Stem Cell-Secreted Allogeneic Elastin-Rich Matrix with Subsequent Decellularization for the Treatment of Critical Valve Diseases in the Young

Figures

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  Figure 1. Developed in-house “torpedo” bioreactor with computational fluid dynamics (CFD; ANSYS Workbench, ANSYS Inc., Canonsburg, PA, USA). (A) A design of our bioreactor with the major parts, including the torpedo and tube inlet and outlet. (B) Bioreactor schematic filled with cell culture media, the torpedo, and a bio-scaffold on the torpedo with seeded human stem cells represented by the green dots. (C) A square waveform was used with a 17% forward and 83% backward flow. This waveform provided a specimen (D) time-averaged wall shear stress (TAWSS) of 3.1 dynes/cm2 and an (E) OSI of ~0.20.

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  Figure 2. ECM quantification of static and dynamic PSIS valves. (A,C) Static PSIS valves were assessed for ECM quantification via spatial intensity maps (B,D–G). Spatial intensity maps for these static PSIS valves at a depth of (A) 16 µm and (C) 304 µm were computed. At the outer surface of the PSIS valve, there was (B) 21% mucins present with no other ECM components. At a deeper depth, closer to the inner surface of the PSIS valve, there was a presence of (D) 9% mucins, (E) 20% collagen, (F) 0.6% elastin and (G) 1% fibrin. (H,M) Dynamic PSIS valves were also assessed for ECM quantification via spatial intensity maps (I–L,N–Q). Spatial intensity maps for these dynamic PSIS vales at a depth of (H) 16 µm and (M) 304 µm were computed. At the outer surface of the PSIS valve, there was (I) 14% mucins, (J) 16% collagen, (K) 1% elastin and (L) 4% fibrin present. At a larger depth, closer to the inner surface of the PSIS valve, there was a presence of (N) 21% mucins, (O) 16% collagen, (P) 9% elastin and (Q) 11% fibrin. Magnification 50×.

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  Figure 3. Elastin immunofluorescent imaging and spatial intensity mapping for: (a) seeding hBMMSCs onto PSIS bio-scaffold substrates, rotisserie cultured for 8 days followed by an additional 14 days of oscillatory flow conditioning (TAWSS of 3.1 dynes/cm2 and an OSI of 0.20), (b) seeding hBMMSCs onto PSIS bio-scaffold substrates and then statically (rotisserie) cultured for 22 days, and (c) the raw PSIS bio-scaffold.

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  Figure 4. ECM quantification of the control (untreated) and decellularized elastin-rich engineered valve tissues. (a) Images of the Movat’s stained control group and (b) of the Movat’s stained decellularized group were assessed for quantification of ECM collagen and elastin spatial intensity maps.

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  Figure 5. Rat VICs collagen secretion quantification and Masson’s Trichrome staining after seeding and subsequent culture for 8 days of static (rotisserie) culture and 7 days of oscillatory flow conditioning (TAWSS of 3.1 dynes/cm2 and an OSI of 0.20; n = 4 specimens/group). (a) Average ± SD of collagen secretion by rat VICs seeded on the elastin-rich engineered ECM versus raw PSIS bio-scaffolds. Masson’s Trichrome histological images of the de novo collagen secretion by rat VICs on the outer substrate surface (sectioned at 16 μm from the outer surface) where the cells were seeded (n = 4/group) on the: (b) engineered elastin-rich engineered ECM and (c) raw PSIS bio-scaffolds.