Porcine Corneas Research Articles

The Resistance of Riboflavin/UV-A Corneal Cross-Linking to Enzymatic Digestion Is Oxygen-Independent.

Corneal cross-linking (CXL) with riboflavin and UV-A induces several effects in the cornea, including biomechanical stiffening, generation of reactive oxygen species, and increased resistance to enzymatic digestion. Whereas the biomechanical stiffening effect is oxygen-dependent, little is known about the effect of oxygen on the resistance to enzymatic digestion. Here, we examined CXL-induced enzymatic resistance in the absence of oxygen. Ex vivo porcine corneas (n = 160) were assigned to 5 groups. Group 1 was the control group (abrasion and riboflavin application). Groups 2 and 3 received accelerated 10 and 15 J/cm2 high-fluence CXL protocols in the presence of oxygen (9'15″ @ 18 mW/cm2 and 8'20″ @ 30 mW/cm2, respectively), whereas groups 4 and 5 received accelerated 10 and 15 J/cm2 high-fluence CXL protocols in the absence of oxygen (oxygen content less than 0.1%). After CXL, corneas were digested in 0.3% collagenase A solution. Mean time until complete dissolution was determined. The mean times to digestion in groups 1 through 5 were 22.31 ± 1.97 hours, 30.78 ± 1.83 hours, 32.22 ± 2.22 hours, 31.38 ± 2.18 hours, and 31.69 ± 2.53 hours, respectively. Experimental CXL groups showed significantly higher (P < 0.001) resistance to digestion than nonirradiated controls. There was no significant difference in time to digestion across all experimental CXL groups, irrespective of fluence delivered or the absence of oxygen. The resistance to digestion in accelerated high-fluence riboflavin/UV-A CXL is oxygen-independent, which is of particular importance when developing future optimized CXL protocols for corneal ectasia and infectious keratitis.

Assessment of performance and safety of Corneal Chamber hypothermic storage medium and PSS-L corneal rinsing solution in human and porcine corneas

PurposeTo prove the safety and performance of the hypothermic corneal storage medium "Corneal Chamber" and the rinsing solution "PSS-L" in support of the new Conformité Européenne (CE) certification process in...

Combining Riboflavin/UV-A Light and Rose Bengal/Green Light Corneal Cross-Linking Increases the Resistance of Corneal Enzymatic Digestion.

The purpose of this study was to determine if concurrent riboflavin/UV-A light (RF/UV-A) and rose Bengal/green light (RB/green) epi-off PACK-CXL enhances corneal resistance to enzymatic digestion compared to separate chromophore/light treatments. Ex vivo porcine corneas were allocated as follows. Group A corneas were soaked with riboflavin (RF) and were either not irradiated (A1, controls) or were irradiated with 10 (A2) or 15 J/cm² (A3) UV-A light at 365nm, respectively. Group B corneas were soaked with RB and either not irradiated (B1, controls) or were illuminated with 10 (B2) or 15 J/cm² (B3) green light at 525nm, respectively. Corneas in group C were soaked with both RF and RB and were either not irradiated (C1, controls) or were subjected to the same session consecutive 10 J/cm2 (C2) or 15 J/cm2 (C3) UV-A and green light exposure. Following treatment, all corneas were exposed to 0.3% collagenase A to assess digestion time until corneal button dissolution. A1 to A3 digestion times were 21.38, 30.5, and 32.25hours, respectively, with A2 and A3 showing increased resistance to A1. B1-3 had digestion times of 31.2, 33.81, and 34.38hours, with B3 resisting more than B1. C1 to C3 times were 33.47, 39.81, and 51.94hours; C3 exhibited superior resistance to C1 and C2 (both P < 0.05). Same-session combined RF/UV-A and RB/green PACK-cross-linking significantly increases corneal enzymatic digestion resistance over standalone treatments. Combining RF-based and RB-based PACK-CXL considerably increases corneal collagenase digestion resistance, potentially minimizing ulcer size in clinical contexts.

