Altered multiaxial mechanical properties of the porcine anterior lens capsule cultured in high glucose

Hyperglycemia can alter the mechanical properties of tissues through the formation of advanced glycation endproducts in matrix proteins that have long half-lives. We used a custom experimental system and subdomain finite element method to quantify alterations in the regional multiaxial mechanical properties of porcine lens capsules that were cultured for 8 or 14 weeks in high glucose versus control media. Findings revealed that high glucose significantly stiffened the capsules in both the circumferential and the meridional directions, but it did not affect the known regional variations in anisotropy. Such information could be important in the design of both improved clinical procedures and intraocular implants for diabetic patients.     

MALDI-MS-imaging of whole human lens capsule

The ocular lens capsule is a smooth, transparent basement membrane that encapsulates the lens and is composed of a rigid network of interacting structural proteins and glycosaminoglycans. During cataract surgery, the anterior lens capsule is routinely removed in the form of a circular disk. We considered that the excised capsule could be easily prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-MSI) analysis. MALDI-MSI is a powerful tool to elucidate the spatial distribution of small molecules, peptides, and proteins within tissues. Here, we apply this molecular imaging technique to analyze the freshly excised human lens capsule en face. We demonstrate that novel information about the distribution of proteins by MALDI-MSI can be obtained from this highly compact connective tissue, having no evident histo-morphological characteristics. Trypsin digestion carried out on-tissue is shown to improve MALDI-MSI analysis of human lens capsules and affords high repeatability. Most importantly, MALDI-MSI analysis reveals a concentric distribution pattern of proteins such as apolipoprotein E (ApoE) and collagen IV alpha-1 on the anterior surface of surgically removed lens capsule, which may indicate direct or indirect effects of environmental and mechanical stresses on the human ocular lens.     

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.     

Mechanical characterization of an unusual elastic biomaterial from the egg capsules of marine snails (Busycon spp.)

Egg capsule material serves as a putative protection mechanism for developing snail embryos facing the perils of the marine environment. We conducted the first quantitative study of this acellular structural protein with the goals of characterizing its chemical and mechanical properties and the relationship of these properties to its biological protective function. We have found that this protein polymer exhibits long-range elasticity with an interesting recoverable yield evidenced by an order of magnitude decrease in elastic modulus (apparent failure) that begins at 3%-5% strain. This material differs significantly from other common structural proteins such as collagen and elastin in mechanical response to strain. Qualitative similarities in stress/strain behavior to keratin, another common structural protein, are more than coincidental when composition and detailed mechanical quantification are considered. This suggests the possibility of alpha-helical structure and matrix organization that might be similar in these two proteins. Indeed, the egg capsule protein may be closely related to vertebrate keratins such as intermediate filaments. We conclude that while this material's bimodal tensile properties may serve as useful protection against the impact loading egg capsules encounter in the intertidal zone, the full biological importance of these capsules is not known.     

Characterizing molecular diffusion in the lens capsule

The lens capsule compartmentalizes the cells of the avascular lens from other ocular tissues. Small molecules required for lens cell metabolism, such as glucose, salts, and waste products, freely pass through the capsule. However, the lens capsule is selectively permeable to proteins such as growth hormones and substrate carriers which are required for proper lens growth and development. We used fluorescence recovery after photobleaching (FRAP) to characterize the diffusional behavior of various sized dextrans (3, 10, 40, 150, and 250 kDa) and proteins endogenous to the lens environment (EGF, γD-crystallin, BSA, transferrin, ceruloplasmin, and IgG) within the capsules of whole living lenses. We found that proteins had dramatically different diffusion and partition coefficients as well as capsule matrix binding affinities than similar sized dextrans, but they had comparable permeabilities. We also found ionic interactions between proteins and the capsule matrix significantly influence permeability and binding affinity, while hydrophobic interactions had less of an effect. The removal of a single anionic residue from the surface of a protein, γD-crystallin [E107A], significantly altered its permeability and matrix binding affinity in the capsule. Our data indicated that permeabilities and binding affinities in the lens capsule varied between individual proteins and cannot be predicted by isoelectric points or molecular size alone.     

