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Marcus Himmler, Daniel Thieme, Florian Kueng, Piotr Stafiej, Dirk Dippold, Dirk W. Schubert, Thomas Armin Fuchsluger; Electrospun PCL-collagen nanofiber membranes as substitutes for posterior lamellar keratoplasty. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4149.
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© ARVO (1962-2015); The Authors (2016-present)
Posterior lamellar keratoplasty (DSAEK, DMEK) currently is the gold standard for treating patients with a corneal endothelial cell pathologies resulting in functional impairment. This surgical approach depends on the availability of human donor corneas. An artificial biomimetic graft carrying human corneal endothelium could minimize this dependency giving access of this surgical method to larger numbers of patients. Thus, waiting lists could be reduced. In this study, electrospun polycaprolactone (PCL)-collagen nanofibers were tissue-engineered with corneal endothelial cells (EC) and analyzed with regard to their potential application as artificial posterior lamellar grafts.
Membranes were produced from nanofibers obtained by electrospinning of a polymeric solution: A blend of PCL (80 kDa) and purified collagen type I from bovine skin was dissolved in diluted acetic acid (90 % AcOH, ratio of 2:1, final concentration of 0,12 g/ml). Nanofibers were electrospun with a voltage of 15 kV and a flow rate of 1 ml/h (distance needle tip counter electrode: 15 cm). The fabricated membranes were characterized regarding fiber diameter and fiber orientation. Human corneal endothelial cells (HCEC-12) were seeded on the membranes in 100 µl culture medium F99 with an initial cell count of 100.000 cells per well in 24 well plates. Evaluation of the cell number was performed with a cell counting kit 8 (CCK-8) and fluorescence microscopy for a period of 20 days. Furthermore, spinning was performed on a dome-shaped mold mimicking the physiological curvature of a human cornea. The grafts were equally studied and characterized.
Electrospinning of PCL-collagen blends provided membranes in a non-woven, fibrous structure with a random fiber orientation and fiber diameter of 236 (±86) nm. With a spinning time of 5 minutes membranes with an average thickness of 10 µm were obtained, a thickness similarly to a Descemet’s membrane. Dome-shaped molded grafts enabled a wrinkle-free attachment on the inner surface of the cornea. Cell adhesion could already be observed 6 hours after cell seeding and proceeding cell proliferation was detected for the period of observation.
Randomly electrospun PCL-collagen nanofibers offer an interesting opportunity to overcome the shortage of posterior lamellar grafts. Further studies are needed to evaluate functional performance in both ex vivo and in vivo settings.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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