Preparation of extracellular matrix of fish swim bladders by decellularization with supercritical carbon dioxide

Abstract

Fish swim bladders used to be considered as byproducts or waste in fishery; however, they are potential materials for biological medicine with abundant collagen. In this work, an efficient noncytotoxic decellularization process using sodium dodecyl sulfate (SDS) ternary system assisted with supercritical carbon dioxide (scCO₂) as the green extraction fluid and ethanol (ET) as the cosolvent has been developed to harvest acellular fish swim bladders (AFSBs). The experimental results show that the tissue treated by SDS assisted with scCO₂ and ethanol at 37 °C and 25 MPa can be decellularized thoroughly and maintains intact fibers and uniform pore distribution, which resulting in a tensile strength of 5.61 MPa and satisfactory biocompatibility. Meanwhile, the residual SDS content in scCO₂/SDS/ET ternary system is 0.0122% which is significantly lower than it in scCO₂/SDS system due to the enhanced mass transfer rate of SDS in tissues by scCO₂ with ethanol. The synergy between SDS and ethanol can enhance the diffusion coefficient and the solubility of SDS in scCO₂, which reduced the contact time between SDS and tissues. Meaningfully, the results obtained in this work can not only provide a novel strategy to produce acellular matrix with superior properties, but also offer a further understanding of the decellularization through scCO₂ extraction processing with the synergy of suitable detergent/cosolvent.

Keywords: Decellularization; Fish swim bladder; Interaction energy; Sodium dodecyl sulfate; Supercritical carbon dioxide.

Fig. 1

Histology of AFSBs specimens: H&E staining sections of (a-1) Native, (a-2) scCO₂/SDS/ET, (a-3) scCO₂/ET, (a-4) scCO₂/SDS, (a-5) SDS-12-24 and (a-6) SDS-12-1. EVG stained sections of (b-1) Native, (b-2) scCO₂/SDS/ET and (b-3) SDS-12-24

Fig. 2

DNA content of AFSBs. Values below 50 represent complete decellularization

Fig. 3

SEM images of Native, scCO₂/SDS/ET and SDS-12-24

Fig. 4

Stress–strain curves from different treatment of Native, scCO₂/SDS/ET, scCO₂/ET and SDS-12-24

Fig. 5

Quantitation of residual SDS: a SDS standard absorbance curve and b residual SDS in samples

Fig. 6

CCK-8 assay results of a cell proliferation on the AFSBs (based 8 mm × 8 mm) on 1, 3, 5, and 7 days, b cell cytotoxicity on 1, 3, and 5 days. The Native group was served as the control group for comparison. All values are the means with ± SD. n = 3 (*p < 0.05)

Fig. 7

a E_SDS-SDS, b E_SDS-CO2, and E_SDS-ET in scCO₂/SDS and scCO₂/SDS/ET systems (kJ/mol), c number of H-bonds between SDS and ethanol molecules, RDF between pairs of d SO₄⁻–SO₄⁻, e SO₄⁻–Na⁺ and f SO₄⁻–CO₂ and SO₄⁻–ethanol in SDS50 and SDS50/ET systems