Hydrogen Sulfide-Releasing Fibrous Membranes: Potential Patches for Stimulating Human Stem Cells Proliferation and Viability under Oxidative Stress

Abstract

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large surface area-to-volume ratio for an optimal release of gaseous signaling molecules. Recently, the antioxidant and anti-inflammatory properties of the endogenous gasotramsmitter hydrogen sulfide (H₂S), as well as its ability to stimulate relevant biochemical processes on the growth of mesenchymal stem cells (MSC), have been investigated. Therefore, in this work, new poly(lactic) acid fibrous membranes (PFM), doped and functionalized with H₂S slow-releasing donors extracted from garlic, were synthetized. These innovative H₂S-releasing mats were characterized for their morphological, thermal, mechanical, and biological properties. Their antimicrobial activity and effects on the in vitro human cardiac MSC growth, either in the presence or in the absence of oxidative stress, were here assessed. On the basis of the results here presented, these new H₂S-releasing PFM could represent promising and low-cost scaffolds or patches for biomedical applications in tissue repair.

Keywords: PLA fibers; antibacterial properties; cytotoxicity; garlic extracts; mesenchymal stem cells; microstructure; organosulfur compounds; thermal and mechanical properties.

Figure 1

Characterization of the GaOS extract. (a) RP-HPLC chromatogram of the GaOS obtained using C₁₈ column at 0.8 mL/min flow rate. The elution was performed with a linear gradient of solv. B (80% CH₃CN, 0.1% TFA). Peaks a and b are characteristic of DADS; (b) H₂S-release by 25 μL of GaOS after 30 min, 2 h and 5 h of incubation at 37 °C in the presence of 1 mM DTT and detected by MB assay. ** p < 0.02.

Figure 2

Microstructural characterization of the neat and GaOS doped PLA fibrous membranes (PFM). (a) SEM micrographs of PFM and PFM+GaOS (left: magnification 1k×, scale bar 50 µm, right: magnification 10k×, scale bar 5 µm); (b) SEM micrograph (magnification 30k×, scale bar 2 µm) and (c) energy dispersive X-ray (EDS) spectrum of PFM+GaOS. The white arrow indicates the area submitted to the EDS microanalysis.

Figure 3

H₂S slow-release and antimicrobial activity of PFM+GaOS. (a) H₂S-release from PFM doped with 10 and 25 μL of GaOS and dried; the values of PFM alone were subtracted. H₂S-release over time from PFM+GaOS, doped with 25 μL of GaOS and dried, after incubation: (b) in 50 mM Tris-HCl buffer, pH 8.0 for 0, 12 h, 2 days; (c) dried in a petri dish at 25 °C for 0, 3 and 6 days; (d) Photo-optical image of the E. coli^AmpR growth in agar-LB medium in the presence of PFM disks (1 cm diameter) doped with 0, 10, 25 μL of GaOS (42 mg/mL).

Figure 4

PFM+GaOS as scaffolds for cMSC cultures. (a) Cell viability of cMSC seeded on PFM disks (0.5 cm of diameter) with 0 μg, 4.2 μg (d.w.) and 25.2 μg (d.w.) of GaOS after 3 days of growth; (b) fluorescence confocal micrographs of cMSC cultured on PFM+GaOS with 4.2 μg (d.w.) of GaOS for 3 days. The nuclei are stained with Hoeschst 33342 (in blue) and the expressions of α-sma (in green) and phalloidin (in red) proteins are detected. Scale bars = 10 μm.

Figure 5

Effect of gaseous release from PFM+GaOS on 2D-culture of cMSC. (a) Photo-optical image of PFM+GaOS on the lids of the petri dishes where 1 × 10⁴ cells/cm² were seeded; (b) pptical micrographs of cMSC after 24 h of cell growth in the presence of PFM+GaOS disk (1 cm of diameter) on the petri dishes-lid and stained with crystal violet; (c) cell viability of cMSC cultures in the presence or in the absence of 100 µM H₂O₂ with PFM, PFM+GaOS, or PFM + Na₂S on the petri dishes-lids. ** p < 0.02; *** p < 0.005.

Figure 6

Microstructural and thermal characterization of functionalized PFM. SEM micrographs of (a) GaOSPFM and (b) DADSPFM (left: magnification 1k×, scale bar 50 µm, right: magnification 10k×, scale bar 5 µm); Differential scanning calorimetry (DSC) curves related to the first (c) and second (d) heating scans of PFM, DADSPFM and GaOSPFM.

Figure 7

Mechanical properties of functionalized PFM. (a) Stress-strain curves of PFM, GaOSPFM, and DADSPFM; (b) SEM micrographs of the related fracture stress-strained surfaces (left: magnification 1k×, scale bar 50 µm, right: magnification 5k×, scale bar 10 µm).

Figure 8

H₂S release from functionalized PFM. H₂S release from (a) GaOSPFM and (b) DADSPFM disks (1 cm of diameter) performed with 1 h of incubation at 37 °C; H₂S release from DADSPFM disks over time, after incubation: (c) in buffer solution (50 mM Tris HCl, pH 8.0) at 37 °C, and (d) dried in petri dish at room temperature for 0, 1, 3, and 7 days. * p < 0.05; ** p < 0.02.

Figure 9

Biological properties of functionalized PFM. Cell viability of cMSC cultured on functionalized PFM: (a) on GaOSPFM and (b) on DADSPFM after 7 and 6 days of growth, respectively; (c) fluorescence micrograph of cMSC seeded on DADSPFM after 6 days and (d) confocal micrograph of cMSC seeded on DADSPFM after 7 days of culture. The nuclei are stained with Hoechst 33342 (in blue) and the phalloidin is in red. Scale bars = 10 µm. * p < 0.05.