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Enzymatic regulation of functional vascular networks using gelatin hydrogels.

ABSTRACT: To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl (gelatin-Ph) hydrogels that can be enzymatically crosslinked, allowing tuning of the storage modulus and the proteolytic degradation rate, for use as injectable hydrogels to support the human progenitor cell-based formation of a stable and mature vascular network. Porcine gelatin-Ph hydrogels were found to be cytocompatible with human blood-derived endothelial colony-forming cells and white adipose tissue-derived mesenchymal stem cells, resulting in >87% viability, and cell proliferation and spreading could be modulated by using hydrogels with different proteolytic degradability and stiffness. In addition, gelatin was extracted from mouse dermis and murine gelatin-Ph hydrogels were prepared. Importantly, implantation of human cell-laden porcine or murine gelatin-Ph hydrogels into immunodeficient mice resulted in the rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, the degree of enzymatic crosslinking of the gelatin-Ph hydrogels could be used to modulate cell behavior and the extent of vascular network formation in vivo. Our report details a technique for the synthesis of gelatin-Ph hydrogels from allogeneic or xenogeneic dermal skin and suggests that these hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues.

SUBMITTER: Chuang CH

PROVIDER: S-EPMC4589259 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Enzymatic regulation of functional vascular networks using gelatin hydrogels.

Chuang Chia-Hui CH Lin Ruei-Zeng RZ Tien Han-Wen HW Chu Ya-Chun YC Li Yen-Cheng YC Melero-Martin Juan M JM Chen Ying-Chieh YC

Acta biomaterialia 20150306

To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl ...[more]

PMID: 25749296

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Project description:Background3D bioprinting cardiac patches for epicardial transplantation are a promising approach for myocardial regeneration. Challenges remain such as quantifying printability, determining the ideal moment to transplant, and promoting vascularisation within bioprinted patches. We aimed to evaluate 3D bioprinted cardiac patches for printability, durability in culture, cell viability, and endothelial cell structural self-organisation into networks.MethodsWe evaluated 3D-bioprinted double-layer patches using alginate/gelatine (AlgGel) hydrogels and three extrusion bioprinters (REGEMAT3D, INVIVO, BIO X). Bioink contained either neonatal mouse cardiac cell spheroids or free (not-in-spheroid) human coronary artery endothelial cells with fibroblasts, mixed with AlgGel. To test the effects on durability, some patches were bioprinted as a single layer only, cultured under minimal movement conditions or had added fibroblast-derived extracellular matrix hydrogel (AlloECM). Controls included acellular AlgGel and gelatin methacryloyl (GELMA) patches.ResultsPrintability was similar across bioprinters. For AlgGel compared to GELMA: resolutions were similar (200-700 μm line diameters), printing accuracy was 45 and 25%, respectively (AlgGel was 1.7x more accurate; p < 0.05), and shape fidelity was 92% (AlgGel) and 96% (GELMA); p = 0.36. For durability, AlgGel patch median survival in culture was 14 days (IQR:10-27) overall which was not significantly affected by bioprinting system or cellular content in patches. We identified three factors which reduced durability in culture: (1) bioprinting one layer depth patches (instead of two layers); (2) movement disturbance to patches in media; and (3) the addition of AlloECM to AlgGel. Cells were viable after bioprinting followed by 28 days in culture, and all BIO X-bioprinted mouse cardiac cell spheroid patches presented contractile activity starting between day 7 and 13 after bioprinting. At day 28, endothelial cells in hydrogel displayed organisation into endothelial network-like structures.ConclusionAlgGel-based 3D bioprinted heart patches permit cardiomyocyte contractility and endothelial cell structural self-organisation. After bioprinting, a period of 2 weeks maturation in culture prior to transplantation may be optimal, allowing for a degree of tissue maturation but before many patches start to lose integrity. We quantify AlgGel printability and present novel factors which reduce AlgGel patch durability (layer number, movement, and the addition of AlloECM) and factors which had minimal effect on durability (bioprinting system and cellular patch content).

Project description:Injectable biomaterials are attractive for soft tissue regeneration because they are handled in a minimally invasive manner and can easily adapt to complex defects. However, inadequate vascularization of the injectable constructs has long been a barrier, leading to necrosis or volume reduction after implantation. In this work, we developed a three-step process to synthesize injectable gelatin-derived hydrogels that are capable of controlling growth factor delivery to induce angiogenesis. In our approach, tyramine was first introduced into gelatin chains to provide enzymatic crosslinking points for gel formation after injection. Next, heparin, a polysaccharide with binding domains to many growth factors, was covalently linked to the tyramine-modified gelatin. Finally, vascular endothelial growth factor (VEGF) was incorporated into the gelatin derivative by binding with the heparin in the gelatin derivative, and an injectable gel with controlled VEGF release was formed by an enzymatic catalytic reaction with hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The gelation time, mechanical properties and degradation of the gel was readily tailored by the gelatin concentration and the ratio of H2O2/HRP. Binding VEGF to heparin stabilizes this growth factor, protects it from denaturation and proteolytic degradation and subsequently prolongs the sustained release. An in vitro release study and bioactivity assay indicated that the VEGF was released in a sustained manner with high bioactivity for over 3 weeks. Furthermore, a chicken chorioallantoic membrane (CAM) assay and animal experiments were performed to evaluate in vivo bioactivity of the VEGF released from the hydrogels. After 5 days of incubation on CAM, the number of blood vessels surrounding the heparin-modified hydrogels was increased by 2.4-fold compared with that of the control group. Deeper and denser cell infiltration and angiogenesis in the heparin-modified gelatin/VEGF gels were observed compared to the controls after being subcutaneously injected in the dorsal side of the mice for 2 weeks. Interestingly, even without the incorporation of VEGF, the heparin-modified gelatin derivative still had the capability to induce angiogenesis to a certain degree. Our results suggest that the gelatin derivative/VEGF is an excellent injectable delivery system for induced angiogenesis of soft tissue regeneration.

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Enzymatic regulation of functional vascular networks using gelatin hydrogels.

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Enzymatic regulation of functional vascular networks using gelatin hydrogels.

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