Poster P4 - Functionalized Leaf-derived Vascular Structures (LeaVS) Modulate In Vitro Skin Regeneration

Introduction: Every year, more than 500,000 Americans are treated for burn injuries and chronic wounds. These injuries often lead to extensive scarring and permanent loss of function. The current standard of care is a split-thickness skin autograft; however, more extensive burns may have insufficient donor sites, requiring an alternative treatment. Dermal analogs have demonstrated some success but require 2-3 weeks for vascular integration. The absence of robust internal vascular networks is typically the cause of graft failure. So, we need to develop implantable skin scaffolds that already contain vascular network structures. The project aims to develop Leaf-derived Vascular Structures (LeaVS) that support skin regeneration and provide provisional vascular support. Previous data showed that decellularized leaf scaffolds retain their vascular networks while maintaining structural integrity. We hypothesize that the surfaces of these LeaVS can be functionalized with fibronectin (FN) to promote keratinocyte attachment, epidermal stratification, and differentiation.

Materials and

Methods: To prepare LeaVS, spinach leaves were subject to cuticle removal, decellularization, and sterilization before being stored at 4C. Keratinocyte attachment assays were performed by seeding normal human epidermal keratinocytes (NHEKs) at 500,000 cells/cm2 on LeaVS or tissue culture polystyrene (TCP) controls treated with 0, 0.01, or 0.1% poly-L-lysine (PLL) as well as 0 or 50 g/mL human fibronectin (FN) for 2 hours. Samples were stained with Hoechst, imaged, and quantified for cell density. Epidermal morphology assessments were performed using leaves treated with 0 or 0.01% PLL as well as 0, 10, 50, or 100 g/mL FN. Neonatal human fibroblasts (NHFs) were seeded at 100,000 cells/cm2 on the underside of the leaves, followed by neonatal human keratinocytes (NHKs) seeded at 1 million cells/cm2 on the topside of the leaves. For decellularized dermis (DED) controls, only NHKs were seeded. Constructs were cultured submerged for 3 days, followed by culture at the air/liquid interface for 7 days. Histological analyses of hematoxylin and eosin (H&E) stained samples were performed to quantify the epidermal thicknesses.

Results, Conclusions, and Discussions: Keratinocyte attachment assays showed that the TCP positive controls were not significantly enhanced by surface treatment. Interestingly, the LeaVS showed decreasing cell attachment as PLL concentration increased. However, the addition of FN to LeaVS resulted in a significant increase in cell density for PLL groups. Untreated LeaVS did not exhibit a significant difference in NHEK attachment compared to the untreated TCP controls. Epidermal morphology experiments demonstrated that LeaVS support the growth of a contiguous layer of keratinocytes with characteristic cobblestone morphology and progressive epithelial stratification, as well as fibroblast attachment. Interestingly, untreated LeaVS had the thickest epithelial layer (~20 m) compared to other treatment conditions and was comparable to the DED positive control (~20 m), although not as thick as native epithelial layers (~80 m), which is expected as it is a fully matured tissue (Figure 1).
These studies describe the strategic functionalization of LeaVS to modulate keratinocyte attachment as well as epidermal stratification and differentiation. We showed that fibronectin conjugated LeaVS displayed promise for supporting cellular attachment and epidermal stratification. We anticipate that these scaffolds will enable the future design of a multifunctional engineered skin substitutes for the treatment of traumatic skin injuries by facilitating rapid tissue vascularization and functional regeneration.

Acknowledgements (Optional): This work is supported by NIH 1R15AR080988.