Interfibrillar Spacing Research Articles

Collagen cross-linking impact on keratoconus extracellular matrix.

BackgroundKeratoconus (KC) is a common multifactorial ectatic corneal disease with unknown onset. KC most commonly appears in adolescence and affects approximately 1:400 people worldwide. Treatment options, for advanced KC cases, are collagen cross-linking (CXL) and corneal transplants. CXL is a new KC treatment that helps arrest the disease. Unfortunately, only a fraction of KC patients will qualify for CXL treatment. Our goal, in this study, was to begin to understand how CXL affects the corneal microenvironment and pave the way towards a more patient-driven CXL treatment.MethodsPrimary human corneal fibroblasts from healthy and KC donors were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added followed by UVA irradiation. Transmission Electron Microscopy (TEM) and western blots were used to assess the effect of CXL on the extracellular matrix (ECM) and the resident cells, pre- and post CXL.ResultsData shows CXL improved lamellar organization showing more organized collagen fibrils decorated with proteoglycans (PGs). The distribution of the collagen fibrils and interfibrillar spacing was also visibly improved, post-CXL. Lumican, mimecan, and decorin were the dominant PGs and were significantly upregulated in post-CXL cultures. ECM degradation proteins, matrix metalloproteinases (MMPs), MMP-1, -3, and -9, but not MMP-2, were significantly downregulated post-CXL. TIMP-1 and -2 were not modulated by CXL.ConclusionThe unknown effects of CXL on the human corneal microenvironment have hampered our ability to make CXL available to all KC patients. Our current study provides a deeper understanding on CXL activity, using our unique 3D in vitro model.

Ultrastructure and 3D transmission electron tomography of collagen fibrils and proteoglycans of swollen human corneal stroma.

The transparency of the cornea is regulated by the unique organization of collagen fibrils (CFs) which is maintained by proteoglycans (PGs). The interlacing of CF lamellae in the anterior stroma provides the biomechanical properties of the cornea. To investigate the alterations of CFs and PGs in the swollen cornea, with special reference to the anterior stroma by using electron microscopy and 3D ultrastructural tomography. Nine healthy normal scleral corneal rings (age from 40 to 65 years) were hydrated individually in deionised water to induce swelling in the cornea. Three of them were hydrated for 2hr whereas the other three were hydrated for 48hr. The remaining three scleral normal corneal rings were used as a control.The corneas were processed for electron microscopy (EM) to study the CFs and PGs. Ultrathin sections were observed using transmission electron microscopy (JOEL 1400) and digital images of CFs, PGs and lamellae were captured using a bottom mounted Quemesa camera and iTEM Soft Imaging System. The software program 'Composer-x64, version 3.4.2.0' was used to construct individual 3D images from 120 digital images taken from -60 to + 60 degree angles. The 3D tomography showed the degeneration of microfibrils within the CFs of the swollen cornea. The CF diameter was significantly reduced and the interfibrillar spacing significantly increased in both the 2hr and 48hr hydrated corneas compared to the normal cornea. Within the hydrated corneas, the CF diameter was smaller and the interfibrillar spacing was increased in the middle and posterior stroma compared to the anterior stroma. The PG area in both the 2hr and the 48hr hydrated cornea was reduced in the anterior stroma, whereas it was increased in middle and posterior stroma compared to the normal cornea. The density of the PGs in both the 2hr and the 48hr samples, was reduced compared to the density of PGs in the normal cornea. The CFs, PGs and lamellae had degenerated, caused by swelling. 3D imaging demonstrated that the impairment of the microfibrils and PGs within the CF, is caused by the excessive hydration or swelling in the anterior as well as in the middle and posterior stroma. The lamellae of the anterior stroma which provides the biomechanical strength in the normal cornea, had degenerated in the swollen corneas due to the presence of the damaged CFs and PGs.