Identification of noncollagenous components of calf lens capsule: evaluation of their adhesion-promoting activity

Extraction of calf anterior and posterior lens capsules with 5 M guanidine HCI resulted in the solubilization of protein (12% of total) with a noncollagenous amino acid composition leaving behind the collagen matrix. Polyacrylamide gel electrophoresis of the solubilized material revealed a number of components, all of which were susceptible to trypsin but resistant to collagenase digestion. Fractionation of the extracted proteins by Sepharose CL-6B filtration as well as by affinity chromatography was undertaken, and laminin, fibronectin, entactin, and beta-crystallin were identified by electrophoresis and solid-phase radioimmunoassays in both anterior and posterior capsules. An entactin (Mr = 150,000), which constituted the most prominent component on electrophoresis, was purified after Sepharose CL-6B filtration by a two-step lectin affinity chromatography procedure, which was based on the failure of this protein to bind to Bandeiraea simplicifolia I but its positive reactivity with wheat germ lectin. Neither the mixture of proteins extracted from lens capsules by guanidine nor fractions prepared therefrom were able to enhance lens epithelial cell attachment to type I or type IV collagen-coated surfaces or to guanidine-prepared lens capsules; adhesion-stimulating activity could not be demonstrated even when cycloheximide-treated cells were employed. Furthermore, the cells were observed to attach as effectively to guanidine-extracted as to native capsules. These observations indicate that noncollagenous proteins are not essential for the in vitro attachment of epithelial cells to lens capsule; it appears that the collagen component itself provides an optimal surface for cell-basement membrane interaction.     

Multiaxial mechanical behavior of the porcine anterior lens capsule

The biomechanics of the lens capsule of the eye is important both in physiologic processes such as accommodation and clinical treatments such as cataract surgery. Although the lens capsule experiences multiaxial stresses in vivo, there have been no measurements of its multiaxial properties or possible regional heterogeneities. Rather all prior mechanical data have come from 1-D pressure–volume or uniaxial force-length tests. Here, we report a new experimental approach to study in situ the regional, multiaxial mechanical behavior of the lens capsule. Moreover, we report multiaxial data suggesting that the porcine anterior lens capsule exhibits a typical nonlinear pseudoelastic behavior over finite strains, that the in situ state is pre-stressed multiaxially, and that the meridional and circumferential directions are principal directions of strain, which is nearly equibiaxial at the pole but less so towards the equator. Such data are fundamental to much needed constitutive formulations.     

FGF-2 release from the lens capsule by MMP-2 maintains lens epithelial cell viability

The lens is an avascular tissue, separated from the aqueous and vitreous humors by its own extracellular matrix, the lens capsule. Here we demonstrate that the lens capsule is a source of essential survival factors for lens epithelial cells. Primary and immortalized lens epithelial cells survive in low levels of serum and are resistant to staurosporine-induced apoptosis when they remain in contact with the lens capsule. Physical contact with the capsule is required for maximal resistance to stress. The lens capsule is also a source of soluble factors including fibroblast growth factor 2 (FGF-2) and perlecan, an extracellular matrix component that enhances FGF-2 activity. Matrix metalloproteinase 2 (MMP-2) inhibition as well as MMP-2 pretreatment of lens capsules greatly reduced the protective effect of the lens capsule, although this could be largely reversed by the addition of either conditioned medium or recombinant FGF-2. These data suggest that FGF-2 release from the lens capsule by MMP-2 is essential to lens epithelial cell viability and survival.     

Developmental acquisition of basement membrane heterogeneity: type IV collagen in the avian lens capsule

To investigate potential heterogeneity and developmental changes in basement membranes during embryogenesis, we performed immunohistochemical analyses on lens capsules in chicken embryos of different ages using domain-specific monoclonal antibodies against type IV collagen. We found that the capsule of the newly formed lens stained uniformly with antibodies against this component of basement membranes, but with increasing age and differentiation of the lens cells the anterior lens capsule remained brightly fluorescent while staining of the posterior capsule became relatively much less intense. This antero- posterior gradient of anti-type IV collagen antibody reactivity demonstrated that developmentally-regulated changes can occur within a single, continuous basement membrane.     

