Age-related enhanced degeneration of bioprosthetic valves due to leaflet calcification, tissue crosslinking, and structural changes.

AIMS: Bioprosthetic heart valves (BHVs), made from glutaraldehyde-fixed heterograft materials, are subject to more rapid structural valve degeneration (SVD) in paediatric and young adult patients. Differences in blood biochemistries and propensity for disease accelerate SVD in these patients, which results in multiple re-operations with compounding risks. The goal of this study is to investigate the mechanisms of BHV biomaterial degeneration and present models for studying SVD in young patients and juvenile animal models. METHODS AND RESULTS: We studied SVD in clinical BHV explants from paediatric and young adult patients, juvenile sheep implantation model, rat subcutaneous implants, and an ex vivo serum incubation model. BHV biomaterials were analysed for calcification, collagen microstructure (alignment and crimp), and crosslinking density. Serum markers of calcification and tissue crosslinking were compared between young and adult subjects. We demonstrated that immature subjects were more susceptible to calcification, microstructural changes, and advanced glycation end products formation. In vivo and ex vivo studies comparing immature and mature subjects mirrored SVD in clinical observations. The interaction between host serum and BHV biomaterials leads to significant structural and biochemical changes which impact their functions. CONCLUSIONS: There is an increased risk for accelerated SVD in younger subjects, both experimental animals and patients. Increased calcification, altered collagen microstructure with loss of alignment and increased crimp periods, and increased crosslinking are three main characteristics in BHV explants from young subjects leading to SVD. Together, our studies establish a basis for assessing the increased susceptibility of BHV biomaterials to accelerated SVD in young patients.

Scleral growth stunting via sub-Tenon injection of cross-linking solutions in live rabbits.

BACKGROUND: Scleral cross-linking is a potential method to inhibit axial elongation of the eye, preventing the progression of pathological myopia. Formaldehyde releasers, which are common preservatives found in cosmetics and ophthalmic solutions, have been shown to be not only effective in cross-linking corneal collagen in vitro and in vivo, but also have minimal toxicity effects on the eye. The present study aims to evaluate the efficacy of scleral cross-linking using sodium hydroxymethylglycinate (SMG) to inhibit eye growth using an in vivo rabbit model. METHODS: A cross-linking solution containing 40 mM SMG was delivered to the sub-Tenon's space behind the equator. The application regimen included a two-quadrant injection performed five times over 2 weeks on New Zealand White rabbits (n=5, group 1), and one-time injection followed for up to 5 days on Dutch-Belted rabbits (n=6, group 2). Group 1 was monitored serially for axial length changes using B-scan ultrasound for 5-6 weeks. Group 2 was injected with a higher viscosity solution formulation. Both groups were evaluated for thermal denaturation temperature changes of the sclera postmortem. RESULTS: Axial growth was limited by 10%-20% following SMG treatment as compared with the untreated eye. Thermal denaturation analysis showed increased heat resistance of the treated eyes in the areas of injection. Overall, the SMG treatment inhibited eye growth with few side effects from the injections. CONCLUSIONS: Cross-linking solutions delivered via sub-Tenon injection provide a potential method for limiting axial length growth in progressive myopia and could be used as a potential treatment for myopia.

Outcomes of four-point suture fixated and two-point sutureless posterior chamber IOLs combined with pars plana vitrectomy.

BACKGROUND: While each scleral fixation method has its own advantages, there is a lack of strong evidence to suggest a superior technique. Advances in cataract surgery expand patient eligibility for successful cataract extraction, benefitting a growing population of pseudophakic patients. However, implantation of secondary intraocular lens (IOL) with compromised anterior or posterior capsule is a more challenging task. Each method of scleral fixation has its own advantages and none of them has strong evidence to be superior. This paper describes postsurgical outcomes of two scleral intraocular(IOL) fixation techniques combined with pars plana vitrectomy(PPV) from a single tertiary referral eye center. METHODS: Patients underwent PPV and IOL implantation with either four-point sutured scleral fixation (Akreos AO60(AK); n = 24) or two-point sutureless flanged intrascleral fixation (CT Lucia(CTL); n = 7). Reports include IOL and sclerotomy placement, fixation techniques, and IOL model. RESULTS: Thirty-one eyes of thirty patients were analyzed. Average change in vision from baseline measurement was LogMAR - 0.68 ± 0.66 and - 0.90 ± 0.63 for AK and CTL groups, respectively. Average postoperative refractive error was - 0.3 ± 1.03 D (AK) and 0.4 ± 0.60 D (CTL). No opacification cases of Akreos lens were found in this study with the longest follow up of 53 months. CONCLUSIONS: Both methods of implantation (sutured and sutureless) could provide good visual and refractive outcomes. Minimal complication rates were reported despite including patients with multiple comorbidities, making both techniques an attractive choice for secondary IOL implantation.

Diagnosing Common Deadly Infections in the Era of COVID-19: A Case Report.