The Relationship Between Mechanical Properties, Ultrastructural Changes, and Intrafibrillar Bond Formation in Corneal UVA/Riboflavin Cross-linking Treatment for Keratoconus.

To determine the relationship between mechanical behavior in cross-linked corneas and changes in the corneal ultrastructure after corneal cross-linking (CXL). Porcine corneas were treated following the "Dresden" protocol, the current gold standard for clinical treatment, consisting of dropwise application of 0.1% riboflavin in 20% dextran followed by 30 minutes of ultraviolet-A (UVA) irradiation. The effect of CXL was assessed using uniaxial tensile testing, transmission electron microscopy, and Fourier transform infrared spectroscopy, with results compared against corneas treated with each of the treatment solution components individually. UVA/riboflavin cross-linked corneas displayed 28% ± 17% increase in the material tangent modulus compared with dextran treatment alone, and altered collagen architecture within the first 300 µm of stromal depth consisting of 5% increase in the thickness of collagen fibrils, no significant changes to interfibrillar spacing, and an 8% to 12% decrease in number of fibrils per unit area. Fourier transform infrared spectroscopy confirmed formation of interfibrillar bonds (P = .012) induced by UVA-mediated CXL. The data support a model wherein collagen fibril diameter and structural density are fundamental parameters in defining tissue stiffening following UVA/riboflavin CXL and provide benchmarks against which modifications to the Dresden CXL protocol can be evaluated. [J Refract Surg. 2018;34(4):264-272.].

Stretch-induced structural evolution of poly (vinyl alcohol) film in water at different temperatures: An in-situ synchrotron radiation small- and wide-angle X-ray scattering study

The structural evolution (not only intra-fibrillar but also inter-fibrillar structures) during extension of poly (vinyl alcohol) (PVA) in water at different temperatures are investigated by in-situ small- and wide-angle X-ray scattering (SAXS and WAXS). The stress-strain curves can be defined into four zones with the onset and the end of stress plateau, and the strain hardening point as the zone boundaries. WAXS results show that stretch induces melting of PVA crystal from zones I to III, while SAXS measurements reveal stretch-induced transformation from initial lamella to nanofibrils starts at the onset of zone II, which proceeds via a melting-reconstruction process instead of direct transformation. In zone III, the nanofibrils increase in content and pack periodically with an average inter-fibrillar spacing of about 14–18 nm. Increasing temperature leads to larger inter-fibrillar spacing and more perfect packing of nanofibrils, which may benefit the production of polarizer with homogeneously structural and optical properties.

Changes in Corneal Biomechanical Properties With Different Corneal Cross-linking Irradiances.

To evaluate whether different corneal cross-linking (CXL) irradiances, all with the same delivered total energy, achieve similar increases in corneal material stiffness. One hundred twenty-six healthy white Japanese rabbits were randomly divided into seven groups (n = 18 each). After removing the epithelium of the left corneas, six groups were exposed to riboflavin (0.22% concentration by volume) and ultraviolet-A (370 nm) at different CXL irradiations, all with the same total dose (5.4 J/cm2), ranging from 3 mW/cm2 for 30 minutes to 90 mW/cm2 for 1 minute. The left corneas of the seventh group were exposed to riboflavin without irradiation. Twelve corneas of each group were prepared for inflation testing, where they were subjected to internal hydrostatic pressure simulating intraocular pressure, whereas the other six specimens were processed for electron microscopy measurements of fibril diameter and interfibrillar spacing. The inverse modeling process was used to estimate the tangent modulus of the tissue, which is considered an accurate measure of the material stiffness. The stiffening effect of CXL decreased when using high irradiation/short duration settings. Compared with the group with no irradiation (NUVA group), the tangent modulus increases reduced from 212.5% in the 3mW/30min group to 196.8% in the 90mW/1min group. These increases were significant (P < .05) in the 3mW/30min and 9mW/10min groups, but became insignificant in other CXL groups. The interfibrillar spacing in the anterior 50 μm of the corneal stroma also reduced with high irradiation/short duration settings, changing from 20.05 ± 1.89 nm in the NUVA group down to 13.06 ± 2.07 and 14.37 ± 1.90 nm in the 3mW/30min and 9mW/10min groups, respectively. These changes were significant (P < .05) between non-adjacent groups, but became non-significant otherwise. The corresponding changes in fibril diameter were small and nonsignificant in all cases (P > .05). Because the effect of CXL in stiffening the tissue and reducing the interfibrillar spacing consistently decreased with reducing the irradiance duration, the Bunsen-Roscoe law may not be readily applicable in the CXL of corneal tissue. [J Refract Surg. 2018;34(1):51-58.].

