Electrospun Chitosan–Polyethylene Oxide/Fibrinogen Scaffolds for Sustained PDGF Delivery and Enhanced Wound Healing

Background

Despite advances in wound care, the translation of bioactive dressings into the clinic remains limited. The wound healing cascade relies on a coordinated interplay of cytokines, immune cells, and matrix proteins. Growth factors, while potent, suffer from rapid clearance and a short half‑life, necessitating repeated dosing that is burdensome for patients. A dressing capable of sustained, localized delivery would dramatically improve outcomes and uptake.

PDGF is a pivotal chemotactic agent that mobilizes neutrophils, monocytes, and fibroblasts, and it modulates matrix deposition and scar formation. The FDA‑approved Regranex® gel (2.2 µg cm⁻²) accelerates healing by ~30 % in diabetic ulcers, yet its daily application is inconvenient. Thus, a sustained‑release system is desirable.

Electrospinning offers a versatile route to produce nanofibrous mats that emulate the native extracellular matrix. Natural polymers such as collagen, elastin, and fibrinogen have inherent bioactivity and affinity for growth factors. However, fibrinogen alone lacks the mechanical strength required for long‑term dressing applications. Chitosan, a deacetylated chitin derivative, provides antimicrobial activity and can be electrospun when blended with PEO. The polycationic nature of CS complicates blending with negatively charged Fb, but a dual‑nozzle electrospinner overcomes electrostatic incompatibilities, enabling uniform composite formation.

Methods

Materials

All reagents (acetic acid, CS, PEO, Fb, HFIP, BSA, DMSO) and cell lines (human dermal fibroblasts PCS‑201‑012) were purchased from Sigma‑Aldrich or ATCC and used as received. Recombinant human PDGF‑BB was sourced from PeproTech.

Scaffold Fabrication

CS‑PEO solutions (5.5 wt %, CS:PEO = 2:1) were prepared in 1 % acetic acid with 0.5 % BSA and 10 % DMSO. Fb was dissolved in a 1:9 (v/v) HFIP/10× EMEM mixture at 110 mg mL⁻¹. PDGF was added immediately before spinning. Dual‑spinneret electrospinning (NE‑1000 syringe pump, 18‑gauge needles) employed 0.7 mL h⁻¹ (CS‑PEO) and 1.0 mL h⁻¹ (Fb) flows, voltages of 28 kV and 22 kV, and a 25 RPM rotating mandrel at 22 cm (CS‑PEO) and 12.5 cm (Fb). Scanning was performed for 3 h at 35–45 % RH.

Morphology

FESEM imaging (Zeiss Sigma VP‑40) of gold‑coated samples quantified fiber diameters (n = 100 per scaffold) via ImageJ. CS‑PEO/Fb fibers averaged 202 ± 113 nm, thinner than CS‑PEO (270 ± 68 nm) and Fb (351 ± 102 nm) alone.

Surface Chemistry

ToF‑SIMS (Bi₃²⁺, 30 kV) mapped characteristic ions (CNO⁻, CN⁻, C₂H₅O⁺) across 50 × 50 µm rasters, confirming uniform CS‑PEO and Fb distribution.

Water Contact Angle

Measured per ASTM D7334‑08 with 2 µL distilled water. CS‑PEO/Fb exhibited a hydrophilic angle of 61.4 ± 7.6°, intermediate between CS‑PEO (44.2 ± 5.1°) and Fb (115.7 ± 16.2°).

Mechanical Testing

Uniaxial tensile tests (Instron E3000, 1 mm min⁻¹) on 40 × 25 mm strips (thickness 94 ± 7 µm) produced stress‑strain curves. CS‑PEO/Fb achieved 0.31 ± 0.02 MPa peak stress, surpassing Fb alone (0.12 ± 0.01 MPa).

Water Vapor Transfer Rate

WVTR was determined per ASTM E96 using silica‑gel desiccants; CS‑PEO/Fb achieved 806.5 ± 56.1 g m⁻² day⁻¹, within the clinically desirable range (≈2 000 g m⁻² day⁻¹).

Cytotoxicity

Indirect WST‑1 assays (ISO 10993‑5) over 120 h showed no significant proliferation inhibition at 48 h, though a modest decline emerged at 72 h, likely due to CS degree of acetylation.

Cell Attachment

Live/Dead and phalloidin–DAPI staining of fibroblasts on scaffold surfaces revealed healthy morphology and robust attachment comparable to fibronectin‑coated controls.

Degradation

Scaffolds incubated in FBM (37 °C, 5 % CO₂) displayed linear mass loss after an initial 1‑h burst, maintaining integrity for ≥48 h.

PDGF Release

ELISA quantified cumulative release: 1.8 ± 0.7, 4.4 ± 1.8, and 11.4 ± 4.8 ng mg⁻¹ at 2, 4, and 8 µg mL⁻¹ loading, respectively. A biphasic pattern released 21.6 % within the first hour.

Fibroblast Migration

ORIS™ migration assays demonstrated that eluates from PDGF‑loaded scaffolds matched the migration induced by a single 50 ng mL⁻¹ PDGF dose, with sustained effects up to 48 h. Fb alone also modestly promoted migration.

Results and Discussion

Dual‑spinneret electrospinning successfully merged CS‑PEO and Fb into a homogeneous nanofibrous matrix, overcoming electrostatic incompatibilities. Fiber diameter reduction in the composite is attributed to increased jet repulsion, extending deposition time and inducing bending instabilities that yield finer fibers.

ToF‑SIMS mapping confirmed uniform surface composition, and water contact angles indicated a hydrophilic surface that discourages bacterial adhesion while supporting protein adsorption. Mechanical testing showed that CS‑PEO integration restores tensile strength suitable for wound dressing, addressing Fb’s inherent weakness.

WVTR measurements placed CS‑PEO/Fb within the therapeutic window for moist wound environments, balancing exudate management and oxygen permeability. Cytotoxicity assays suggested that any residual HFIP or high DA CS concentrations were minimal; the scaffold’s biological safety profile is acceptable for clinical translation.

PDGF release profiles exhibited a desirable biphasic pattern: an early burst delivering ~21 % of the dose, followed by a controlled, linear release sustaining biologically relevant concentrations over 48 h. This sustained delivery negates the need for daily dressing changes required by Regranex®, potentially improving patient compliance.

Functional assays confirmed that released PDGF retained chemotactic activity, achieving fibroblast migration equivalent to a single high‑dose treatment. The additive effect of Fb’s intrinsic chemotactic properties further enhances cellular recruitment, suggesting a synergistic mechanism.

Conclusion

Electrospun CS‑PEO/Fb nanofibrous scaffolds combine the antimicrobial and mechanical advantages of chitosan with the bioactivity of fibrinogen, while enabling sustained, functional PDGF delivery. Their nanoscale architecture, appropriate WVTR, and mechanical robustness render them promising candidates for next‑generation wound dressings that can accelerate fibroblast recruitment and healing without the inconvenience of repeated growth‑factor applications.