The COVID-19 pandemic has challenged clinicians to recognize COVID-19 as one of the diagnostic explanation for common presentations, including fever, cough, and shortness of breath. Latent tuberculosis is responsible for 80% of active tuberculosis cases in the United States, and presentation can vary from asymptomatic to disseminated disease. This potential diagnosis should be thoroughly investigated in foreign-born patients in US hospitals, regardless of travel history and presenting symptoms. We report a patient diagnosed with postpartum disseminated tuberculosis with hematogenous spread to the fetus.

Topical Corneal Cross-Linking Solution Delivered Via Corneal Reservoir in Dutch-Belted Rabbits.

Purpose: A topical corneal cross-linking solution that can be used as an adjunct or replacement to standard photochemical cross-linking (UV-riboflavin) methods remain an attractive possibility. Optimal concentration and delivery method for such topical corneal stabilization in the living rabbit eye were developed. Methods: A series of experiments were carried out using Dutch-belted rabbits (3 months old, weighing 1.0-1.5 kg) and topical cross-linking solutions (sodium hydroxymethylglycinate) (10-250 mM) delivered via corneal reservoir. The application regimen included a one-time 30-minute application (10-40 mM sodium hydroxymethylglycinate) as well as a once per week 5-minute application (250 mM sodium hydroxymethylglycinate) for 7 weeks. Animals were evaluated serially for changes in IOP, pachymetry, epithelial integrity, and endothelial cell counts. Keratocyte changes were identified using intravital laser scanning confocal microscopy. Post mortem efficacy was evaluated by mechanical inflation testing. Results: Overall, there were very few differences observed in right eye treated versus left eye controls with respect to intraocular pressure, pachymetry, and endothelial cell counts, although 30-minute cross-linking techniques did cause transient increases in thickness resolving within 7 days. Epithelial damage was noted in all of the 30-minute applications and fully resolved within 72 hours. Keratocyte changes were significant, showing a wound healing pattern similar to that after riboflavin UVA photochemical cross-linking in rabbits and humans. Surprisingly, post mortem inflation testing showed that the lower concentration of 20 mM delivered over 30 minutes showed the most profound stiffening/strengthening effect. Conclusions: Topical cross-linking conditions that are safe and can increase corneal stiffness/strength in the living rabbit eye have been identified. Translational Relevance: A topical corneal cross-linking solution delivered via corneal reservoir is shown to be both safe and effective at increasing tissue strength in living rabbit eyes and could now be tested in patients suffering from keratoconus and other conditions marked by corneal tissue weakness.

Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis.

BACKGROUND: Corneal infections with antibiotic-resistant microorganisms are an increasingly difficult management challenge and chemically or photochemically cross-linking the cornea for therapy presents a unique approach to managing such infections since both direct microbial pathogens killing and matrix stabilization can occur simultaneously. The present study was undertaken in order to compare the anti-microbial efficacy, in vitro, of 5 candidate cross-linking solutions against 5 different microbial pathogens with relevance to infectious keratitis. METHODS: In vitro bactericidal efficacy studies were carried out using 5 different FARs [diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), 2-nitro-1-propanol (NP)] against 5 different microbial pathogens including two antibiotic-resistant species [methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa (PA), and Candida albicans (CA)]. Standard in vitro antimicrobial testing methods were used. RESULTS: The results for MSSA were similar to those for MRSA. DAU, DMDM, and SMG all showed effectiveness with greater effects generally observed with longer incubation times and higher concentrations. Against MRSA, 40 mM SMG at 120 min showed a > 95% kill rate, p < 0.02. Against VRE, 40 mM DAU for 120 min showed a > 94% kill rate, p < 0.001. All FARs showed bactericidal effect against Pseudomonas aeruginosa, making PA the most susceptible of the strains tested. Candida showed relative resistance to these compounds, requiring high concentrations (100 mM) to achieve kill rates greater than 50%. CONCLUSION: Our results show that each FAR compound has different effects against different cultures. Our antimicrobial armamentarium could potentially be broadened by DAU, DMDM, SMG and other FARs for antibiotic-resistant keratitis. Further testing in live animal models are indicated.

Development of a topical tissue cross-linking solution using sodium hydroxymethylglycinate (SMG): viscosity effect.

