Search

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Orton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polyethylene glycol (PEG)-based conformal coating (CC) encapsulation of transplanted islets is a promising β cell replacement therapy for the treatment of type 1 diabetes without chronic immunosuppression because it minimizes capsule thickness, graft volume, and insulin secretion delay. However, we show here that our original CC method, the direct method, requiring exposure of islets to low pH levels and inclusion of viscosity enhancers during coating, severely affected the viability, scalability, and biocompatibility of CC islets in nonhuman primate preclinical models of type 1 diabetes. We therefore developed and validated in vitro and in vivo, in several small- and large-animal models of type 1 diabetes, an augmented CC method-emulsion method-that achieves hydrogel CCs around islets at physiological pH for improved cytocompatibility, with PEG hydrogels for increased biocompatibility and with fivefold increase in encapsulation throughput for enhanced scalability.

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., Competing Interests: AAT and AAS are inventors of the IP used in the studies and licensed to Sernova. AAT and AAS have received payment from Sernova related to the IP and stand to gain royalties from commercialization of the IP. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Toni, Stock, Devaux, Gonzalez, Nunez, Rubanich, Safley, Weber, Ziebarth, Buchwald and Tomei.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

Purpose: The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics., Methods: The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XII-deficient mice (Col12a1-/-). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy., Results: Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1-/- corneas. The difference between these two groups was significant (P < 0.001, t-test)., Conclusions: Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.

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 (τ 1/2 ), 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, τ 1/2 =17.26±4.840 s, D=0.17±0.05 μm 2 /s), serum albumin (MW=66.5 kDa, τ 1/2 =18.45±6.110 s, D=0.17±0.06 μm 2 /s), or ceruloplasmin (MW=120 kDa, τ 1/2 =36.57±5.660 s, D=0.08±0.01 μm 2 /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 (P albumin : 0.09-0.71, P ceruloplasmin : 0.42-0.95, P transferrin : 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., (Copyright © 2019 Molecular Vision.)

Islet transplantation within mechanically stable microcapsules offers the promise of long-term diabetes reversal without chronic immunosuppression. Reinforcing the ionically gelled network of alginate (ALG) hydrogels with covalently linked polyethylene glycol (PEG) may create hybrid structures with desirable mechanical properties. This report describes the fabrication of hybrid PEG-ALG interpenetrating polymer networks and the investigation of microcapsule swelling, surface modulus, rheology, compression, and permeability. It is demonstrated that hybrid networks are more resistant to bulk swelling and compressive deformation and display improved shape recovery and long-term resilience. Interestingly, it is shown that PEG-ALG networks behave like ALG during microscale surface deformation and small amplitude shear while exhibiting similar permeability properties. The results from this report's in vitro characterization are interpreted according to viscoelastic polymer theory and provide new insight into hybrid hydrogel mechanical behavior. This new understanding of PEG-ALG mechanical performance is then linked to previous work that demonstrated the success of hybrid polymer immunoisolation devices in vivo., Competing Interests: Conflict of Interest A.A.T. is a co-inventor of intellectual property used in the study and may gain royalties from future commercialization of the technology licensed to Converge Biotech Inc. A.A.T. and V.M. are stock option holders in Converge Biotech, licensee of some of the intellectual property used in this study.

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.

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 (O 2 ) 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/cm 2 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/cm 2 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 O 2 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 O 2 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 O 2 . Conventional CXL increases corneal stiffness more than HI CXL both with and without O 2 enrichment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: This study presents a method to quantify micro-stiffness variations in experimental arteriovenous fistulae (AVF)., Methods: AVF created by anastomosing the superficial epigastric vein to the femoral artery in Sprague-Dawley rats were allowed to remodel for 21 days before being harvested and preserved in culture medium. A custom atomic force microscope was used to measure microvascular stiffness (Young's modulus) in three areas of the AVF: the inflow artery, the juxta-anastomotic area, and the outflow vein. Morphometric measurements and collagen and elastin contents were also determined., Results: Atomic force microscopy indentation revealed an increased stiffness in the juxta-anastomotic area of the AVF compared to the outflow vein and inflow artery. The juxta-anastomotic area was also significantly stiffer than the contralateral vein. The lack of elasticity (higher Young's modulus) of the juxta-anastomotic region was associated with a thicker vascular wall that was rich in collagen but poor in elastin., Conclusions: This study demonstrates for the first time the feasibility of using atomic force microscopy to measure local stiffness variations in experimental AVF. This technique could be instrumental in advancing our understanding of how micro-spatial organization of the AVF wall determines the overall biomechanical performance of this type of vascular access.

