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Ghosh and Yi
structures. Isolated tissue-like generating materials require could be used to form effective and sustainable bioinks .
[32]
only small, non-sacrificial donations from the parent plant. Whole organ perfusion decellularization procedures can be
Cells can then be collected, maintained, and expanded modified for the synthesis of diverse plant tissues [33-35] . For
several folds from these biological starters to produce vast the preparation of spinach (Spinacia oleracea) leaf-based
volumes of plant matter. decellularized extracellular matrix (dECM) scaffolds,
Compared to current procedures, the suggested isolated leaves are first cannulated through the petiole, while parsley
plant tissue-like production process promises numerous (Petroselinum crispum) stems are cannulated through the
major benefits. For instance, reducing waste related to basipetal end of the stem. A series of hexane treatments
the biomaterial manufacturing process by producing only (98%, mixed isomers) and 1 × phosphate-buffered saline
useful plant components (e.g., secondary xylem or wood) (PBS) solution are then used to remove the cuticles
[32]
rather than undesirable or unusable plant parts (e.g., leaves, surrounding the leaf epithelial tissues on the plants .
small twigs, roots, or bark). In vitro plant substrate growth The cannulas are perfused with 10% sodium dodecyl
can better control cellular composition, thereby improving sulfate in deionized water for 5 days before perfusion with
the product’s material characteristics and chemical make- 0.1% Triton X-100 in a 10% sodium chlorite bleach and
up tunability. Furthermore, with the controlled deposition of deionized water solution for 48 h at a constant pressure
[32]
gel-scaffold material, the macroscopic substrate architecture of 152 mmHg . Cannulation and perfusion can also
may change to better fit application-specific needs, thereby be used to decellularize the leaves of sweet wormwood
allowing the creation of structures in a near-final form (Artemisia annua) and hairy roots of peanuts (Arachis
through casting or 3D bioprinting and minimizing waste hypogaea) using the same solutions described above.
further. In addition, novel methods or analysis can be easily After decellularization, tissues can be stored at 4°C in
[32]
replicated using a 3D printer, which is a key advantage of sterile deionized water for up to 2 weeks .
3D printing in plant research . These dECM processes could help clarify the
[31]
This suggests that bioprinted plant constructs could natural structural organization of higher plants, such as the
be useful replacements for complex living systems. transportation of nutrients through vascular tissues (xylem
These constructs may aid in the resolution of unanswered and phloem) from any part of the plant, such as the transfer
[32]
biological questions, with potential for use as learning of water from roots to the stem and leaves, and vice versa .
tools in plant research. Cellular components, such as the Spinach leaves have been used as a model biomaterial for
CW and mitochondria, are dynamic entities ; hence, dECM-based natural scaffolds due to their availability,
[19]
understanding these dynamics is essential to determine the intricate vascular network pattern, density, and petiole with
morphophysiological response of cells to various growth a large diameter, which simplifies the cannulation process.
conditions. This can be easily accomplished using 3D The use of this type of process as a bioink preparation
bioprinting, while the layer-by-layer printing method can medium remains uncommon for plant printing but has
be useful for studying subcellular and molecular dynamics attracted attention in the field of green bioprinting, as
within plant cells. Plant roots are highly capable of soil a result researchers are attempting to develop several
penetration as well as water and nutrient uptake; thus, a methodologies to understand plant cellular dynamics, cell to
comprehensive understanding of plant roots could help cell translocation, leaf venation system, plant–environment
with agricultural and environmental challenges such as interactions, and the plant immune system. New bioink
the sustainability of soil resources. Plant physiological processes for printing plant cells on a large scale in a simple
factors and external stimuli are considered in current and quick manner are also being developed.
studies on the expansion of below-ground root clusters, The field of green bioprinting is vast with new
plant root elongation, and soil penetration. However, the discoveries being reported that require multiple studies
root architecture and geometry, which are important for the to cover. Therefore, the primary goal of this review paper
movement of the root apex, have not been studied to analyze is to address various aspects of existing bioinks and their
root penetration performance. Bioprinted root-based 3D use in plant bioprinting.
models or biorobots may help clarify the geometrical and 2. Classification of bioinks
mechanical properties of root analogs . Decellularized
[19]
spinach leaves, for example, can help explain leaf venation, Bioinks are classified into two main categories: scaffold-
while the chemical analysis of decellularized leaves can be based bioinks and scaffold-free bioinks. In bioprinting,
useful for learning about the components present in plant scaffolds are fibrous, porous, or permeable three-
leaves. Decellularization processes are used to create pre- dimensional (3D) biomaterials that allow biological
vascularized, acellular tissue engineering scaffolds from liquids and gases to pass through while facilitating cell
a wide range of plant tissues. The rapid development of interaction, viability, and extracellular matrix (ECM)
different plant species and their abundance provides several deposition with minimal inflammation and toxicity while
cost effective and long-lasting scaffold biomaterials, which biodegrading at a controlled rate.

International Journal of Bioprinting (2022)–Volume 8, Issue 4 175