Effect of accelerated high-fluence riboflavin and rose bengal-mediated corneal cross-linking on resistance to enzymatic digestion

PurposeThis study evaluated the effect of high-fluence accelerated corneal cross-linking on the resistance to enzymatic digestion, assessing two chromophore/light combinations: riboflavin/UV-A light (RF/UV-A) and rose bengal/green light (RB/green).MethodsFreshly prepared ex-vivo porcine corneas (n = 189) were divided into 8 groups groups. Group A corneas were unirradiated controls without chromophore soaking (A0), or soaked with riboflavin (A1) or rose bengal (A2). Group B corneas underwent accelerated epi-off RF/UV-A CXL at fluences of 5.4 J/cm² (B1), 10 J/cm² (B2), or 15 J/cm² (B3). Group C corneas underwent accelerated epi-off RB/green CXL at fluences of either 10 J/cm² (C1) or 15 J/cm² (C2). Following CXL, all corneas were digested in 0.3% collagenase-A solution, and the time until complete dissolution was measured.ResultsNon-irradiated controls exposed to RF and RB enhanced corneal resistance to collagenase digestion, with RB having a stronger effect than RF. RF/UV-A-treated corneas showed significantly increased digestion resistance with increasing fluence levels. RB/green-treated corneas displayed enhanced digestion resistance with each increase in fluence up to 10 J/cm²; a 15 J/cm² fluence yielded similar digestion resistance times to a 10 J/cm² fluence, suggesting a plateau effect in accelerated RB/green CXL protocols.ConclusionsWhen compared to standard-fluence treatments, high-fluence accelerated epi-off CXL using both riboflavin and rose bengal significantly increases resistance to enzymatic digestion. The optimal settings for clinical protocols might be 15 J/cm² (30 mW/cm² for 8 min 20 s) for RF/UV-A and 10 J/cm² (15 mW/cm² for 11 min 7 s) for RB/Green Light.

Evaluation of the Possible Influence of Povidone Iodine (PVI) Solution and Polyhexanide (PHMB) on Wound Healing in Corneal Epithelial Regeneration.

This study investigates the possible toxic effects of the preoperative antiseptic substances povidone iodine (PVI) and polyhexanide (PHMB; Serasept® 2) on wound healing in ophthalmology. To assess this impact, human telomerase-immortalized corneal epithelial (hTCEpi) cells and human telomerase-immortalized conjunctival epithelial (hCjE) cells were exposed to 1% and 5% PVI or 0.04% PHMB for different periods to evaluate the cytotoxicity of these two antiseptics. Furthermore, the toxicity of these antiseptics was investigated in a human tissue-specific corneal epithelial construct and porcine eye culture model. The results reveal the high cytotoxicity of PVI and PHMB in the hTCEpi and hCjE in monolayer cell culture models, independent of the incubation time and concentration of these substances. However, after hTCEpi cell differentiation into a tissue-specific corneal epithelial construct, contact with these antiseptics for the relevant preoperative time did not alter cPARP1 or Ki67 expression. Furthermore, the wound-healing process in the porcine cornea was not significantly influenced after incubation with these antiseptics. In summary, corneal and conjunctival epithelial cell lines are very sensitive to PVI and PHMB, whereas no significant alterations were found in intact tissue-specific corneal epithelial constructs or porcine corneas. Therefore, we could not identify PVI and PHMB as reasons for postoperative eye irritation.

Matrix hyaluronic acid and bilayer poly-hydroxyethyl methacrylate-hyaluronic acid films as potential ocular drug delivery platforms

This study aimed to design hydrogel based films comprising hyaluronic acid (HA) to overcome limitations of currently used eye drops. Timolol-loaded crosslinked (X2) HA-based and bilayer (B2) (pHEMA/PVP-HA-based layers) films were designed and characterized. The films were transparent (UV, visual observation) with crosslinked (<80 %) films showing lower light transmittance than bilayer (>80 %) films. X2 showed significantly higher swelling capacity, tensile strength and elastic modulus (5491.6 %, 1539.8 Nmm−2, 1777.2 mPa) than B2 (1905.0 %, 170.0N mm−2, 67.3 mPa) respectively. However, X2 showed lower cumulative drug released and adhesive force (27.3 %, 6.2 N) than B2 (57.5 %, 8.6 N). UV sterilization did not significantly alter physical properties, while SEM and IR microscopy showed smooth surface morphology and homogeneous drug distribution. Timolol permeation (EpiCorneal™/porcine cornea) depended on the film matrix with erodible films showing similar permeation to commercial eyedrops. Drug permeation for porcine cornea (X2 = 549.0.2, B2 = 312.1 μgcm−2 h−1) was significantly faster than EpiCorneal™ (X2 = 55.2, B2 = 37.6 μgcm−2 h−1), but with a linear correlation between them. All the selected optimized films showed acceptable compatibility (MTT assay) with both HeLa cells and EpiCorneal™. In conclusion, crosslinked and bilayer HA based films showed ideal characteristics suitable for potential ocular drug delivery, though further work is required to further optimize these properties and confirm their efficacy including in vivo tests.

