PAS glycoprotein staining of control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).

<p>All scale bars represent 200 µm, with exception of the insert representing a higher magnification image (scale bar = 800 µm).</p

Average results of the evaluation of decellularization efficiency, semi-quantitative analysis of tissue structure and quantitative analysis of fibrillar and non-fibrillar ECM components in control non-decellularized intestine and in all decellularization global groups.

<p>Nuclear cell removal and the staining signal for picrosirius, Gomori reticulin, PAS and Alcian blue correspond to percentages in reference of control non-decellularized intestine samples and are expressed as means plus/minus standard deviations. Tissue DNA quantification is shown as nanograms of DNA per milligram of dry weight of tissue. HE results are shown as median values and quartiles Q1–Q3 and correspond to the following classification: (0) highly organized tissue, (1) low levels of disorganization, (2) intermediate levels of disorganization, (3) high tissue disorganization. The analysis of tissue structure by SEM (interfibrillar spaces and collagen fibers) is shown in a scale ranging between (−) (small and regular interfibrillar spaces and highly disorganized fibers and disrupted three-dimensional structure) and (+++) (very large and irregular interfibrillar spaces and highly organized fibers and adequate three-dimensional structure) as defined in the methods section.</p

Alcian blue proteoglycan staining of control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).

<p>All scale bars represent 200 µm, with exception of the insert representing a higher magnification image (scale bar = 800 µm).</p

Statistical <i>p</i> values corresponding to the comparison of the results obtained for each decellularization method and for each technique.

<p>Global decellularization groups were compared with the control non-decellularized samples and with the rest of global groups. Specific decellularization groups were compared with controls and with the other specific groups corresponding to the same decellularization agent. All comparisons were carried out by using the Student <i>t</i> test except the HE results, which were compared with the Mann-Whitney non-parametric test. Significant <i>p</i> values are labeled with “a”, whereas marginally significant <i>p</i> values are labeled with “b”.</p

Picrosirius collagen fibers staining of control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).

<p>All scale bars represent 200 µm, with exception of the insert representing a higher magnification image (scale bar = 800 µm).</p

Photographic images of control and decellularized SI samples using a patterned surface to estimate transparency.

<p>Images correspond to control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).</p

Scanning electron microscopy (SEM) images of control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).

<p>Scale bar represents 50 µm.</p

Gomori reticulin staining of control SI samples (A), and tissues decellularized with 1.5 M NaCl (B), 3 M NaCl (C), 5 M NaCl (D), 0.1% SDS (E), 0.3% SDS (F), 0.6% SDS (G), 0.1% triton X-100 (H), 0.3% triton X-100 (I), 0.6% triton X-100 (J), 10 min SC (K), 20 min SC (L), 30 min SC (M), 10 min UV (N), 20 min UV (O) and 30 min UV (P).

<p>All scale bars represent 200 µm, with exception of the insert representing a higher magnification image (scale bar = 800 µm).</p

Light transmittance analysis of control and decellularized SI samples.

<p>Panel A corresponds to samples decellularized using chemical agents, whereas results of the application of physical decellularization methods are shown in panel B. Non-decellularized intestine and native porcine cornea are shown as controls.</p

Histograms representing the efficiency of cell removal and tissue structure preservation as determined by DAPI (A), residual DNA quantification (B), picrosirius collagen staining (C), Gomori reticulin (D), PAS glycoprotein staining (E) and alcian blue proteoglycan staining (F).

<p>Bars correspond to average values ± standard deviations.</p