Effect of compound treatments on mouse lens viscoelasticity.

Previous studies have shown that pharmaceutical agents such as lipoic acid have the ability to soften the lens, presenting a promising avenue for treating presbyopia. One obstacle encountered in the preclinical stage of such agents is the need for precise measurements of lens elasticity in experimental models. This study aimed to evaluate the effects of 25-hydroxycholesterol, lipoic acid, and obeticholic acid on the viscoelastic properties of mouse lenses using a custom-built elastometer system. Data were acquired on lenses from C57BL/6J female mice from two age groups: young (age: 8-10 weeks) and old (age: 32-43 weeks). OD lenses were used as the control and OS lenses were treated. Control lenses were immersed in Dulbecco's Modified Eagle Medium (DMEM) and treatment lenses were immersed in a compound solution containing 25-hydroxycholesterol (5 young and 5 old), lipoic acid at 2.35 mM (5 young and 5 old), lipoic acid at 0.66 mM (5 old), or obeticholic acid (5 old) at 37 °C for 18 h. After treatment, the mouse lenses were placed in a DMEM-filled chamber within a custom-built elastometer system that recorded the load and lens shape as the lens was compressed by 600 µm at a speed of 50 µm/s. The load was continuously recorded during compression and during stress-relaxation. The compression phase was fit with a linear function to quantify lens stiffness. The stress-relaxation phase was fit with a 3-term exponential relaxation model providing relaxation time constants (t1, t2, t3), and equilibrium load. The lens stiffness, time constants and equilibrium load were compared for the control and treated groups. Results revealed an increase in stiffness with age for the control group (young: 1.16 ± 0.11 g/mm, old: 1.29 ± 0.14 g/mm) and relaxation time constants decreased with age (young: t1 = 221.9 ± 29.0 s, t2 = 24.7 ± 3.8 s, t3 = 3.12 ± 0.87 s, old: t1 = 183.0 ± 22.0 s, t2 = 20.6 ± 2.6 s and t3 = 2.24 ± 0.43 s). Among the compounds tested, only 25-hydroxycholesterol produced statistically significant changes in the lens stiffness, relaxation time constants, and equilibrium load. In conclusion, older mouse lenses are stiffer and less viscous than young mouse lenses. Notably, no significant change in lens stiffness was observed following treatment with lipoic acid, contrary to previous findings.

Glaucomatous aqueous humor vesicles are smaller and differ in composition compared to controls.

Cells communicate with each other using vesicles of varying sizes, including a specific repertoire known as exosomes. We isolated aqueous humor (AH)-derived vesicles using two different methods: ultracentrifugation and an exosome isolation kit. We confirmed a unique vesicle size distribution in the AH derived from control and primary open-angle glaucoma (POAG) patients using various techniques, including Nanotracker, dynamic light scattering, atomic force imaging, and electron microscopy. Bonafide vesicle and/or exosome markers were present by dot blot in both control and POAG AH-derived vesicles. Marker levels differed between POAG and control samples, while non-vesicle negative markers were absent in both. Quantitative labeled (iTRAQ) proteomics showed a reduced presence of a specific protein, STT3B, in POAG compared to controls, which was further confirmed using dot blot, Western blot, and ELISA assays. Along the lines of previous findings with AH profiles, we found vast differences in the total phospholipid composition of AH vesicles in POAG compared to controls. Electron microscopy further showed that the addition of mixed phospholipids alters the average size of vesicles in POAG. We found that the cumulative particle size of type I collagen decreased in the presence of Cathepsin D, which normal AH vesicles were able to protect against, but POAG AH vesicles did not. AH alone had no effect on collagen particles. We observed a protective effect on collagen particles with an increase in artificial vesicle sizes, consistent with the protective effects observed with larger control AH vesicles but not with the smaller-sized POAG AH vesicles. Our experiments suggest that AH vesicles in the control group provide greater protection for collagen beams compared to POAG, and their increased vesicle sizes are likely contributing factors to this protection.

Collagen XIV Is an Intrinsic Regulator of Corneal Stromal Structure and Function.

