A Magnetic Actuator Device for Fully Automated Blinking in Total Bidirectional Eyelid Paralysis: First Proof of Concept in a Human Participant.

Purpose: Currently, no solution exists to restore natural eyelid kinematics for patients with complete eyelid paralysis due to loss of function of both the levator palpebrae superioris and orbicularis oculi. These rare cases are prone to complications of chronic exposure keratopathy which may lead to corneal blindness. We hypothesized that magnetic force could be used to fully automate eyelid movement in these cases through the use of eyelid-attached magnets and a spectacle-mounted magnet driven by a programmable motor (motorized magnetic levator prosthesis [MMLP]). Methods: To test this hypothesis and establish proof of concept, we performed a finite element analysis (FEA) for a prototype MMLP to check the eyelid-opening force generated by the device and verified the results with experimental measurements in a volunteer with total bidirectional eyelid paralysis. The subject was then fitted with a prototype to check the performance of the device and its success. Results: With MMLP, eye opening was restored to near normal, and blinking was fully automated in close synchrony with the motor-driven polarity reversal, with full closure on the blink. The device was well tolerated, and the participant was pleased with the comfort and performance. Conclusions: FEA simulation results conformed to the experimentally observed trend, further supporting the proof of concept and design parameters. This is the first viable approach in human patients with proof of concept for complete reanimation of a bidirectionally paretic eyelid. Further study is warranted to refine the prototype and determine the feasibility and safety of prolonged use. Translational Relevance: This is first proof of concept for our device for total bidirectional eyelid paralysis.

The prophylactic value of TNF-α inhibitors against retinal cell apoptosis and optic nerve axon loss after corneal surgery or trauma.

BACKGROUND AND PURPOSE: Late secondary glaucoma is an often-severe complication after acute events like anterior segment surgery, trauma and infection. TNF-α is a major mediator that is rapidly upregulated, diffusing also to the retina and causes apoptosis of the ganglion cells and degeneration of their optic nerve axons (mediating steps to glaucomatous damage). Anti-TNF-α antibodies are in animals very effective in protecting the retinal cells and the optic nerve-and might therefore be useful prophylactically against secondary glaucoma in future such patients. Here we evaluate (1) toxicity and (2) efficacy of two TNF-α inhibitors (adalimumab and infliximab), in rabbits by subconjunctival administration. METHODS: For drug toxicity, animals with normal, unburned corneas were injected with adalimumab (0.4, 4, or 40 mg), or infliximab (1, 10, or 100 mg). For drug efficacy, other animals were subjected to alkali burn before such injection, or steroids (for control). The rabbits were evaluated clinically with slit lamp and photography, electroretinography, optical coherence tomography, and intraocular pressure manometry. A sub-set of eyes were stained ex vivo after 3 days for retinal cell apoptosis (TUNEL). In other experiments the optic nerves were evaluated by paraphenylenediamine staining after 50 or 90 days. Loss of retinal cells and optic nerve degeneration were quantified. RESULTS: Subconjunctival administration of 0.4 mg or 4.0 mg adalimumab were well tolerated, whereas 40.0 mg was toxic to the retina. 1, 10, or 100 mg infliximab were also well tolerated. Analysis of the optic nerve axons after 50 days confirmed the safety of 4.0 mg adalimumab and of 100 mg infliximab. For efficacy, 4.0 mg adalimumab subconjunctivally in 0.08 mL provided practically full protection against retinal cell apoptosis 3 days following alkali burn, and infliximab 100 mg only slightly less. At 90 days following burn injury, control optic nerves showed about 50% axon loss as compared to 8% in the adalimumab treatment group. CONCLUSIONS: Subconjunctival injection of 4.0 mg adalimumab in rabbits shows no eye toxicity and provides excellent neuroprotection, both short (3 days) and long-term (90 days). Our total. accumulated data from several of our studies, combined with the present paper, suggest that corneal injuries, including surgery, might benefit from routine administration of anti-TNF-α biologics to reduce inflammation and future secondary glaucoma.

An Adjustable Magnetic Levator Prosthesis for Customizable Eyelid Reanimation in Severe Blepharoptosis II: Randomized Evaluation of Angular Translation.

