AB022.Biosynthetic implants for corneal regeneration in patients at high risk of rejecting donor transplantation

Background:Patients with inflammation or severe corneal pathology are often at high risk of rejecting the human donor corneas that they receive during transplantation.Our goal was to determine whether cell-free implants incorporating phosphorylcholine-based polymer,2-methacryloyloxyethyl phosphorylcholine(MPC),which has inflammation suppressing properties,can support repair and regeneration of ulcerated,high-risk corneas.Methods:Interpenetrating networks of recombinant human collagen and MPC(RHC-MPC)were fabricated into corneal implants in a certified and monitored cleanroom.An open-label,first-in-human observational study was conducted following ISO 14971 and 14155:2011,the Declaration of Helsinki and relevant laws of Ukraine and India,after respective ethical approval and trial registration.Seven unilaterally blind patients,aged 36 to 76 years old,diagnosed with conditions putting them at high risk of rejecting conventional corneal transplantation,and capable of providing informed written consent were grafted and followed up for an average of two years.However,RHC like native collagen is a large macromolecule and difficult to process and is non-customisable.Hence,small,customisable analogs to collagen comprising collagen-like peptides(CLP)conjugated to polyethylene glycol(PEG)were developed.These CLP-PEG analogs like RHC were combined with MPC into implants and tested in mini-pig models with alkali burned corneas,a high-risk condition.Results:One patient had an unrelated infection that necessitated re-grafting and was excluded from the study.The RHC-MPC implants in the remaining six patients were stably integrated throughout the entire observational period.There was regeneration of the cornea epithelium and stroma.Significant vision improvement was observed in in patients with damaged corneas due to infection.By two weeks post-operation,RHC-MPC implanted corneas of patients with active ulcers were free from the symptoms of pain,irritation and photophobia.Over the two-year follow-up period,sensitivity to touch improved,suggesting that the implants were able to promote nerve regeneration.The results seen in the clinical trial were reproduced in corneal grafts comprising MPC and the CLP-PEG collagen analog.Both CLP-PEG-MPC and control CLP-PEG only implants promoted regeneration of corneal epithelium,stroma and nerves.However,the alkali-burned corneas grafted with CLP-PEG-MPC implants retained the thickness of their healthy contralateral controls.Control corneas with CLP-PEG implants without the MPC however,were significantly thicker.Conclusions:These results demonstrate that collagen or its synthetic analog comprising CLP-PEG,can promote regeneration.The incorporation of MPC appears to suppress inflammation in pathologies that constitute conditions with a high-risk for graft rejection.Regeneration was able to occur in inflamed corneas as evidenced by CLP-PEG only grafts,but long-term follow-up is needed to determine if the chronic inflammation may influence graft failure over time.

AB085.E-beam sterilization of recombinant human collagen-phosphorylcholine corneal implants for transplantation

Background:The sterilization of corneal implants composed of carbodiimide crosslinked recombinant human collagen type III(RHCIII)and phosphorylcholine polymers(RHCIII-MPC)is constrained by the biochemical properties of RHCIII.Early human trials used 1%chloroform in 0.1 M phosphate buffered saline(C-PBS),but require a stringent wash procedure with antibiotics to remove the chloroform.Irradiation with gamma or electron-beam(e-beam)allows a chemical-free sterilization method,but may result in crosslinking or denaturation.Here,electron-beam irradiation is evaluated as a sterilization method for RHCIII-MPC implants.Methods:Dose-finding study:RHCIII-MPC were cast in round,350µm thick,12 mm diameter molds for corneal implants and 0.5 mm thick dumbbell-shaped molds for mechanical testing.The hydrogels received an irradiation dose of 17,19,or 21 kGy and unirradiated controls were stored in C-PBS,n=3 per group.The hydrogels were tested for sterility and endotoxin,optical and mechanical properties,biodegradation,free radicals,and cell compatibility.Clinical evaluation in rabbits:RHCIII-MPC implants were e-beamed at 17 kGy or kept in C-PBS.One implant from each group was implanted into the right cornea of each rabbit by deep anterior lamellar keratoplasty,n=4 animals per group.Animals underwent preoperative and 6-month post-operative in vivo confocal microscopy(IVCM)to check nerve count and ingrowth of keratocytes.Corneal grafts and controls were assessed via histology and immunohistochemistry.Results:Dose finding study:hydrogels were sterile at all irradiation doses with no evidence of free radicals.There were no significant differences in optical or mechanical properties between the treatment groups and controls.All hydrogels supported cell growth.The 19 and 21 kGy implants had high collagenase degradation for 21 hours until they stabilized,whereas the 17 kGy and C-PBS implants had gradual degradation until 48 hours.Clinical results:the rabbits did not experience post-surgical inflammatory reactions and full epithelial coverage of the implants occurred within the first week of surgery for all animals.Mild neovascularization occurred in all animals,but resolved by 6-month follow-up.A mild 0.5-1.0 grade subepithelial haze was observed in all rabbits,but the implanted grafts remained transparent.Re-innervation occurred in all grafts with no significant differences between sterilization methods.All regenerated corneas had mucin production and were positive for cytokeratin 3 and 12.Grafted and control corneas were negative for macrophages and blood vessels.Conclusions:E-beam sterilization is a safe and effective form of sterilization for RHCIII-MPC implants.