Collagen XIV is poorly characterized in the body, and the current knowledge of its function in the cornea is limited. The aim of the current study was to elucidate the role(s) of collagen XIV in regulating corneal stromal structure and function. Analysis of collagen XIV expression, temporal and spatial, was performed at different postnatal days (Ps) in wild-type C57BL/6 mouse corneal stromas and after injury. Conventional collagen XIV null mice were used to inquire the roles that collagen XIV plays in fibrillogenesis, fibril packing, and tissue mechanics. Fibril assembly and packing as well as stromal organization were evaluated using transmission electron microscopy and second harmonic generation microscopy. Atomic force microscopy was used to assess stromal stiffness. Col14a1 mRNA expression was present at P4 to P10 and decreased at P30. No immunoreactivity was noted at P150. Abnormal collagen fibril assembly with a shift toward larger-diameter fibrils and increased interfibrillar spacing in the absence of collagen XIV was found. Second harmonic generation microscopy showed impaired fibrillogenesis in the collagen XIV null stroma. Mechanical testing suggested that collagen XIV confers stiffness to stromal tissue. Expression of collagen XIV is up-regulated following injury. This study indicates that collagen XIV plays a regulatory role in corneal development and in the function of the adult cornea. The expression of collagen XIV is recapitulated during wound healing.

Measuring the effects of postmortem time and age on mouse lens elasticity using atomic force microscopy.

The mouse lens is frequently used both in vivo and ex vivo in ophthalmic research to model conditions affecting the human lens, such as presbyopia. The mouse lens has a delicate structure which is prone to damage and biomechanical changes both before and after extraction from the whole globe. When not properly controlled for, these changes can confound the biomechanical analysis of mouse lenses. In this study, atomic force microscopy microindentation was used to assess changes in the Young's Modulus of Elasticity of the mouse lens as a function of mouse age and postmortem time. Old mouse lenses measured immediately postmortem were significantly stiffer than young mouse lenses (p = 0.028). However, after 18 h of incubation, there was no measurable difference in lens stiffness between old and young mouse lenses (p = 0.997). This demonstrates the need for careful experimental control in experiments using the mouse lens, especially regarding postmortem time.

Endogenous ocular lipids as potential modulators of intraocular pressure.

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open-angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function.

Porous Silicon Nanoparticles Embedded in Poly(lactic-co-glycolic acid) Nanofiber Scaffolds Deliver Neurotrophic Payloads to Enhance Neuronal Growth.

Scaffolds made from biocompatible polymers provide physical cues to direct the extension of neurites and to encourage repair of damaged nerves. The inclusion of neurotrophic payloads in these scaffolds can substantially enhance regrowth and repair processes. However, many promising neurotrophic candidates are excluded from this approach due to incompatibilities with the polymer or with the polymer processing conditions. This work provides one solution to this problem by incorporating porous silicon nanoparticles (pSiNPs) that are pre-loaded with the therapeutic into a polymer scaffold during fabrication. The nanoparticle-drug-polymer hybrids are prepared in the form of oriented poly(lactic-co-glycolic acid) nanofiber scaffolds. We test three different therapeutic payloads: bpV(HOpic), a small molecule inhibitor of phosphatase and tensin homolog (PTEN); an RNA aptamer specific to tropomyosin-related kinase receptor type B (TrkB); and the protein nerve growth factor (NGF). Each therapeutic is loaded using a loading chemistry that is optimized to slow the rate of release of these water-soluble payloads. The drug-loaded pSiNP-nanofiber hybrids release approximately half of their TrkB aptamer, bpV(HOpic), or NGF payload in 2, 10, and >40 days, respectively. The nanofiber hybrids increase neurite extension relative to drug-free control nanofibers in a dorsal root ganglion explant assay.

Age-dependency of molecular diffusion in the human anterior lens capsule assessed using fluorescence recovery after photobleaching.

