Inverse opal substrate-loaded mesenchymal stem cells contribute to decreased myocardial remodeling after transplantation into acute myocardial infarction mice

Abstract

Background: The two-dimensional incubation method is now the most commonly method for mesenchymal stem cell (MSC) production. however, gene expression and secretion of growth factors are relatively low; thus, the transplanted cells cannot be effectively utilized for potential clinical applications after acute myocardial infarction (AMI).

Objectives: We aimed to investigate whether our newly made substrates of inverse opal with specific surface microstructures for MSC culturing can increase the viability of the cells and can contributes to decreased myocardial remodeling after transplanted to AMI mice.

Methods: The inverse opal structure is fabricated by the convenient bottom-up approach of the self-assembly of colloidal nanoparticles. Mouse-derived MSCs were then cultured on the substrates when expanded at different times to investigate the cell growth status including morphology. Then the inverse opal substrates loaded MSCs were transplanted to AMI mice, cardiomyocyte apoptosis and LV remodeling were further compared. To explore the possible mechanisms of curation, the secretions and viability of MSCs on substrates were determined using mice ELISA kits and JC-1 mitochondrial membrane potential assay kits respectively at normal and hypoxic conditions.

Results: 6 times expanded inverse opals allowed greatly the orderly growth of MSCs as compared to four (34% ± 10.6%) and two (20%±7.2%) times expanded as well as unexpanded (13%±4.1%) (P<0.001). Nearly 90% of MSCs showed orientation angle intervals of less than 30° when at the 6X expanded (89.6%±25%) compared to the percent of cells with 30°-60° (8.7%±2.6%) or ≥60° (1.7%±1.0%) orientation angle (P<0.001). After inverse opal loaded MSCs transplanted to AMI mice, greatly decreased apoptosis of cardiomyocytes (20.45%±8.64% vs.39.63%±11.71%, P<0.001) and infarction area (5.87±2.18 mm² vs 9.31±3.11 mm², P<0.001) were identified. In the end, the viability of inverse opal loaded MSCs determined by membrane potential (P<0.001) and the secretion of growth factors including VEGF-α, SDF-1 and Ang-1 (P<0.001) were both confirmed significantly higher than that of the conventional culture in petri dish.

Conclusion: The structure of inverse opal can not only adjust the arrangement of MSCs but also contribute to its orientated growth. Inverse opal loaded MSCs transplantation extremely curbed myocardial remodeling, the underlying mechanisms might be the high viability and extremely higher secretions of growth factors of MSCs as devoted by this method.

Keywords: AMI; MSCs; inverse opal.

Figure 1

Characterization of inverse opal substrates. Notes: (A) Schematic diagram of the preparation of the expanded inverse opal substrate. (B) Photos and SEM images of SiO₂, inverse opal scaffold expanded by 1–6 times (a–d) and the corresponding pattern on SEM (e–h). The double-sided arrows indicate the direction of expansion. Scale bars in SEM images are 2 µm. (C) PSI value calculation, where A is the area and P is the perimeter of the micropores. Usually, the PSI ranges from 1 (a perfect circle) to 0 (a straight line), which reveals the degree of elongation of a nanoscale pore. Abbreviations: SEM, scanning electron microscopy; PSI, pore shape index.

Figure 2

MTT assays of MSCs cultured on different expanded inverse opal substrates. Note: Control 0.77±0.29/OD490 nm vs 1×, 0.71±0.33/OD490 nm vs 2×, 0.82±0.24/OD490 nm vs 4×, 0.69±0.27/OD490 nm vs 6×, 0.83±0.37/OD490 nm. Abbreviation: MSC, mesenchymal stem cell.

Figure 3

The novel expanded inverse opal subtrate allows orderly growth of MSCs. Notes: (A) Light microscopy images of MSCs. Schemes of MSCs cultured on a gradient-expanded inverse opal substrate (a); light microscopy images of MSCs cultured on different expanded inverse opal substrates (b–e). (B) Fluorescence microscopy images of MSCs cultured on a gradient-expanded inverse opal substrate from unexpanded portion to six × of expanded portion (a–d); the double-sided arrows indicate the direction of expansion. Scale bar =200 µm. (C) Orientation angle frequency distribution of MSCs cultured on different portions of the expanded inverse opal substrate after 7 days. ^*P<0.05 vs the corresponding control group,^** vs 1× and 2×, ^*** vs 1×, 2× and 4×. Abbreviation: MSCs, mesenchymal stem cells.

Figure 4

Expanded inverse opal makes a certain orientation angle with growing MSCs. Notes: (A) Schematic diagram of the orientation angle of the cells and the expanded scaffolds. (B–E) Percentage of orientated MSCs was calculated at 1×, 2×, 4× and 6× respectively, 500 cells in total were measured on each portion. ^*P<0.05 vs the corresponding control group of ≥60°, ^**P<0.05 vs the corresponding control group of both ≥60° and 30°–60°. Abbreviation: MSCs, mesenchymal stem cells.

Figure 5

Inverse opal-loaded MSC transplantation contributes to decreased apoptosis and attenuated myocardial remodeling (n=12). Notes: (A, B) Image of immunofluorescent staining for TUNEL (apoptotic cardiomyocyte) in the infarct border zone 3 days after MSCs transplantation was shown and compared. (C–E) Infarct size at baseline (day 1) and at day 14 as well as the thickness of the LV posterior walls at day 21 in the two groups. The red arrows point to the LVPW at day 21 and the green dashs point to the infarct size. The data represent the mean ± SD. ^*P<0.05 vs the corresponding control group. Abbreviations: MSCs, mesenchymal stem cells; LVPW, left ventricular posterior wall.

Figure 6

Secretion of growth factors by MSCs and cell viability analysis. Notes: (A–C) SDF-1, VEGF-α, and Ang-1 derived from MSCs grown on the substrates of 6× expanded inverse opal (substrates) or unexpanded substrates (control group) were analyzed by ELISA kits. (D) Mitochondrial membrane potential of MSCs (calculated by PE/FITC) on the 6× expanded substrates and the control group under normal and hypoxia conditions. (E) Representative test results shown by FACSCalibur cytometer analysis. ^*P<0.05 vs the corresponding control group in normal conditions; ^#P<0.05 vs the corresponding control group in hypoxic conditions. Abbreviations: Ang-1, angiopoietin-1; FITC, fluorescein isothiocyanate; MSCs, mesenchymal stem cells; PE, phycoerythrin; SDF-1, stromal cell-derived factor 1; VEGF-α, vascular endothelium growth factor-α.

Figure 6

Secretion of growth factors by MSCs and cell viability analysis. Notes: (A–C) SDF-1, VEGF-α, and Ang-1 derived from MSCs grown on the substrates of 6× expanded inverse opal (substrates) or unexpanded substrates (control group) were analyzed by ELISA kits. (D) Mitochondrial membrane potential of MSCs (calculated by PE/FITC) on the 6× expanded substrates and the control group under normal and hypoxia conditions. (E) Representative test results shown by FACSCalibur cytometer analysis. ^*P<0.05 vs the corresponding control group in normal conditions; ^#P<0.05 vs the corresponding control group in hypoxic conditions. Abbreviations: Ang-1, angiopoietin-1; FITC, fluorescein isothiocyanate; MSCs, mesenchymal stem cells; PE, phycoerythrin; SDF-1, stromal cell-derived factor 1; VEGF-α, vascular endothelium growth factor-α.