Supercritical carbon dioxide decellularization of plant material to generate 3D biocompatible scaffolds

Abstract

The use of plant-based biomaterials for tissue engineering has recently generated interest as plant decellularization produces biocompatible scaffolds which can be repopulated with human cells. The predominant approach for vegetal decellularization remains serial chemical processing. However, this technique is time-consuming and requires harsh compounds which damage the resulting scaffolds. The current study presents an alternative solution using supercritical carbon dioxide (scCO2). Protocols testing various solvents were assessed and results found that scCO2 in combination with 2% peracetic acid decellularized plant material in less than 4 h, while preserving plant microarchitecture and branching vascular network. The biophysical and biochemical cues of the scCO2 decellularized spinach leaf scaffolds were then compared to chemically generated scaffolds. Data showed that the scaffolds had a similar Young’s modulus, suggesting identical stiffness, and revealed that they contained the same elements, yet displayed disparate biochemical signatures as assessed by Fourier-transform infrared spectroscopy (FTIR). Finally, human fibroblast cells seeded on the spinach leaf surface were attached and alive after 14 days, demonstrating the biocompatibility of the scCO2 decellularized scaffolds. Thus, scCO2 was found to be an efficient method for plant material decellularization, scaffold structure preservation and recellularization with human cells, while performed in less time (36 h) than the standard chemical approach (170 h).

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

“…Thus, optimization and alternative protocols have been explored (Table 1). Amazon sword [55], Anthurium [35], Anthurium (queen) [35], Apple interior [24,32,33,53,56], Asparagus [56], Bamboo [31,35], Basil [55], Broccoli stem [52,56], Cabbage [57], Calathea zebrina [35], Carrot [52,56], Celery [53,56,58], Cucumber [56], Ficus hispida [59], Garcinia [59], Green onion [56,60], Impatiens capensis [35], Jujube [52], Leek [61], Lucky bamboo [55], Orchid pseudobulb [35], Pachira aquatica [59], Parsley stem [34,58], Peanut hairy root [34], Persimmon [52], Potato [56], Solenostemon "wasabi" [35], Spinach [34,54,55,58,60,62,63], Sweet yellow bell pepper [52], Sweet wormwood …”

“…Additional clearing with surfactant might be needed to remove debris [1] Supercritical Fluid (scCO 2 ) [58] scCO 2 (2500 psi at 33 As the chemical approach utilizes strong detergent agents, this processing can often be harsh on the resulting scaffold by degrading proteins, damaging ultrastructure, and leaving behind a toxic reside [1]. Thus, optimization and alternative protocols have been explored (Table 1).…”

“…Recently, Harris et al introduced plant material decellularization by supercritical fluid technology (i.e., compressed (supercritical) carbon dioxide, (scCO 2 )) [58]. ScCO 2 presented an alternative option to decellularization, as the compressed carbon dioxide can penetrate dense material and act as a powerful solvent [66,67] with gas-like transport properties, liquid-like density, and lack of surface tension [68].…”

“…One advantage of using cellulose-based vegetal material as a scaffold is the ability to build on this tissue construct. It has been demonstrated that a variety of cell types, including human endothelial cells [34,57,62], human dermal fibroblasts (HDFs) [35,58,62,65], human skeletal myoblasts [56], human cancer cell lines [32,55,70], human aortic smooth muscle cells [64], mesenchymal stem cells [34,35,54,64], human-induced pluripotent stem cells (hiPSC) [52,63], and hiPSC-derived cardiomyocytes (hiPSC-CM) [34], as well as mouse fibroblasts [32,53,64] and mouse myoblasts [32,56] (Figure 3a,b), can attach and survive on a variety of decellularized plant scaffolds for periods of several weeks. (c,d) Osteoblastic differentiation of hiPSCs on 3D plant scaffold.…”

Current Advances in the Development of Decellularized Plant Extracellular Matrix

“…This transfer of ScCO 2 reduces the intracellular pH through the formation of carbonic acid, which leads to disruption of cell metabolism and removal of essential enzymes. In addition, the combined application of supercritical carbon dioxide and co-solvents (like peracetic acid) can significantly improve the efficiency of decellularization ( Harris et al, 2021 ).…”