Purpose: To quantify the partition coefficient and the diffusion coefficient of metal-carrier proteins in the human lens capsule as a function of age. Methods: Whole lenses from human donors were incubated overnight in a solution of fluorescently labeled transferrin, albumin, or ceruloplasmin. In the central plane of the capsule thickness, fluorescence recovery after photobleaching (FRAP) experiments were conducted to measure the diffusion of the protein within the lens capsule. The anterior portion of the lens was recorded before the FRAP experiments to locate the boundaries of the anterior lens capsule and to measure the partition coefficient of the labeled proteins. The partition coefficient (P), the time to half maximum recovery of the fluorescent intensity (τ1/2), and the diffusion coefficient (D) for each protein were analyzed as a function of donor age. Results: There was no statistically significant relationship between the half maximum recovery time or the diffusion coefficient and age for transferrin (molecular weight [MW]=79.5 kDa, τ1/2=17.26±4.840 s, D=0.17±0.05 µm2/s), serum albumin (MW=66.5 kDa, τ1/2=18.45±6.110 s, D=0.17±0.06 µm2/s), or ceruloplasmin (MW=120 kDa, τ1/2=36.57±5.660 s, D=0.08±0.01 µm2/s). As expected, the larger protein (ceruloplasmin) took longer to recover fluorescent intensity due to its slower movement within the lens capsule. The partition coefficient statistically significantly increased with age for each protein (Palbumin: 0.09-0.71, Pceruloplasmin: 0.42-0.95, Ptransferrin: 0.19-1.17). Conclusions: The diffusion of heavy-metal protein carriers within the anterior lens capsule is not dependent on age, but it is dependent on the size of the protein. The permeability of the lens capsule to these heavy-metal protein carriers increases with age, suggesting that there will be a higher concentration of heavy metals in the older lens. This behavior may favor the formation of cataract, because heavy metals enhance protein oxidation through the Fenton reaction.

Micromorphology analysis of the anterior human lens capsule.

PURPOSE: This study aimed to quantify the three-dimensional micromorphology of the surface of the human lens capsule as a function of age. METHODS: Imaging experiments were conducted on whole human lenses received from eight human cadavers (donor age range: 30-88 years). Imaging was performed with an atomic force microscope (AFM) in contact mode in fluid. The porosity and surface roughness were quantified from the height images obtained. A novel approach, based on stereometric and fractal analysis of three-dimensional surfaces developed for use in conjunction with AFM data, was also used to analyze the surface microtexture as a function of age. RESULTS: The AFM images obtained depict a highly ordered fibrous structure at the surface of the lens capsule, although the overall structure visually changes with age. Porosity and roughness were quantified for each image and analyzed as a function of donor age. The interfibrillar spacing revealed an increasing trend with age, although this result was not significant (p = 0.110). The root mean square (RMS) deviation and average deviation significantly decreased with increasing age (p<0.001 for both). The fractal analysis provided quantitative values for 29 amplitude, hybrid, functional, and spatial parameters. All the hybrid parameters decreased with age, although not significantly. Of the functional parameters, the surface bearing index increased significantly with age (p = 0.017) and the summit height exhibited a decreasing trend with age (p = 0.298). Of the spatial parameters, the dominant radial wavelength trend moved toward an increase with age (p = 0.103) and the cross-hatch angle tended toward a decrease with age (p = 0.213). CONCLUSIONS: Significant changes in the three-dimensional surface microtexture of the human lens capsule were found with age, although more experiments on a larger dataset are needed to conclude this with certainty. The analyzed AFM images demonstrate a fractal nature of the surface, which is not considered in classical surface statistical parameters. The surface fractal dimension may be useful in ophthalmology for quantifying human lens architectural changes associated with different disease states to further our understanding of disease evolution.

Corneal elasticity after oxygen enriched high intensity corneal cross linking assessed using atomic force microscopy.

