Effect Of High Oxygen Concentration On Corneal Collagen Crosslinking

Crosslinking in human connective tissue may occur according to a natural pattern during aging.Inter-and intra-molecular crosslinks are essential for many biological processes.Collagen crosslinking can be induced by various methods,such as non-enzymatic glycosylation,UV irradiation with or without photosensitizer,and aldehyde reactions.Crosslinking technology has been widely used in medicine;crosslinked bioprostheses have been shown to reduce antigenicity,improve mechanical properties,and improve resistance to recipient tissue degradation.Crosslinking has also been used extensively in the treatment of progressive keratoconus(KC).KC is a non-inflammatory disease characterized by localized thinning of the central or paracentral cornea and subsequent conical protrusion.This disease primarily occurs in adolescence and can lead to severe irregular corneal astigmatism and high myopia,resulting in vision deterioration.The effect of wearing frame glasses is inadequate after correction.In later stages,KC may progress to acute hydrops which can cause serious vision damage due to the formation of scars under the epithelium and considerable impact on the patient’s quality of life.Currently,there are many clinical methods to treat KC,such as wearing rigid contact lenses,using intrastromal corneal ring segments,lamellar or penetrating keratoplasty,and riboflavin/ultraviolet A corneal crosslinking(CXL),which has been shown to improve corneal biomechanical strength.Among these,conventional CXL is currently the only minimally invasive and effective surgical method to prevent the progression of KC.Sporl et al.(1997)first proposed that corneal collagen crosslinking can change the biomechanical properties of the cornea,thus providing a possibility for conservative treatment of KC.In 2003,Wollensak et al.first published a clinical report on KC treated by corneal collagen crosslinking,suggesting that this therapy not only effectively prevents corneal dilation and reduces the demand for penetrating keratoplasty in KC patients,but also has the benefits of minimal trauma,simple treatment and relative safety.To shorten the time required for CXL treatment,reduce intraoperative and postoperative complications of CXL,and improve the comfort of patients,several improved crosslinking models such as rapid CXL and transepithelial CXL have been developed successively in clinical practice.However,whether these models can achieve the same surgical effect as conventional CXL is disputed.Therefore,conventional CXL is still regarded by many clinicians as the best method of crosslinking.In more than 10 years of widespread clinical application,the treatment of patients with KC has achieved significant results,yet in the clinical setting,we find that some KC cases cannot be effectively controlled after conventional CXL treatment.CXL uses 370 nm ultraviolet-A(UVA)and the photosensitizer riboflavin to induce formation of new connections in the corneal collagen fibers,thereby improving the corneal biomechanical properties.In addition to requiring UVA and the photosensitizer riboflavin,effective crosslinking also requires oxygen.In an aerobic environment,the interaction between UVA and riboflavin leads to the production of reactive oxygen species(ROS),which further activate the normal lysyl oxidase pathway and lead to the formation of covalent connections between collagen fibers.In CXL,especially in the early stages of crosslinking,oxygen consumption in the cornea is very fast and recovery is very slow;consequently,oxygen may be a limiting factor.Therefore,we speculated that providing sufficient oxygen during the operation may improve the effect of CXL,especially for patients with KC that cannot be controlled by conventional CXL without oxygen enrichment.The first part of this study was an animal experiment.By detecting the Young’s modulus of the porcine cornea after conventional CXL with oxygen enrichment,we explored the effects of high oxygen concentration on corneal biomechanical strength after CXL.The second part of this study was a prospective randomized controlled study.By evaluating the effectiveness and safety of conventional CXL treatment in advanced KC with oxygen enrichment,we were able to investigate the effect of high oxygen concentration on conventional CXL treatment of progressive KC,and the feasibility of clinical application of this new method.Part Ⅰ Corneal Biomechanical Evaluation After Conventional Corneal Crosslinking with Oxygen EnrichmentPurposeTo assess corneal biomechanical changes after conventional corneal crosslinking(CXL),with and without oxygen enrichment.MethodsSixty fresh porcine corneas were randomly divided into group 1(control),group 2(conventional CXL),and group 3(conventional CXL in a high-oxygen environment during ultraviolet A[UVA]irradiation).Corneal crosslinking(CXL):Prior to treatment,the corneal epithelium was removed with a scalpel,within a central 9 mm diameter area.After saturation with a 0.1%isotonic riboflavin solution every 3 minutes for 30 minutes,the epithelium-off corneas from groups 2 and 3 were irradiated with UVA with a 5 cm working distance for 30 minutes,with continued application of riboflavin drops every 5 minutes.During UVA irradiation,the corneas in group 3 were exposed to a constant 4 L per minute,moistened pure oxygen gas stream,but suspended for 20 seconds after application of the riboflavin drops.In this study we made the oxygen supply by delivering the oxygen with a vacuum suction ring inverted over the porcine eye,to keep the cornea in a relatively closed chamber.Pure oxygen was delivered onto the center area of the cornea through the pipeline of the vacuum