Regional multiaxial mechanical properties of the porcine anterior lens capsule

The lens capsule of the eye plays fundamental biomechanical roles in both normal physiological processes and clinical interventions. There has been modest attention given to the mechanical properties of this important membrane, however, and prior studies have focused on 1-D analyses of the data. We present results that suggest that the porcine anterior lens capsule has a complex, regionally dependent, nonlinear, anisotropic behavior. Specifically, using a subdomain inverse finite element method to analyze data collected via a new biplane video-based test system, we found that the lens capsule is nearly isotropic (in-plane) near the pole but progressively stiffer in the circumferential compared to the meridional direction as one approaches the equator. Because the porcine capsule is a good model of the young human capsule, there is strong motivation to determine if similar regional variations exist in the human lens capsule for knowledge of such complexities may allow us to improve the design of surgical procedures and implants.     

Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine)

In general, microcapsules prepared from alginate and polycations lack mechanical strength because the interaction between alginate and polycations is ionic instead of covalent, which represents a much stronger bond. To increase the mechanical strength of the capsule, we prepared photosensitive microcapsules that could form covalent bonds between polymers in the capsular membrane by light irradiation. Two types of photosensitive poly(allylamine), with 5% and 10% of amino groups modified by alpha-phenoxycinnamylidene acetylchloride, were synthesized. Both photopolymers exhibited an absorption maximum at 325 nm and were capable of crosslinking upon light exposure. These photosensitive polymers were used for the preparation of microcapsules. The capsules formed from this photosensitive poly(allylamine) and alginate were strengthened significantly by light irradiation. Only 28% of the microcapsules prepared from the 5%-modified photopolymer fractured after 48 h of shaking at 150 rpm. This fracture percentage is much lower when compared with the 60% of capsules fractured when prepared from the untreated poly(allylamine). By using poly(allylamine) at 10% modification, the mechanical strength was improved only slightly, with 26% of capsules fractured. Analysis of the permeability test indicated that the photo-crosslinked capsular membrane was freely permeable to cytochrome c and myoglobin, but less permeable to serum albumin. The encapsulation method was used to entrap and culture IW32 mouse leukemia cells. The cells proliferated to a density of about 1.1 x 10(7) cells/mL in the capsules after 7 days of cultivation. Concurrently, the concentration of erythropoietin in the microcapsules increased to 800 mU/mL. This new encapsulation technique has great potential in the application of a bioindustrial cell-culturing process.     

Collagen IV in the developing lens capsule.

The lens capsule is a specialized thickened basement membrane that completely surrounds the lens and provides anchoring sites for zonules, the filamentous bodies that suspend the lens. Like other basement membranes, the lens capsule contains collagen IV, which is a family of six polypeptides, subunits alpha1(IV)-alpha6(IV), each of which is encoded by a distinct gene. We have investigated the presence of collagen IV subunits in the developing lens capsule by using confocal immunohistochemistry and antibodies against each of the six collagen IV subunits. In murine embryos, subunits alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) were detected in the basement membrane surrounding the lens vesicle, and they persisted in the capsule until adulthood. In contrast, neither collagen alpha3(IV) nor alpha4(IV) was detected in the lens capsule until 2 weeks postnatal. Similarly, we detected no collagen alpha3(IV) or alpha4(IV) in lens capsules of 54-day human embryos, while collagen alpha3(IV) and alpha4(IV) were detected in adult humans. Thus, in the lens capsule, there is a developmental shift in detectable collagen IV subunits; early in development we observed subunits alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV), which is consistent with the presence of fibrillar [alpha1alpha1alpha2] and elastic [alpha5alpha5alpha6] protomers, but later in development components of the more cross-linked [alpha3alpha4alpha5] protomer appear. An elastic lens capsule may be necessary in order to accommodate rapid lens growth in early development, whereas later in development a stronger, more cross-linked capsule may be necessary in order to tolerate the stress caused by postnatal accommodation and disaccommodation of the lens.     