Purpose: Examine the effect of force modulation via angular translation of a static magnetic field for customizable treatment of severe blepharoptosis. Methods: Prototype adjustable-force magnetic levator prostheses (aMLP) consisted of a spectacle-mounted magnet in rotatable housing and small eyelid-attached magnets embedded in a biocompatible polymer. Interpalpebral fissure (IPF) of 17 participants with severe blepharoptosis was continuously measured for one minute at five spectacle magnet angles, with order randomized and participant and data analyst masked. The hypothesis that angular position affected opening IPF (o-IPF), minimum blink IPF (m-IPF), and comfort ratings (1-10) was tested. Results: The aMLP improved o-IPF from 4.5 mm without the device to 6.2 mm on the lowest force setting (P < 0.001) and 7.1 mm on the highest setting (P < 0.001) and allowed for complete volitional blink regardless of setting (average m-IPF 0.4 mm and no change with aMLP; P = 0.76). Spontaneous blink without the device (2.0 mm) was affected on the highest force setting (m-IPF 3.9 mm; P < 0.001) but only marginally so on the lowest setting (3.0 mm; P = 0.06). Comfort (7.6/10) did not vary with the angle (P > 0.36). Profile analysis found substantial individual responses to angle (P < 0.001), confirming the value of customization. Conclusions: Angular translation provided adjustable force, which had a statistically and clinically meaningful impact on eye opening and the completeness of the spontaneous blink. This quantitative evidence supports continued use of the angular translation mechanism for force adjustment in the customizable magnetic correction of severe blepharoptosis. Translational Relevance: Evidence for the benefit of customizable magnetic force via angular translation in a larger sample of participants than reported previously.

Clinical Report: Clinician Feedback on the Magnetic Levator Prosthesis.

SIGNIFICANCE: Stakeholder engagement has been identified by national health organizations as a crucial step to successful translation of new health care treatments. In this clinical report, clinician-stakeholder feedback is presented for the magnetic levator prosthesis (MLP), a promising noninvasive spectacle device that restores eyelid motility with magnetic force. PURPOSE: This study aimed to evaluate MLP clinical need and translational barriers. METHODS: Ten vision rehabilitation optometrists who attended an educational presentation on the MLP and participated in a hands-on workshop in the fitting of a patient were invited to complete an anonymous online survey. Ten multiple-choice items gathered data on estimated patient need, current approaches, main barriers to MLP, temporary versus chronic use, cost barriers, and need for insurance coverage. Open fields allowed for additional comments. RESULTS: Nine of 10 specialists completed the survey. Of those, seven answered that they could potentially see at least 1 to 5 patients for ptosis management within a year. The most common ptosis management options reported were the ptosis crutch, taping the eyelid open, and oxymetazoline drops, all with six responses each. Seven clinicians indicated that cost was a main concern. If cost to patient was not a barrier, all indicated they would be at least somewhat likely to try the MLP (1) for temporary management of ptosis, (2) as a pre-surgical trial, and (3) for long-term management of ptosis, with more selecting extremely likely and very likely than somewhat likely. Main comments were expressing enthusiasm for the technology and that it would be more appealing for patients if covered by insurance. CONCLUSIONS: This clinical report suggests that the main barriers to clinical success of the MLP may be cost and insurance coverage, appearance of the device, and self-application. Possible solutions are cost-benefit analysis research, engineering efforts to reduce spectacle magnet size and improve the ease of eyelid magnet application.

CSF1R inhibition is not specific to innate immune cells but also affects T-helper cell differentiation independently of microglia depletion.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissue-resident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation. In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data.

An Adjustable Magnetic Levator Prosthesis for Customizable Eyelid Re-Animation in Severe Blepharoptosis: Design and Proof-of-Concept.

