Decellularised tissues obtained by a CO2-philic detergent and supercritical CO2

Abstract

Tissue decellularisation has gained much attention in regenerative medicine as an alternative to synthetic materials. In decellularised tissues, biological cues can be maintained and provide cellular environments still unmet by synthetic materials. Supercritical CO (scCO ) has recently emerged as a promising alternative decellularisation technique to aggressive detergents; in addition, scCO provides innate sterilisation. However, to date, decellularisation with scCO is limited to only a few tissue types with low cellular density. In the current study, a scCO technique to decellularise high density tissues, including articular cartilage, tendon and skin, was developed. Results showed that most of the cellular material was removed, while the sample structure and biocompatibility was preserved. The DNA content was reduced in cartilage, tendon and skin as compared to the native tissue. The treatment did not affect the initial tendon elastic modulus [reduced from 126.35 ± 9.79 MPa to 113.48 ± 8.48 MPa (p 〉 0.05)], while it reduced the cartilage one [from 12.06 ± 2.14 MPa to 1.17 ± 0.34 MPa (p 〈 0.0001)]. Interestingly, cell adhesion molecules such as fibronectin and laminin were still present in the tissues after decellularisation. Bovine chondrocytes were metabolically active and adhered to the surface of all decellularised tissues after 1 week of cell culture. The developed method has the potential to become a cost-effective, one-step procedure for the decellularisation of dense tissues.

Decellularization of tissues and organs

“…On the other hand; the most significant property of this fluid is that the transport coefficients 1 . The temperature and pressure of the supercritical fluid are higher than its critical point in which liquid and gas phases cannot be distinguished 3 . Particularly, this fluid has a unique property that can be behaved like a liquid-like density and a gas-like diffusivity/viscosity for transportation.…”

“…However, maintaining the complexity of the cell microenvironment causes many difficulties in the use of synthetic scaffolds. Therefore, studies on the extracellular matrix (ECM) are increasing dramatically in all areas of the world 3 .…”

Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes

“…Detergents have also been shown to reduce levels of beneficial growth factors in some tissue 23 . Moreover, residual surfactants are cytotoxic 24 , yielding detrimental effects on the subsequent recellularization.As an alternative to detergents, supercritical carbon dioxide (scCO 2 ) has been proposed as a safer, non-toxic and non-residual technology to obtain bio-ECMs from multiple tissues, including porcine heart valve, equine tendon, porcine esophagus, bovine cartilage, human skin, porcine aorta, and human amniotic membrane 17,21,[25][26][27][28][29][30] . The vast majority of these studies have revealed no or a very moderate influence of pressurized carbon dioxide on ECM structure and mechanical properties.…”

Decellularized Scaffolds for Skin Repair and Regeneration

“…On human skin, processing with supercritical CO 2 after a hypertonic 1 M NaCl exposure has produced results similar to those with SDS in terms of cellular content removal (DNA level post-treatment <30 ng/mg of dried ECM) [91]. Small fractures reducing global ECM density were detected, but the fibronectin network seems to be unaffected.…”

“…To create hypertonic conditions, NaCl is commonly dissolved at 1 M to weaken cell membranes, as reported in the literature and in patented protocols to process fibroblast sheets, fish skin, and human skin [83,86,89]. Hypertonic NaCl also has been used to dissociate epidermis and dermis [90,91]. A hypertonic solution composed of 1 M KI has been used to process mouse skin [87].…”