Tuning Recombinant Perlecan Domain V to Regulate Angiogenic Growth Factors and Enhance Endothelialization of Electrospun Silk Vascular Grafts.

Synthetic vascular grafts are used to bypass significant arterial blockage when native blood vessels are unsuitable, yet their propensity to fail due to poor blood compatibility and progressive graft stenosis remains an intractable challenge. Perlecan is the major heparan sulfate (HS) proteoglycan in the blood vessel wall with an inherent ability to regulate vascular cell activities associated with these major graft failure modes. Here the ability of the engineered form of perlecan domain V (rDV) to bind angiogenic growth factors is tuned and endothelial cell proliferation via the composition of its glycosaminoglycan (GAG) chain is supported. It is shown that the HS on rDV supports angiogenic growth factor signaling, including fibroblast growth factor (FGF) 2 and vascular endothelial growth factor (VEGF)165, while both HS and chondroitin sulfate on rDV are involved in VEGF189 signaling. It is also shown that physisorption of rDV on emerging electrospun silk fibroin vascular grafts promotes endothelialization and patency in a murine arterial interposition model, compared to the silk grafts alone. Together, this study demonstrates the potential of rDV as a tunable, angiogenic biomaterial coating that both potentiates growth factors and regulates endothelial cells.

Biomaterials containing extracellular matrix molecules as biomimetic next-generation vascular grafts.

The performance of synthetic biomaterial vascular grafts for the bypass of stenotic and dysfunctional blood vessels remains an intractable challenge in small-diameter applications. The functionalization of biomaterials with extracellular matrix (ECM) molecules is a promising approach because these molecules can regulate multiple biological processes in vascular tissues. In this review, we critically examine emerging approaches to ECM-containing vascular graft biomaterials and explore opportunities for future research and development toward clinical use.

Gas-modulating microcapsules for spatiotemporal control of hypoxia.

Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 14% with deviations from homeostasis impacting regulation of various physiological processes. In this work, we developed an approach to encapsulate enzymes at high loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix-embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, cancer spheroids, and intestinal organoids. Varying capsule placement, media formulation, and timing of replenishment yields tunable oxygen gradients, with concurrent spatial growth and morphogenesis in a single well. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourages neovascularization, providing scope for topical treatments or hydrogel wound dressings. This platform can be used in a variety of formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platform's simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any in vitro or in vivo format, with scope for inclusion in biomedical materials for treating injury or disease.

Biomaterials directed activation of a cryostable therapeutic secretome in induced pluripotent stem cell derived mesenchymal stromal cells.

Mesenchymal stem cell therapy has suffered from wide variability in clinical efficacy, largely due to heterogeneous starting cell populations and large-scale cell death during and after implantation. Optimizing the manufacturing process has led to reproducible cell populations that can be cryopreserved for clinical applications. Nevertheless, ensuring a reproducible cell state that persists after cryopreservation remains a significant challenge, and is necessary to ensure reproducible clinical outcomes. Here we demonstrate how matrix-conjugated hydrogel cell culture materials can normalize a population of induced pluripotent stem cell derived mesenchymal stem cells (iPSC-MSCs) to display a defined secretory profile that promotes enhanced neovascularization in vitro and in vivo. Using a protein-conjugated biomaterials screen we identified two conditions-1 kPa collagen and 10 kPa fibronectin coated polyacrylamide gels-that promote reproducible secretion of pro-angiogenic and immunomodulatory cytokines from iPSC-MSCs that enhance tubulogenesis of endothelial cells in Geltrex and neovascularization in chick chorioallantoic membranes. Using defined culture substrates alone, we demonstrate maintenance of secretory activity after cryopreservation for the first time. This advance provides a simple and scalable approach for cell engineering and subsequent manufacturing, toward normalizing and priming a desired cell activity for clinical regenerative medicine.

Silk fibroin photo-lyogels containing microchannels as a biomaterial platform for in situ tissue engineering.

The biophysical properties of biomaterials are key to directing the biological responses and biomaterial integration and function in in situ tissue engineering approaches. We present silk photo-lyogels, a biomaterial format fabricated using a new combinatorial approach involving photo-initiated crosslinking of silk fibroin via di-tyrosine bonds followed by lyophilization to generate 3D, porous lyogels showing physical properties distinct to those of lyophilized silk sponges or silk hydrogels. This fabrication approach allowed introduction of microchannels into 3D constructs via biofabrication approaches involving silk crosslinking around an array of 3D printed photocurable resin pillars to generate parallel channels or around a 3D printed sacrificial thermosensitive gel to generate interconnected channels in a rapid manner and without the need for chemical modification of silk fibroin. The presence of interconnected microchannels significantly improved migration of endothelial cells into 3D photo-lyogels in vitro, and tissue infiltration, photo-lyogel integration, and vascularization when implanted in vivo in a mouse subcutaneous model. Taken together, these findings demonstrate the feasibility and utility of a new combinatorial fabrication approach for generation of silk biomaterials that support cell interactions and implant integration for in situ tissue engineering approaches.

A Biomimetic Approach toward Enhancing Angiogenesis: Recombinantly Expressed Domain V of Human Perlecan Is a Bioactive Molecule That Promotes Angiogenesis and Vascularization of Implanted Biomaterials.

Angiogenic therapy involving delivery of pro-angiogenic growth factors to stimulate new blood vessel formation in ischemic disease is promising but has seen limited clinical success due to issues associated with the need to deliver supra-physiological growth factor concentrations. Bio-inspired growth factor delivery utilizing the native growth factor signaling roles of the extracellular matrix proteoglycans has the potential to overcome many of the drawbacks of angiogenic therapy. In this study, the potential of the recombinantly expressed domain V (rDV) of human perlecan is investigated as a means of promoting growth factor signaling toward enhanced angiogenesis and vascularization of implanted biomaterials. rDV is found to promote angiogenesis in established in vitro and in vivo angiogenesis assays by potentiating endogenous growth factor signaling via its glycosaminoglycan chains. Further, rDV is found to potentiate fibroblast growth factor 2 (FGF2) signaling at low concentrations that in the absence of rDV are not biologically active. Finally, rDV immobilized on 3D porous silk fibroin biomaterials promotes enhanced vascular ingrowth and integration of the implanted scaffolds with the surrounding tissue. Together, these studies demonstrate the important role of this biologically active perlecan fragment and its potential in the treatment of ischemia in both native and bioengineered tissues.