The Emerging Role of Decellularized Plant-Based Scaffolds as a New Biomaterial

Abstract

The decellularization of plant-based biomaterials to generate tissue-engineered substitutes or in vitro cellular models has significantly increased in recent years. These vegetal tissues can be sourced from plant leaves and stems or fruits and vegetables, making them a low-cost, accessible, and sustainable resource from which to generate three-dimensional scaffolds. Each construct is distinct, representing a wide range of architectural and mechanical properties as well as innate vasculature networks. Based on the rapid rise in interest, this review aims to detail the current state of the art and presents the future challenges and perspectives of these unique biomaterials. First, we consider the different existing decellularization techniques, including chemical, detergent-free, enzymatic, and supercritical fluid approaches that are used to generate such scaffolds and examine how these protocols can be selected based on plant cellularity. We next examine strategies for cell seeding onto the plant-derived constructs and the importance of the different functionalization methods used to assist in cell adhesion and promote cell viability. Finally, we discuss how their structural features, such as inherent vasculature, porosity, morphology, and mechanical properties (i.e., stiffness, elasticity, etc.) position plant-based scaffolds as a unique biomaterial and drive their use for specific downstream applications. The main challenges in the field are presented throughout the discussion, and future directions are proposed to help improve the development and use of vegetal constructs in biomedical research.

Keywords: biomaterial; cellulose; decellularization; plant-based scaffolds; tissue engineering.

Figure 1

(a) Spinach leaf decellularization by serial chemical treatment. Perfusion of sodium dodecyl sulfate (SDS) causes the leaf to lose chlorophyll, while the bleach solution is used to remove any residual plant content and flush debris from the scaffold. Reproduced from Gershlak et al. [34]. (b) To visually demonstrate decellularization efficiency, scanning electron microscopy (SEM) images of fresh and chemically decellularized spinach leaf scaffolds revealed that the fullness of the fresh leaf was lost after decellularization. Cells were removed, revealing micro-vessel ultrastructure and plant features such as the cell wall and guard cells of the stomata. Data generated by authors for illustrative purposes for this review. (c) SEM images of scaffold architecture reveal decellularized apple tissue generates a three-dimensional scaffold. Reproduced from Modulevsky et al. [32]. (d) Various decellularized vegetal tissues’ pore size found in the ideal range for TE. Reproduced from Lee et al. [52].

Figure 2

Green dashed line indicates current proposed quantitative threshold for decellularized animal tissues to be 50 ng of DNA/mg of tissue. Native plant materials such as lucky bamboo stems or celery stalks naturally fall below this level; standards should be modified to be more conducive to the extensive plant kingdom. Data generated by authors for illustrative purposes for this review.

Figure 3

(a) Example of lung epithelial cells (nuclei stained with DAPI) seeded on the surface of a decellularized spinach leaf scaffold. (b) Brightfield image of plant scaffold alone shows plant features such as stomata (red arrow) and confirms cell attachment with the presence of cell shape imprints (white arrow) in the scaffold and points of cell attachment (blue arrow). Data generated by authors for illustrative purposes for this review using epifluorescence microscopy. (c,d) Osteoblastic differentiation of hiPSCs on 3D plant scaffold. Phase contrast images, Alizarin Red S stain and von Kossa stain before and after differentiation. Levels of osteocalcin and type I collagen mRNA expressed by hiPSCs before and after osteoblastic differentiation and expression levels of OCT4, OCN, and SOST mRNA after osteoblastic differentiation. Reproduced, from Lee et al. [52].

Figure 4

Commonly used biofunctionalization agents for promoting cell attachment to the hydroxyl groups of the cellulose-based scaffold.

Figure 5

The vein network of vascular plant tissues, such as spinach leaves, tapers, and branches similar to that found in a mammalian network (a). Reproduced from Gershlak et al. [34]. Vascular networks of (b-i) spinach, (b-ii) lemon, and (b-iii) amazon sword plant leaves display various tapered patterns, including reticulate, parallel, or pinnate designs, respectively. Topographical images were obtained by authors for illustrative purposes for this review using a tactile sensor pad imaged with a GelSight, Inc., Benchtop System.

Figure 6

Young’s modulus of vegetal tissues before (native) and after decellularization. Graph reproduced, in part, from Lacombe et al. [55].

Figure 7

Correlation of mechanical properties between decellularized plant-based scaffold and human tissue.

Figure 8

Decellularized vegetal scaffold topography. Each scaffold displays a different surface morphology, such as seen in the (a) apple hypanthium, (b) celery stalk, (c) aquatic plant leaf surface, (d) wheatgrass stem sheath, (e) hybrid cherry tomato plant leaf, or (f) curly parsley stem. Such topography can differ within a plant, as seen in the green onion’s leaf (g) exterior and (h) interior tissue. Data generated by authors for illustrative purposes for this review.

Figure 9

(a,b) Fibroblast cells pattern themselves around the topography of the stem of a queen anthurium. Reproduced from Fontana et al. [35]. (c) Cells align horizontally along pattern found in a wasabi plant stem. Reproduced from Fontana et al. [35].