Evaluation of Supercritical CO2-Assisted Protocols in a Model of Ovine Aortic Root Decellularization

Abstract

One of the leading trends in the modern tissue engineering is the development of new effective methods of decellularization aimed at the removal of cellular components from a donor tissue, reducing its immunogenicity and the risk of rejection. Supercritical CO₂ (scCO₂)-assisted processing has been proposed to improve the outcome of decellularization, reduce contamination and time costs. The resulting products can serve as personalized tools for tissue-engineering therapy of various somatic pathologies. However, the decellularization of heterogeneous 3D structures, such as the aortic root, requires optimization of the parameters, including preconditioning medium composition, the type of co-solvent, values of pressure and temperature inside the scCO₂ reactor, etc. In our work, using an ovine aortic root model, we performed a comparative analysis of the effectiveness of decellularization approaches based on various combinations of these parameters. The protocols were based on the combinations of treatments in alkaline, ethanol or detergent solutions with scCO₂-assisted processing at different modes. Histological analysis demonstrated favorable effects of the preconditioning in a detergent solution. Following processing in scCO₂ medium provided a high decellularization degree, reduced cytotoxicity, and increased ultimate tensile strength and Young's modulus of the aortic valve leaflets, while the integrity of the extracellular matrix was preserved.

Keywords: aortic valve; biomaterials; biomedical engineering; decellularization; supercritical CO2.

Figure 1

Histological study of native (A1,A2) and decellularized ovine aortic roots under hematoxylin-eosin staining. Decellularization protocols included scCO₂-assisted processing at the pressure of 15 MPa (B1,B2) and 25 MPa (C1,C2) with the use of ethanol as a co-solvent; combinations of alkaline treatment with scCO₂-assisted processing at the pressure of 10 MPa (D1, D2) and 15 MPa (E1,E2); detergents alone (F1,F2) or with following scCO₂ treatment at the pressure of 15 MPa (G1,G2) and 25 MPa (H1,H2). Three layers are indicated in aortic valve leaflets: ventricularis (V), fibrosa (F) and spongiosa (S). Additionally, three layers are indicated in aortic walls: intima (I), media (M) and adventitia (A). Typical accumulations of cells are shown with asterisks (*, see Supplementary Figure S1 for enlarged images).

Figure 2

Collagen content in aortic valve leaflets and walls of native (A1–4) and decellularized ovine aortic roots. Decellularization protocols included scCO₂-assisted processing at the pressure of 15 MPa (B1–4) and 25 MPa (C1–4) with the use of ethanol as a co-solvent; combinations of alkaline treatment with scCO₂-assisted processing at the pressure of 10 MPa (D1–4) and 15 MPa (E1–4); detergents alone (F1–4) or with following scCO₂ treatment at the pressure of 15 MPa (G1–4) and 25 MPa (H1–4). The collagen fibers were stained red with Picrosirius red. Dashed lines mark the areas where the 400× images were acquired. 100× scale bar = 100 µm; 400× scale bar = 300 µm.

Figure 3

Elastin content in aortic valve leaflets and walls of native (A1–4) and decellularized ovine aortic roots. Decellularization protocols included scCO₂-assisted processing at the pressure of 15 MPa (B1–4) and 25 MPa (C1–4) with the use of ethanol as a co-solvent; combinations of alkaline treatment with scCO₂-assisted processing at the pressure of 10 MPa (D1–4) and 15 MPa (E1–4); detergents alone (F1–4) or with following scCO₂ treatment at the pressure of 15 MPa (G1–4) and 25 MPa (H1–4). Weigert staining. Dashed lines mark the areas where the 400× images were acquired. 100× scale bar = 100 µm; 400× scale bar = 300 µm.

Figure 4

Mechanical characteristics and cytotoxicity of ovine aortic roots decellularized in a 0.5% sodium dodecyl sulfate/0.5% sodium deoxycholate detergent solution (SDS/SD) and after the subsequent processing in scCO₂ medium (T = 37 °C, P = 25 MPa, t = 3 h). (A). Mechanical characteristics established by atomic force microscopy. Differences between the groups were analyzed by one-way Brown-Forsythe ANOVA followed by a post-hoc Tukey test. (B). [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) test for the estimation of cytotoxicity towards 3T3 murine fibroblasts. (C). Confocal laser scanning microscopy of human umbilical cord multipotent mesenchymal stromal cells stained with calcein-AM (green) and ethidium homodimer-1 (red) after 48 h of culturing on the sample surfaces and a glass coverslip (control). Scale bar = 100 µm.

Figure 5

Scanning electron microscopy of ovine aortic roots decellularized in a 0.5% SDS/0.5% SD detergent solution (SDS/SD) and after the subsequent processing in scCO₂ medium (T = 37 °C, P = 25 MPa, t = 3 h). Scale bar = 10 μm.