Cell-assembled extracellular matrix (CAM): a human biopaper for the biofabrication of pre-vascularized tissues able to connect to the host circulation in vivo

Abstract

When considering regenerative approaches, the efficient creation of a functional vasculature, that can support the metabolic needs of bioengineered tissues, is essential for their survival after implantation. However, it is widely recognized that the post-implantation microenvironment of the engineered tissues is often hypoxic due to insufficient vascularization, resulting in ischemia injury and necrosis. This is one of the main limitations of current tissue engineering applications aiming at replacing significant tissue volumes. Here, we have explored the use of a new biomaterial, the cell-assembled extracellular matrix (CAM), as a biopaper to biofabricate a vascular system. CAM sheets are a unique, fully biological and fully human material that has already shown stable long-term implantation in humans. We demonstrated, for the first time, the use of this unprocessed human ECM as a microperforated biopaper. Using microvalve dispensing bioprinting, concentrated human endothelial cells (30 millions ml−1) were deposited in a controlled geometry in CAM sheets and cocultured with HSFs. Following multilayer assembly, thick ECM-based constructs fused and supported the survival and maturation of capillary-like structures for up to 26 d of culture. Following 3 weeks of subcutaneous implantation in a mice model, constructs showed limited degradative response and the pre-formed vasculature successfully connected with the host circulatory system to establish active perfusion.This mechanically resilient tissue equivalent has great potential for the creation of more complex implantable tissues, where rapid anastomosis is sine qua non for cell survival and efficient tissue integration.

Bioprinting of pre-vascularized constructs for enhanced in vivo neo-vascularization

“…A large number of hCD31-positive vessels were identified within the µVP region of the implant, and some vessels with relatively large lumens (yellow arrows in the fluorescent image) were located in the same positions with vessel-like structures including red blood cells (H&E image). These blood-perfused vessel structures are expected to result from the vascular integration between the engineered and host vasculatures reported in various studies [13,14,[36][37][38][39]. We also confirmed the in vivo potency of µVP located nearby the AV bundle by observing the vessel-like structures composed of implanted EC.…”

“…A pre-vascularization strategy is reportedly beneficial for inducing in vivo vascularization in implanted constructs [11][12][13][14][15][16][17][18][19][36][37][38][39]. Recently, the implantation of scaffolds with aligned vascular patterns has been reported to promote graft vascularization and lead to enhanced viability and functionality of the engrafted parenchymal cells (e.g.…”

“…We developed a method for printing high density µVP with mature capillaries (figures 2(a)-(f)). Several studies have used EC and ADSC (or other tissuederived mesenchymal stem cells) co-cultures for in vitro vascular formation and introduced the positive aspects of in vivo vascularization into these prevascularized grafts [13,14,[24][25][26][36][37][38][39]. Baranski et al [13] demonstrated that the formation of highdensity µVPs with in vitro cultures can promote host vessel infiltration into the grafts.…”

Oxygen-Generating Scaffolds for Cardiac Tissue Engineering Applications

“…Maintaining high cell viability within artificial constructs proves to be an obstacle for developing 3D organ-scale tissue constructs due to diffusion limitations for oxygen and nutrients beyond the 300 μm range. − Notably, the cardiac tissue is a high oxygen demanding tissue, consuming up to 70 mL O 2 /min/100 g of oxygen during strenuous activity . Therefore, oxygen availability in cardiac tissue regeneration is vital to facilitating optimum growth and function and preventing hypoxia-induced necrosis. , The host′s blood is the primary source of oxygen and nutrients to the encapsulated cells in engineered tissue constructs. ,, Upon in vivo implantation, a tissue-engineered construct can take up to 4 or 5 weeks to effectively integrate with the host′s vasculature. − In recognition of the long integration period, researchers are increasingly seeking to develop approaches to improve vascularization of tissue constructs. − Nevertheless, attempts solely to improve vascularization do not address the lack of immediate oxygen required for maintaining cell viability and function to ensure the clinical success of the tissue-engineered constructs. − Consequently, there have been efforts to develop biomaterials with immediate oxygen provision and release within the cellular microenvironment as a potential solution for the high oxygen demand of the surrounding tissues until optimum vascularization can occur. , The encapsulation of cells within such oxygen-generating biomaterial scaffolds has garnered a significant scientific incentive. ,− …”