Using decellularized grafted leaves as tissue engineering scaffolds for mammalian cells

Abstract

Inadequate availability of organs and tissues for transplantation is a major problem. While offering an alternative, development of engineered tissue constructs containing a functional vascular network that allows delivery of nutrients and especially O₂ remains a challenge. Using native vasculature, especially microvasculature, of a tissue or an organ offers potential to overcome this shortcoming. Decellularized animal organs retain the extracellular matrix (ECM) scaffold and native vascular networks but are challenging to source. A low cost, more readily available, and consistent source of tissues and organs is required. We investigated a plant scaffold model that would mimic input (arterial)-output (venous) flow by grafting two Aptenia cordifolia leaves together with opposite facing petioles. The abaxial epidermis of the top leaf and the adaxial epidermis of the bottom leaf were removed; wounds were soaked for 20 s in 0.1-mg L⁻¹ naphthaleneacetic acid (NAA) and 1-mg L⁻¹ benzylaminopurine (BAP) before horizontally appressing the wounded side of each leaf together. Leaves were decellularized 2 to 4 wk after grafting, lyophilized, and stored at room temperature. Prior to use, grafts were sterilized by ethylene oxide and rehydrated. Structural connections between the leaves were visualized histologically in thin sections using hematoxylin and eosin (H&E) and toluidine blue stains. Ponceau Red dye and red blood cells were perfused into the grafted leaves through one petiole of one leaf after decellularization to observe flow from input petiole into the first and then the second of the grafted leaves prior to exiting via the output petiole. Grafts were recellularized with the green fluorescent protein (GFP) expressing human breast cancer line MDA-MB231 and cell attachment and morphology observed 7 d post cell seeding. Results suggest that these grafted leaves provided an input-output cell-compatible vascularized scaffold as a possible bioscaffold for engineering tissues for transplantation.