(I-331) Enabling patterned neovascularization using granular hydrogel scaffolds

Current techniques for accelerating neovascularization use hydrogel scaffolds. Due to their extracellular matrix mimetic and mechanical properties, these scaffolds provide a pro-angiogenic environment. However, a serious limitation of bulk hydrogels is that they are nanoporous, inhibiting degradation-independent cell infiltration into the scaffold. Granular hydrogel scaffolds (GHS) are a promising technology that enable a precisely engineered interconnected porosity, which may overcome this barrier. Current techniques, using bulk hydrogel scaffolds, lead to slow and random neovascularization. To combat these issues, we propose a technique that combines surgical micropuncture (MP) with GHS. These combined approaches enhance cellular extravasation and provide a specifically engineered interconnected porosity, enabling the acceleration and precise patterning of new microvascular network formation.

Gelatin methacryloyl (GelMA) was synthesized from gelatin Type A derived from porcine skin and methacrylic anhydride (Sigma) following a previously published method.[1] To fabricate monodispersed droplets of three distinct sizes (small, medium, and large), high-throughput step-emulsification microfluidic devices were utilized, with droplet size controlled by step sizes of around 8, 27, or 60 µm for small, medium, or large droplets, respectively.[2,3] The aqueous phase comprised a 10% w/v GelMA solution in DPBS, containing 0.1% w/v of a photoinitiator, while the oil phase consisted of Novec 7500 engineering fluid with a biocompatible surfactant (2% v/v). Physical crosslinking at 4 °C overnight converted the droplets into microgels. Subsequently, GelMA microgels of different sizes were assembled together and exposed to light (395-405 nm, 15 mw/cm2, 60 s) to photo-crosslink and form the GHS. Characterization of the GHS involved measuring pore size using fluorescein isothiocyanate-dextran (FITC-Dextran, Mw ~ 2 MDa) through fluorescent microscopy and assessing mechanical properties using compression and oscillatory rheology tests. For precision MP, rat hindlimb artery and vein were subjected to MP according to the established protocol.[4] Evaluation of neovascularized network formation included immunostaining of CD31 and demonstration of perfusability (angiogram), followed by artificial intelligent (AI)-assisted quantification of vascular density, vessel diameter, total tube length, number of branches, and intercapillary distance.[5]

GHS were constructed from physically crosslinked monodisperse microgels of three different sizes: small (~29 µm, GHS-S), medium (~81 µm, GHS-M), and large (~173 µm, GHS-L). The sizes of these microgels were quantified using a MATLAB code. GHS pores were controlled by microgel size, which were imaged in 3D, showing an equivalent median pore diameter of 10, 20, 45 µm for GHS-S, GHS-M, GHS-L, respectively. Void fraction ~25% was not significantly different between conditions. Mechanical characterizations, including compression tests and oscillatory shear frequency sweeps, showed that GHS-M and GHS-S had significantly higher compressive and storage moduli than GHS-L, with all GHS groups attaining significantly lower moduli when compared with the bulk hydrogel control group. In vivo implantation in the rat hindlimb model with and without MP was conducted on four study groups (GHS-S, GHS-M, GHS-L, and bulk hydrogel). The scaffolds were explanted after 7 days, and fluorescence images of CD31-labeled cells were captured. AI-based quantification showed that GHS-S and GHS-M had significantly higher mean vascular density compared with the bulk hydrogel. GHS-M had a larger total tube length than all other groups. The vessel diameter did not change significantly among groups.

In conclusion, our study introduces a novel hybrid microsurgical-biomaterial approach using GHS to overcome limitations of current hydrogels to accelerate and pattern the formation of new blood vessels. This innovative combination of MP technique with GHS holds significant promise in accelerating and precisely guiding the formation of microvascular networks. The results underscore the potential of this approach to revolutionize current clinical treatments and pave the way for advancements in neovascularization. By establishing a new platform for translational research in regenerative engineering and reconstructive surgery, this study may open exciting opportunities for further developments in soft tissue repair and regeneration.

[1]          Z. Ataie, A. Jaberi, S. Kheirabadi, A. Risbud, A. Sheikhi, J. Vis. Exp. 2022, 190, e64829.

[2]          Z. Ataie, S. Kheirabadi, J. W. Zhang, A. Kedzierski, C. Petrosky, R. Jiang, C. Vollberg, A. Sheikhi, Small 2022, 18, 2202390.

[3]          J. M. de Rutte, J. Koh, D. Di Carlo, Adv. Funct. Mater. 2019, 29, 1.

[4]          P. C. Hancock, S. V. Koduru, M. Sun, D. J. Ravnic, Microvasc. Res. 2021, 134, 104121.

[5]          D. J. Ravnic, X. Jiang, T. Wolloscheck, J. P. Pratt, H. Huss, S. J. Mentzer, M. A. Konerding, Microvasc. Res. 2005, 70, 90.