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

Purpose: To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM)., Methods: Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-μm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-μm/s and 15-μm/s indentation rates. Young's modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy., Results: The force curves were obtained at stromal depths ranging from 59 to 218 μm. At the tissue level, Young's modulus (ES) showed a steep decrease at approximately 140-μm stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Young's modulus (EC) was significantly greater than at the tissue level; EC decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma., Conclusions: The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials.

The objective of this study is to further investigate the ultrastructural details of the surface of Bowman's membrane of the human cornea, using atomic force microscopy (AFM) images. One representative image acquired of Bowman's membrane of a human cornea was investigated. The three-dimensional (3-D) surface of the sample was imaged using AFM in contact mode, while the sample was completely submerged in optisol solution. Height and deflection images were acquired at multiple scan lengths using the MFP-3D AFM system software (Asylum Research, Santa Barbara, CA), based in IGOR Pro (WaveMetrics, Lake Oswego, OR). A novel approach, based on computational algorithms for fractal analysis of surfaces applied for AFM data, was utilized to analyze the surface structure. The surfaces revealed a fractal structure at the nanometer scale. The fractal dimension, D, provided quantitative values that characterize the scale properties of surface geometry. Detailed characterization of the surface topography was obtained using statistical parameters, in accordance with ISO 25178-2: 2012. Results obtained by fractal analysis confirm the relationship between the value of the fractal dimension and the statistical surface roughness parameters. The surface structure of Bowman's membrane of the human cornea is complex. The analyzed AFM images confirm a fractal nature of the surface, which is not taken into account by classical surface statistical parameters. Surface fractal dimension could be useful in ophthalmology to quantify corneal architectural changes associated with different disease states to further our understanding of disease evolution.

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.

Vascular disease is one of the leading causes of death worldwide. Vascular repair, essential for tissue maintenance, is critically reduced during vascular disease and aging. Efficient vascular repair requires functional adult stem cells unimpaired by aging or mutation. One protein candidate for reducing stem cell?mediated vascular repair is progerin, an alternative splice variant of lamin A. Progerin results from erroneous activation of cryptic splice sites within the LMNA gene, and significantly increases during aging. Mutations triggering progerin overexpression cause the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS), in which patients die at approximately 13-years of age due to atherosclerosis-induced disease. Progerin expression affects tissues rich in cells that can be derived from marrow stromal cells (MSCs. Studies using various MSC subpopulations and models have led to discrepant results. Using a well-defined, immature subpopulation of MSCs, Marrow Isolated Adult Multilineage Inducible (MIAMI) cells, we find progerin significantly disrupts expression and localization of self-renewal markers, proliferation, migration, and membrane elasticity. One potential treatment, farnesyltransferase inhibitor, ameliorates some of these effects. Our results confirm proposed progerin-induced mechanisms and suggest novel ways in which progerin disturbs critical stem cell functions collectively required for proper tissue repair, offering promising treatment targets for future therapies.

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., (Copyright 2014, SLACK Incorporated.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: To quantify the cut quality of lamellar dissections made with the femtosecond laser using atomic force microscopy (AFM)., Setting: Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA., Design: Experimental study., Methods: Experiments were performed on 3 pairs of human cadaver eyes. The cornea was thinned to physiologic levels by placing the globe, cornea side down, in 25% dextran for 24 hours. The eyes were reinflated to normal pressures by injecting a balanced salt solution into the vitreous cavity. The eyes were placed in a holder, the epithelium was removed, and the eyes were cut with a Visumax femtosecond laser. The energy level was 180 nJ for the right eye and 340 nJ for the left eye of each pair. The cut depths were 200 μm, 300 μm, and 400 μm, with the cut depth maintained for both eyes of each pair. A 12.0 mm trephination was then performed. The anterior portion of the lamellar surface was placed in a balanced salt solution and imaged with AFM. As a control, the posterior surface was placed in 2% formalin and imaged with environmental scanning electron microscopy (SEM). Four quantitative parameters (root-mean-square deviation, average deviation, skewness, kurtosis) were calculated from the AFM images., Results: From AFM, the 300 μm low-energy cuts were the smoothest. Similar results were seen qualitatively in the environmental SEM images., Conclusion: Atomic force microscopy provided quantitative information on the quality of lamellar dissections made using a femtosecond laser, which is useful in optimizing patient outcomes in refractive and lamellar keratoplasty surgeries., (Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