Optical coherence elastography measures the biomechanical properties of the ex vivo porcine cornea after LASIK.

The biomechanical impact of refractive surgery has long been an area of investigation. Changes to the cornea structure cause alterations to its mechanical integrity, but few studies have examined its specific mechanical impact. To quantify how the biomechanical properties of the cornea are altered by laser assisted in situ keratomileusis (LASIK) using optical coherence elastography (OCE) in ex vivo porcine corneas. Three OCE techniques, wave-based air-coupled ultrasound (ACUS) OCE, heartbeat (Hb) OCE, and compression OCE were used to measure the mechanical properties of paired porcine corneas, where one eye of the pair was left untreated, and the fellow eye underwent LASIK. Changes in stiffness as a function of intraocular pressure (IOP) before and after LASIK were measured using each technique. ACUS-OCE showed that corneal stiffness changed as a function of IOP for both the untreated and the treated groups. The elastic wave speed after LASIK was lower than before LASIK. Hb-OCE and compression OCE showed regional changes in corneal strain after LASIK, where the absolute strain difference between the cornea anterior and posterior increased after LASIK. The results of this study suggest that LASIK may soften the cornea and that these changes are largely localized to the region where the surgery was performed.

Efficacy of enzyme‑induced collagen crosslinking on porcine cornea.

The purpose of the present study was to investigate the effect of a new crosslinking (CXL) method, induced by enzymes, on porcine corneas. Corneal strip (10x3 mm) pairs obtained from 60 fresh porcine eyes were harvested and divided into four groups, Groups A-D. Each pair of corneal strips was incised from the central part of the same cornea; one was incubated in transglutaminase (Tgase) solution (microbial Tgase 2 produced by tissue engineering) and the other remained untreated as a control. CXL strips of Groups A-D were incubated with 2, 1, 0.5 and 0.25 U/ml Tgase solution, respectively at 37˚C for 30 min. After that, tensile strain measurements were performed for all strips. One cornea from each group was chosen randomly for hematoxylin and eosin, and Masson staining to identify histological morphology changes. The elastic modulus of treated corneas of Groups A-D were 6.56±2.93, 4.72±1.29, 5.24±2.13 and 3.48±1.60 MPa (mean ± SD), respectively at a strain of 20%, and had a 66, 43, 36 and -6% increase compared with those of their control strips. Compared with the control strips, the elastic modulus of the treated strips significantly increased in Groups A-C. The central corneal thickness of the treated corneas in Groups A-D were 1.54±0.14, 1.41±0.15, 1.47±0.11 and 1.43±0.13 µm, respectively; however, there was not a statistically significant difference compared with the control group. No reduction in corneal transparency was observed, and no obvious abnormalities were found in corneal morphology. CXL mediated by enzymes can lead to a notable enhancement in the biomechanical characteristics of the cornea while maintaining its structural integrity. Enzyme-induced CXL could be a new generation CXL method for strengthening the cornea.

A pilot study for decellularizing human and porcine cornea for future use in corneal regeneration

Purpose: To effectively decellularize human and porcine corneas for future use in corneal regeneration by using an already established decellularization technique being efficient in decellularizing blood vessels.Methods: One human and 4 porcine corneas were decellularized by using various washing steps with 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propanesulfonate (CHAPS) detergent, Benzonase, Ethylenediaminetetraacetic acid (EDTA), 5% Dextran or 5% D‐Mannitol. The quality of the decellularization process was assessed by quantitative and qualitative measurement of DNA content, glycosaminoglycans (GAGs), immunofluorescent staining for Collagen I, V and keratocan, and Transmission Electron Microscopy (TEM) to observe the structure of the collagen fibrils was used.Results: The decellularization process showed 99,94% and 99,52% DNA content reduction in the human and porcine corneas when using Dextran, respectively, while more DNA remained in the porcine corneas when D‐Mannitol was used. Similar pattern was observed in the preservation of GAGs, with the highest amount being found in the human cornea, and less in the porcine corneas. H&amp;E and Immunofluorescent staining showed no presence of cell nuclei when compared to the control native corneas, while Alcian blue staining qualitatively confirmed the presence of GAGs. Collagen I, V and keratocan were still present in the decellularized corneas when compared to the native ones, while TEM microscopy further confirmed the similar patterns of the collagen fibrils in the decellularized‐, compared to the native‐ corneas.Conclusions: This pilot study showed that our decellularization method is effective in decellularizing human and porcine corneas, with very high amount of DNA being removed from the corneas, while the GAGs were preserved to an acceptable extent, and the structure and pattern of the collagen fibrils maintained.

