Accelerating patterned neovascularization via combining surgical micropuncture and granular hydrogel scaffolds

Introduction:: Reconstructive surgery often employs hydrogel scaffolds to enable repair and regeneration of soft tissue loss via providing a niche for revascularization. However, slow and random vascularization may limit the efficacy of this technique, resulting in suboptimal repair or treatment failure. In response to this challenge, we developed a novel approach to hasten and pattern microvascular network formation. We hypothesize that by combining a surgical micropuncture (MP) technique and granular hydrogel scaffolds (GHS) with tailored interstitial void space architecture, microvascular networks are rapidly formed and precisely guided. This approach may have the potential to shift current clinical treatment paradigms and scale up regenerative engineering platforms.


Materials and Methods:: Gelatin methacryloyl (GelMA) was synthesized using gelatin Type A obtained from porcine skin and methacrylic anhydride (Sigma), following a previously published protocol.^[1] High-throughput step-emulsification microfluidic devices were used to fabricate monodispersed droplets of three different sizes (small, medium, and large), with the size of droplets being governed by the step size (8, 27, or 60 µm for small, medium, or large droplets, respectively).^[2,3] A 10% w/v GelMA solution in DPBS, including 0.1% w/v of a photoinitiator, was used as the aqueous phase, while Novec 7500 engineering fluid, including a biocompatible surfactant (2% v/v), was used as the oil phase. The droplets were then converted to microgels via physical crosslinking at 4°C. GelMA microgels of three different sizes were packed and exposed to light (395-405 nm, 15 mw/cm², 60 s) to crosslink and form GHS. GHS were characterized by measuring pore size using fluorescein isothiocyanate-dextran (FITC-Dextran, Mw ~ 2 MDa) followed by fluorescence microscopy, and their mechanical properties were assessed using compression and oscillatory rheology tests. A precision MP was conducted in rat hindlimb artery and vein according to our established protocol.^[4] The neovascularized network formation was assessed using CD31 staining and via perfusability tests (angiogram),^[5] followed by the artificial intelligence (AI) assisted quantification of vascular density, vessel diameter, total tube length, number of branches, and intercapillary distance. A one-way or two-way analysis of variance (ANOVA) was performed with Tukey’s posthoc multiple comparison test. The significance was identified as non-significant (ns) p>0.05, *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001.

Results, Conclusions, and Discussions:: Results and discussion:

Figure 1A shows monodisperse GelMA droplets with three different sizes: small (~29 µm), medium (~81 µm), or large (~173 µm). Droplets were converted to microgels via physical (thermal) crosslinking at 4°C. GHS scaffolds were fabricated using small (GHS-S), medium (GHS-M), or large (GHS-L) microgels (Figure 1B) with a similar void fraction (~20-25%, Figure 1C) but a different median pore equivalent diameter (Figure 1D). Mechanical characterizations, such as compression test and oscillatory frequency sweep, showed that GHS-L had significantly lower compressive and storage moduli than GHS-S and GHS-M. Additionally, all GHS groups had significantly lower compressive and storage moduli compared with the bulk (conventional) hydrogel control group (Figures 1E-F). Precision MP was performed with four different groups (GHS-S, GHS-M, GHS-L, and bulk hydrogel), along with non-MP controls for each group. After 7 days, the scaffolds were explanted, and fluorescence images of CD31-labeled cells were acquired (Figure 1G). AI-based quantification showed that GHS-S and GHS-M had significantly higher mean vascular density compared with their bulk counterparts (Figure 1H). The average vessel diameter was almost similar among the groups (Figure 1I), but the total tube length of branches was higher in MP GHS-M compared with all other groups (Figure J). Figure 1K shows that intercapillary distances were regulated using different microgel building blocks.

Conclusions:

In conclusion, the hybrid microsurgical-bioengineering approach using GHS holds promise for overcoming limitations in reconstructive surgery and regenerative engineering. The optimum scaffold microarchitecture was identified in GHS-M, which significantly promoted vascular network formation. Overall, we envision that hybridizing MP and GHS may provide unique opportunities for engineering neovascularization, enabling a new platform for translational research in reconstructive surgery and regenerative engineering.

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References (Optional): : References:

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