In Vitro Evaluation Of Human Cornea And Sclera Resistance Against Enzymatic Degradation After Crosslinking Alone And In Combination With Protease Inhibitors

to evaluate the resistance against enzymatic degradation of human cornea buttons and human scleral strips after crosslinking alone, crosslinking plus incubation with active principles showing antiprotease activity and principles incubation alone.

Siena Crosslinking Center, Monteriggioni, Siena, Italy and Biotecnica Associates Laboratory, Castelfidardo, Ancona, Italy  

24 Eye-bank human corneas with their scleral ring were used. 12 corneal buttons 6 mm in diameter, 500 ±27µm in thickness and 12 scleral strips 6 mm L x 2 mm W were selected. Study groups included controls (Ctrl), crosslinking (CXL-t), N-Acetil-Cysteine-Polygonum capsulatum (NAC-Pc) alone, NAC-Pc pre-CXL, NAC-Pc post-CXL, NAC-Pc pre and post-CXL incubated for 10 min with phosphate buffered saline (PBS) solution (Ctrl), NAC-Pc solution (3.6 g of a mixture with 400 mg N-acetylcysteine (NAC) and 200 mg Polygonum cuspidatum (Pc) dry extract in 25 ml of deionized water). The samples were incubated at 25°C ± 2°C/60% ± 5% RH in sterile multiwells, containing 2 ml of collagenase solution in 100 ml PBS pH 7.5.

Control corneas dissolved into 7 days (±3 days) and the scleral strips into 16 days (±3 days). After CXL alone, the digestion time was double for corneas and significantly extended for scleral strips, 14 and 24 days on average ± (3 days) respectively.. The group with NAC-Pc pre/post-CXL incubation demonstrated the higher prolongation of digestion time, by 7 days more for corneal buttons to 5 days further for scleral strips (±2 days). The increase in protein concentration in the buffer solution was proportional to the sample’s diameter reduction, with a significant difference between the control and treatment groups at each time point. 

The study confirms the utility of crosslinking in increasing the resistance to enzymatic digestion of corneal and scleral collagen, but also demonstrates the potentiality of adjuvant active principles with antiprotease capacity, further increasing corneal and scleral resistance in vitro. Clinical studies are required to confirm this evidence, paving the way to the use adjuvant therapies in combination with crosslinking and alone for supporting structural resistance of the corneal and scleral wall that could be very useful in the prevention of corneal ectasias and myopic progression.