suction ring.Biomechanical measurements;After CXL,the 5×14 mm corneal strips with approximately 1.0 mm of sclera remnants were extracted from the corneas from 12 to 6’o clock.The corneal strips were then clamped horizontally between the jaws of a biomaterial-testing device.The distance between the two clamps was 8 to 10 mm.Each of the specimens was loaded,and unloaded under a constant velocity of 2 mm/min for 7 cycles.A linearly increasing strain was then carried out at a rate of 2 mm/min until the specimen ruptured.The value for stress(s)was obtained using the equation σ=F/5 t，where t is the corneal thick-ness.The related strain(e)value was obtained using the equation s=AL/L0.Young’s modulus(E)was calculated using the equation E=dσ/dε=A B exp(B×ε).with the slope of the stress-strain graph at 4%,6%,and 8%strain.The CCT was determined via ultrasound pachymetry.Results1.After de-epithelization,CCT readings were 771±55 μ.m,780±70 μm,and 804 ±66 μm in groups 1,2,and 3,respectively.After CXL,the mean CCT reading were 749±67 μm and 764±63 μm in groups 2 and 3,respectively.2.At 4%strain,the average Young’s modulus was measured at 0.68±0.20 MPa for group 1,1.01±0.23 MPa for group 2 and 1.12±0.24 MPa for group 3.Group 1 exhibited a lower Young’s modulus compared with groups 2 and 3,which was statistically significant(P<0.05).However,there was no difference in the Young’s modulus between groups 2 and 3(P>0.05)(Fig.3).3.At 6%，and 8%strain,measurements revealed that the average Young’s modulus was 0.97±0.21 MPa and 1.29±0.26 MPa for group 1,1.35±0.25 MPa and 1.72±0.45 MPa for group 2,and 1.64±0.44 MPa and 2.20±0.74 MPa for group 3,respectively.The lower values for group 1 were statistically significant,when compared with groups 2 and 3 at 6%and 8%strain(P<0.05).Moreover,the values for group 3 were statistically higher than group 2(P<0.05).Conclusions1.Increasing oxygen concentration during UVA irradiation may improve the efficacy of conventional CXL.2.The results of the present study merit further investigation,including the clinical application of this procedure on patients who have shown poor results with the conventional CXL surgery.Part Ⅱ Conventional Corneal Crosslinking with Oxygen Enrichment for the Treatment of Progressive Keratoconus:A Clinical StudyPurposeTo evaluate the efficacy and safety of conventional corneal crosslinking(CXL)with oxygen enrichment for the treatment of progressive keratoconus.MethodsThis prospective randomized controlled study included 30 eyes from 22 patients diagnosed with progressive keratoconus.Eyes were randomized(1:1)either to receive conventional CXL(Group 1)or conventional CXL with oxygen enrichment(Group 2).Surgical technique:After topical anesthesia,a central 9.0 mm diameter area of the corneal epithelium was removed by mechanical debridement.Subsequently,0.1%isotonic riboflavin(in instances when the thinnest corneal thickness(TCT)was greater than 400 μm)or 0.1%hypotonic riboflavin(when TCT was lesser than 400μm)was applied to the denuded stroma every 3 minutes for 30 minutes.After 30 minutes of riboflavin application,we used a slitlamp with a blue filter to confirm the presence of riboflavin in the anterior chamber,and then irradiated the cornea with UVA(370 nm,3 mW/cm2)for 30 minutes.During UVA irradiance,riboflavin drops were applied every 5 minutes,and Group 2 corneas were also exposed to a moistened stream of pure oxygen(4 L/min).This gas stream was suspended for approximately 20 seconds after the application of riboflavin drops,to mitigate the risk of the gas stream accelerating the rupture of the riboflavin film and affecting the absorption of riboflavin by the corneal stroma.To ensure uniformity and stability of the oxygen supply,the gas was delivered using a vacuum suction ring inverted over the cornea,producing a relatively closed chamber.In this protocol,oxygen travels through the pipeline of the vacuum suction ring to the cornea.The bandage soft contact lens and antibiotic eyedrop were used after CXL.Baseline,3-month,and 6-month uncorrected visual acuity(UCVA),best corrected visual acuity(BCVA),maximum keratometry(Kmax),mean keratometry(Kmean),thinnest corneal thickness(TCT),and endothelial cell density(ECD)were evaluated.Changes in these parameters with respect to baseline measurements were compared between the two groups to determine differences in efficacy and safety.Results1.A total of 21 patients(29 eyes)were included in the statistical analysis.1 patient(1 eye)in group 2 was excluded due to lack of follow-up data.Corneal epithelium recovered well in both groups,and no serious complications occurred after treatment.2.Both groups showed improved UCVA and BCVA values at 6 months postoperatively compared with baseline measurements,with the difference in UCVA reaching statistical significance(P<0.05).3.Both groups showed a significant decrease in Kmax from baseline to 6 months postoperatively(P<0.05).A reduction in Kmean was also seen in both groups;although this was not statistically significant at any follow-up time point in Group 1(P>0.05),the Kmean values in Group 2 at 6 months postoperatively were significantly lower than baseline values(P<0.05).4.No significant changes were observed in TCT and ECD compared to baseline values(P>0.05).5.There was no significant difference between the two groups in the changes in Kmax,Kmean,UCVA,BCVA,TCT,or ECD at any follow-up time point(P>0.05).Conclusions1.In this study,conventional CXL with oxygen enrichment provided a safe option for keratoconus treatment and partially prevented disease progression.2.Our results also provide a basis for a further investigation into the application of this procedure in patients who have shown poor results with conventional CXL surgery.