Macromolecular organization of basement membranes. Characterization and comparison of glomerular basement membrane and lens capsule components by immunochemical and lectin affinity procedures

The macromolecular components of bovine glomerular basement membrane (GBM) and lens capsules (anterior and posterior) solubilized by sequential extractions with denaturing agents were quantitated and characterized by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography with the help of immunochemical techniques. Laminin, entactin, fibronectin, and heparan sulfate proteoglycan were primarily recovered (over 80%) from both basement membranes in a guanidine HCl extract which contained only a limited amount of the total protein (10-14%); most of the remainder of these noncollagenous components could be solubilized by the guanidine in the presence of reducing agent. Although a portion of the Type IV collagen could be obtained by these treatments, effective extraction of this protein depended on exposure to sodium dodecyl sulfate under reducing conditions. Immunoblot analysis revealed a remarkably similar pattern for GBM and lens capsule Type IV collagens with prominent bands of Mr = 390,000, 210,000, and 190,000 being evident. Fibronectin was present in much greater amounts in GBM than lens capsule while the reverse was true for entactin. In both GBM and lens capsules, the entactin (Mr = 150,000) exceeded laminin; the latter protein on immunoblotting was found to contain primarily the alpha-subunit (Mr = 200,000). The size of the heparan sulfate proteoglycan from anterior (Mr = 400,000) and posterior lens capsule (Mr greater than 500,000) was substantially larger than that from GBM (Mr = 200,000). During DEAE-cellulose chromatography under nonreducing conditions in a denaturing solvent, a portion of the Type IV collagen coeluted with the proteoglycan from these membranes. Considerable Bandeiraea simplicifolia I binding activity (alpha-D-galactose specific) was observed in GBM and lens capsule extracts and column fractions which could not be accounted for by laminin alone. Several components which reacted with this lectin were seen on transblots and among these Type IV collagen was identified. In contrast to the basement membranes from bovine tissues, the constituents from human GBM did not react with the B. simplicifolia I lectin.     

Influence of oxidation state on iron binding by Bacillus licheniformis capsule.

We examined ferric (Fe3+) and ferrous (Fe2+) iron binding by the anionic gamma-glutamyl capsule polymer of Bacillus licheniformis ATCC 9945. The addition of FeCl3 to B. licheniformis capsule under aerobic conditions resulted in flocculation due to the capsule-induced formation of amorphous, rust-colored ferrihydrite. Significant binding of iron, which could be attributed to binding by both the anionic capsule and the ferrihydrite precipitate, occurred. In contrast, the addition of FeCl2 to B. licheniformis capsule under anaerobic conditions resulted in significantly less iron being bound and no color change or flocculation occurring. Capsule-bound ferric iron could be partially released upon addition of several reducing agents. From these observations, it can be concluded that the oxidation state of iron significantly influences its tendency to be bound by anionic bacterial polymers such as capsules.     

Evidence for collagen molecular orientation in basement membranes

Summary The following basement membranes (BMs) from representative species of the main vertebrate classes were studied by the Picrosirius-polarization method: lens capsule, Reichert's membrane and glomerular BMs. A distinct birefringence was consistently observed in all BMs from all species studied by this method. The results reported provide a strong evidence for collagen macromolecular orientation in BMs. Heparitin sulphate was the only glycosaminoglycan detected in dog lens capsules.     

Influence of oxidation state on iron binding by Bacillus licheniformis capsule.

We examined ferric (Fe3+) and ferrous (Fe2+) iron binding by the anionic gamma-glutamyl capsule polymer of Bacillus licheniformis ATCC 9945. The addition of FeCl3 to B. licheniformis capsule under aerobic conditions resulted in flocculation due to the capsule-induced formation of amorphous, rust-colored ferrihydrite. Significant binding of iron, which could be attributed to binding by both the anionic capsule and the ferrihydrite precipitate, occurred. In contrast, the addition of FeCl2 to B. licheniformis capsule under anaerobic conditions resulted in significantly less iron being bound and no color change or flocculation occurring. Capsule-bound ferric iron could be partially released upon addition of several reducing agents. From these observations, it can be concluded that the oxidation state of iron significantly influences its tendency to be bound by anionic bacterial polymers such as capsules.     

Basement membrane glycosaminoglycans: Examination of several membranes and evaluation of the effect of sonic treatment

The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment.     

