Repurposing Laboratory Plastic into Functional Fibrous Scaffolds via Green Electrospinning for Cell Culture and Tissue Engineering Applications

Abstract

Cell culture for tissue engineering is a global and flexible research method that relies heavily on plastic consumables, which generates millions of tons of plastic waste annually. Here, we develop an innovative sustainable method for scaffold production by repurposing spent tissue culture polystyrene into biocompatible microfiber scaffolds, while using environmentally friendly solvents. Our new green electrospinning approach utilizes two green, biodegradable and low-toxicity solvents, dihydrolevoglucosenone (Cyrene) and dimethyl carbonate (DMC) to process laboratory cell culture petri dishes into polymer dopes for electrospinning. Scaffolds produced from these spinning dopes, produced both aligned and non-aligned microfiber configurations, were examined in detail. The scaffolds exhibited mechanical properties comparable to cancellous bones whereby aligned scaffolds achieved an ultimate tensile strength (UTS) of 4.58 ± 0.34 MPa and a Young's modulus of 11.87 ± 0.54 MPa, while the non-aligned scaffolds exhibited a UTS of 4.27 ± 0.92 MPa and a Young's modulus of 20.37 ± 4.85. To evaluate their potential for cell-culture, MG63 osteoblast-like cells were seeded onto aligned and non-aligned scaffolds to assess their biocompatibility, cell adhesion, and differentiation, where the cell viability, DNA content, and proliferation were monitored over 14 days. DNA quantification demonstrated an eight-fold increase from 0.195 μg/mL (day 1) to 1.55 μg/mL (day 14), with a significant rise in cell metabolic activity over 7 days, and no observed cytotoxic effects. Confocal microscopy revealed elongated cell alignment on aligned fiber scaffolds, while rounded, disoriented cells were observed on non-aligned fiber scaffolds. Alizarin Red staining and calcium quantification confirmed osteogenic differentiation, as evidenced by mineral deposition on the scaffolds. This research therefore demonstrates the feasibility of this new method to repurpose laboratory polystyrene waste into sustainable cell culture tissue engineering scaffolds using eco-friendly solvents. Such an approach provides a route for cell culture for tissue engineering related activities to transition towards more sustainable and environmentally conscious scientific practices, thereby aligning with the principles of a circular economy.

Keywords: biomaterials; cell-scaffold interaction; electrospinning; green solvent; sustainable tissue engineering; tissue engineering.

1

(A) Schematic diagram illustrating the experimental procedure for fabricating polystyrene electrospun scaffolds using a green solvent and recycled Petri dish material, with fibers collected onto a rotating drum collector. (B) Photograph of the fabricated scaffold. (C) SEM micrograph of the scaffold showing nonaligned fibers. (D) SEM micrograph of the scaffold showing aligned fibers. (E) Cross-sectional SEM image of the scaffold, with a zoomed-in view highlighting the morphology of a single fiber.

2

Comparative analysis of polystyrene sources and scaffold properties:(A) FTIR spectra of PS from recycled Petri dishes, pellets, PS bulk material, and green scaffolds (B) DSC characterization of fibers and bulk materials (C) TGA and DTG thermal stability: (D) Stress–strain behavior of aligned vs nonaligned fiber scaffolds (mean ± SD).

3

Comprehensive analysis of fiber morphology and orientation in PS green scaffolds: SEM images showcasing (A) nonaligned and (B) aligned fibers with 400 diameter measurements per sample, Probability density distributions of fiber diameters for (C) nonaligned and (D) aligned fibers. Fiber orientation visualizations highlighting (E) nonaligned and (F) aligned fibers. Rose plots displaying orientation distributions for (G) nonaligned and (H) aligned fibers.

4

Evaluation of MG63 cell behavior seeded on aligned and nonaligned fiber scaffolds and control (A) MG63 cell density obtained via PrestoBlue over 7 days, highlighting cell viability and proliferation trends. (B) DNA concentration quantified via PicoGreen assay at days 0, 7, and 14, with black brackets showing within-group comparisons across days and red brackets standing for within-day comparisons across groups. asterisks denote statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001). Confocal images (cell tracker staining) on day 7 illustrate cell morphology and distribution for (C) control, (D) nonaligned fibers, and (E) aligned fibers.

5

(A) Quantification of the extracted alizarin from the stained sample converted into concentration (μg/mL) across six experimental groups measured at days 1, 7, 14, and 21. Experimental groups include AF Exp (Aligned Fiber Experimental), No-AF Exp (Non-Aligned Fiber Experimental), AF OST diff (Aligned Fiber Osteogenic Differentiation), No-AF Ost diff (Non-Aligned Fiber Osteogenic Differentiation), Control EXP (Control Experimental), and Control Ost diff (Control Osteogenic Differentiation). Error bars represent standard deviations. Statistical significance was assessed using Tukey’s HSD test. Brackets indicate significant differences between pairs of groups (p ≤ 0.05), with bracket positions staggered for clarity. (B) Brightfield microscopy of the Alizarin Red staining of undifferentiated and differentiated cells seeded on the scaffold and control at day 1 and day 14.