Ultraviolet-A absorbance analysis in thin porcine corneas pre-and post-crosslinking.

To determine the absorbance coefficient of the thin porcine cornea to ultraviolet-A radiation (365 nm) submitted for crosslinking. This in vitro, benchtop experiment using cadaver tissue study analyzed 12 porcine corneal lamellas, which were obtained using a microkeratome after mechanical de-epithelization and separated into three thickness groups: 180, 300, and 360 μm. The corneal thickness values were measured by anterior-segment optical coherence tomography. All lamellas had ultraviolet-A (365 nm) absorbance measured with a 96-well plate spectrophotometer using an ultraviolet transparent microplate before riboflavin instillation and preand post-crosslinking according to the Dresden protocol. The ultraviolet absorbance profiles of the 180, 300, and 360 μm groups were obtained as α-coefficients of 12.85, 76.55, and 120.27, respectively. A theoretical formula was calculated though a statistical analysis that demonstrated the correlation between stromal lamellar thickness and ultraviolet absorbance. Corneal thickness and ultraviolet-A spectral absorbance of corneal lamellas showed linear correlation. These findings can potentially contribute to the optimization of ultraviolet-A application during crosslinking, making the treatment of corneas with thickness <400 μm safe and personalized energy delivery for each corneal thickness.

Structure and properties of the acellular porcine cornea irradiated with 60Co-γ and electron beam and its histocompatibility.

Acellular porcine cornea (APC) has been used in corneal transplantation and treatment of the corneal diseases. Sterilization is a key step before the application of graft, and irradiation is one of the most commonly used methods. In this paper, APC was prepared by the physical freeze-thawing combined with biological enzymes, and the effects of the electron beam (E-beam) and cobalt 60 (60Co-γ) at the dose of 15 kGy on the physicochemical properties, structure, immunogenicity, and biocompatibility of the APC were investigated. After decellularization, the residual DNA was 20.86 ± 1.02 ng/mg, and the α-Gal clearance rate was more than 99%. Irradiation, especially the 60Co-γ, reduced the cornea's transmittance, elastic modulus, enzymatic hydrolysis rate, swelling ratio, and cross-linking degree. Meanwhile, the diameter and spacing of the collagen fibers increased. In the rat subcutaneous implantation, many inflammatory cells appeared in the unirradiated APC, while the irradiated had good histocompatibility, but the degradation was faster. The lamellar keratoplasty in rabbits indicated that compared to the E-beam, the 60Co-γ damaged the chemical bond of collagen to a larger extent, reduced the content of GAGs, and prolonged the complete epithelization of the grafts. The corneal edema was more serious within 1 month after the surgery. After 2 months, the thickness of the APC with the two irradiation methods tended to be stable, but that in the 60Co-γ group became thinner. The pathological results showed that the collagen structure was looser and the pores were larger, indicating the 60Co-γ had a more extensive effect on the APC than the E-beam at 15 kGy.

Suitability of Slaughterhouse-Acquired Pig Eyes as Model Systems for Refractive Ultraviolet and Infrared Femtosecond Laser Research