Purpose: Blepharoptosis is a common oculoplastic condition causing incomplete opening of the upper eyelid. Surgical approaches, the mainstay for correction, often fail to improve blink function. The purpose of this study was to develop a nonsurgical treatment option for severe ptosis that allows blink re-animation. Methods: Magnetic force required to perform blink re-animation was characterized by evaluation of eye-opening and closing using inter-palpebral fissure (IPF) outcomes with various combinations of eyelid array and box magnets. Optimal size of the spectacle magnet that achieved forces required for optimal blink dynamics was selected using simulation. The adjustable magnetic levator prosthesis (aMLP) included an eyelid array magnet and an adjustable rotating spectacle magnet that allowed change in the magnetic direction, thus changing the net magnetic interactive force between the magnets. The clinical feasibility of aMLP in improving eye opening without limiting eye closing was evaluated in patients with ptosis through a proof-of-concept study using IPF and comfort outcomes. Results: Optimal eye opening and closing was achieved by a magnet-array combination providing 45 grams of surface force (gF) in the tested ptosis population. The aMLP was able to modulate eye opening and closing with change in rotation of the spectacle magnet in two patients with ptotis. The best fitting of an aMLP improved IPF opening without limiting eye closing and with good comfort reported. Conclusions: Preliminary results suggest that the an aMLP can correct ptosis without adversely affecting blink function. Further evaluation in a larger patient population is warranted. Translational Relevance: A nonsurgical, proof of concept, adjustable magnetic treatment option for blink re-animation in patients with severe ptosis is presented.

Opposing Roles of Blood-Borne Monocytes and Tissue-Resident Macrophages in Limbal Stem Cell Damage after Ocular Injury.

Limbal stem cell (LSC) deficiency is a frequent and severe complication after chemical injury to the eye. Previous studies have assumed this is mediated directly by the caustic agent. Here we show that LSC damage occurs through immune cell mediators, even without direct injury to LSCs. In particular, pH elevation in the anterior chamber (AC) causes acute uveal stress, the release of inflammatory cytokines at the basal limbal tissue, and subsequent LSC damage and death. Peripheral C-C chemokine receptor type 2 positive/CX3C motif chemokine receptor 1 negative (CCR2+ CX3CR1-) monocytes are the key mediators of LSC damage through the upregulation of tumor necrosis factor-alpha (TNF-α) at the limbus. In contrast to peripherally derived monocytes, CX3CR1+ CCR2- tissue-resident macrophages have a protective role, and their depletion prior to injury exacerbates LSC loss and increases LSC vulnerability to TNF-α-mediated apoptosis independently of CCR2+ cell infiltration into the tissue. Consistently, repopulation of the tissue by new resident macrophages not only restores the protective M2-like phenotype of macrophages but also suppresses LSC loss after exposure to inflammatory signals. These findings may have clinical implications in patients with LSC loss after chemical burns or due to other inflammatory conditions.

Sustained Inhibition of VEGF and TNF-α Achieves Multi-Ocular Protection and Prevents Formation of Blood Vessels after Severe Ocular Trauma.

PURPOSE: This study aimed to develop a clinically feasible and practical therapy for multi-ocular protection following ocular injury by using a thermosensitive drug delivery system (DDS) for sustained delivery of TNF-α and VEGF inhibitors to the eye. METHODS: A thermosensitive, biodegradable hydrogel DDS (PLGA-PEG-PLGA triblock polymer) loaded with 0.7 mg of adalimumab and 1.4 mg of aflibercept was injected subconjunctivally into Dutch-belted pigmented rabbits after corneal alkali injury. Control rabbits received 2 mg of IgG-loaded DDS or 1.4 mg of aflibercept-loaded DDS. Animals were followed for 3 months and assessed for tolerability and prevention of corneal neovascularization (NV), improvement of corneal re-epithelialization, inhibition of retinal ganglion cell (RGC) and optic nerve axon loss, and inhibition of immune cell infiltration into the cornea. Drug-release kinetics was assessed in vivo using an aqueous humor protein analysis. RESULTS: A single subconjunctival administration of dual anti-TNF-α/anti-VEGF DDS achieved a sustained 3-month delivery of antibodies to the anterior chamber, iris, ciliary body, and retina. Administration after corneal alkali burn suppressed CD45+ immune cell infiltration into the cornea, completely inhibited cornea NV for 3 months, accelerated corneal re-epithelialization and wound healing, and prevented RGC and optic nerve axon loss at 3 months. In contrast, anti-VEGF alone or IgG DDS treatment led to persistent corneal epithelial defect (combined: <1%; anti-VEGF: 15%; IgG: 10%, of cornea area), increased infiltration of CD45+ immune cells into the cornea (combined: 28 ± 20; anti-VEGF: 730 ± 178; anti-IgG: 360 ± 186, cells/section), and significant loss of RGCs (combined: 2.7%; anti-VEGF: 63%; IgG: 45%) and optic nerve axons at 3 months. The aqueous humor protein analysis showed first-order release kinetics without adverse effects at the injection site. CONCLUSIONS: Concomitant inhibition of TNF-α and VEGF prevents corneal neovascularization and ameliorates subsequent irreversible damage to the retina and optic nerve after severe ocular injury. A single subconjunctival administration of this therapy, using a biodegradable, slow-release thermosensitive DDS, achieved the sustained elution of therapeutic levels of antibodies to all ocular tissues for 3 months. This therapeutic approach has the potential to dramatically improve the outcomes of severe ocular injuries in patients and improve the therapeutic outcomes in patients with retinal vascular diseases.

