Contributions of supercritical fluid technology for advancing decellularization and postprocessing of viable biological materials

Duarte, Marta M.M.F.; Silva, Inês V.; Eisenhut, Tony; Bionda, Nina; Duarte, Ana Rita C.; Oliveira, Ana L.

doi:10.1039/d1mh01720a

Cited by 23 publications

(12 citation statements)

References 194 publications

(367 reference statements)

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“…dsDNA values were higher than the arbitrarily definition of decellularization of 50 ng dsDNA/mg dry scaffold for all scCO 2 methods. Changes in the duration of the decellularization process and addition of co-solvents might help to reduce DNA content in scCO 2 processed scaffolds even more [22] . Previous studies indicated that complete removal of chondrocytes might be undesirable: immunogenicity is low and the recellularization process afterwards is challenging.…”

Section: Discussionmentioning

confidence: 99%

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

Wit

Dis

Bertrand³

et al. 2023

Acta Biomaterialia

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Section: Discussionmentioning

confidence: 99%

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

Wit

Dis

Bertrand³

et al. 2023

Acta Biomaterialia

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“…While it is unclear which exact mechanisms occur for collagen in bone, it appears that the increasing SCF‐ethanol treatment time may control whether the ethanol impact on bone's material property is reversible or not after rehydration. Finally, despite ethanol as additive in the SCF‐treatment process significantly improving the fracture toughness, it is important to note that its efficacy as a sterilant additive to achieve the required SAL of 10 −6 is lower than additives such as peracetic acid or hydrogen peroxide 93‐96 …”

Section: Discussionmentioning

confidence: 99%

“…Finally, despite ethanol as additive in the SCF-treatment process significantly improving the fracture toughness, it is important to note that its efficacy as a sterilant additive to achieve the required SAL of 10 À6 is lower than additives such as peracetic acid or hydrogen peroxide. [93][94][95][96]…”

Section: Nalla Et Al Have Shown That Initiation and Growth Toughness ...mentioning

confidence: 99%

Effect of gamma irradiation and supercritical carbon dioxide sterilization with Novakill™ or ethanol on the fracture toughness of cortical bone

Shin,

Pelletier,

Lovric

et al. 2023

J Biomed Mater Res

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Sterilization of structural bone allografts is a critical process prior to their clinical use in large cortical bone defects. Gamma irradiation protocols are known to affect tissue integrity in a dose dependent manner. Alternative sterilization treatments, such as supercritical carbon dioxide (SCCO2), are gaining popularity due to advantages such as minimal exposure to denaturants, the lack of toxic residues, superior tissue penetration, and minor impacts on mechanical properties including strength and stiffness. The impact of SCCO2 on the fracture toughness of bone tissue, however, remains unknown. Here, we evaluate crack initiation and growth toughness after 2, 6, and 24 h SCCO2‐treatment using Novakill™ and ethanol as additives on ~11 samples per group obtained from a pair of femur diaphyses of a canine. All mechanical testing was performed at ambient air after 24 h soaking in Hanks' balanced salt solution (HBSS). Results show no statistically significant difference in the failure characteristics of the Novakill™‐treated groups whereas crack growth toughness after 6 and 24 h of treatment with ethanol significantly increases by 37% (p = .010) and 34% (p = .038), respectively, compared to an untreated control group. In contrast, standard 25 kGy gamma irradiation causes significantly reduced crack growth resistance by 40% (p = .007) compared to untreated bone. FTIR vibrational spectroscopy, conducted after testing, reveals a consistent trend of statistically significant differences (p < .001) with fracture toughness. These trends align with variations in the ratios of enzymatic mature to immature crosslinks in the collagen structure, suggesting a potential association with fracture toughness. Additional Raman spectroscopy after testing shows a similar trend with statistically significant differences (p < .005), which further supports that collagen structural changes occur in the SCF‐treated groups with ethanol after 6 and 24 h. Our work reveals the benefits of SCCO2 sterilization compared to gamma irradiation.

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“…Other co-solvents have been posed as potential alternatives to enhance scCO 2 efficiency [37]. One such is Dehypon ® , a compound with a molecular weight higher than ethanol that could therefore act as a stronger enhancer of the SCF's density [38].…”

Section: Discussionmentioning

confidence: 99%

Bioinks Enriched with ECM Components Obtained by Supercritical Extraction

et al. 2022

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Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell’s own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO2 protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extract’s composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.

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Contributions of supercritical fluid technology for advancing decellularization and postprocessing of viable biological materials

Abstract: This review summarizes relevant literature on scCO2-assisted decellularization, and discusses major topics dominating the field, such as mechanism of action, effect of cosolvent and operational parameters, and range of results across different works.

Cited by 23 publications

References 194 publications

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

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

Effect of gamma irradiation and supercritical carbon dioxide sterilization with Novakill™ or ethanol on the fracture toughness of cortical bone

Bioinks Enriched with ECM Components Obtained by Supercritical Extraction

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