Poster BB9 - Engineering Microvascular Network with Polystyrene Microtubes Embedded Hybrid Hydrogels

Introduction: Microvascular engineering aims to fabricate functional microvascular networks for applications in regenerative medicine and tissue engineering. Lack of adequate micro-vascularization constrains the size and lifespan of engineered tissues, as well as the viability of transplanted tissue in vivo. Current technologies that mimic microvascular networks rely on spontaneous angiogenesis and vasculogenesis [2][3]. However, they lack precise control over key aspects of vascularization including size, density, and connectivity. Developing hierarchical vasculature, consisting of diverse vessels distributed across multiple levels, remains challenging. Therefore, we introduce a hybrid hydrogel matrix with polystyrene microtubes (PS-MTs) to engineer Microvascular Network (MVNs) development, aiming for better control in microvessel structural features.

Materials and

Methods: Polystyrene microtubes (PS-MTs) were fabricated using an electrospinning platform equipped with concentric core-sheath spinnerets utilizing polyethylene oxide and polystyrene dissolved in dichloromethane solutions. Following electrospinning, the microtubes were immersed in water to remove soluble PEO, air-dried, and sterilized in ethanol. The microtubes were ultrasonically fragmented to sizes suitable for embedding on the chip. The microtube morphology was characterized using SEM and optical microscopy. RFP-expressing Human Umbilical Vein Endothelial Cells (RFP-HUVECs) and human fibroblasts were co-cultured in a hybrid fibrin hydrogel embedded with PS-MTs for seven days within a microfluidic chip to create microvascular networks (MVNs) under interstitial fluid flow conditions. The biocompatibility of the polymer microtubes was assessed by comparing PS-MTs with other biocompatible polymers using a cell viability (CCK8) assay, and a Lactate Dehydrogenase (LDH) assay was conducted to evaluate cell viability during on-chip culture with PS-MTs embedded in hydrogels. Our study investigated the effect of PS-MTs on microvascular networks by assessing both morphological and functional characteristics. Morphological evaluation involved analyzing fluorescence and confocal microscopy images with ImageJ software to measure the structural features of the vascular network. Functional assessment was conducted by measuring the perfusibility and permeability using fluorescent microspheres and dextran solutions, respectively. These methods allowed for a detailed investigation of the potential of PS-MTs to support vascular network formation and function in a controlled 3D environment.

Results, Conclusions, and Discussions: nitially, a biocompatibility assay of the polymer microtube was conducted using the CCK-8 assay, which showed that PS-MTs supported 90% cell viability in comparison to biocompatible polymers such as PLA (98 %) and PCL (78 %). This finding led to further testing of cell viability within the hybrid hydrogel embedded with PS-MTs on the chip. We observe PS-MTs exhibited no detrimental effects on cell viability evidenced by consistent LDH assay results. The morphological analysis highlights that modulating PS-MT density within hydrogels influences the resulting vascular networks. Higher PS-MT densities (>4%) led to significant enhancements in vessel area percentage and average branch diameter. The average branch diameter in these higher density groups has a significant increase to 115.15 ± 28.07μm from 59.16 ± 9.42μm in controls. These changes also impacted network functionality, with improved perfusion and stronger microvascular barriers compared to low density ( < 4%) and control groups evidenced by the perfusion and permeability test through the formed vessel network.
he hybrid PS-MTs-fibrin hydrogel demonstrated excellent biocompatibility. Cocultures of vascular cells embedded in the hybrid hydrogels sustained on chip and formed microvascular networks by day 7. Incorporating fragmented polystyrene microtubes in hydrogel effectively enhances the microvascular network formation, resulting in increased vessel diameter and vascular area. Functional analysis reveals enhanced perfusion and reduced permeability of microvascular networks in hybrid hydrogels incorporated with high polystyrene microtube density, indicating improved functionality of MVNs. The hybrid hydrogel exhibits significant promise as a versatile tool for precisely controlling the diameter of engineered microvessels. It facilitates the construction of hierarchical vasculature for studying the 3D organs and tissues in vitro.

Acknowledgements (Optional): This research is funded by Dr. Ying Wang’s & Dr. Yingge Zhou’s startup fund.

Reference:
[1] O’Connor, C. et al. Engineering the multiscale complexity of vascular networks. Nat Rev Mater 7, 702–716 (2022).
[2] Z. Wan et al. A Robust Method for Perfusable Microvascular Network Formation In Vitro. Small Methods 2022, 6,200143
[3]Hajal, C., Offeddu, G.S., Shin, Y. et al. Engineered human blood–brain barrier microfluidic model for vascular permeability analyses. Nat Protoc 17, 95–128 (2022).