The purpose of this study was to assess anterior and mid corneal stromal elasticity after high intensity (HI) corneal cross linking (CXL), with and without oxygen (O2) enrichment, and compare these results to conventional CXL. Experiments were performed on 25 pairs of human cadaver eyes, divided into four different groups. Group 1 included corneas that did not receive treatment and served as controls; Group 2 included corneas that received conventional CXL treatment (Dresden Protocol: corneal epithelial debridement, 30 min of riboflavin pretreatment followed by 30 min of exposure to 3 mW/cm2 of ultraviolet light); Group 3 included corneas that received HI CXL treatment (corneal epithelial debridement, 30 min of riboflavin pretreatment followed by 3 min of exposure to 30mW/cm2 of ultraviolet light); and Group 4 included corneas that received the same treatment as Group 3, except that they were enriched with oxygen (4 L per minute pure O2 gas stream) during ultraviolet irradiation. In each group, corneas were subdivided to assess anterior stromal elasticity and mid stromal elasticity. Corneal stromal elasticity was quantified using Atomic Force Microscopy (AFM) through micro-indentation. Young's modulus for the anterior corneal stroma was 14.5 ± 6.0 kPa, 80.7 ± 44.6 kPa, 36.6 ± 10.5 kPa, and 30.6 ± 9.2 kPa, for groups 1, 2, 3 and 4 respectively. Young's modulus for the mid corneal stroma was 5.8 ± 2.0 kPa, 20.7 ± 4.3 kPa, 12.1 ± 4.9 kPa, and 11.7 ± 3.7 kPa, for groups 1, 2, 3 and 4, respectively. In the anterior stromal region, conventional CXL demonstrated a significantly different result from the control, whereas the two HI CXL protocols were not significantly different from the control. There were no statistical differences between the two HI CXL protocols, although only the HI CXL protocol with O2 enrichment was significantly different from the conventional CXL group. In the mid stromal region, once again only conventional CXL demonstrated a significantly different result from the control. There were no statistical differences between the two HI CXL protocols, and both HI CXL protocols were significantly different from the conventional CXL group. Oxygen enriched HI CXL seems to offer similar changes in corneal elasticity when compared to HI CXL without the presence O2. Conventional CXL increases corneal stiffness more than HI CXL both with and without O2 enrichment.

Lens capsule structure assessed with atomic force microscopy.

PURPOSE: To image the ultrastructure of the anterior lens capsule at the nanoscale level using atomic force microscopy (AFM). METHODS: Experiments were performed on anterior lens capsules maintained in their in situ location surrounding the lens from six human cadavers (donor age range: 44-88 years), four cynomolgus monkeys (Macaca fascicularis age range: 4.83-8.92 years), and seven pigs (<6 months). Hydration of all samples was maintained using Dulbecco's Modified Eagle Medium (DMEM). Whole lenses were removed from the eye and placed anterior side up in agarose gel before gel hardening where only the posterior half of the lens was contained within the gel. After the gel hardened, the Petri dish was filled with DMEM until the point where the intact lens was fully submerged. AFM was used to image the anterior lens surface in contact mode. An integrated analysis program was used to calculate the interfibrillar spacing, fiber diameter, and surface roughness of the samples. RESULTS: The AFM images depict a highly ordered fibrous structure at the surface of the lens capsule in all three species. The interfibrillar spacing for the porcine, cynomolgus monkey, and human lens capsules was 0.68±0.25, 1.80±0.39, and 1.08±0.25 µm, respectively. In the primate, interfibrillar spacing significantly decreased linearly as a function of age. The fiber diameters ranged from 50 to 950 nm. Comparison of the root mean square (RMS) and average deviation demonstrate that the surface of the porcine lens capsule is the smoothest, and that the human and cynomolgus monkey capsules are significantly rougher. CONCLUSIONS: AFM was successful in providing high-resolution images of the nanostructure of the lens capsule samples. Species-dependent differences were observed in the overall structure and surface roughness.

Corneal stromal elasticity and viscoelasticity assessed by atomic force microscopy after different cross linking protocols.

The purpose of this study was to evaluate elasticity and viscoelasticity in the anterior and deeper stromal regions of the cornea after cross linking with three different protocols using atomic force microscopy (AFM) through indentation. A total of 40 porcine corneas were used in this study and were divided into 4 groups (10 corneas per group): control (no treatment), Dresden (corneal epithelial debridement, riboflavin pretreatment for 30 min and a 3mw/cm(2) for 30 min UVA irradiation), accelerated (corneal epithelial debridement, riboflavin pretreatment for 30 min and a 30mw/cm(2) for 3 min UVA irradiation), and genipin (corneal epithelial debridement and submersion of anterior surface in a 1% genipin solution for 4 h). Elasticity and viscoelasticity were quantified using AFM through indentation for all corneas, for the anterior stroma and at a depth of 200 µm. For the control, Dresden, accelerated, and genipin groups, respectively, the average Young's modulus for the anterior stromal region was 0.60 ± 0.58 MPa, 1.58 ± 1.04 MPa, 0.86 ± 0.46 MPa, and 1.71 ± 0.51 MPa; the average for the 200 µm stromal depth was 0.08 ± 0.06 MPa, 0.08 ± 0.04 MPa, 0.08 ± 0.04 MPa, and 0.06 ± 0.01 MPa. Corneas crosslinked with the Dresden protocol and genipin were significantly stiffer than controls (p < 0.05) in the anterior region only. For the control, Dresden, Accelerated, and genipin groups, respectively, the average calculated apparent viscosity for the anterior stroma was 88.2 ± 43.7 kPa-s, 8.3 ± 7.1 kPa-s, 8.1 ± 2.3 kPa-s, and 9.5 ± 3.8 kPa-s; the average for the 200 µm stromal depth was 35.0 ± 3.7 kPa-s, 49.6 ± 35.1 kPa-s, 42.4 ± 17.6 kPa-s, and 41.8 ± 37.6 kPa-s. All crosslinking protocols resulted in a decrease in viscosity in the anterior region only (p < 0.05). The effects of cross-linking seem to be limited to the anterior corneal stroma and do not extend to the deeper stromal region. Additionally, the Dresden and genipin protocols seem to produce a stiffer anterior corneal stroma when compared to the accelerated protocol.

