Engineering Highly Porous Granular Hydrogels to Support Microvascular Network Formation

Introduction: Granular hydrogels have emerged as a class of biomaterial scaffolds with great potential in regenerative medicine and tissue engineering. These materials consist of packed hydrogel microparticles, which can be easily injected or extruded forming a stabilized scaffold after deposition that can be stabilized through subsequent crosslinking. These particle-based materials inherently include interconnected micropores in the spaces between particles, and this porosity can greatly improve cell migration and proliferation. However, while increasing porosity and larger pore sizes in granular hydrogels have been shown to improve cellular infiltration, more and larger pores in granular hydrogels are also associated with decreased packing density and reduced points of contact or interaction between particles. Consequently, reductions in mechanical integrity are often associated with increasing porosity. Here, we address this challenge by demonstrating 1) the fabrication of granular scaffolds using sacrificial hydrogel microparticles to establish high-porosity and 2) the stabilization of the high-porosity scaffolds via high-aspect ratio electrospun hydrogel fibers included within the scaffolds. Towards creating vascularized biomaterials, we include endothelial cells within these scaffolds and observe the formation endothelial networks that might ultimately be used to support engineered tissues or tissue regeneration in systems based on fiber-reinforced granular hydrogel scaffolds.

Materials and

Methods: We fabricated granular scaffolds using multiple hydrogel microparticle populations: one formed from norbornene-modified hyaluronic acid (norHA) as a scaffold-forming microparticle component and another microparticle population formed from gelatin that served as a sacrificial component within the granular material. Scaffolds were formed by combining the component materials, inducing interparticle crosslinking, and melting away the gelatin particles through incubation at 37C. Microparticles with varying norHA concentrations and sizes were fabricated to quantify the effect of microparticle mechanics and sizes on the flow behavior of the granular hydrogel prior to crosslinking and mechanical properties afterwards. Varying ratios of norHA and gelatin microparticle populations were combined and the effects on porosity were assessed. Electrospun norHA fibers were incorporated in scaffolds at 5-10 vol% and their effects on scaffold stability and porosity were measured. Porosity was characterized from 2D confocal images as well as using LOVAMAP, software engineered to enable 3D reconstructions of microporous scaffolds and to analyze pore spaces. Endothelial cells (HUVECs) and mesenchymal stromal cells (MSCs) were incoporated into the granular hydrogels at a 10:1 ratio prior to crosslinking and gelatin particle removal. After scaffold stabilization and melting gelatin, cells were cultured over weeks in stable scaffolds. Viability was measured over time and as a function of extrusion of the scaffolds prior to crosslinking. Cells within scaffolds were stained for actin (phalloidin) and endothelial cell phenotypes (CD31) to assess the fates of the HUVEC:MSC cocultures within the scaffolds.

Results, Conclusions, and Discussions: Measurements of scaffold flow and mechanical properties were performed on nine different norHA microgel populations: small (~10 μm diameter), medium (~25 μm), and large (~50 μm) each at 2 wt%, 3 wt% and 4 wt% norHA concentrations. Storage moduli of the macroscale granular materials were observed to be dependent on norHA concentration, but not particle size; yield stress varied with both particle size (increasing yield stress with decreasing diameters) and norHA concentration (increasing with norHA concentration). The inclusion of gelatin microgels was observed to yield continuous void spaces rather than discrete pockets when gelatin microparticle sizes were equal to or smaller than norHA particle size. The porosities seen in the final scaffolds was observed to correspond predictably to the ratio of gelatin:norHA microgel volume in the initial granular system. We were able to establish scaffolds with up to 50% porosity using sacrificial gelatin hydrogel microparticles. When electrospun norHA fibers were incorporated in scaffolds at 5-10 vol.%, highly porous scaffolds retained their structure over a period of 28 days. Pore spaces within the scaffolds were seen – in both characterization via 2D confocal slices as well as 3D reconstructions in LOVAMAP – to become more interconnected with one another and the exterior of the scaffold as porosity increased. The inclusion of fibers was not observed to significantly decrease porosity, and it did not increase the mechanical properties of the macroscale granular hydrogel except in the highest porosity (50% porosity) tested. The cell containing granular hydrogel system could be extruded from a needle and then crosslinked to form a scaffold, with post-injection viability >80% over days in culture. Endothelial cells were observed via CD31 to form extensive, robust 3D systems that suggest the potential to engineer microvasculature within these scaffolds. Overall, this study demonstrates the development and characterization of a stable, highly porous granular scaffold system with high processibility and cytocompatibility. The system has potential for engineering highly porous and vascularized systems for regenerative medicine and tissue engineering, and the inclusion of fibers within particle-based hydrogel system may enable advances in the design and application of these rapidly emerging systems.

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