Purpose To elucidate whether it is feasible to use porcine eyes from scalded, abattoir-acquired animals for refractive femtosecond laser research. Methods An infrared laser (FS 200) and an ultraviolet laser (prototype version) were tested for their applicability on scalded pig eyes. Fifty porcine eyes were divided into two equally-sized groups and assigned to either the infrared or the ultraviolet laser. Both laser groups were comprised of five subgroups of n = 5 eyes each. Group A: non-scalded eyes (negative control); group B: eyes taken from tunnel-scalded animals; group C1: eyes taken from tank-scalded animals without opaque corneal lesion; group C2: eyes taken from animals with opaque corneal lesion; group D: eyes scalded in toto in the laboratory (positive control). In each group the lasers were employed to create a stromal flap. The quality of the laser cuts and the resulting flap beds, as well as of the porcine corneas themselves, was examined by anterior segment optical coherence tomography and scanning electron microscopy. Results All scalded specimens exhibited substantial corneal swelling, most pronounced in group C2. After ultraviolet laser application, the tank- and tunnel-scalded samples displayed marked irregularities and an increased degree of surface roughness in the flap beds. After infrared laser application, this was only the case in the tank-scalded specimens. Conclusion It is not recommended to use eyes taken from scalded pigs for ultraviolet femtosecond laser experiments. For infrared femtosecond lasers, eyes taken from tunnel-scalded animals may represent an acceptable alternative, if non-scalded eyes are not available.

Simultaneous tensile and shear measurement of the human cornea in vivo using S0- and A0-wave optical coherence elastography

Understanding corneal stiffness is valuable for improving refractive surgery, detecting corneal abnormalities, and assessing intraocular pressure. However, accurately measuring the elastic properties, specifically the tensile and shear moduli that govern mechanical deformation, has been challenging. To tackle this issue, we have developed guided-wave optical coherence elastography that can simultaneously excite and analyze symmetric (S0) and anti-symmetric (A0) elastic waves in the cornea at around 10 kHz frequencies, enabling us to extract tensile and shear properties from measured wave dispersion curves. We verified the technique using elastomer phantoms and ex vivo porcine corneas and investigated the dependence on intraocular pressure using acoustoelastic theory that incorporates corneal tension and a nonlinear constitutive tissue model. In a pilot study involving six healthy human subjects aged 31 to 62, we measured shear moduli (Gzx) of 94±20 kPa (mean±standard deviation) and tensile moduli (Exx) of 4.0±1.1 MPa at central corneas. Our preliminary analysis of age-dependence revealed contrasting trends: -8.3±4.5 kPa/decade for shear and 0.30±0.21 MPa/decade for tensile modulus. This OCE technique has the potential to become a highly useful clinical tool for the quantitative biomechanical assessment of the cornea. Statement of SignificanceThis article reports an innovative elastography technique using two guided elastic waves, demonstrating the measurement of both tensile and shear moduli in human cornea in vivo with unprecedented precision. This technique paves the way for comprehensive investigations into corneal mechanics and holds clinical significance in various aspects of corneal health and disease management.

PLGA Nanoparticles Containing Natural Flavanones for Ocular Inflammation.

Flavanones are natural compounds that display anti-inflammatory activity. The aim of this work was to prepare PLGA nanoparticles (NPs) containing natural flavanones I ((2S)-5,7-dihydroxy-6-methyl-8-(3-methyl-2-buten-1-il)-2-phenyl-2,3-dihydro-4H-1-Benzopyran-4-one) and II (2S)-5,7-dihydroxy-2-(4'-methoxyphenyl)-6-methyl-8-(3-methyl-2-buten-1-yl)-2,3-dihydro-4H-1-Benzopyran-4-one) (NP I and NP II, respectively) so as to evaluate their potential for topical anti-inflammatory ocular therapy. An in silico study was carried out using the Molinspiration® and PASS Online web platforms before evaluating the in vitro release study and the ex vivo porcine cornea and sclera permeation. The HPLC analytical method was also established and validated. Finally, the in vitro anti-inflammatory efficacy of NPs was studied in the HCE-2 model. The flavanones I and II could be released following a kinetic hyperbolic model. Neither of the two NPs was able to permeate through the tissues. NP I and NP II were found to be respectful of any changes in the tissues' morphology, as evidenced by histological studies. In HCE-2 cells, NP I and NP II were not cytotoxic at concentrations up to 25 µM. NP I showed higher anti-inflammatory activity than NP II, being able to significantly reduce IL-8 production in LPS-treated HCE-2 cells. In summary, ocular treatment with NP I and NP II could be used as a promising therapy for the inhibition of ocular inflammation.

Optomechanical assessment of photorefractive corneal cross-linking via optical coherence elastography.