Impact of Hydration Media on Ex Vivo Corneal Elasticity Measurements.

OBJECTIVES: To determine the effect of hydration media on ex vivo corneal elasticity. METHODS: Experiments were conducted on 40 porcine eyes retrieved from an abattoir (10 eyes each for phosphate-buffered saline (PBS), balanced salt solution, Optisol, 15% dextran). The epithelium was removed, and the cornea was excised with an intact scleral rim and placed in 20% dextran overnight to restore its physiological thickness. For each hydration media, corneas were evenly divided into two groups: one with an intact scleral rim and the other without. Corneas were mounted onto a custom chamber and immersed in a hydration medium for elasticity testing. Although in each medium, corneal elasticity measurements were performed for 2 hr: at 5-min intervals for the first 30 min and then 15-min intervals for the remaining 90 min. Elasticity testing was performed using nanoindentation with spherical indenters, and Young modulus was calculated using the Hertz model. Thickness measurements were taken before and after elasticity testing. RESULTS: The percentage change in corneal thickness and elasticity was calculated for each hydration media group. Balanced salt solution, PBS, and Optisol showed an increase in thickness and Young moduli for corneas with and without an intact scleral rim. Fifteen percent dextran exhibited a dehydrating effect on corneal thickness and provided stable maintenance of corneal elasticity for both groups. CONCLUSIONS: Hydration media affects the stability of corneal thickness and elasticity measurements over time. Fifteen percent dextran was most effective in maintaining corneal hydration and elasticity, followed by Optisol.

Surface quality of human corneal lenticules after SMILE assessed using environmental scanning electron microscopy.

PURPOSE: To assess the cut quality of the anterior and posterior surfaces of intrastromal refractive lenticules removed during small incision lenticule extraction (SMILE). METHODS: The VisuMax femtosecond laser (500 kHz; Carl Zeiss Meditec, Dublin, CA) was used to perform SMILE on 8 eyes of 5 individuals to correct only myopia (no cylinder). The cut energy index was 26 (equivalent to an energy of 130 nJ) with a 2.5 × 2.5 µm spot/track separation. The lenticule diameter was 6.5 mm with a minimum edge thickness of 15 µm and the cap diameter was 7.3 mm with an intended thickness of 120 µm. After laser treatment, the lenticule was loosened with a spatula and removed with forceps. The extracted lenticules were placed in 2% formalin and sent for imaging with an environmental scanning electron microscope. Images of the anterior and posterior surfaces of the lenticules were obtained at multiple magnifications (100×, 250×, and 500×). Surface quality was evaluated by an investigator who specializes in electron microscopy using three criteria: overall surface regularity, percent of surface irregularity, and position of irregular area. RESULTS: Both the anterior and posterior surfaces of the extracted lenticules were smooth and absent of surface irregularities. The cut edges also appeared uniform. Jagged edges were seen in several images, but were clearly caused by the forceps during extraction. CONCLUSIONS: Using the VisuMax laser to perform SMILE produces smooth cuts absent of surface irregularities.

Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis.

The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

Enhancing Rose Bengal penetration in ex vivo human corneas using iontophoresis.

