multicell type cardiac organoids.

A peptide-modified chitosan–collagen hydrogel for cardiac cell culture and delivery

Myocardial infarction (MI) results in the death of cardiomyocytes (CM) followed by scar formation and pathological remodeling of the heart. We propose that chitosan conjugated with the angiopoietin-1 derived peptide, QHREDGS, and mixed with collagen I forms a thermoresponsive hydrogel better suited for the survival and maturation of transplanted cardiomyocytes in vitro compared to collagen and chitosan-collagen hydrogels alone. Conjugation of QHREDGS peptide to chitosan does not interfere with the gelation, structure or mechanical properties of the hydrogel blends. The storage modulus of 2.5 mg ml(-1) 1:1 mass:mass (m:m) chitosan-collagen was measured to be 54.9 ± 9.1 Pa, and the loss modulus 6.1±0.9 Pa. The dose-response of the QHREDGS peptide was assessed and it was found that CMs encapsulated in High-peptide gel (651 ± 8 nmol peptide ml-gel(-1)) showed improved morphology, viability and metabolic activity in comparison to the Low-peptide (100 ± 30 nmol peptide ml-gel(-1)) and Control (No Peptide) groups. Construct (CMs in hydrogel) functional properties were not significantly different between the groups; however, the success rate of obtaining a beating construct was improved in the hydrogel with the High amount of QHREDGS peptide immobilized compared to the Low and Control groups. Subcutaneous injection of hydrogel (Control, Low and High) with CMs in the back of Lewis rats illustrated its ability to localize at the site of injection and retain cells, with CM contractile apparati identified after seven days. The hydrogel was also able to successfully localize at the site of injection in a mouse MI model.

Biphasic Electrical Field Stimulation Aids in Tissue Engineering of Multicell-Type Cardiac Organoids

The main objectives of current work were (1) to compare the effects of monophasic or biphasic electrical field stimulation on structure and function of engineered cardiac organoids based on enriched cardiomyocytes (CM) and (2) to determine if electrical field stimulation will enhance electrical excitability of cardiac organoids based on multiple cell types. Organoids resembling cardiac myofibers were cultivated in Matrigel-coated microchannels fabricated of poly(ethylene glycol)-diacrylate. We found that field stimulation using symmetric biphasic square pulses at 2.5 V/cm, 1 Hz, 1 ms (per pulse phase) was an improved stimulation protocol, as compared to no stimulation and stimulation using monophasic square pulses of identical total amplitude and duration (5 V/cm, 1 Hz, 2 ms). This was supported by the highest success rate for synchronous contractions, low excitation threshold, the highest cell density, and the highest expression of Connexin-43 in the biphasic group. Subsequently, enriched CM were seeded on the networks of (1) cardiac fibroblasts (FB), (2) D4T endothelial cells (EC), or (3) a mixture of FB and EC that were precultured for 2 days prior to the addition of enriched CM. Biphasic field stimulation was also effective at improving electrical excitability of these cardiac organoids by improving the three-dimensional organization of the cells, increasing cellular elongation and enhancing Connexin-43 presence.

Defining conditions for covalent immobilization of angiogenic growth factors onto scaffolds for tissue engineering.

Rapid vascularization of engineered tissues in vitro and in vivo remains one of the key limitations in tissue engineering. We propose that angiogenic growth factors covalently immobilized on scaffolds for tissue engineering can be used to accomplish this goal. The main objectives of this work were: (a) to derive desirable experimental conditions for the covalent immobilization of vascular endothelial growth factor (VEGF) and angiopoietin‐1 (Ang1) on porous collagen scaffolds; and (b) to determine whether primary endothelial cells respond to these scaffolds with covalently immobilized angiogenic factors. VEGF and Ang1 were covalently immobilized onto porous collagen scaffolds, using 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemistry. To improve covalent immobilization conditions: (a) different reaction buffers [phosphate‐buffered saline (PBS), distilled water, or 2‐(N‐morpholino)ethanesulphonic acid (MES)] were used; and (b) step immobilization was compared to bulk immobilization. In step immobilization, growth factors are applied after EDC activation of the scaffold, while in bulk immobilization, reagents are simultaneously applied to the scaffold. PBS as the reaction buffer resulted in higher amounts of VEGF and Ang1 immobilized (ELISA), higher cell proliferation rates (XTT) and increased lactate metabolism compared to water and MES as the reaction buffers. Step immobilization in PBS buffer was also more effective than bulk immobilization. Immobilized growth factors resulted in higher cell proliferation and lactate metabolism compared to soluble growth factors used at comparable concentrations. Tube formation by CD31‐positive cells was also observed in collagen scaffolds with immobilized VEGF or Ang1 using H5V and primary rat aortic endothelial cells but not on control scaffolds. Copyright

