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Biomaterials

Biomaterials - Poster Session C

Poster C3 - Influence of Porosity on Vascularization of Granular Scaffolds

Friday, October 25, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Granular hydrogels have emerged as promising scaffolds for tissue engineering applications as a result of their many advantages over traditional bulk 3-D hydrogel structures. Due to their porous architecture, they allow for greater diffusion of nutrients, cell invasion, and tissue formation [2],[5]. Granular hydrogels have also been shown to minimize foreign body response [6]. While prior studies have shown that granular scaffolds allow for blood vessel invasion in vivo [2], little has been done to actively promote enhanced vascularization in granular materials. Previous work has demonstrated that the porosity of bulk hydrogels can influence vascularization potential [1],[3]. As porosity is of critical importance in granular materials, we aimed to understand how the shape and size of pores within granular scaffolds influence vessel morphogenesis.

Materials and

Methods:  We used previously established methods to generate poly(ethylene glycol) microgels that could be assembled into granular scaffolds via strain-promoted azide-alkyne click (SPAAC) chemistry [5],[7]. Microgel populations contained either an excess of azide or DBCO functional groups that permitted secondary annealing. Microgels of homogeneous shape (spherical) were fabricated via batch emulsification in which precursor components were added to a continuously stirring hexanes bath and subsequently polymerized. Microgels of heterogenous shape were fabricated via bulk hydrogel fragmentation in which precursor solutions were polymerized in a syringe barrel then extruded through progressively smaller needles down to 27 gauge. Phase images were used to quantify microgel size distribution prior to microgel jamming using centrifugation to form granular scaffolds. For cellular scaffolds, RGD was included in the microgel precursor solutions, and endothelial cells (HUVEC) and stromal cells (BM-hMSC) were dispersed throughout the microgels at a concentration of 4M/mL total cells (in a 1:1 ratio) prior to jamming. In some cases, an interstitial matrix (fibrin, 2.5 mg/mL) was added to support cell migration and assembly throughout the scaffolds. After 7 days of culture in fully supplemented EGM2, scaffolds were fixed and stained with ulex europaeus agglutinin I (UEA; endothelial cells), DAPI (nuclei), and phalloidin (F-actin) before fluorescent imaging was conducted to assess cell morphology.

Results, Conclusions, and Discussions: Both fabrication methods resulted in microgels spanning a wide range of diameters. Compared to the fragmented microgels, batch emulsified microgels were spherical and less heterogeneous in size (PDIazide = 0.184, PDIDBCO = 0.209) spanning from ~50-500 µm (Fig. 1 A, B). As expected, fragmented microgels were more heterogenous in both shape and size (PDIazide = 0.816, PDIDBCO = 0,480) with diameters ranging from ~30-600 µm (Fig. 1C, D). Granular scaffolds comprised of fragmented microgels were significantly stiffer than those comprised of batch emulsified microgels (G’Frag = 1596.09 ± 256.11 Pa, G’Batch = 718.98 ± 76.30 Pa; p < 0.01). This is likely the result of fragmented microgels packing more tightly due to the presence of small, non-uniformly shaped fragments, leaving little to no pore space compared to a batch-emulsified scaffold which possessed spherical microgels (Fig. 1G-H).

In both scaffolds, MSCs spread along the surface of the microgels; however, there was minimal evidence of endothelial cell assembly in either scaffold (Fig. 1I, J). Fibrin was incorporated into the interstitial space to provide endothelial cells with a supportive matrix for migration and assembly within the porous architecture of the scaffolds. When fibrin was incorporated into the scaffolds after jamming, we observed evidence of vessel morphogenesis in the scaffolds comprised of batch emulsified microgels (Fig. 1K). These vessel-like structures were observed along the surface of the spherical microgels. Despite the presence of fibrin, endothelial cells within the scaffolds containing fragmented microgels appeared to be unable to migrate throughout the scaffold and resulted in aggregates of endothelial cells but the lack of vessel-like structures (Fig. 1L).

Thus far, this study has demonstrated the importance of porosity for vascularization of granular scaffolds. Scaffolds with larger pores promoted cell migration and endothelial cell assembly; however, this was only in the presence of interstitial fibrin. Future studies will formulate softer and degradable granular scaffolds. We will further investigate pore size by creating microgels via microfluidic droplet generation which creates relatively homogeneous microgels and subsequent pores. Through these studies we will determine the optimal shape and size of pores to promote greater vascularization of granular scaffolds.

Acknowledgements (Optional):  This research was performed at the University of Colorado Boulder through participation in its Young Scholars Summer Research Program, with support from the National Science Foundation and the CU Department of Chemical and Biological Engineering.

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