Aim: Rose Bengal photodynamic antimicrobial therapy (RB-PDAT) has poor corneal penetration, limiting its efficacy against acanthamoeba keratitis (AK). Iontophoresis enhances corneal permeation of charged molecules, piquing interest in its effects on RB in ex vivo human corneas. Methods: Five donor whole globes each underwent iontophoresis with RB, soaking in RB, or were soaked in normal saline (controls). RB penetration and corneal thickness was assessed using confocal microscopy. Results: Iontophoresis increased RB penetration compared with soaking (177 ± 9.5 µm vs. 100 ± 5.7 µm, p < 0.001), with no significant differences in corneal thickness between groups (460 ± 87 µm vs. 407 ± 69 µm, p = 0.432). Conclusion: Iontophoresis significantly improves RB penetration and its use in PDAT could offer a novel therapy for acanthamoeba keratitis. Further studies are needed to validate clinical efficacy.

The study aimed to improve a new treatment for eye infections known as photodynamic antimicrobial therapy. It investigated whether the use of electricity through a technique called iontophoresis could help a chemical called Rose Bengal go deeper into the eye in order to target more severe infections. The iontophoresis machine was custom built, with patient-contacting components 3D printed. The experiments were performed using donated human eye tissue and found that iontophoresis significantly improved the penetration depth of Rose Bengal as compared with the current technique of only soaking the eye in Rose Bengal.

Anterior and posterior corneal stroma elasticity assessed using nanoindentation.

Corneal biomechanics is an essential parameter for developing diagnostic and treatment methods of corneal-related diseases. It is widely accepted that corneal mechanical strength stems from the stroma's collagenous composition. However, more comprehensive insight into the mechanical properties within the stroma is needed to improve current corneal diagnostic and treatment techniques. The purpose of this study was to perform elasticity characterization of anterior and posterior stromal regions of human corneas using atomic force microscopy (AFM). Nine pairs of human whole globes were placed in 20% Dextran solution, cornea side down, to restore the corneal thickness to physiological levels (400-600 µm). The epithelium and Bowman's membrane were removed from all eyes. Anterior stromal AFM elasticity testing was then performed on left (OS) eyes. Additional stroma was removed from right (OD) eyes to allow posterior stromal measurements at a depth of 50% of the original thickness. All experiments were performed with corneas submerged in 15% Dextran to maintain corneal hydration. The results of the study showed that the Young's modulus of elasticity of the anterior stroma (average: 281 ± 214 kPa; range: 59-764 kPa) was significantly higher than that of the posterior stroma (average: 89.5 ± 46.1 kPa; range: 29-179 kPa) (p = 0.014). In addition, a linear relationship was found between the posterior stromal elasticity and anterior stromal elasticity (p = 0.0428). On average, the elasticity of the posterior stroma is 39.3% of the anterior stroma. In summary, there appears to be an elasticity gradient within the corneal stroma, which should be considered in the design and development of corneal diagnostic and treatment methods to enhance efficacy.

Anterior and posterior corneal stroma elasticity after corneal collagen crosslinking treatment.

The purpose of this project was to assess anterior and posterior corneal stromal elasticity after corneal collagen cross linking (CXL) treatment in human cadaver eyes using Atomic Force Microscopy (AFM) through indentation. Twenty four human cadaver eyes (12 pairs) were included in this study and divided into 2 groups (6 pairs per group). In both groups, the left eye (OS) served as a control (no riboflavin or CXL treatment was performed) and the right eye (OD) underwent CXL treatment (30 min of riboflavin pretreatment followed by 30 min of exposure to 3 mW/cm(2) of ultraviolet light). In group 1, the anterior stroma was exposed by manual delamination of approximately 50 µm of the corneal stroma including Bowman's membrane. In group 2, the posterior stroma was exposed by delamination of the anterior 50% of the corneal stroma including Bowman's membrane. Delamination was performed after crosslinking treatment in the case of the treated eyes. In all eyes, the stromal elasticity was quantified using AFM through indentation. Young's modulus of elasticity for the anterior cornea (group 1) was 245.9 ± 209.1 kPa (range: 82.3-530.8 kPa) for the untreated control eyes, and 467.8 ± 373.2 kPa (range: 157.4-1126 kPa) for the CXL treated eyes. Young's modulus for the posterior cornea (group 2) was 100.2 ± 61.9 kPa (range: 28.1-162.6 kPa) for the untreated control eyes and 66.0 ± 31.8 kPa (range: 31.3-101.7 kPa) for the CXL treated eyes. Young's modulus of the anterior stroma significantly increased after CXL treatment (p = 0.024), whereas the posterior stroma did not demonstrate a significant difference in Young's modulus after CXL treatment (p = 0.170). The anterior stroma was stiffer than the posterior stroma for both the control and CXL treatment groups (p = 0.077 and p = 0.023, respectively). Our findings demonstrate that stiffness of the anterior corneal stroma after CXL treatment seems to increase significantly, while the posterior stroma does not seem to be affected by CXL.

