3D printed recombinant collagen type III (RHC III) scaffolds and their potential for corneal tissue engineering

Introduction: Corneal visual impairment is the 4th cause of blindness globally (5.1%), according to the WHO1, and currently the only treatment is surgical intervention using a human donor cornea, a procedure known as penetrating keratoplasty. The greatest challenge facing the treatment of corneal visual impairment is the global lack of donor corneas. The use of 3D printing in tissue engineering (TE) is a way of producing customized scaffolds, offering a tantalising prospect in the treatment of corneal visual impairment. This work studies the potential of 3D printing for producing recombinant collagen type III (RHC III) scaffolds for corneal tissue engineering. Methods: RHC III scaffolds were formed by nebulizing a 3.4mg/ml RHC III solution in 10mM HCL and accelerating it onto a substrate using nitrogen gas. Layers were formed with assumed thickness of approximately 3µm and repeated 2, 10, or 29 times to achieve thicknesses of up to 90µm. Via this method, patterns were imparted on the sample surface in the direction of printing, and spiral patterns were used. Collagen scaffolds were then crosslinked using EDC [1-ethyl-3-(3-diaminopropyl)carbodiimide] and NHS (N-hydroxysuccinimide) at a molar ratio of 1:1. The thickness of 10- and 29-layer samples was measured using Optical Coherence Tomography (OCT). Corneal MSCs were isolated through collagenase digestion (4 hours) of the stroma. Cells were cultivated in DMEM + 10% FBS. First passage MSCs were seeded onto the collagen scaffold samples consisting of 2 layers (n=4), 10 layers (n=4) and 29 layers (n=4) of RHC III at a density of 100 cells/sample. For controls, cells were seeded on glass (n=3) and plastic (n=3). At day 23, scaffolds were processed for immunocytochemistry (n=9) and inspected via Scanning Electron Microscopy (SEM) (n=3). Results & Discussion: OCT thickness measurements showed a mean thickness of 28 ± 9µm for 10-layer samples and 87 ± 23µm for 29-layer samples. Light microscopy showed that corneal MSCs proliferated on all printed RHC III scaffolds, regardless of the number of layers. SEM showed the circular print pattern of the RHC III. With immunocytochemistry, we observed collagen type III, and MSCs were observed throughout the samples, indicating penetration into the RHCIII along with superficial growth, which was also observed. The MSCs cytoskeleton stained positive for phalloidin, and we observed alignment of the superficial cells following the direction of the collagen in the SEM images. Conclusions: These results demonstrate that 3D printing can be used to produce RHC III scaffolds which are suitable as a substrate for cultivating corneal MSCs. 1 World Health Organization, Causes of Blindness and Visual Impairment, http://www.who.int/blindness/causes/en/, accessed 30/11/2016.status: publishe