Poster AA16 - Implantable 3D Bioengineered Constructs with Perfusable Capillary Networks Mimicking Native Vasculature

Introduction: Attaining vascularization in engineered 3D constructs at the anatomical scale is crucial for ensuring a sufficient supply of nutrients and oxygen post-implantation. However, existing pre-vascularized 3D tissues are confined to micro-scale hydrogels, lacking the complexity of native extracellular matrix (ECM) environments. There is a pressing need for anatomical scale perfusable constructs to advance translational applications. To bridge this gap, we introduce an innovative approach utilizing ECM scaffolds derived from human dermal fibroblast, pre-vascularized via human mesenchymal stem cells (hMSCs) and Human Umbilical Vein Endothelial Cells (HUVECs) co-culturing under optimal oxygen conditions. These pre-vascularized scaffolds boast a dense microvascular network, serving as foundational elements for bioengineering intricate 3D tissues. Our research aims to assess the patency, immunogenicity, and innate immune response to these engineered constructs in vivo, employing macrophage co-culture and subcutaneous implantation in the RNU nude rat model, respectively.

Materials and

Methods: Decellularized hDF-ECM scaffolds underwent pre-vascularization via hMSC and HUVECs co-culturing. To mimic innate immune responses towards the pre-vascularized constructs, PMA-stimulated THP-1-derived macrophages were cultured on these pre-vascularized constructs for seven days. The microvascular networks were characterized as in-vitro-like via z-stack Confocal Imaging stained with CD31. Proteins and RNA isolated from the co-cultured constructs were analyzed with western blotting, cytokine array, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To determine the perfusability of the constructs in vivo, hMSC/EC/ECM or hMSC/ECM constructs were implanted in the subcutaneous space of RNU nude rats. Before euthanization, human-specific lectin (UEA-1) was injected into the rat heart, and tissue samples were collected on days 3, 7, 14, and 28 for immunofluorescence and histological analysis.

Results, Conclusions, and Discussions: Characteristics of the microvascular network (vessel integrity, tubular, and lumen structure) were evaluated in vitro with macrophage co-culture using IF staining (Fig. 1(IA-B)). Notably, pre-vascularized constructs subcutaneous implantation exhibited viability and formed a functional anastomosis with host vasculature within 3 days (Fig. 1(IIA-B)). These completely biological and pre-vascularized engineered constructs remain viable and form functional anastomosis after in vivo implantation. These pre-vascularized constructs constituting immunomodulatory hMSC efficiently mitigated prolonged inflammatory response in vivo. Macrophage recruitment consistently diminished one week post-implantation, suggesting the constructs’ hypoimmunogenic properties. A completely biological and native tissue architecture mimicking pre-vascularized 3D engineered tissue constructs was fabricated that remain viable and form functional anastomosis after in vivo implantation in RNU nude rats. Overall, these constructs have a great potential to serve as building blocks to fabricate anatomical scale large-scale 3D tissue for diverse tissue engineering applications.

Acknowledgements (Optional): We also acknowledge Texas A&M University Microscopy and Imaging Center Core Facility (RRID:SCR_022128), and the Integrated Microscopy and Imaging Laboratory at Texas A&M College of Medicine (RRID:SCR_021637) for providing microscopy resources. This study was supported by the National Institutes of Health (R01HL146652 and R15CA202656) and the National Science Foundation (1703570, 2106048) to Feng Zhao.