Characterization of Trabecular Meshwork Mechanical Property Modulation After Application of Lipids.

Treatment of lipids endogenous to the aqueous humor of the eye could serve as a potential therapy to slow the progression of glaucoma. Herein, we describe the method to treat trabecular meshwork samples in vitro with lipids and characterize changes in the samples' stiffness.

Biomechanical properties of porcine meniscus as determined via AFM: Effect of region, compartment and anisotropy.

The meniscus is a fibrocartilaginous tissue that plays an essential role in load transmission, lubrication, and stabilization of the knee. Loss of meniscus function, through degeneration or trauma, can lead to osteoarthritis in the underlying articular cartilage. To perform its crucial function, the meniscus extracellular matrix has a particular organization, including collagen fiber bundles running circumferentially, allowing the tissue to withstand tensile hoop stresses developed during axial loading. Given its critical role in preserving the health of the knee, better understanding structure-function relations of the biomechanical properties of the meniscus is critical. The main objective of this study was to measure the compressive modulus of porcine meniscus using Atomic Force Microscopy (AFM); the effects of three key factors were investigated: direction (axial, circumferential), compartment (medial, lateral) and region (inner, outer). Porcine menisci were prepared in 8 groups (= 2 directions x 2 compartments x 2 regions) with n = 9 per group. A custom AFM was used to obtain force-indentation curves, which were then curve-fit with the Hertz model to determine the tissue's compressive modulus. The compressive modulus ranged from 0.75 to 4.00 MPa across the 8 groups, with an averaged value of 2.04±0.86MPa. Only direction had a significant effect on meniscus compressive modulus (circumferential > axial, p = 0.024), in agreement with earlier studies demonstrating that mechanical properties in the tissue are anisotropic. This behavior is likely the result of the particular collagen fiber arrangement in the tissue and plays a key role in load transmission capability. This study provides important information on the micromechanical properties of the meniscus, which is crucial for understanding tissue pathophysiology, as well as for developing novel treatments for tissue repair.

Parallel Evaluation of Polyethylene Glycol Conformal Coating and Alginate Microencapsulation as Immunoisolation Strategies for Pancreatic Islet Transplantation.

Pancreatic islet transplantation improves metabolic control and prevents complications in patients with brittle type 1 diabetes (T1D). However, chronic immunosuppression is required to prevent allograft rejection and recurrence of autoimmunity. Islet encapsulation may eliminate the need for immunosuppression. Here, we analyzed in parallel two microencapsulation platforms that provided long-term diabetes reversal in preclinical T1D models, alginate single and double capsules versus polyethylene glycol conformal coating, to identify benefits and weaknesses that could inform the design of future clinical trials with microencapsulated islets. We performed in vitro and in vivo functionality assays with human islets and analyzed the explanted grafts by immunofluorescence. We quantified the size of islets and capsules, measured capsule permeability, and used these data for in silico simulations of islet functionality in COMSOL Multiphysics. We demonstrated that insulin response to glucose stimulation is dependent on capsule size, and the presence of permselective materials augments delays in insulin secretion. Non-coated and conformally coated islets could be transplanted into the fat pad of diabetic mice, resulting in comparable functionality and metabolic control. Mac-2+ cells were found in conformally coated grafts, indicating possible host reactivity. Due to their larger volume, alginate capsules were transplanted in the peritoneal cavity. Despite achieving diabetes reversal, changes in islet composition were found in retrieved capsules, and recipient mice experienced hypoglycemia indicative of hyperinsulinemia induced by glucose retention in large capsules as the in silico model predicted. We concluded that minimal capsule size is critical for physiological insulin secretion, and anti-inflammatory modulation may be beneficial for small conformal capsules.