Accelerated and patterned neovascularization using surgically engineered granular hydrogel scaffolds

Introduction: Reconstructive surgery relies heavily on hydrogel scaffolds to facilitate tissue repair and regeneration. Traditional bulk hydrogels, however, suffer from slow and random vascularization, significantly impeding effective tissue healing. We address these limitations by integrating microsurgical techniques with granular hydrogel scaffolds (GHS) to promote rapid and patterned vascularization. This hybrid approach aims to overcome the limitations of conventional bulk scaffolds by guiding microvascular network formation, potentially shifting clinical treatment paradigms in regenerative medicine.

Materials and

Methods: Gelatin methacryloyl (GelMA) was synthesized using the reaction of gelatin with methacrylic anhydride, followed by purification and lyophilization. GelMA droplets with three varying sizes were produced using high-throughput step-emulsification microfluidic devices, creating monodispersed droplets that were subsequently crosslinked physically at 4°C to form microgels. These microgels were then packed into GHS and photocrosslinked upon light exposure (395-400 nm wavelength, 15 mw/cm2, 2 min) with tailored interstitial void spaces to support vascular network formations. We also examined the GHS mechanical properties and their interaction with cells through rheological/compression assessments and biological analyses, respectively. Micropunctures (MP) were applied to rat hindlimb via making small perforations in the artery and vein, then GHS were placed directly onto the perforated blood vessels, facilitating the integration and growth of vascular structures. The vascular development evaluation was conducted using immunostaining for endothelial (CD31), pericyte (NG-2), arteriovenous (Ephrin B2 and EphB4), and macrophage (F4/80) markers. Additionally, the perfusability of vascularized construct was shown using angiogram, followed by artificial intelligence-assisted analysis to quantitatively assess vascular density, loop formation, and the stability of the formed networks over 7 and 28 days.

Results, Conclusions, and Discussions: The application of GHS in micropunctured vasculature significantly enhanced microvascular density and facilitated the formation of structured vascular loops, as opposed to the random and sparse networks associated with bulk hydrogels. Statistical analyses confirmed the superiority of the GHS approach in terms of both immediate and long-term vascularization metrics. Moreover, the scaffolds supported a dynamic cellular environment conducive to vascular maturation, characterized by an influx of macrophages and pericytes. These cells play pivotal roles in stabilizing the nascent vasculature and transitioning from an inflammatory to a regenerative microenvironment. Longitudinal studies showed that the GHS not only promoted faster vascular integration but also ensured the persistence and functionality of these networks, highlighting the potential of this method to significantly enhance soft tissue reconstruction and regeneration. This study presents a novel microsurgical and bioengineering strategy that significantly improves the outcomes of reconstructive surgery by enabling rapid, directed vascularization. Integrating GHS with targeted microsurgical techniques is an advancement in regenerative engineering, which may offer new therapeutic avenues for clinical applications.

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