Controlled release of thymosin β4 using collagen–chitosan composite hydrogels promotes epicardial cell migration and angiogenesis

Rapid vascularization at the infarcted site is crucial for cardiac repair following myocardial infarction. Thymosin β4 (Tβ4), a 43-amino acid peptide, is both angiogenic and cardioprotective. Tβ4 in soluble form was previously shown to promote cell migration from quiescent adult cardiac explants. Here we developed a collagen-chitosan hydrogel for the encapsulation of Tβ4, which allowed its controlled release over 28days to elicit localized and prolonged effects. Contrastingly, Tβ4 was fully released over 3days when encapsulated in collagen-only hydrogels due to charge repulsion and lack of interconnected pores as shown by SEM. The charge of encapsulated molecules affected their release from collagen-chitosan hydrogels. While the release of neutral polyalanine was size-controlled diffusion, that of negatively-charged Tβ4 and positively-charged polylysine was affected by electrostatic interactions of peptides with collagen/chitosan molecules. Hydrogels with encapsulated Tβ4 significantly increased cell migration and outgrowth of CD31-positive capillaries from mouse and rat epicardial explants in vitro, compared to Tβ4-free and soluble controls. Potential advantage of Tβ4 over commonly-used angiogenic growth factors is that it can induce recruitment and differentiation of both endothelial and smooth muscle cells necessary for vascular stability. Importantly, Tβ4-encapsulated collagen-chitosan hydrogels promoted angiogenesis in vivo upon subcutaneous injection, compared to collagen-only hydrogels.

Engineered cardiac tissues.

Cardiac tissue engineering offers the promise of creating functional tissue replacements for use in the failing heart or for in vitro drug screening. The last decade has seen a great deal of progress in this field with new advances in interdisciplinary areas such as developmental biology, genetic engineering, biomaterials, polymer science, bioreactor engineering, and stem cell biology. We review here a selection of the most recent advances in cardiac tissue engineering, including the classical cell-scaffold approaches, advanced bioreactor designs, cell sheet engineering, whole organ decellularization, stem cell-based approaches, and topographical control of tissue organization and function. We also discuss current challenges in the field, such as maturation of stem cell-derived cardiac patches and vascularization.

Endothelial cells guided by immobilized gradients of vascular endothelial growth factor on porous collagen scaffolds

A key challenge in tissue engineering is overcoming cell death in the scaffold interior due to the limited diffusion of oxygen and nutrients therein. We here hypothesize that immobilizing a gradient of a growth/survival factor from the periphery to the center of a porous scaffold would guide endothelial cells into the interior of the scaffold, thus overcoming a necrotic core. Proteins were immobilized by one of three methods on porous collagen scaffolds for cardiovascular tissue engineering. The proteins were first activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/sulfo N-hydroxysuccinimide and then applied to the scaffold by one of three methods to establish the gradient: perfusion (the flow method), use of a source and a sink (the source-sink method) or by injecting 5 μl of the solution at the center of the scaffold (point source method). Due to the high reproducibility and ease of application of the point source method it was further used for VEGF-165 gradient formation, where an ~2 ng ml(-1) mm(-1) gradient was formed in a radial direction across a scaffold, 12 mm in diameter and 2.5mm thick. More endothelial cells were guided by the VEGF-165 gradient deep into the center of the scaffold compared with both uniformly immobilized VEGF-165 (with the same total VEGF concentration) and VEGF-free controls. All scaffolds (including the controls) yielded the same number of cells, but notably the VEGF-165 gradient scaffolds demonstrated a higher cell density in the centre. Thus we concluded that the VEGF-165 gradient promoted the migration, but not proliferation, of cells into the scaffold. These gradient scaffolds provide the foundation for future in vivo tissue engineering studies.