Incidence and risk factors for glaucoma development and progression after corneal transplantation.

OBJECTIVE: To assess the cumulative incidence and risk factors for glaucoma development and progression within 1-2 years following corneal transplant surgery. DESIGN: Retrospective cohort study. METHODS: Patients undergoing penetrating keratoplasty (PK), deep anterior lamellar keratoplasty (DALK), Descemet stripping endothelial keratoplasty (DSEK), Descemet membrane endothelial keratoplasty (DMEK), Boston keratoprosthesis type I (KPro) implantation, or endothelial keratoplasty (DSEK or DMEK) under previous PK (EK under previous PK) at one academic institution with at least 1 year of follow-up were included. Primary outcome measures were cumulative incidence of glaucoma development and progression after corneal transplant, in patients without and with preoperative glaucoma, respectively. Risk factors for glaucoma development and progression were also assessed. RESULTS: Four hundred and thirty-one eyes of 431 patients undergoing PK (113), DALK (17), DSEK (71), DMEK (168), KPro (35) and EK under previous PK (27) with a mean follow-up of 22.9 months were analyzed. The 1-year cumulative incidence for glaucoma development and progression was 28.0% and 17.8% in patients without and with preoperative glaucoma, respectively. In a Cox proportional hazards analysis, DSEK surgery, KPro implantation, average intraocular pressure (IOP) through follow-up and postoperative IOP spikes of ≥30 mmHg were each independently associated with glaucoma development or progression (p < 0.04 for all). CONCLUSIONS: A significant proportion of patients developed glaucoma or exhibited glaucoma progression within 1 year after corneal transplantation. Patient selection for DSEK may partly explain the higher risk for glaucoma in these patients. Postoperative IOP spikes should be minimized and may indicate the need for co-management with a glaucoma specialist.

Feasibility of Magnetic Levator Prosthesis Frame Customization Using Craniofacial Scans and 3-D Printing.

Purpose: To determine the feasibility of a custom frame generation approach for nonsurgical management of severe blepharoptosis with the magnetic levator prosthesis (MLP). Methods: Participants (n = 8) with severe blepharoptosis (obscuring the visual axis) in one or both eyes who had previously been using a non-custom MLP had a craniofacial scan with a smartphone app to generate a custom MLP frame. A magnetic adhesive was attached to the affected eyelid. The custom MLP frame held a cylindrical magnet near the eyebrow above the affected eyelid, suspending it in the magnetic field while still allowing blinking. The spectacle magnet could be rotated manually, providing adjustable force via angular translation of the magnetic field. Fitting success and comfort were recorded, and interpalpebral fissure (IPF) was measured from video frames after 20 minutes in-office and one-week at-home use. Preference was documented, custom versus non-custom. Results: Overall, 88% of patients (7/8) were successfully fitted with a median 9/10 comfort (interquartile 7-10) and median ptosis improvement of 2.3 mm (1.3-5.0); P = 0.01). Exact binomial testing suggested, with 80% power, that the true population success rate was significantly greater than 45% (P = 0.05). Five participants took the custom MLP home for one week, with only one case of mild conjunctival redness which resolved without treatment. Highest to lowest force modulation resulted in a marginally significant median IPF adjustment of 1.5 mm (0.8 to 2.7; P = 0.06). All preferred the custom frame. Conclusions: The three-dimensional custom MLP frame generation approach using a smartphone app-based craniofacial scan is a feasible approach for clinical deployment of the MLP. Translational Relevance: First demonstration of customized frame generation for the MLP.

Circulating inflammatory monocytes oppose microglia and contribute to cone cell death in retinitis pigmentosa.

