O32 - Leaf-Derived Vascular Scaffolds (LeaVS) Functionalized with Pro-Angiogenic Factors Support Endothelial Recruitment

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

LB

Leah Braun

Student
George Mason University, Virginia, United States

Co-Author(s)

BS

Bryanna Samolyk

PhD Candidate
Worcester Polytechnic Institute, United States
Catherine F. Whittington

Associate Professor
Worcester Polytechnic Institute, United States
GG

Glenn Gaudette

Chair of the Department of Engineering Professor
Boston College, United States
Ad

Antonio CF dos Santos

Research Assistant Professor
Boston College, United States

Last Author(s)

GP

George Pins

Professor
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Primary Investigator(s)

GP

George Pins

Professor
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Co-Author(s)

Jeannine Coburn, Associate Professor

Associate Professor
Worcester Polytechnic Institute
Worcester, Massachusetts, United States

Introduction: : More than 6.5 million Americans suffer from chronic wounds annually, a number that is on the rise due to an aging population and an increasing rate of diabetes [1]. To restore quality of life and reduce mortality, engineered skin substitutes are often necessary. However, currently available options have insufficient vascular networks, which can lead to graft failure [2]. Previous work has shown that decellularized spinach leaves (leaf-derived vascular scaffolds, LeaVS) are a promising alternative, as their vascular structures are retained and similar to that of human skin [3]. With the novel contribution of functionalization with pro-angiogenic factors, we hypothesize that neovascular network formation in LeaVS can be enhanced. The aim of this work is to evaluate the adsorption rate, release kinetics, and endothelial cell recruitment of functionalized LeaVS.

Materials and

Methods: : LeaVS were prepared through a decellularization protocol previously described [4]. For the assessment of adsorption rate and release kinetics, 8 mm diameter LeaVS were submerged in 0.1 mg/mL 40 kDa FITC-dextran or 10 mg/mL BSA, respectively, for 1, 2, or 24 hours. The LeaVS were then briefly washed to remove non-adhered molecules and submerged in 0.2 mL PBS. For adsorption studies, LeaVS were immediately imaged, and relative fluorescence units were quantified with ImageJ. For release kinetic studies, 0.03 mL of solution was removed and replaced with fresh PBS at 0.5, 1, 2, 4, 24, and 45 hours. Protein concentrations were analyzed with a BCA kit. To evaluate endothelial cell recruitment toward functionalized LeaVS, Transwell migration assays were used. Human dermal microvascular endothelial cells (HMVECs; Lonza) were starved in 0.1% FBS EBM-2 for 16-18 hours. Sterile 8 mm diameter lyophilized LeaVS were hydrated with 0.015 mL solutions containing 0.3 nmol sphingosine-1-phosphate (S1P; Fisher) or 15 ng vascular endothelial growth factor A (VEGF-A; Fisher). Solutions were allowed to absorb for 2 hours, following which the functionalized LeaVS were washed with DPBS and placed in a 24 well-plate. For each well, 5×10^(4 )HMVECs in 0.1 mL serum-reduced medium were seeded in an 8 µm pore size Transwell above the functionalized LeaVS and 0.6 mL serum-reduced medium was added to the lower wells. After a 4-hour migration period, HMVECs were fixed with 4% paraformaldehyde and stained with Hoechst. ImageJ was used to count nuclei of migrated cells on the basal membrane.

Results, Conclusions, and Discussions:: FITC-dextran studies showed that LeaVS have a non-uniform adsorption profile across their surface and that an adsorption period of 24 hours significantly increases adsorption compared to 1- or 2-hour periods (Figure 1A, B). This data was further supported by the BSA release results, with the 24-hour adsorption condition releasing 58 µg BSA after 45 hours compared to 21 and 37 µg in the 1- and 2-hour conditions, respectively (Figure 1C). Transwell migration results showed that S1P functionalized LeaVS induced a 7-fold increase in migration compared to nonfunctionalized controls, while VEGF-A functionalization only resulted in a 2-fold increase (Figure 1D). This demonstrates that S1P functionalization is a strong candidate to maximize endothelial migration to LeaVS, which is a vital step toward their revascularization. Future studies should not only evaluate endothelial migration into LeaVS, but also covalent functionalization methods capable of supporting pro-angiogenic factor release over a longer period of time.

Acknowledgements and/or References (Optional): : This work was supported by NSF REU grant EEC2150076 and NIH grant 1R15AR080988.

[1] C. K. Sen, "Human wound and its burden: Updated 2020 compendium of estimates," Adv. Wound Care, vol. 10, no. 5, pp. 281–292, May 2021, doi: 10.1089/wound.2021.0026.
[2] Q. H. Phua, H. A. Han, and B-S. Soh, “Translational stem cell therapy: vascularized skin grafts in skin repair and regeneration,” J. Transl. Med., vol. 19, p. 83, 2021, doi: 10.1186/s12967-021-02752-2.
[3] J. R. Gershlak et al., "Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds," Biomaterials, vol. 125, pp. 13–22, May 2017, doi: 10.1016/j.biomaterials.2017.02.011.
[4] J. D. Jones, A. S. Rebello, and G. R. Gaudette, "Decellularized spinach: An edible scaffold for laboratory-grown meat," Food Bioscience, vol. 41, p. 100986, Mar. 2021, doi: 10.1016/j.fbio.2021.100986.