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

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).