Bioengineered corneal tissue for minimally invasive vision restoration in advanced keratoconus in two clinical cohorts
Immediately post-operatively, minipigs were placed on a ventilator and once spontaneous breathing resumed, 0.05 mg kgintravenous buprenorphine was given as an analgesic. Post-operatively, topical anesthetic eye drops were again instilled, followed by a combination topical corticosteroid-antibiotic given three times daily the first post-operative week and twice daily during the following three weeks. Post-operative analgesia consisted of intramuscular injection of 0.
Six months after surgeries, minipigs were placed under general anesthesia as described above, and we performed in vivo examinations and photo documentation in operated eyes. Examinations consisted of digital photography , anterior segment optical coherence tomography and in vivo confocal microscopy using a previously described microscopy protocol
. Following examinations and data collection, minipigs were euthanized while under general anesthesia and deep sedation, by intramuscular injection of 7 mg kgHistology and Immunohistochemistry Following euthanasia, rat tissue and porcine corneas were dissected under an operating microscope, embedded in optimal cutting temperature compound, and snap-frozen in liquid nitrogen. Corneas were stored at −80 °C until further use. For histology, rat and pig tissue were thawed, fixed in 4% paraformaldehyde solution, embedded in paraffin and sectioned to a thickness of 4 µm, followed by staining with hematoxylin and eosin .
Ethical permission was granted for the pilot studies to treat up to 20 subjects with advanced keratoconus at each site , based on the ability to detect adverse events in 10% of cases. We report results of 24-month follow-up of the first 12 patients treated in Iran and the first 8 patients treated in India . For the results reported here, clinical data collection occurred during February 2017–January 2020 in Iran and during November 2016–March 2020 in India.
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