Reusable biosorbents in capsules from zoogloea ramigera cells for cadmium removal

A biosorbent was prepared by immobilizing and culturing Zoogloea ramigera cells in calcium alginate capsules to high density. The biosorbent (the cell and its exopolysaccharide "Zooglan") along with the [calcium] alginate is known to be responsible for cadmium removal. The dry weight of the biosorbent reached 107 g/L after 3 days of cultivation and 220 g/L after 5 days based on the core volume of a 2.0-mm diameter capsule used. The biosorbents were completely contained in the core of the capsule where the cells grew preferentially near the shell of the capsules while the polymer distributed homogeneously in the core. The specific cadmium uptake by the capsule biosorbent was 1.9 mg/g adsorbent at an initial cadmium concentration of 3 mg/L. This is 1.24 times more than the specific cadmium uptake by the 1.8-mm beads prepared under a comparable condition. The capsules crosslinked with 1% triethylene tetramine and 1% glutamic dialdehyde solutions were superior to the uncrosslinked capsules in mechanical strength. The crosslinked capsules maintained their mechanical strength and adsorption/desorption capacity even after 30 cycles of repeated use. Copyright 1999 John Wiley & Sons, Inc.     

A structural constitutive model for the human lens capsule

Published data on the mechanical performance of the human lens capsule when tested under uniaxial and biaxial conditions are reviewed. It is concluded that two simple phenomenological constitutive models (namely a linear elastic model and a Fung-type hyperelastic model) are unable to provide satisfactory representations of the mechanical behaviour of the capsule for both of these loading conditions. The possibility of resolving these difficulties using a structural constitutive model for the capsule, of a form that is inspired by the network of collagen IV filaments that exist within the lens capsule, is explored. The model is implemented within a rectangular periodic cell. Prescribed stretches are imposed on the periodic cell and the network is allowed to deform in a non-affine manner. The performance of the constitutive model correlates well with previously published test data. One possible application of the model is in the development of a multi-scale analysis of the mechanics of the human lens capsule.     

Investigating the effects of membrane deformability on artificial capsule adhesion to the functionalized surface

Understanding, manipulating and controlling cellular adhesion processes can be critical in developing biomedical technologies. Adhesive mechanisms can be used to the target, pattern and separate cells such as leukocytes from whole blood for biomedical applications. The deformability response of the cell directly affects the rolling and adhesion behavior under viscous linear shear flow conditions. To that end, the primary objective of the present study was to investigate numerically the influence of capsule membrane’s nonlinear material behavior (i.e. elastic-plastic to strain hardening) on the rolling and adhesion behavior of representative artificial capsules. Specifically, spherical capsules with radius of \(3.75\, \upmu \hbox {m}\) were represented using an elastic membrane governed by a Mooney–Rivlin strain energy functions. The surfaces of the capsules were coated with P-selectin glycoprotein-ligand-1 to initiate binding interaction with P-selectin-coated planar surface with density of \(150\,\upmu \hbox {m}^{-2}\) under linear shear flow varying from 100 to \(400\,\hbox {s}^{-1}\). The numerical model is based on the Immersed Boundary Method for rolling of deformable capsule in shear flow coupled with Monte Carlo simulation for receptor/ligand interaction modeled using Bell model. The results reveal that the mechanical properties of the capsule play an important role in the rolling behavior and the binding kinetics between the capsule contact surface and the substrate. The rolling behavior of the strain hardening capsules is relatively smoother and slower compared to the elastic-plastic capsules. The strain hardening capsules exhibits higher contact area at any given shear rate compared to elastic-plastic capsules. The increase in contact area leads to decrease in rolling velocity. The capsule contact surface is not in complete contact with the substrate because of thin lubrication film that is trapped between the capsule and substrate. This creates a concave shape on the bottom surface of the capsule that is referred to as a dimple. In addition, the present study demonstrates that the average total bond force from the capsules lifetime increases by 37 % for the strain hardening capsules compared to elastic-plastic capsules at shear rate of \(400\,\hbox {s}^{-1}\). Finally, the model demonstrates the effect of finite membrane deformation on the coupling between hydrodynamic and receptor/ligand interaction.