Y15 - Biotextile Scaffolds for Cornea Tissue Engineering

Introduction: : Corneal opacity is a leading cause of blindness worldwide. Artificial corneal implants often fail due to poor host integration and neovascularization causing opacity. Melt electrowriting (MEW), a polymer fiber-weaving technology, offers high-resolution (i.e., fiber diameters below 50 microns) and high accuracy fabrication of textile scaffolds. We hypothesize that cell-free scaffolds will not fully regenerate the cornea. Therefore, we also hypothesize that these scaffolds may show better outcomes when seeded with corneal fibroblasts.

Materials and

Methods: : Porcine corneal fibroblasts (pCFs) were isolated and expanded in Dulbecco’s Modified Eagle Medium. Fibroblasts were seeded on five MEW-fabricated poly-(ε-caprolactone) (PCL) scaffolds (n = 6); one unseeded scaffold served as a negative control. MEW scaffold fibers with a diameter of 22.7 ± 2.8 µm were deposited in a 0°/90° orientation to create 50 µm pores. L929 (mouse) fibroblasts served as positive viability (i.e., scaffold biocompatibility) controls. PrestoBlue metabolic assays were conducted on Days 1 and 3. DAPI was used for nuclear staining, and Alexa Fluor 488-phalloidin for actin visualization. An Olympus FV3000 confocal microscope (Olympus, Japan) and Gen5 software from Agilent BioTek (Santa Clara, CA, USA) were used for quantitative imaging and viability assessment. L929 mouse fibroblasts were tested separately on identical 50 µm MEW scaffolds to evaluate scaffold biocompatibility per ISO 10993-5 guidance.

Results, Conclusions, and Discussions:: Porcine corneal fibroblasts exhibited PrestoBlue fluorescence of 1789.8 ± 83.5 RFU (n=6) on Day 1 and 561.8 ± 49.0 RFU (n=3) on Day 3. The decline in metabolic activity likely reflects a transition from early proliferation to a more quiescent, structurally integrated state. This interpretation is supported by actin staining, which revealed elongated cytoskeletal architecture and directional alignment along scaffold fibers by Day 3. L929 fibroblasts seeded on MEW scaffolds with 50 µm pores exhibited PrestoBlue fluorescence of 1623.8 ± 137.7 RFU on Day 1 and 2287.7 ± 141.7 RFU on Day 3 (n = 6 each); representative actin alignment at 8 hours is shown in Fig. 1, and at 6 hours in Fig. 2. Data variability was reduced by excluding outliers beyond ±2 standard deviations and applying percent coefficient of variation (%CV) thresholds to ensure consistent fluorescence measurements. DAPI-based quantification identified 13.6 ± 6.4 cells/mm² on Day 1 and 11.5 ± 5.7 cells/mm² on Day 3 for pCFs. The modest decrease in detectable nuclei is attributed to cellular elongation and alignment along the fibers, which reduces nuclear visibility in 2D confocal imaging. Actin-rich cytoskeletal projections spanning scaffold pores were also observed, indicating active migration and scaffold integration. A prototype of a MEW-fabricated 3D curved scaffold designed to conform to corneal topography is shown in Fig. 3, illustrating the feasibility of printing anatomically relevant geometries for future tissue integration.
MEW-fabricated PCL textile scaffolds demonstrated strong biocompatibility, with 50-micron pores facilitating the alignment and viability of both porcine and L929 fibroblasts. These results confirm scaffold support for stromal fibroblast survival, attachment, and cytoskeletal organization across both experimental and control cell lines. Early curved printing results (Fig. 3) emphasize progress toward geometries mimicking the native corneal dome, reinforcing MEW’s promise in regenerating anatomically relevant stromal structures. Ongoing work will evaluate time-to-confluence metrics and optimized curvature fidelity for future in vivo applications.

Acknowledgements and/or References (Optional)::