Bioinks Enriched with ECM Components Obtained by Supercritical Extraction

Abstract

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.

Effect of sterilization processes on alginate/gelatin inks for three-dimensional printing

“…All tests were set at 37°C and were repeated twice to ensure data reproducibility. One hertz constant frequency and shear rate from 1 s -1 to 100 s -1 were used to obtain shear viscosity curves, which were fitted to Cross model for shear thinning fluids (Equation I) [ 35 , 36 ] :…”

Biomaterials of human source for 3D printing strategies

“…An alternative to these strategies is the inclusion of external crosslinking agents for improved printability. The extensive literature on crosslinking agent mechanisms [175], allowed for the application of less cytotoxic solutions on human-derived materials such as thrombin (that can induce reticulation on fibrinogen) [123,197,199], and calcium ions (that enable reticulation of anionic alginate) [180,245]. Another property that is trending among researchers is the use of temperature for the reticulation of matrices derived from decellularization procedures [25,118,242,243].…”

“…In these cases, the use of an external support bath is required (section 3.1) [8,250]. The used support baths/layers for 3D printing of human matrices were composed either of gelatin or agarose microparticles and demonstrated having an additional role in diffusing ions for additional filament crosslinking [245].…”