Distinct reorganization of collagen architecture in lipopolysaccharide-mediated premature cervical remodeling.

Previous work has identified divergent mechanisms by which cervical remodeling is achieved in preterm birth (PTB) induced by hormone withdrawal (mifepristone) or lipopolysaccharide (LPS). Our current study aims to document how collagen architecture is modified to achieve premature cervical remodeling in mice treated with LPS as a model of infection-induced inflammation. Cervices were collected on gestation day (d) 15 from mice with premature cervical ripening induced by LPS and compared to d15 and d18 controls as well as a hormone withdrawal PTB model. Second harmonic generation (SHG) and electron microscopy were utilized for visualization of collagen morphology and ultrastructure. LPS-mediated premature cervical ripening is characterized by unique structural changes in collagen fiber morphology. LPS treatment increased the interfibrillar spacing of collagen fibrils. A preferential disruption of collagen fiber architecture in the subepithelial region compared to midstroma region was evidenced by increased pores lacking collagen signal in SHG images in the LPS-treated mice. Coinciding with this alteration, the infiltration of neutrophils was concentrated in the subepithelial stromal region as compared to midstromal region implicating the potential role of immune cells to extracellular matrix reorganization in inflammation-induced preterm cervical ripening. The current study demonstrates a preferential disorganization of collagen interfibrillar spacing and collagen fiber structure in LPS-mediated ripening.

The structural response of the cornea to changes in stromal hydration.

The primary aim of this study was to quantify the relationship between corneal structure and hydration in humans and pigs. X-ray scattering data were collected from human and porcine corneas equilibrated with polyethylene glycol (PEG) to varying levels of hydration, to obtain measurements of collagen fibril diameter, interfibrillar spacing (IFS) and intermolecular spacing. Both species showed a strong positive linear correlation between hydration and IFS2 and a nonlinear, bi-phasic relationship between hydration and fibril diameter, whereby fibril diameter increased up to approximately physiological hydration, H = 3.0, with little change thereafter. Above H = 3.0, porcine corneas exhibited a larger fibril diameter than human corneas (p < 0.001). Intermolecular spacing also varied with hydration in a bi-phasic manner but reached a maximum value at a lower hydration (H = 1.5) than fibril diameter. Human corneas displayed a higher intermolecular spacing than porcine corneas at all hydrations (p < 0.0001). Human and porcine corneas required a similar PEG concentration to reach physiological hydration, suggesting that the total fixed charge that gives rise to the swelling pressure is the same. The difference in their structural responses to hydration can be explained by variations in molecular cross-linking and intra/interfibrillar water partitioning.

The Structural Role of Elastic Fibers in the Cornea Investigated Using a Mouse Model for Marfan Syndrome.

