Melt electro-written scaffolds with box-architecture support orthogonally oriented collagen

Abstract

Melt Electro-Writing (MEW) is a state-of-the-art technique that supports fabrication of 3D, precisely controlled and reproducible fiber structures. A standard MEW scaffold design is a box-structure, where a repeat layer of 90° boxes is produced from a single fiber. In 3D form (i.e., multiple layers), this structure has the potential to mimic orthogonal arrangements of collagen, as observed in the corneal stroma. In this study, we determined the response of human primary corneal stromal cells and their deposited fibrillar collagen (detected using a CNA35 probe) following 6-weeks in vitro culture on these box-structures made from poly(ε-caprolactone). Comparison was also made to glass substrates (topography-free) and electrospun poly(ε-caprolactone) fibers (aligned topography). Cell orientation and collagen deposition were non-uniform on glass substrates. Electrospun scaffolds supported an excellent parallel arrangement of cells and deposited collagen to the underlying architecture of aligned fibers, but there was no evidence of bidirectional collagen. In contrast, MEW scaffolds encouraged the formation of a dense, interconnected cellular network and deposited fibrillar collagen layers with a distinct orthogonal-arrangement. Collagen fibrils were particularly dominant through the middle layers of the MEW scaffolds’ total thickness and closer examination revealed these fibrils to be concentrated within the pores’ central regions. With the demand for donor corneas far exceeding the supply - leaving many with visual impairment - the application of MEW as a potential technique to recreate the corneal stroma with spontaneous, bidirectional collagen organization warrants further study.

Melt electrowritten scaffold architectures to mimic vasculature mechanics and control neo-tissue orientation

“…F-actin staining demonstrated that the chosen scaffold architectures can tune the cell spatial arrangement, in agreement with previous in vitro studies [28][40] [51][53] [60]. A square-shape pore was responsible for an orthogonally oriented distribution of the cell cytoskeleton [61][62], while the use of diamond-like pores allowed for a preferential cell organisation. The narrower the printed WA the higher the obtained alignment of cell along the major diagonal demonstrating the contact guidance provided by the MEW framework.…”

Detection of Limbal Stem Cells Adhered to Melt Electrospun Silk Fibroin and Gelatin-Modified Polylactic Acid Scaffolds

“…Corneal stroma replacement was also suggested to be grown onto melt electrowritten PCL with an orthogonal 3D arrangement, showing deposited collagen fibrils of the corneal stromal cells laid entirely within and across the open pores of the scaffold. This confirmed crucial topographical cues for cell support since collagen was laid only in the uniaxial direction of the aligned fibers in the case of solution electrospun scaffolds [ 23 ]. A structure similar to the native cornea was also successfully fabricated by 3D printing of a composite ink of gelatin methacrylate modified by hyaluronic acid.…”

Fibrin gel enhanced trilayer structure in cell‐cultured constructs

“…Cells cultured on electrospun substrates typically deposit ECM materials over the surface of the electrospun fibers. However, collagen fibrils produced by the cells can be formed between the electrospun fibers because of their relatively high mechanical properties compared with other ECM components, such as GAG (Bosworth et al, 2021; Szewczyk et al, 2018). Thus, SEM imaging at high magnification showed that collagen fibrils were formed in both cell‐cultured constructs between electrospun fibers (Figure 5a,b).…”