Retinitis pigmentosa (RP) is an intractable inherited disease that primarily affects the rods through gene mutations followed by secondary cone degeneration. This cone-related dysfunction can lead to impairment of daily life activities, and ultimately blindness in patients with RP. Paradoxically, microglial neuroinflammation contributes to both protection against and progression of RP, but it is unclear which population(s) - tissue-resident microglia and/or peripheral monocyte-derived macrophages (mφ) - are implicated in the progression of the disease. Here we show that circulating blood inflammatory monocytes (IMo) are key effector cells that mediate cone cell death in RP. Attenuation of IMo and peripherally engrafted mφ by Ccl2 deficiency or immune modulation via intravenous nano-particle treatment suppressed cone cell death in rd10 mice, an animal model of RP. In contrast, the depletion of resident microglia by a colony-stimulating factor 1 receptor inhibitor exacerbated cone cell death in the same model. In human patients with RP, IMo was increased and correlated with disease progression. These results suggest that peripheral IMo is a potential target to delay cone cell death and prevent blindness in RP.

A One-Step Electrochemical Aptasensor Based on Signal Amplification of Metallo Nanoenzyme Particles for Vascular Endothelial Growth Factor.

In this study, a one-step electrochemical aptasensor was developed to detect the biomarker vascular endothelial growth factor (VEGF), an important protein in the pathogenesis of many retinal diseases, including age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion. The aptamer has a good affinity and can rapidly identify and capture VEGF based on its unique structure. We designed a VEGF aptasensor based on the aptamer recognition and complex metallo nanoenzyme particles as an electron exchange center and bridge between capture DNA and electrode. The aptamers maintained the hairpin structure to avoid nonspecific surface adsorption and expose the capture sequence outwards when the target was inexistent. Conversely, the aptamers opened the hairpin structure to release space to accomplish binding between VEGF and DNA, resulting in increased impedance. The performance of the electrochemical aptasensor is detected by electrochemical impedance spectroscopy (EIS). The limit of detection by EIS was as low as 8.2 pg ml-1, and the linear range was 10 pg ml-1-1 µg ml-1. The electrochemical aptasensor also showed high specificity and reproducibility.

The Prevalence of Autoimmune Diseases in Patients with Primary Open-Angle Glaucoma Undergoing Ophthalmic Surgeries.

PURPOSE: To assess the prevalence of autoimmune disease (AiD) in patients with primary open-angle glaucoma (POAG) undergoing ophthalmic surgery. DESIGN: Retrospective, cross-sectional study. PARTICIPANTS: Patients with POAG undergoing any ophthalmic surgery and control subjects undergoing cataract surgery at the Massachusetts Eye and Ear from March 2019 to April 2020. METHODS: All available medical records with patient demographics, ocular, and medical conditions were reviewed. Differences in AiD prevalence were assessed and adjusted for covariates using multiple logistic regression. Additionally, a subgroup analysis comparing the POAG patients with and without AiD was performed. MAIN OUTCOME MEASURES: To assess the prevalence of AiD based on the American Autoimmune Related Diseases Association list. RESULTS: A total of 172 patients with POAG and 179 controls were included. The overall prevalence of AiD was 17.4% in the POAG group and 10.1% in the controls (P = 0.044); 6.4% of POAG patients and 3.4% of controls had more than 1 AiD (P = 0.18). The most prevalent AiDs in POAG group were rheumatoid arthritis (4.6%) and psoriasis (4.1%), which were also the most common in controls (2.8% each). In a fully adjusted multiple logistic regression analysis accounting for steroid use, having an AiD was associated with 2.62-fold increased odds of POAG relative to controls (95% confidence interval, 1.27-5.36, P = 0.009); other risk factors for POAG derived from the analysis included age (odds ratio [OR], 1.04, P = 0.006), diabetes mellitus (OR, 2.31, P = 0.008), and non-White ethnicity (OR, 4.75, P < 0.001). In a case-only analysis involving the eye with worse glaucoma, there was no statistical difference in visual field mean deviation or retinal nerve fiber layer (RNFL) thickness in POAG patients with AiD (n = 30) and without AiD (n = 142, P > 0.13, for both). CONCLUSIONS: A higher prevalence of AiD was found in POAG patients compared with control patients undergoing ophthalmic surgery. The presence of AiD was associated with increased risk for POAG after adjusting for covariates. Additional factors may have prevented a difference in RNFL thickness in POAG patients with and without AiD. Autoimmunity should be explored further in the pathogenesis of POAG.

