Functional fabrication of recombinant human collagen–phosphorylcholine hydrogels for regenerative medicine applications

The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes of the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500 μm thick RHCIII-MPC constructs comprising over 85% water, are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, as compared to the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30 μm wide fibronectin stripes enhanced cell attachment and showed highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas. We thank Dr. Chyan-Jang Lee for establishing the GFP-HCEC cell line used for this study, and Ms. Kimberley Merrett for assistance in characterization of the hydrogels. We also thank Dr. Sadhana Kulkani and David Priest, University of Ottawa Eye Institue, for assistance with the laser cutting study; and Dr. Joanne M. Hackett (currently at Cambridge University Health Partners) for assistance with preliminary cell culture/biocompatibility studies during optimization of the RHCIII-MPC hydrogels. We thank Johannes Junger and Michael Baumann, MLase AG, for help with the UV crosslinking, and the Medical Devices Bureau, Health Canada, for use of the SEM system. We gratefully acknowledge funding from an NSERC-CIHR Canada Collaborative Health Research Project grant (M.G.) and subsequent funding for an EU Nanomedicine ERAnet project "I-CARE" to M.G., R.V. and MLase AG, through the Swedish Research Council, Research Council of Lithuania and VDI Germany, respectively.