The purpose of this project is to measure the elasticity of the human and non-human primate lens capsule at the microscopic scale using Atomic Force Microscopy (AFM). Elasticity measurements were performed using AFM on the excised anterior lens capsule from 9 cynomolgus monkey (5.9-8.0 years), 8 hamadryas baboon (2.8-10.1 years), and 18 human lenses (33-79 years). Anterior capsule specimens were obtained by performing a 5 mm continuous curvilinear capsulorhexis and collecting the resulting disk of capsular tissue. To remove the lens epithelial cells the specimen was soaked in 0.1% trypsin and 0.02% EDTA for 5 min, washed, and placed on a Petri dish and immersed in DMEM. Elasticity measurements of the capsule were performed with a laboratory-built AFM system custom designed for force measurements of ophthalmic tissues. The capsular specimens were probed with an AFM cantilever tip to produce force-indentation curves for each specimen. Young's modulus was calculated from the force-indentation curves using the model of Sneddon for a conical indenter. Young's modulus of elasticity was 20.1-131 kPa for the human lens capsule, 9.19-117 kPa for the cynomolgus lens capsule, and 13.1-62.4 kPa for the baboon lens capsule. Young's modulus increased significantly with age in humans (p = 0.03). The age range of the monkey and baboon samples was not sufficient to justify an analysis of age dependence. The capsule elasticity of young humans (<45 years) was not statistically different from that of the monkey and baboon. In humans, there is an increase in lens capsule stiffness at the microscale that could be responsible for an increase in lens capsule bulk stiffness., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose. To characterize the age dependence of shape, refractive power, and refractive index of isolated lenses from nonhuman primates. Methods. Measurements were performed on ex vivo lenses from cynomolgus monkeys (cyno: n = 120; age, 2.7-14.3 years), rhesus monkeys (n = 61; age, 0.7-13.3 years), and hamadryas baboons (baboon: n = 16; age, 1.7-27.3 years). Lens thickness, diameter, and surface curvatures were measured with an optical comparator. Lens refractive power was measured with a custom optical system based on the Scheiner principle. The refractive contributions of the gradient, the surfaces, and the equivalent refractive index were calculated with optical ray-tracing software. The age dependence of the optical and biometric parameters was assessed. Results. Over the measured age range isolated lens thickness decreased (baboon: -0.04, cyno: -0.05, and rhesus: -0.06 mm/y) and equatorial diameter increased (logarithmically for the baboon and rhesus, and linearly for cyno: 0.07 mm/y). The isolated lens surfaces flattened and the corresponding refractive power from the surfaces decreased with age (-0.33, -0.48, and -0.68 D/y). The isolated lens equivalent refractive index decreased (only significant for the baboon, -0.001 D/y), and as a result the total isolated lens refractive power decreased with age (baboon: -1.26, cyno: -0.97, and rhesus: -1.76 D/y). Conclusions. The age-dependent trends in the optical and biometric properties, growth, and aging, of nonhuman primate lenses are similar to those of the pre-presbyopic human lens. As the lens ages, the decrease in refractive contributions from the gradient refractive index causes a rapid age-dependent decrease in maximally accommodated lens refractive power.

Purpose: To determine whether changes in elastic properties of the lens capsule ex vivo with age contribute to the forces necessary for accommodation., Methods: Postmortem human (n = 22; age average: 41 +/- 17 years; range: 6-7) and cynomolgus monkey (n = 19; age average: 7.7 +/- 1.8 years, range: 4.2-10) tissues including the lens, capsule, zonules, ciliary body, and sclera were mounted in an optomechanical lens-stretching system. Starting at 0 load, the sclera was symmetrically stretched to 2 mm in 0.25-mm steps at a speed of 0.1 mm x s(-1). The load and lens diameter were measured at each step. The lens contents were removed through a mini-capsulorhexis. The stretching cycles were repeated on the empty capsular bag. The forces necessary to stretch the natural lens and empty bag were quantified as a function of age and compared., Results: The force needed to stretch the empty lens capsule was independent of age (human, 2.6-34.9 g/mm [25.2-342.7 mN/mm]; monkey, 8.2-21.3 g/mm [80.3-208.6 mN/mm]). The ratio of the force necessary to stretch the empty lens capsule to the force necessary to stretch the natural lens decreased with age in the human and monkey lenses (P = 0.003, P = 0.72, respectively)., Conclusions: The mechanical properties of the empty lens capsule assessed ex vivo in a lens stretcher remain constant with age, suggesting that the changes in elasticity of the lens capsule do not play a significant role in presbyopia. In young eyes, the lens capsule determines the force necessary to stretch the whole lens. The age-related increase in force needed to stretch the lens is due to changes in the lens contents.

