Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.

Critical limb ischemia impairs circulation to the extremities, causing pain, disrupted wound healing, and potential tissue necrosis. Therapeutic angiogenesis seeks to repair the damaged microvasculature directly to restore blood flow. In this study, we developed modular, micro-scale constructs designed to possess robust handling qualities, allow in vitro pre-culture, and promote microvasculature formation. The microbead matrix consisted of an agarose (AG) base to prevent aggregation, combined with cell-adhesive components of fibrinogen (FGN) and/or hydroxyapatite (HA). Microbeads encapsulating a co-culture of human umbilical vein endothelial cells (HUVEC) and fibroblasts were prepared and characterized. Microbeads were generally 80–100 microns in diameter, and the size increased with the addition of FGN and HA. Addition of HA increased the yield of microbeads, as well as the homogeneity of distribution of FGN within the matrix. Cell viability was high in all microbead types. When cell-seeded microbeads were embedded in fibrin hydrogels, HUVEC sprouting and inosculation between neighboring microbeads were observed over seven days. Pre-culture of microbeads for an additional seven days prior to embedding in fibrin resulted in significantly greater HUVEC network length in AG+HA+FGN microbeads, as compared to AG, AG+HA or AG+FGN microbeads. Importantly, composite microbeads resulted in more even and widespread endothelial network formation, relative to control microbeads consisting of pure fibrin. These results demonstrate that AG+HA+FGN microbeads support HUVEC sprouting both within and between adjacent microbeads, and can promote distributed vascularization of an external matrix. Such modular microtissues may have utility in treating ischemic tissue by rapidly re-establishing a microvascular network.

Bardet–Biedl syndrome (BBS) is a hereditary genetic disorder that results in numerous clinical manifestations including olfactory dysfunction. Of at least 21 BBS‐related genes that can carry multiple mutations, a pathogenic mutation, BBS1M390R, is the single most common mutation of clinically diagnosed BBS outcomes. While the deletion of BBS‐related genes in mice can cause variable penetrance in different organ systems, the impact of the Bbs1M390R mutation in the olfactory system remains unclear. Using a clinically relevant knock‐in mouse model homozygous for Bbs1M390R, we investigated the impact of the mutation on the olfactory system and tested the potential of viral‐mediated, wildtype gene replacement therapy to rescue smell loss. The cilia of olfactory sensory neurons (OSNs) in Bbs1M390R/M390R mice were significantly shorter and fewer than those of wild‐type mice. Also, both peripheral cellular odor detection and synaptic‐dependent activity in the olfactory bulb were significantly decreased in the mutant mice. Furthermore, to gain insight into the degree to which perceptual features are impaired in the mutant mice, we used whole‐body plethysmography to quantitatively measure odor‐evoked sniffing. The Bbs1M390R/M390R mice showed significantly higher odor detection thresholds (reduced odor sensitivity) compared to wild‐type mice; however, their odor discrimination acuity was still well maintained. Importantly, adenoviral expression of Bbs1 in OSNs restored cilia length and re‐established both peripheral odorant detection and odor perception. Together, our findings further expand our understanding for the development of gene therapeutic treatment for congenital ciliopathies in the olfactory system.