3D-printed Nano-structural Poly(ε-caprolactone) (PCL) Scaffolds for Corneal Stromal Regeneration

Abstract

Purpose : Regeneration of corneal stroma is believed to be among the greatest challenges in corneal tissue engineering. The aim of this study is to investigate the effect of a 3D-printed nano-structural poly(ε-caprolactone) (PCL) scaffold on the material-cellular interaction, and to evaluate the efficiency for the corneal stroma regeneration in vitro.

Methods : Two nano-structural scaffolds of different design and fiber distribution were fabricated by melt electrospinning 3D printing method using PCL. Human keratocytes were isolated from collagenase-digested limbal stromal tissue and seeded on the 3D-printed PCL scaffold. Cell morphology on scaffolds was studied by confocal microscopy, and cell proliferation was analyzed by PrestoBlue assay. Extracellular matrix(ECM) produced by keratocytes on the scaffolds was characterized by scanning electron microscope(SEM) and two-photon fluorescent microscopy.

Results : The 3D-printed PCL scaffolds exhibited different micro-structure under SEM. As revealed in Figure.1, scaffold on the left presented offset fiber distribution, while the other one showed a fiber distribution at regular intervals. SEM and two-photon fluorescent microscopy demonstrated that keratocytes tended to grow along the fibrous network, and spread well on both scaffolds. Collagenous ECM produced by cells on the regular-fibrous scaffold was more abundant than on the offset-fibrous scaffold, forming a relatively thicker cross-section (Figure 2). Compared to a collagen fibrillar network secreted on regular-fibrous scaffolds, a lamellar ultrastructural ECM composed of aligned collagen was remodeled on offset-fibrous scaffolds, which was similar to native cornea stroma. Immunofluorescence staining showed that regular-fibrous scaffolds induced less secretion of type I collagen (COLI) and type V collagen (COLV) than offset-fibrous scaffolds.

Conclusions : Our study showed that the fiber composition and topography of 3D-printed nanofibrous PCL scaffolds have a significant effect on the cell behavior through the material-cellular interaction. The refined design of nano-structure by melt electrospinning method stimulates self-organization and self-adaption of ECM, which helps mimicking native tissue microenvironments and offering the potential as cornea stroma regeneration substitutes.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.