Critical media attributes in E-beam sterilization of corneal tissue.

When ionizing irradiation interacts with a media, it can form reactive species that can react with the constituents of the system, leading to eradication of bioburden and sterilization of the tissue. Understanding the media's properties such as polarity is important to control and direct those reactive species to perform desired reactions. Using ethanol as a polarity modifier of water, we herein generated a series of media with varying relative polarities for electron beam (E-beam) irradiation of cornea at 25 kGy and studied how the irradiation media's polarity impacts properties of the cornea. After irradiation of corneal tissues, mechanical (tensile strength and modulus, elongation at break, and compression modulus), chemical, optical, structural, degradation, and biological properties of the corneal tissues were evaluated. Our study showed that irradiation in lower relative polarity media improved structural properties of the tissues yet reduced optical transmission; higher relative polarity reduced structural and optical properties of the cornea; and intermediate relative polarity (ethanol concentrations = 20-30% (v/v)) improved the structural properties, without compromising optical characteristics. Regardless of media polarity, irradiation did not negatively impact the biocompatibility of the corneal tissue. Our data shows that the absorbed ethanol can be flushed from the irradiated cornea to levels that are nontoxic to corneal and retinal cells. These findings suggest that the relative polarity of the irradiation media can be tuned to generate sterilized tissues, including corneal grafts, with engineered properties that are required for specific biomedical applications. STATEMENT OF SIGNIFICANCE: Extending the shelf-life of corneal tissue can improve general accessibility of cornea grafts for transplantation. Irradiation of donor corneas with E-beam is an emerging technology to sterilize the corneal tissues and enable their long-term storage at room temperature. Despite recent applications in clinical medicine, little is known about the effect of irradiation and preservation media's characteristics, such as polarity on the properties of irradiated corneas. Here, we have showed that the polarity of the media can be a valuable tool to change and control the properties of the irradiated tissue for transplantation.

Toward electron-beam sterilization of a pre-assembled Boston keratoprosthesis.

PURPOSE: To evaluate the effects of electron-beam (E-beam) irradiation on the human cornea and the potential for E-beam sterilization of Boston keratoprosthesis (BK) devices when pre-assembled with a donor cornea prior to sterilization. METHODS: Human donor corneas and corneas pre-assembled in BK devices were immersed in recombinant human serum albumin (rHSA) media and E-beam irradiated at 25 kGy. Mechanical (tensile strength and modulus, and compression modulus), chemical, optical, structural, and degradation properties of the corneal tissue after irradiation and after 6 months of preservation were evaluated. RESULTS: The mechanical evaluation showed that E-beam irradiation enhanced the tensile and compression moduli of human donor corneas, with no impact on their tensile strength. By chemical and mechanical analysis, E-beam irradiation caused a minor degree of crosslinking between collagen fibrils. No ultrastructural changes due to E-beam irradiation were observed. E-beam irradiation slightly increased the stability of the cornea against collagenase-induced degradation and had no impact on glucose diffusion. The optical evaluation showed transparency of the cornea was maintained. E-beam irradiated corneal tissues and BK-cornea pre-assembled devices were stable for 6 months after room-temperature preservation. CONCLUSIONS: E-beam irradiation generated no detrimental effects on the corneal tissues or BK-cornea pre-assembled devices and improved native properties of the corneal tissue, enabling prolonged preservation at room temperature. The pre-assembly of BK in a donor cornea, followed by E-beam irradiation, offers the potential for an off-the-shelf, ready to implant keratoprosthesis device.

Combined blockade of complement C5 and TLR co-receptor CD14 synergistically inhibits pig-to-human corneal xenograft induced innate inflammatory responses.

