In vitro model assesses the susceptibility of polymeric scaffolds for material-driven heart valve regeneration to calcification

Figures

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  Fig. 1. Study Design. A Within the in vitro model, pVICs are seeded on fibrous, electrospun polymer scaffolds in Transwell inserts and cultured for 21 days in calcification-permitting medium (CM). B Cultured constructs are evaluated for cytotoxicity, calcification, and collagen formation after 3 weeks of culture. C The application of the model has been tested in two ways: (I) three materials used for in situ tissue engineering are selected and thoroughly analyzed before cell seeding and after cell culture. They are compared to a clinically used material, glutaraldehyde treated bovine pericardium. (II) An applicational pilot evaluated the calcification potential of constructs cultured under cyclic stretch. pVICS, porcine valvular interstitial cells; DMEM, Dulbecco’s modified eagle medium; FBS, heat-inactivated fetal bovine serum; PS, penicillin/streptomycin; β-GP, β-glycerophosphate; SEM, scanning electron microscopy; AFM, atomic force microscopy; DSC, differential scanning calorimetry; GPC, gel permeation chromatography; LDH, lactate dehydrogenase; IF, immunofluorescence; PCL, polycaprolactone; PCL-BU, bisurea chain extended PCL; PC-BU, bisurea chain extended polycarbonate

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  Fig. 2. Fibrous material scaffolds allow for cell integration throughout the scaffold to form constructs that can be cultured for up to 21 days and show calcification potential. A Cells cultured for 7 days in FIB medium (right) showed lower expression of α-SMA (green) and calponin (yellow), as well as a more round-shaped morphology, compared to cells cultured in NM (left). B Expression of vimentin increased and α-SMA decreased after 7 days of culture in FIB medium (n = 2), compared to NM (n = 4). C Transwell system used for suspension scaffold culture and × 7000 zoom of scanning electron microscopy images of PCL scaffold as used within the model. D Cell integration into the scaffold after 21 days of culture (PCL Example from Experiment 3) shows calcification (pink) and F-actin positive (cyan) cells on the top (left), as well as within the bottom (right) of the scaffold, signifying cell integration throughout the scaffold. Further, a close relation between cells and scaffold fibers (white shadows), particularly at the bottom of the scaffold. NM, normal medium; FIB, fibroblast inactivation medium; α-SMA, α-smooth muscle cell actin; F-actin, filamentous actin

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  Fig. 3. Application I: HVTE material constructs show significantly less calcification compared to pericardial patch constructs after 21 days of culture. A Calcification imaging using a hydroxyapatite-tracing calcium probe (pink) showed an increase in calcification within the pericardial patch control group (bottom row), compared to the three HVTE materials (top 3 rows). HVTE materials showed varying results between experiments, with an increase in calcification in PCL within experiments 1 and 2 (top row, first two columns), and an even distribution of calcification in experiments 3 and 4 (right three columns) between the HVTE materials. B Quantification of imaging for calcification per cell count confirmed the results shown in A. C Presto blue analysis showed no significant difference in cell activity between groups after 21 days of culture. D LDH assay results showed 8–9% cell death directly after cell-seeding onto HVTE scaffolds and 3% after seeding onto pericardial patch tissue, with a significant decrease in cytotoxicity within all groups to 2.5–4% after 21 days of culture. Represented as mean (line) + standard deviation (dotted line) E Calcium quantification using a cresolphthalein…

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  Fig. 4. Correlation of cellularity, collagen formation, and calcification shows cell-dependent calcification in HVTE constructs but not in pericardial tissue, and equal cell-dependent collagen formation within HVTE constructs. A Bare, acellular scaffolds cultured for 21 days in CM showed no calcification (pink) in HVTE materials (top three), but significant calcification in pericardial tissue (bottom). Representative maximum intensity projections. B Calcification in HVTE constructs (top three) was colocalized with F-actin (cyan) positive cell locations, whereas in pericardial tissue, calcification was found in F-actin positive and negative areas (bottom). Representative single z-slice images. C Collagen staining (green) in combination with nuclei (dark blue) and calcification (pink) showed more aligned, dense collagen in pericardial tissue compared to HVTE constructs, and calcification particles seemed not to be integrated into the collagen layer. Representative single z-slice images. D Calcium assay quantification showed similar calcium levels between pericardial tissue, independent of cell seeding, and E image calcium quantification showed an increase in calcium particles within bare,…

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  Fig. 5. Scaffold properties do not explain inter-experimental differences in material calcification. A Scaffolds were evaluated using scanning electron microscopy (SEM) to show fiber diameter and morphology. B Fiber diameter quantification showed a similar fiber distribution, with a significantly higher fiber diameter of PC-BU in the first three experiments. C Molecular number was equally distributed within PCL and PCL-BU materials throughout experiments (n = 1). D Molecular weight of PCL was slightly higher within experiment 3 and otherwise equally distributed throughout experiments (n = 1). E Fiber-reduced modulus evaluation showed a higher reduced modulus of PCL fibers compared to PCL-BU and PC-BU, with a slight increase within experiments 2 and 3. F AFM evaluated fiber morphology showed similar fiber characteristics compared to SEM evaluation. G DSC data showed a single melting peak in PCL and a double peak in H PCL-BU as well as I PC-BU that remained unchanged between experiments. PCL, polycaprolactone; PCL-BU, bisurea chain extended PCL; PC-BU, bisurea chain extended polycarbonate; SEM, scanning electron microscopy; GPC, gel permeation chromatography; Mn, molecular number; Mw,…

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  Fig. 6. Application III: application of cyclic strain to pVIC-loaded HVTE constructs does not significantly change their calcification potential. A, B Cyclic strain analysis of PCL-BU (A) and PC-BU (B) scaffolds in the x-direction at day 0 (before the start of the experiment, top), in the y-direction at day 0, and the maximum and minimum x-directional strain of cell-cultured scaffolds at day 21 of the experiment show an x-directional strain at day 0 within both materials and material fatigue over time, as indicated by a negative minimum strain at day 21 within PCL-BU material (A, bottom). C, D The maximum strain applied to the constructs was 10–15% and did not change throughout the 21-day culture period. The minimum strain stayed at 0% within cell-free scaffolds. Within cell-loaded constructs, a minimum strain of − 0.5% was found within PCL-BU scaffolds at day 21. E Calcium imaging shows no significant differences in calcification between constructs in strained or static conditions. F DNA assay showed a trending but insignificant increase in DNA in stretched materials. G Calcium assay showed no significant difference between strained and unstrained materials. H Hyp assay shows no…