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Tissue Engineering

Tissue Engineering - Poster Session A

Poster L5 - Determining Suitable Leaf Species for Perfusion of Leaf-Derived Vascular Scaffolds

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  In the United States alone, nearly 500,000 Americans are treated annually for burns with 40,000 requiring hospitalization and advanced treatment. The healthcare costs associated with these burns are between $2-3 billion USD annually with many needing skin grafts or bioengineered skin substitutes. Treating full-thickness burns is vital for preventing infection, water loss, scarring, and death in severe cases. The standard of care is a split-thickness skin autograft, but due to limited tissue availability and donor site morbidity, a bioengineered skin substitute may be necessary. Current bioengineered skin substitutes lack vasculature necessary to enhance graft take. Since oxygen and nutrient diffusion is limited to 200 µm from capillaries, vasculature is necessary to support new skin ingrowth. Decellularized leaf scaffolds possess vasculature mimicking the structural architecture of human skin and they have the potential to function as inexpensive bioengineered skin substitutes, since cellulose is biocompatible and elicits a minimal inflammatory response. Previous work utilized spinach leaves as decellularized scaffolds, however different leaf types may possess characteristics for enhancing endothelial cell perfusion throughout the vasculature of the leaf. As such, the goal of this study is to evaluate several different leaf types for their decellularization ability, their perfusable vascular network morphology, as well as their xylem diameters.

Materials and

Methods:  Leaves for decellularization analyses were selected based on criteria including dicot vasculature, the presence of a petiole, and the ability to withstand the decellularization process without degrading. Leaves were quantified for vascular density of perfusable vascular structures as well as xylem diameter. Leaves selected for decellularization include baby kale (Brassica oleracea), leatherleaf viburnum (Viburnum rhytidophyllum), sweet basil (Ocimum basilicum), Chicago hardy fig (Ficus carica), sage (Salvia officinalis), and spinach (Spinacia oleracea) as a control. To decellularize the leaves, a series of three rinses alternating between hexanes and deionized water were performed to remove the waxy cuticle of the leaf. Leaves were then placed in 50 mL conical tubes containing 1% w/v sodium dodecyl sulfate solution on a rocker for 5 days, with daily solution changes. Next, the leaves were switched to a 1% Tween 20, 3% bleach solution for 3-21 days with daily solution changes until colorless. Decellularized leaves were rinsed with deionized water before storing at 4 °C. To examine the vasculature of the decellularized leaves, samples were dried for a minimum of 90 minutes on a benchtop before submerging the petiole in red dye. Vessel Analysis (ImageJ plugin) was used to analyze vascular density. To determine average Feret diameters of the xylem in the petioles for each leaf type, a portion of each sample was embedded in paraffin, sectioned, and imaged via phase contrast microscopy followed by image analysis using a custom ImageJ macro to isolate xylem features from the surrounding tissue morphology.

Results, Conclusions, and Discussions: All leaves were durable enough to withstand the decellularization process, but the basil leaves were the most fragile. Following decellularization, the dye perfusion test was performed, and vascular density of each leaf was analyzed. Spinach had a mean vascular density of 26.1 ± 2.4%; sweet basil, 20.3 ± 3.9%; Chicago hardy fig, 25.3 ± 2.8%; baby kale, 20.7 ± 3.5%; and sage, 16.0 ± 0.6% with n = 2 samples per leaf. Vascular density could not be analyzed for leatherleaf viburnum as the dye perfused uniformly as a front through the leaf instead of flowing only through the vasculature with sage exhibiting a similar perfusion pattern where vasculature was only visible in the tip. The spinach leaves had the greatest mean vascular density followed by Chicago hardy fig. Analysis of the petiole cross-sections revealed spinach had mean xylem Feret diameter of 13.0 ± 2.4 µm; sweet basil, 25.6 ± 0.7 µm; Chicago hardy fig, 20.5 ± 2.32 µm; baby kale, 17.4 ± 1.3 µm; leatherleaf viburnum 16.4 µm; and sage, 17.7 µm with n = 1- 3 samples per leaf. Of the leaves analyzed, basil had the largest mean xylem Feret diameter, followed by the fig leaves; all leaves had a greater mean xylem Feret diameter than spinach. Based on the results of this study, Chicago hardy fig and sweet basil possess vascular density closest to spinach, mean Feret xylem diameters greater than spinach, and have potential to function as decellularized scaffolds for seeding endothelial cells through the petiole. Additionally, leatherleaf viburnum and sage may be useful in drug delivery to a graft because of the uniform perfusion pattern observed during the dye perfusion test.

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