Hyperviscosity agents are commonly used in ophthalmic formulations for improving corneal drug penetration by increasing tissue contact time. One such viscosity agent is hydroxypropyl methylcellulose (HPMC). HPMC has been used in riboflavin solutions for photochemical UVA cross-linking (CXL). Sodium hydroxymethylglycinate (SMG) is a small molecule formaldehyde releaser that can function as a therapeutic tissue cross-linker for corneal and scleral applications. The present study was undertaken in order to study formulation factors using HPMC and SMG that could positively influence the cross-linking effect in these ocular tissues. Formulations containing 10 mM SMG and 100 mM sodium bicarbonate were prepared with varying HPMC concentrations from 0 to 4.4%. Their cross-linking effects on porcine and rabbit eyes were measured using differential scanning calorimetry (DSC), expressed as the change/difference in melting temperature (ΔTm) compared with the control. SMG in 4.4% HPMC solution resulted in ΔTm of 6.3 ± 1.21, while other concentration showed no differences in Tm shift on porcine cornea. In ex vivo rabbit cornea, there was a trend toward an increasing cross-linking effect with higher viscosity albeit mild differences. While a significant Tm shift was observed in porcine and rabbit sclera, there was no difference in effect of cross-linking between four HPMC concentrations. Increasing the HPMC concentration does not negatively affect the cross-linking efficacy attributed by SMG and could still be a positive cross-linking enhancer by virtue of increasing tissue contact time in a dynamic biological system. This information will be useful for planning further animal and human studies.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon's space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Antimicrobial Studies Using the Therapeutic Tissue Cross-Linking Agent, Sodium Hydroxymethylglycinate: Implication for Treating Infectious Keratitis.

Purpose: Our recent studies raise the possibility of using sodium hydroxymethylglycinate (SMG), for pharmacologic therapeutic tissue cross-linking (TXL) of the cornea. The present study was performed to evaluate the antimicrobial effects of SMG for potential use in treating infectious keratitis. Methods: In initial (group 1) experiments, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (PA) were treated with SMG (10-40 mM) for 10 to 120 minutes. In group 2 experiments, MRSA, PA, Candida albicans (CA), and vancomycin-resistant Enterococcus (VRE) were treated with SMG (20-200 mM) for 30 minutes. In group 2 experiments, BSA and neutralizing buffer were added to provide a proteinaceous medium, and to ensure precise control of SMG exposure times, respectively. SMG effectiveness was quantitated based on pathogen growth following a 24- to 48-hour incubation period. Results: In group 1 experiments, as expected, time- and concentration-dependent bactericidal effects were noted using MSSA. In addition, the effect of SMG (40 mM) was greatest against MSSA (99.3%), MRSA (96.0%), and PA (97.4%) following a 2-hour exposure with lesser effects following 30- and 10-minute exposures. In group 2 experiments, concentration-dependent bactericidal effects were confirmed for MRSA (91%), PA (99%), and VRE (55%) for 200-mM SMG with 30-minute treatment. SMG was not as effective against CA, with a maximum kill rate of 37% at 80 mM SMG. Conclusions: SMG solution exhibits a dose-dependent bactericidal effect on MSSA, MRSA, and PA, with milder effects on VRE and CA. These studies raise the possibility of using SMG TXL for the treatment of infectious keratitis.

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera.

Purpose: Second harmonic generation signals (SHG) are emitted preferentially from collagenous tissue structures and have been used to evaluate photochemically-induced (CXL) crosslinking changes in the cornea. Since therapeutic tissue crosslinking (TXL) using sodium hydroxymethylglycinate (SMG) of the sclera is a potential treatment for high myopia, we explored the use of SHG microscopy to evaluate the effects. Methods: Single sub-Tenon's (sT) injections (400 µL) using SMG (40-400 mM) were made at the equatorial 12 o'clock position of the right eye of cadaveric rabbit heads (n = 16 pairs). After 3.5 hours, confocal microscopy (CM) was performed using 860 nm two-photon excitation and 400 to 450 nm emission. Pixel density and fiber bundle "waviness" analyses were performed on the images. Crosslinking effects were confirmed using thermal denaturation (Tm) temperature. Comparison experiments with riboflavin photochemical crosslinking were done. Results: Therapeutic tissue crosslinking localization studies indicated that crosslinking changes occurred at the site of injection and in adjacent sectors. Second harmonic generation signals revealed large fibrous collagenous bundled structures that displayed various degrees of waviness. Histogram analysis showed a nearly 6-fold signal increase in 400 mM SMG over 40 mM. This corresponded to a ΔTm = 13°C for 400 mM versus ΔTm = 4°C for 40 mM. Waviness analysis indicated increased fiber straightening as a result of SMG CXL. Conclusions: Second harmonic generation signal intensity and fiber bundle waviness is altered by scleral tissue crosslinking using SMG. These changes provide insights into the macromolecular changes that are induced by therapeutic crosslinking technology and may provide a method to evaluate connective tissue protein changes induced by scleral crosslinking therapies.

Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy.

PURPOSE: To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea. METHODS: Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm. RESULTS: The ΔTm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 ± 0.9°C, 1.3 ± 0.5°C, and 1.1 ± 0.5°C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 ± 1.7% (control) and 8.9 ± 11.1% (CXL) (P = 0.390); 1 ± 0.2% (control) and 19.5 ± 32.2% (1/3 max SMG) (P = 0.426); and 2.7 ± 2.4% (control) and 2.8 ± 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows: 2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max. CONCLUSIONS: Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.