Purpose: Corneal cross-linking (CXL) has recently been used with promising results to positively affect corneal refractive power in the treatment of hyperopia and mild myopia. However, understanding and predicting the optomechanical changes induced by this procedure are challenging. Methods: We applied ambient pressure modulation based optical coherence elastography (OCE) to quantify the refractive and mechanical effects of patterned CXL and their relationship to energy delivered during the treatment on porcine corneas. Three different patterned treatments were performed, designed according to Zernike polynomial functions (circle, astigmatism, coma). In addition, three different irradiation protocols were analyzed: standard Dresden CXL (fluence of 5.4J/cm2), accelerated CXL (fluence of 5.4J/cm2), and high-fluence CXL (fluence of 16.2J/cm2). The axial strain distribution in the stroma induced by ocular inflation (Δp = 30mmHg) was quantified, maps of the anterior sagittal curvature were constructed and cylindrical refraction was assessed. Results: Thirty minutes after CXL, there was a statistically significant increase in axial strain amplitude (p < 0.050) and a reduction in sagittal curvature (p < 0.050) in the regions treated with all irradiation patterns compared to the non-irradiated ones. Thirty-6hours later, the non-irradiated regions showed compressive strains, while the axial strain in the CXL-treated regions was close to zero, and the reduction in sagittal curvature observed 30minutes after the treatment was maintained. The Dresden CXL and accelerated CXL produced comparable amounts of stiffening and refractive changes (p = 0.856), while high-fluence CXL produced the strongest response in terms of axial strain (6.9‰ ± 1.9‰) and refractive correction (3.4 ± 0.9 D). Tripling the energy administered during CXL resulted in a 2.4-fold increase in the resulting refractive correction. Conclusion: OCE showed that refractive changes and alterations in corneal biomechanics are directly related. A patient-specific selection of both, the administered UV fluence and the irradiation pattern during CXL is promising to allow customized photorefractive corrections in the future.

Molecular design of an ultra-strong tissue adhesive hydrogel with tunable multifunctionality.

Designing adhesive hydrogels with optimal properties for the treatment of injured tissues is challenging due to the tradeoff between material stiffness and toughness while maintaining adherence to wet tissue surfaces. In most cases, bioadhesives with improved mechanical strength often lack an appropriate elastic compliance, hindering their application for sealing soft, elastic, and dynamic tissues. Here, we present a novel strategy for engineering tissue adhesives in which molecular building blocks are manipulated to allow for precise control and optimization of the various aforementioned properties without any tradeoffs. To introduce tunable mechanical properties and robust tissue adhesion, the hydrogel network presents different modes of covalent and noncovalent interactions using N-hydroxysuccinimide ester (NHS) conjugated alginate (Alg-NHS), poly (ethylene glycol) diacrylate (PEGDA), tannic acid (TA), and Fe3+ ions. Through combining and tuning different molecular interactions and a variety of crosslinking mechanisms, we were able to design an extremely elastic (924%) and tough (4697kJ/m3) multifunctional hydrogel that could quickly adhere to wet tissue surfaces within 5s of gentle pressing and deform to support physiological tissue function over time under wet conditions. While Alg-NHS provides covalent bonding with the tissue surfaces, the catechol moieties of TA molecules synergistically adopt a mussel-inspired adhesive mechanism to establish robust adherence to the wet tissue. The strong adhesion of the engineered bioadhesive patch is showcased by its application to rabbit conjunctiva and porcine cornea. Meanwhile, the engineered bioadhesive demonstrated painless detachable characteristics and in vitro biocompatibility. Additionally, due to the molecular interactions between TA and Fe3+, antioxidant and antibacterial properties required to support the wound healing pathways were also highlighted. Overall, by tuning various molecular interactions, we were able to develop a single-hydrogel platform with an "all-in-one" multifunctionality that can address current challenges of engineering hydrogel-based bioadhesives for tissue repair and sealing.

A predictive model of UV-A-riboflavin crosslinking treatment on porcine corneas

The crosslinking technique (CXL) is an effective low-risk therapeutic treatment of keratoconus and other ectatic disorders of the human cornea. The effect of corneal CXL is to increase the stiffness of the stroma to prevent the progression of the cornea distortion. Several clinical and experimental studies have shown that the stiffening effects predominantly localize on the anterior portion of the stroma and that the in-depth stiffening distribution is highly dependent on the duration of treatment. Yet, how the stiffening effects distribute through the cornea thickness as a function of the treatment duration is an open question. Here, we propose an analytical model of the stiffening profile due to CXL treatment as a function of the irradiation time. We consider linear and nonlinear variations of the crosslinking effects across the thickness and implement them into a finite element model of the porcine cornea. We present a time-dependent in-depth stiffening profile that allows us to predict the post-operative cornea response to physiological intraocular pressure for different irradiation times. We anticipate that this predictive model will support the development of patient specific three-dimensional models that will allow clinicians to design customized CXL treatment, thus enhancing treatment outcomes.