PurposeThe presence of fibrillin-rich elastic fibers in the cornea has been overlooked in recent years. The aim of the current study was to elucidate their functional role using a mouse model for Marfan syndrome, defective in fibrillin-1, the major structural component of the microfibril bundles that constitute most of the elastic fibers.MethodsMouse corneas were obtained from animals with a heterozygous fibrillin-1 mutation (Fbn1+/−) and compared to wild type controls. Corneal thickness and radius of curvature were calculated using optical coherence tomography microscopy. Elastic microfibril bundles were quantified and visualized in three-dimensions using serial block face scanning electron microscopy. Transmission electron microscopy was used to analyze stromal ultrastructure and proteoglycan distribution. Center-to-center average interfibrillar spacing was determined using x-ray scattering.ResultsFbn1+/− corneas were significantly thinner than wild types and displayed a higher radius of curvature. In the Fbn1+/− corneas, elastic microfibril bundles were significantly reduced in density and disorganized compared to wild-type controls, in addition to containing a higher average center-to-center collagen interfibrillar spacing in the center of the cornea. No other differences were detected in stromal ultrastructure or proteoglycan distribution between the two groups. Proteoglycan side chains appeared to colocalize with the microfibril bundles.ConclusionsElastic fibers have an important, multifunctional role in the cornea as highlighted by the differences observed between Fbn1+/− and wild type animals. We contend that the presence of normal quantities of structurally organized elastic fibers are required to maintain the correct geometry of the cornea, which is disrupted in Marfan syndrome.

Disorders of the extracellular matrix in epithelial-stromal and stromal corneal dystrophies

The human cornea is rich in extracellular matrix. The stroma constitutes the main thickness of the cornea, which consists of collagens and proteoglycans mainly. The epithelial-stromal and stromal dystrophies of the cornea are either autosomal dominant or recessive inherited disorders, which are unrelated to inflammation or trauma. The diseases can manifest in each layer of the cornea, but in most cases the corneal stroma is affected. Generally, they develop in childhood or young adulthood but the diagnosis is only possible when clinical signs (epithelial erosions, decreased visual acuity, photophobia) develop. The different protein aggregates (hyaline, amyloid, crystalline) deposited in the corneal layers result in mild or advanced corneal opacity and loss of the corneal transparency due to disorganisation of the extracellular matrix. In some of the corneal dystrophies the keratane sulphate proteoglycan looses its function which results in a loss of the regular interfibrillar spacing. Due to the severe corneal opacity patients may need corneal transplantation. Orv. Hetil., 2016, 157(33), 1299-1303.

Collagen Fibrils and Proteoglycans of Macular Dystrophy Cornea: Ultrastructure and 3D Transmission Electron Tomography.

We report the ultrastructure and 3D transmission electron tomography of collagen fibrils (CFs), proteoglycans (PGs), and microfibrils within the CF of corneas of patients with macular corneal dystrophy (MCD). Three normal corneas and three MCD corneas from three Saudi patients (aged 25, 31, and 49 years, respectively) were used for this study. The corneas were processed for light and electron microscopy studies. 3D images were composed from a set of 120 ultrastructural images using the program "Composer" and visualized using the program "Visuliser Kai". 3D image analysis of MCD cornea showed a clear organization of PGs around the CF at very high magnification and degeneration of the microfibrils within the CF. Within the MCD cornea, the PG area in the anterior stroma was significantly larger than in the middle and posterior stroma. The PG area in the MCD cornea was significantly larger compared with the PG area in the normal cornea. The CF diameter and inter-fibrillar spacing of the MCD cornea were significantly smaller compared with those of the normal cornea. Ultrastructural 3D imaging showed that the production of unsulfated keratin sulfate (KS) may lead to the degeneration of micro-CFs within the CFs. The effect of the unsulfated KS was higher in the anterior stroma compared with the posterior stroma.

Structure of Collagen fibril and proteoglycans at different depth of corneal stroma of CXL treated rats

