Geometry influences inflammatory host cell response and remodeling in tissue-engineered heart valves in-vivo

Figure 1. First- and second-generation TEM-based TEHVs. (A) Schematic of the cross-section of a first-generation TEHV comprising the nitinol stent, the valve wall, leaflet, and the characteristic acute angle of the hinge region (dotted square)6. Image was created using the software Tinkercad under Creative Commons license Non-Commercial 3.0. (B) Macroscopic appearance of a first-generation TEHV after in-vitro culture. (C) In the first-generation TEHV design the leaflets are exposed to compressive strain (dark blue areas in the coaptation and hinge regions) in radial direction. Image was re-adapted from Sanders et al.8 under the Creative Commons license 4.0. (D) Schematic of the cross-section of a second-generation TEHV7, characterized by a more profound hinge region (dotted square). Image was created using the software Tinkercad under Creative Commons license Non-Commercial 3.0. (E) Macroscopic appearance of a second-generation TEHV after in-vitro culture performed with an insert to impose valve geometry8. (F) In second-generation TEHV design radial tissue compression was limited to the hinge region, thereby not affecting the leaflets coaptation area. Image was re-adapted from Sanders et…

Figure 2. Hinge region of first-generation TEHVs at different time-points. Representative images of the H&E staining performed on the cross section of the valve. (A–D) 5 × magnification pictures of the hinge region of the valve (scale bars 500 μm). (E–H) 20 × magnification pictures of the hinge region marked by the dotted rectangles in panels (A–D) (scale bars 100 μm). (I–L) 40 × magnification images of the hinge region marked by dotted rectangles in panels (E–H) (scale bars 50 μm). Presence of contractile cells is detected with immunohistochemical images at 8 (N), 16 (O), and 24 (P) weeks. α-SMA+cells are already detected in the hinge area at the earliest time point considered and, over time, these cells are also present in the leaflet. Acute control valve (M) does not present α-SMA+cells. Scale bars: 500 μm, zoom: 5 × . Presence of T-lymphocytes is detected with immunohistochemical images at 8 (R), 16 (S), and 24 (T) weeks. Acute control valve (Q) does not present T-lymphocytes. T-lymphocytes cluster around polymer remnants or in follicles (yellow arrow). Scale bars: 100 μm, zoom: 10 × . In panel I arrow points at leukocytes, star symbol indicates fibrin, and triangle marks erythrocytes.…

Figure 3. Analysis of cell phenotype for the hinge area of first-generation TEHVs. Immunofluorescence images at the hinge area stained for M1 (red) and M2 (green) macrophages at 8 (A), 16 (B), and 24 (C) weeks follow-up. (D) Total cell number quantification in the wall and hinge region of first-generation TEHVs is showing an abundant cell presence indicating a high ongoing inflammatory response. (E) Quantification of M1 and M2 macrophages in the hinge region at the different time-points considered shows a prevalence of M1 over M2 subpopulation, with the number of M1 macrophages gradually decreasing over time while M2 macrophages is increasing. For immunofluorescence pictures: scale bars: 100 μm, zoom 1 × . For every measurement, cell number is normalized per μm3.

Figure 4. Tissue geometry of first-generation TEHVs. (A) Quantification of leaflet lengths from histological sections of first-generation TEHVs shows gradual leaflet shortening over time. (B) Quantification of hinge thickness from histological sections of first-generation TEHVs demonstrates increased values over time. Acute control TEHVs represent the initial leaflet length and hinge thickness of acutely-implanted valves. Measurements are expressed as average value ± standard deviation.

Figure 5. Hinge region of second-generation TEHVs at different time-points. Representative images of the H&E staining performed on the cross section of the valve. (A–C) 5 × magnification pictures of the hinge region of the valve (scale bars 500 μm). (D–F) 20 × magnification pictures of the hinge region marked by the dotted rectangles in panels (A–C) (scale bars 100 μm). (G–I) 40 × magnification of the hinge region marked by dotted rectangles in panels (D–F) (scale bars 50 μm). In panel (F), SP points at a hole left by a suture point. In panel (G), arrow points at leukocytes and triangle indicates erythrocytes. Immunohistochemical images of the hinge area of second-generation TEHVs stained for α-SMA at 8 weeks (K), and 52 weeks (L) follow-up. For this novel valve geometry, few to none α-SMA+ cells are detected in the hinge area or in the leaflet at the time-point considered. Acute control valve (G) does not present α-SMA+ cells. Scale bars: 500 μm, zoom: 5 × . Presence of T-lymphocytes is detected with immunohistochemical images at 8 (N) and 52 (O) weeks. Scale bars: 100 μm, zoom: 10 × .

Figure 6. Analysis of cell phenotype for the hinge area of second-generation TEHVs. Immunofluorescence images at the hinge area stained for M1 (green) and M2 (red) macrophages at 8 weeks (A) and 52 weeks (B) follow-up. (C) Total cell number quantification in the wall and hinge region of second-generation TEHVs. (D) Quantification of M1 and M2 macrophages in the hinge region at the different time-points considered shows a mild prevalence of M1 over M2 positive cells. For immunofluorescence pictures: scale bars: 100 μm, zoom 1 × . For every measurement, cell number is normalized per μm3.

Figure 7. Tissue geometry of second-generation TEHVs. (A) Quantification of leaflet lengths from histological sections of second-generation TEHVs. (B) Quantification of hinge thickness from histological sections of second-generation TEHVs. Acute TEHVs represent the initial leaflet length and hinge thickness of acutely-implanted valves. Measurements are expressed as average value ± standard deviation.

Figure 8. Summary of host cell infiltration behavior in response to the geometries of first- and second-generation TEM-based TEHVs. (A) We hypothesize that first-generation TEHVs presented a non-physiological geometry, which hindered the physiological formation of blood vortexes (arrows), thereby favoring the deposition of erythrocytes and leukocytes in the hinge region shortly after implantation. At 8 weeks follow-up, first-generation TEHVs showed sustained short-term performance with high expression of M1 macrophages and α-SMA+ cells markers. These inflammatory cells were mostly localized in the implanted TEM material, while few to no cells were detected in the newly formed pannus at the hinge area. Endothelialization was incomplete at this stage. After 24 weeks follow-up, first-generation TEHVs exhibited moderate to severe insufficiency, with a chronic inflammation and extensive maladaptive remodeling of the TEM material and pannus. This maladaptive remodeling was associated with evident hinge region thickening, abundant M1 and M2 macrophages, and α-SMA+ cells expression. Image was created using the software Tinkercad under Creative Commons license Non-Commercial 3.0 and Servier Medical…