Preclinical evaluation of the safety and effectiveness of a new bioartificial cornea.

Cross-linking agents are frequently used to restore corneal properties after decellularization, and it is especially important to select an appropriate method to avoid excessive cross-linking. In addition, how to promote wound healing and how to improve scar formation require further investigation. To ensure the safety and efficacy of animal-derived products, we designed bioartificial corneas (BACs) according to the criteria for Class III medical devices. Our BACs do not require cross-linking agents and increase mechanical strength via self-cross-linking of aldehyde-modified hyaluronic acid (AHA) and carboxymethyl chitosan (CMC) on the surface of decellularized porcine corneas (DPCs). The results showed that the BACs had good biocompatibility and transparency, and the modification enhanced their antibacterial and anti-inflammatory properties in vitro. Preclinical animal studies showed that the BACs can rapidly regenerate the epithelium and restore vision within a month. After 3 months, the BACs were gradually filled with epithelial, stromal, and neuronal cells, and after 6 months, their transparency and histology were almost normal. In addition, side effects such as corneal neovascularization, conjunctival hyperemia, and ciliary body hyperemia rarely occur in vivo. Therefore, these BACs show promise for clinical application for the treatment of infectious corneal ulcers and as a temporary covering for corneal perforations to achieve the more time.

Automated Detection of Corneal Edema With Deep Learning-Assisted Second Harmonic Generation Microscopy

Second Harmonic Generation Microscopy (SHG) is widely acknowledged as a valuable non-linear optical imaging tool, its contrast mechanism providing the premises to non-invasively identify, characterize, and monitor changes in the collagen architecture of tissues. However, the interpretation of SHG data can pose difficulties even for experts histopathologists, which represents a bottleneck for the translation of SHG-based diagnostic frameworks to clinical settings. The use of artificial intelligence methods for automated SHG analysis is still in an early stage, with only few studies having been reported to date, none addressing ocular tissues yet. In this work we explore the use of three Deep Learning models, the highly popular InceptionV3 and ResNet50, alongside FLIMBA, a custom developed architecture, requiring no pre-training, to automatically detect corneal edema in SHG images of porcine cornea. We observe that Deep Learning models building on different architectures provide complementary results for the classification of cornea SHG images and demonstrate an AU-ROC = 0.98 for their joint use. These results have potential to be extrapolated to other diagnostics scenarios, such as automated extraction of hydration level of cornea, or identification of corneal edema causes, and thus pave the way for novel methods for precision diagnostics of the cornea with Deep-Learning assisted SHG imaging.

Tissue Ablation with Multi‐Millimeter Depth and Cellular‐Scale Collateral Damage by a Femtosecond Mid‐Infrared Laser Tuned to the Amide‐I Vibration

AbstractLaser ablation of bio‐tissues is the key technology of future surgeries, owing to the merits of submicron accuracy, non‐contact operation, and precision control assisted by automation and computer intelligence. Excellent efficiency, minimal collateral damage, biological safety, and tissue selectivity are the ideal parameters for delicate surgical applications. Here, by exploring the mid‐infrared (MIR) resonant ablation and the femtosecond cold processing, tissue ablation with multi‐millimeter depth and cellular collateral damage, enabled by a tabletop femtosecond MIR optical parametric amplifier operating at the amide‐I resonant wavelength of 6.1 µm, is demonstrated. Remarkably, the collateral damage is observed to be 15, &lt;4, and &lt;1 µm in the porcine cornea, sclera, and articular cartilage, respectively, with multi‐millimeter incision depth. In addition, preliminary proof‐of‐concept experiments of tissue‐selective ablation, microchannels on cartilage for drug delivery, laser glaucoma, keratotomy, neurologic tissue incision, dentin deep crater formation, and hypertrophic scarring ablation are demonstrated, which pave the way for high‐precision surgical applications with a tabletop solution.