Going green and mimetic: new ECM-based hydrogel for meniscus regeneration using supercritical CO2-assisted decellularization

Meniscal injuries and its subsequent progression to osteoarthritis represent a major clinical challenge, as current treatments often fail to achieve effective and complete functional restoration. An alternative regenerative approach is tissue engineering using decellularized extracellular matrix (dECM), which aims to restore meniscal structure and function. Porcine meniscus, an abundant byproduct of meat production, offers a readily available source for this purpose. Unlike conventional decellularization methods that are harsh, time-consuming, and detrimental to extracellular matrix (ECM) integrity, a detergent-free decellularization protocol utilizing supercritical carbon dioxide (scCO2) fluid was developed. This sustainable and minimally-invasive process was employed in cyclic pressurization-depressurization dynamics and scCO2 fluid was applied for sterilization by incorporating oxidative additives. Quantitative and qualitative analyses using quantification extraction kits and Fourier transform infrared (FTIR) spectroscopic analysis, presented as principal component analysis (PCA) and loading plots through a Machine Learning Toolbox, demonstrated substantial preservation of key ECM components. Sterilization efficiency was also confirmed using turbidity tests. dECM residual DNA content results fell within the range of general benchmark of 50ng/mg of dry tissue and the endotoxin levels, recently recognized as critical cause of host responds and regenerative outcomes [1], were below 20 endotoxin units (EU). The dECM showed cytocompatibility in vitro and an injectable hydrogel was successfully developed. The dECM hydrogel presented adhesive properties and was optimized to be used as a bioink for 3D bioprinting approaches.