Purpose: To demonstrate the feasibility of measuring the elasticity of intact crystalline lenses using atomic force microscopy (AFM)., Methods: AFM elasticity measurements were performed on intact lenses from 18 fresh cynomolgus monkey cadaver eyes (4-10 years old, <1 day postmortem) that had been left attached to their zonule-ciliary body-sclera framework. The eyes were prepared by bonding a plastic ring on the sclera after removal of the conjunctival, adipose, and muscle tissues. The posterior pole was sectioned, with the excess vitreous removed, and the eye's anterior section was placed on a Teflon slide to protect the posterior pole of the lens. The cornea and iris were then sectioned. The lens-zonule-ciliary body-sclera section was then placed in a Petri dish filled with balanced salt solution in an AFM system designed for force measurements. Next, the central pole of the anterior surface of the intact lens was probed with the AFM cantilever tip. The recorded AFM cantilever deflection-indentation curves were used to derive force-indentation curves for the lens after factoring out the deflection of the cantilever on a hard surface. Young's modulus of the lens was calculated from the force-indentation relation using the Hertz model., Results: Young's modulus was 1,720+/-880 Pa (range: 409-3,210 Pa) in the 18 cynomolgus monkey lenses., Conclusions: AFM can be used to provide measurements of the elasticity of the whole lens including the capsule. Values obtained using AFM on cynomolgus monkey lenses are similar to published values obtained using dynamic mechanical analysis on young human lenses.

Purpose: The purpose of this study was to assess the condition of human lenses (obtained from an eye bank) and of fresh monkey lenses, and to determine the effects of maintaining these lenses in various liquid preservation media., Methods: Freshly excised human and monkey lenses were maintained for 5 h in one of four solutions (Balanced Saline Solution [BSS], Ringer's Solution, Dulbecco's Modified Eagle Medium with Ham's F-12 [DMEM/F-12/F-12], and Tissue Culture Medium 199 [TC-199]) using a custom-designed, temperature-regulated testing cell. A modified optical comparator and digital camera were used to photograph magnified lens profiles and measure lens diameter and thickness. Lens volume was then calculated assuming rotational symmetry about the optical axis., Results: Seven of the 33 human lenses exhibited extensive swelling and separation of the capsule from the lens cell mass prior to the incubation. During incubation, for 12/22 of the remaining human and 27/27 of the monkey lenses, thickness increased by 1.0-1.8%, diameter decreased by 0.7-1.6% and the volume was essentially unchanged. Substantial swelling and capsular separation were observed in 10 of the 22 human lenses, 7/10 for those maintained in salt solutions, and 3/12 for those in tissue culture media. Lens volumes increased by an average of 6.8%, due to an 8.7% increase in the thickness, while the diameter decreased by 0.9%. These changes appeared to be independent of postmortem time and donor age., Conclusions: Culture media are more effective than simple salt solutions in maintaining lens physical integrity during short-term incubations. Substantial uptake of water, accompanied by separation of the capsule from the lens cell mass, occurs at various stages during storage and experimental manipulations in >60% of human lenses obtained from the eye bank. Data obtained with such lenses will not be representative of the true ex vivo state. It is recommended that lenses be assessed to determine if swelling has taken place before acceptance of data.

Purpose: To measure interspecies thickness differences in the central anterior and posterior capsules of postmortem crystalline lenses, by a technique that maintains the anatomic integrity of the lens., Methods: Central capsule thickness was measured with a custom-built, noncontact optical system, using a focus detection technique. Anterior and posterior lens capsule thickness measurements were performed on 22 human, 29 monkey, and 34 New Zealand White rabbit intact postmortem lenses in situ. Eyes were prepared for optical measurements by bonding a PMMA ring to the sclera in the region of the ciliary body after the conjunctiva, adipose, and muscle tissues were removed. The posterior pole was removed by making a circumferential incision through the sclera approximately 7 mm posterior to the limbus. Excess vitreous was removed to expose the posterior capsule surface, and the eye assembly was placed on a Teflon slide. The cornea and iris were sectioned to expose the anterior capsule surface. After the experiments, the lenses were excised, placed in 10% buffered formalin, and prepared for histology. Lens capsule thickness was measured from the histologic slides and compared to the optical, Results: results. Central anterior lens capsule thickness was 8.2 +/- 5.5 (human), 7.5 +/- 4.4 (monkey), and 10.7 +/- 4.2 (rabbit) microm optically and 12.4 +/- 2.5 (human), 10.7 +/- 3.7 (monkey), and 10.4 +/- 2.0 (rabbit) microm histologically. Central posterior capsule thickness was 6.3 +/- 2.2 (human), 5.9 +/- 1.7 (monkey), and 7.8 +/- 2.3 (rabbit) microm optically and 4.1 +/- 1.5 (human), 3.5 +/- 1.6 (monkey), and 4.7 +/- 2.5 (rabbit) microm histologically., Conclusions: The central anterior and posterior lens capsule thicknesses do not appear to vary considerably among human, rabbit, and monkey eyes. There were significant differences between optical in situ measurements and histology, which indicates that histologic preparation may affect lens capsule thickness.