Comparative in vitro and in vivo assessment of three experimental extracellular matrix meshes for soft tissue remodeling

Figures

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  Fig. 1. Surface and ultrastructural analysis of ECM meshes. Surface morphology was assessed using high-resolution optical microscopy (A–D), and ultrastructure was examined by scanning electron microscopy, SEM (E–H). The Composite mesh (A, E) displays a compact, integrated structure. Human (B, F) and porcine (C, G) acellular dermis meshes show a fragmented, flaky collagen morphology. Porcine SIS (D, H) possesses a characteristic porous, interwoven collagen fiber network. Scale bars: 500 μm (E), 50 μm (F–H). Magnification times: 20x (A), 280x (B), 280x (C), 820x (D).

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  Fig. 2. In vitro enzymatic degradation kinetics of ECM meshes. Mesh strips (2 cm × 1 cm; initial dry weight W0) were incubated in 19.2 U/mL collagenase I (PBS, pH 7.4) at 37 °C with agitation (60 rpm). Enzyme solution was refreshed daily. Residual mass (W1) was measured after 3, 7, and 10 days.

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  Fig. 3. Quantification of immunogenic residues in decellularized ECM meshes. (A) α-Gal epitope content in PAD and SIS meshes. Values represent residual α-Gal relative to untreated porcine raw tissues (n = 3). Clearance efficiency was calculated via competitive ELISA inhibition assays. (B) Residual DNA content across ECM meshes. Linear standard curve (λ-DNA; y = 0.0172x - 0.9801, R2 = 0.9994) and PicoGreen quantification show DNA residues below ISO 10993–18 thresholds. Data are presented as mean ± SD (n = 3).

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  Fig. 4. Protein concentration detection in components of ECM meshes using BCA kits.

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  Fig. 5. Cytotoxicity evaluation on L929 cell line by the extract of ECM meshes. (A) The average cell viability of L929 cells with different kinds of mesh extracts on 1×10^5/mL cell concentration during 72 h incubation in the MTT method (n = 6). (B) The dynamic cell index from different kinds of mesh extracts on 1 × 105/mL cell concentration during 72 h incubation in the RTCA method (n = 4). Record impedance signals every 20 min in the first 12 frames (4 h) and record every 15 min in the following 80 frames (20 h). The RTCA software provides the CI value based on the impedance signal. Data are presented as mean ± SD.

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  Fig. 6. Chemotaxis effect evaluation of ECM mesh components on BALB/c 3T3 cells. Load Bal/b/c 3T3 cells into the top well of the transwell chamber in a quantity of 3 × 104 cells per well. Add each group of ECM mesh gel, precipitation and supernatant samples at the bottom and then cultivate under the condition of 37 °C and 5% CO2 for 3 h and stain migrating cells with DAPI. Observe and count cells under a microscope. Results showed that the inhibitory effect of SIS mesh hydrogel on cell chemotaxis were significantly lower than that of HAD mesh hydrogel and PAD mesh hydrogel. The inhibitory effect of PAD mesh supernatant on cell chemotaxis was significantly lower than that of HAD mesh supernatant. Data are presented as mean ± SD (**p = 0.000036 for HAD hydrogel vs DMEM, **p = 0.000032 for HAD supernatant vs DMEM, **p = 0.000038 for HAD pellet vs DMEM, **p = 0.00045 for PAD hydrogel vs DMEM, **p = 0.000037 for PAD supernatant vs DMEM, **p = 0.000054 for PAD pellet vs DMEM, **p = 0.000054 for SIS hydrogel vs DMEM, **p = 0.00037 for SIS supernatant vs DMEM, **p = 0.000046 for SIS pellet vs DMEM., n = 3).

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  Fig. 7. Local tissue reaction and degradation of ECM meshes (40 ×). Implant ECM meshes and negative controls into the back muscle tissue of rabbits, respectively. After 1 week, 4 week, 13 week and 26 week implantation, take the implanted samples to perform the mesh degradation and histopathological analysis. Local reaction scoring after implantation was perform according to the GB/T 16,886.6–2022 standards. Subtract the average value of the control from the average value of the test to determine the reactivity level. (A–D) 1 w, 4 w, 13 w and 26 w after PAD mesh implantation, (E–H) 1 w, 4 w, 13 w and 26 w after HAD mesh implantation, (I–L) 1 w, 4 w, 13 w and 26 w after SIS mesh implantation, (M–P) 1 w, 4 w, 13 w and 26 w after the composite mesh implantation, (Q–T) 1w, 4 w, 13 w and 26 w after the HDPE control implantation. (U) The average score for the PAD mesh, HAD mesh, SIS mesh, Composite mesh and HDPE negative control based on the semi-quantitative scoring criteria (n = 12). Scale bars: 20 μm (A–T).

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  Fig. 8. ECM inflammation induced during mesh degradation and the polarization of macrophages. Protein expression levels of inflammatory cytokines in rat serum, (A) Rat abdominal wall injury repair model was established for 4 weeks, (B) Rat abdominal wall injury repair model for 13 weeks (C) Rat abdominal wall damage repair model for 26 weeks; Gene expression of inflammatory-associated cytokines, (D) A rat abdominal wall injury repair model was established for 4 weeks, (E) Establishment of the rat abdominal wall injury repair model for the first 13-week (F) rat abdominal wall injury repair model for the next 26 weeks; Immuno-histochemical analysis of the expression of macrophage surface molecules in rat abdominal wall tissue, (G & H) Expression of CD86 + M1 type macrophages in the extract, (I & J) Expression of CD206 + M2 type macrophages in the extract. Scale bars: 50 μm (H&G).

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  Fig. 9. Collagen remodeling in rat abdominal wall defects. (A) Sirius Red staining of collagen types, (B) Masson’s trichrome staining of collagen fibers, (C) H&E staining showing macrophage infiltration. Scale: 20 × /40 × as indicated. Scale bars: 100 μm (A), 50 μm (B–C).