D11 - Functionalized Leaf-derived Vascular Structures (LeaVS) Enhance Keratinocyte Culture

Introduction: : It is estimated that 160,000 Americans are treated for full-thickness skin wounds annually [1]. 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 long-term goal of this 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 [2]. Towards this, we hypothesize that the surfaces of these LeaVS functionalized with fibronectin (FN) and/or poly-L-lysine (PLL) will promote keratinocyte attachment and spreading.

Materials and

Methods: : To prepare LeaVS, spinach leaves were subject to cuticle removal, decellularization, and sterilization before being stored in DI water at 4C, using methods described previously [3]. LeaVS were then treated with 0 or 0.01% PLL overnight as well as 0, 10, 50, or 100 g/mL human FN (Sigma-Aldrich) overnight, followed by two PBS rinses. Neonatal human keratinocytes (NHKs) were then seeded at 500,000 cells/cm2 on the treated LeaVS or on decellularized dermis (DED, positive control) and cultured for 3 days in keratinocyte medium [4]. Samples were then stained with Hoechst and phalloidin (Invitrogen), imaged, and quantified for cell surface coverage percentage. To validate FN coating of LeaVS surfaces, 1 mg of human FN was fluorescently labeled with AlexaFluor 594 (antibody labeling kit, Invitrogen), then coated on the LeaVS at 0, 10, 50, or 100 g/mL overnight, followed by two PBS rinses. An unlabeled 100 g/mL FN group was also coated as a negative control. Samples were imaged, and relative fluorescence units (RFU) were quantified.

Results, Conclusions, and Discussions:: The keratinocyte coverage results showed that the 50 and 100 g/mL FN coated LeaVS samples, as well as the bare LeaVS group exhibited comparable coverage to DED controls (Figure 1). Interestingly, most of the PLL coated LeaVS samples showed significantly decreased cell coverage compared to DED controls. Only PLL samples coated with 100 g/mL FN were equivalent to the DED group. The higher FN concentration may have compensated for the negative effect of the PLL on keratinocyte attachment in this case. In addition to the quantification of surface coverage, we qualitatively observed keratinocyte morphology. We saw that the FN only LeaVS groups support the growth of a contiguous layer of keratinocytes with characteristic cobblestone morphology, while the PLL LeaVS groups exhibit some evidence of a discontinuous keratinocyte layer. In addition, we confirmed dose-dependent FN coatings on the LeaVS using fluorescently labeled human FN.
These studies describe the strategic functionalization of LeaVS to modulate keratinocyte attachment and surface coverage. We showed that fibronectin conjugated LeaVS displayed promise for supporting cellular attachment and characteristic morphologies. 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 and/or References (Optional):: This work is supported by NIH 1R15AR080988.

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