Inadequate supplies of donor corneas have evoked an escalating interest in corneal xenotransplantation. However, innate immune responses contribute significantly to the mechanism of xenograft rejection. We hypothesized that complement component C5 and TLR co-receptor CD14 inhibition would inhibit porcine cornea induced innate immune responses. Therefore, we measured cytokine release in human blood, induced by three forms of corneal xenografts with or without inhibitors. Native porcine cornea (NPC) induced interleukins (IL-1ß, IL-2, IL-6, IL-8, IL-1ra), chemokines (MCP-1, MIP-1α, MIP-1ß) and other cytokines (TNF, G-CSF, INF-γ, FGF-basic). Decellularized (DPC) and gamma-irradiated cornea (g-DPC) elevated the release of those cytokines. C5-blockade by eculizumab inhibited all the cytokines except G-CSF when induced by NPC. However, C5-blockade failed to reduce DPC and g-DPC induced cytokines. Blockade of CD14 inhibited DPC-induced cytokines except for IL-8, MCP-1, MIP-1α, and G-CSF, while it inhibited all of them when induced by g-DPC. Combined blockade of C5 and CD14 inhibited the maximum number of cytokines regardless of the xenograft type. Finally, by using the TLR4 specific inhibitor Eritoran, we showed that TLR4 activation was the basis for the CD14 effect. Thus, blockade of C5, when combined with TLR4 inhibition, may have therapeutic potential in pig-to-human corneal xenotransplantation. STATEMENT OF SIGNIFICANCE: Bio-engineered corneal xenografts are on the verge of becoming a viable alternative to allogenic human-donor-cornea, but the host's innate immune response is still a critical barrier for graft acceptance. By overruling this barrier, limited graft availability would no longer be an issue for treating corneal diseases. We showed that the xenograft induced inflammation is initiated by the complement system and toll-like receptor activation. Intriguingly, the inflammatory response was efficiently blocked by simultaneously targeting bottleneck molecules in the complement system (C5) and the TLR co-receptor CD14 with pharmaceutical inhibitors. We postulate that a combination of C5 and CD14 inhibition could have a great therapeutic potential to overcome the immunologic barrier in pig-to-human corneal xenotransplantation.

Electron Beam Sterilization of Poly(Methyl Methacrylate)-Physicochemical and Biological Aspects.

Electron beam (E-beam) irradiation is an attractive and efficient method for sterilizing clinically implantable medical devices made of natural and/or synthetic materials such as poly(methyl methacrylate) (PMMA). As ionizing irradiation can affect the physicochemical properties of PMMA, understanding the consequences of E-beam sterilization on the intrinsic properties of PMMA is vital for clinical implementation. A detailed assessment of the chemical, optical, mechanical, morphological, and biological properties of medical-grade PMMA after E-beam sterilization at 25 and 50 kiloGray (kGy) is reported. Fourier transform infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry studies indicate that E-beam irradiation has minimal effect on the chemical properties of the PMMA at these doses. While 25 kGy irradiation does not alter the mechanical and optical properties of the PMMA, 50 kGy reduces the flexural strength and transparency by 10% and 2%, respectively. Atomic force microscopy demonstrates that E-beam irradiation reduces the surface roughness of PMMA in a dose dependent manner. Live-Dead, AlamarBlue, immunocytochemistry, and complement activation studies show that E-beam irradiation up to 50 kGy has no adverse effect on the biocompatibility of the PMMA. These findings suggest that E-beam irradiation at 25 kGy may be a safe and efficient alternative for PMMA sterilization.

Photo-cross-linked Gelatin Glycidyl Methacrylate/N-Vinylpyrrolidone Copolymeric Hydrogel with Tunable Mechanical Properties for Ocular Tissue Engineering Applications.

Corneal transplantation is currently the primary treatment for corneal blindness. However, severe global scarcity of donor corneas is driving the scientific community to find novel solutions. One potential solution is to replace the damaged tissue with a biocompatible artificial cornea. Here, gelatin glycidyl methacrylate (GM) and N-vinylpyrrolidone (VP) were cocrosslinked to afford a hybrid bicomponent copolymeric hydrogel with excellent mechanical, structural, and biological properties. Our studies showed that the GM/VP ratio can be adjusted to generate a construct with high tensile modulus and strength of 1.6 and 1.0 MPa, respectively, compared to 14 and 7.5 MPa for human cornea. The construct can tolerate up to 22.4 kPa pressure before retention sutures can tear through it. Due to the presence of a synthetic component, it has a significantly higher stability against collagenase induced degradation, yet it is biocompatible and promotes cellular adhesion, proliferation, and migration under in vitro settings.