Scaffold-based tissue engineering: Supercritical carbon dioxide as an alternative method for decellularization and sterilization of dense materials

Development of ready-to-use biomaterials and scaffolds is vital for further advancement of scaffold-based tissue engineering in clinical practice. Scaffolds need to mimic 3D ultrastructure, have adequate mechan-ical strength, are biocompatible, non-immunogenic and need to promote tissue regeneration in vivo. Al-though decellularization of native tissues seems promising to deliver scaffolds that meet these criteria, adequate decellularization of hard, poorly penetrable and poorly diffusible tissues remains challenging whilst being a very time-consuming process.In this study, a method to decellularize hard, dense tissues using supercritical carbon-dioxide pre-ceded by a freeze/thaw cycle and followed by several washing steps is presented, demonstrating de-cellularisation efficiency and substantially reduced production/handling time. Additionally, supercritical carbon-dioxide treatment was used as sterilization method, further reducing the time required to produce the final scaffold. Histological evaluation showed that, after fine-tuning of the process, a partially acel-lular scaffold was obtained, with preservation of glycosaminoglycans and collagen fibers, albeit that the amount of residual dsDNA was still higher then chemically decellularized tissue. Biomechanical properties of the scaffold were similar to the native, non-decellularized tissue. After sterilization with supercritical carbon-dioxide the simulated functional outcome was more similar to native trachea, when compared to sterilization using gamma irradiation.Thus, decellularization and sterilization using supercritical carbon-dioxide with washing steps is an effective method for dense cartilaginous materials, and tuneable to meet different demands in other ap-plications, but further optimization may be required.Statement of significanceFurther advancement of the use of tissue engineered tracheal constructs is restricted by the lack of the ideal scaffold. Decellularized trachea is considered a promising scaffold, but the hard, poorly diffusible tissue remains challenging while forming a very time consumable process. Decellularization using super-critical carbon dioxide (scCO2) seems promising, resulting in efficient removal of cellular material while reducing production and handling time. Addition of scCO2 as a sterilization method resulted in further time reduction while improving functional outcome in comparison with traditional sterilization meth-ods. This study presents an promising alternative method for decellularization and sterilization of dense materials, which can be tuned to meet different demands in other applications.(c) 2022 The Authors. Published by Elsevier Ltd on behalf of Acta Materialia Inc.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )

Automated summary

The development of ready-to-use biomaterials and scaffolds is crucial for the advancement of scaffold-based tissue engineering in clinical practice. Decellularization of hard tissues using supercritical carbon-dioxide followed by washing steps is an effective method that reduces handling time and preserves important tissue components. This alternative method shows promise for decellularization and sterilization of dense materials.