AbstractPurpose In the present study we assess the effects of collagen cross‐linking on ultrastructure organization of collagen fibrils (CF) and proteoglycans (PGs) in a rat model.Methods The corneas were treated with a standard cross linking (CXL) method with isotonic riboflavin. Scelral‐corneal rings were removed one week after the treatment. All samples were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy.Results The CXL rat corneal stroma thickness increased compared to the normal rat corneal stromal thickness. The collagen fibril diameter (29.28±3.42nm) in the anterior stroma of CXL rats was larger compared to the CF diameter (27.76±3.77nm) of the anterior stroma of control rats. Within the CXL rat corneal stroma, the CF diameter (29.28±3.42nm) and the interfibrillar spacing (43.01±3.32nm) in the anterior stroma were larger compared to the diameter (26.07±2.8nm) and interfibrillar spacing (41.27±3.52nm) in the posterior stroma. The PGs mean area was larger in the posterior stroma (260.1±282.395nm2) than the PGs mean area in the anterior stroma (209.98±177.0nm2) within the corneal stroma of the CXL rat.Conclusion The present study demonstrates that CXL leads to an increase in the CF diameter and interfibrilar spacing and reduces the PGs area in the anterior stroma of the CXL rats. The changes in the collagen fibril diameter could be due to an increase in the spacing between the microfibrils within the collagen fibrils. The increase in interfibrillar spacing could be due to an increase in hydration in the corneal stroma.

A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea

This study investigated changes in collagen fibril architecture and the sulphation status of keratan sulphate (KS) glycosaminoglycan (GAG) epitopes from central to peripheral corneal regions. Freshly excised adult bovine corneal tissue was examined as a function of radial position from the centre of the cornea outwards. Corneal thickness, tissue hydration, hydroxyproline content, and the total amount of sulphated GAG were all measured. High and low-sulphated epitopes of keratan sulphate were studied by immunohistochemistry and quantified by ELISA. Chondroitin sulphate (CS) and dermatan sulphate (DS) distributions were observed by immunohistochemistry following specific enzyme digestions. Electron microscopy and X-ray fibre diffraction were used to ascertain collagen fibril architecture. The bovine cornea was 1021±5.42μm thick at its outer periphery, defined as 9–12mm from the corneal centre, compared to 844±8.10μm at the centre. The outer periphery of the cornea was marginally, but not significantly, more hydrated than the centre (H=4.3 vs. H=3.7), and was more abundant in hydroxyproline (0.12 vs. 0.06mg/mg dry weight of cornea). DMMB assays indicated no change in the total amount of sulphated GAG across the cornea. Immunohistochemistry revealed the presence of both high- and low-sulphated epitopes of KS, as well as DS, throughout the cornea, and CS only in the peripheral cornea before the limbus. Quantification by ELISA, disclosed that although both high- and low-sulphated KS remained constant throughout stromal depth at different radial positions, high-sulphated epitopes remained constant from the corneal centre to outer-periphery, whereas low-sulphated epitopes increased significantly. Both small angle X-ray diffraction and TEM analysis revealed that collagen fibril diameter remained relatively constant until the outer periphery was reached, after which fibrils became more widely spaced (from small angle x-ray diffraction analysis) and of larger diameter as they approached the sclera. Depth-profiled synchrotron microbeam analyses showed that, at different radial positions from the corneal centre outwards, fibril diameter was greater superficially than in deeper stromal regions. The interfibrillar spacing was also higher at mid-depth in the stroma than it was in anterior and posterior stromal regions. Collagen fibrils in the bovine cornea exhibited a fairly consistent spacing and diameter from the corneal centre to the 12mm radial position, after which a significant increase was seen. While the constancy of the overall sulphation levels of proteoglycans in the cornea may correlate with the fibrillar architecture, there was no correlation between the latter and the distribution of low-sulphated KS.

Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 1: chemical and physical substrate analysis

BackgroundThere is considerable interest in the conversion of lignocellulosic biomass to liquid fuels to provide substitutes for fossil fuels. Pretreatments, conducted to reduce biomass recalcitrance, usually remove at least some of the hemicellulose and/or lignin in cell walls. The hypothesis that led to this research was that reactor type could have a profound effect on the properties of pretreated materials and impact subsequent cellulose hydrolysis.ResultsCorn stover was dilute-acid pretreated using commercially relevant reactor types (ZipperClave® (ZC), Steam Gun (SG) and Horizontal Screw (HS)) under the same nominal conditions. Samples produced in the SG and HS achieved much higher cellulose digestibilities (88% and 95%, respectively), compared to the ZC sample (68%). Characterization, by chemical, physical, spectroscopic and electron microscopy methods, was used to gain an understanding of the effects causing the digestibility differences. Chemical differences were small; however, particle size differences appeared significant. Sum-frequency generation vibrational spectra indicated larger inter-fibrillar spacing or randomization of cellulose microfibrils in the HS sample. Simons’ staining indicated increased cellulose accessibility for the SG and HS samples. Electron microscopy showed that the SG and HS samples were more porous and fibrillated because of mechanical grinding and explosive depressurization occurring with these two reactors. These structural changes most likely permitted increased cellulose accessibility to enzymes, enhancing saccharification.ConclusionsDilute-acid pretreatment of corn stover using three different reactors under the same nominal conditions gave samples with very different digestibilities, although chemical differences in the pretreated substrates were small. The results of the physical and chemical analyses of the samples indicate that the explosive depressurization and mechanical grinding with these reactors increased enzyme accessibility. Pretreatment reactors using physical force to disrupt cell walls increase the effectiveness of the pretreatment process.

Effect of mechanical disruption on the effectiveness of three reactors used for dilute acid pretreatment of corn stover Part 2: morphological and structural substrate analysis

BackgroundLignocellulosic biomass is a renewable, naturally mass-produced form of stored solar energy. Thermochemical pretreatment processes have been developed to address the challenge of biomass recalcitrance, however the optimization, cost reduction, and scalability of these processes remain as obstacles to the adoption of biofuel production processes at the industrial scale. In this study, we demonstrate that the type of reactor in which pretreatment is carried out can profoundly alter the micro- and nanostructure of the pretreated materials and dramatically affect the subsequent efficiency, and thus cost, of enzymatic conversion of cellulose.ResultsMulti-scale microscopy and quantitative image analysis was used to investigate the impact of different biomass pretreatment reactor configurations on plant cell wall structure. We identify correlations between enzymatic digestibility and geometric descriptors derived from the image data. Corn stover feedstock was pretreated under the same nominal conditions for dilute acid pretreatment (2.0 wt% H2SO4, 160°C, 5 min) using three representative types of reactors: ZipperClave® (ZC), steam gun (SG), and horizontal screw (HS) reactors. After 96 h of enzymatic digestion, biomass treated in the SG and HS reactors achieved much higher cellulose conversions, 88% and 95%, respectively, compared to the conversion obtained using the ZC reactor (68%). Imaging at the micro- and nanoscales revealed that the superior performance of the SG and HS reactors could be explained by reduced particle size, cellular dislocation, increased surface roughness, delamination, and nanofibrillation generated within the biomass particles during pretreatment.ConclusionsIncreased cellular dislocation, surface roughness, delamination, and nanofibrillation revealed by direct observation of the micro- and nanoscale change in accessibility explains the superior performance of reactors that augment pretreatment with physical energy.

Molecular and chemical investigations and comparisons of biomaterials for ocular surface regeneration

This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo-scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin-eosin, Masson's trichrome (for total collagen), and picrosirius-red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively.

Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

Post-laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post-LASIK cornea of two patients. Two normal corneas (age 24 and 37 years old) and two post-LASIK ectaic corneas from two patients (A and B) were studied. The "patient A" (age 27 years) underwent penetrating keratoplasty and "patient B" (age 31 years) underwent deep-anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three-dimensional (3D) reconstruction were taken by using the software "Recorder" and using a bottom mounted camera "Quemesa" attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using "Visual Kai" software. In the post-LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman"s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowman's layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post-LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post-LASIK cornea. Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post-LASIK cornea. The CFs diameter and interfibrillar spacing decreased.

Structural and Biomechanical Effects of Photooxidative Collagen Cross-Linking with Photosensitizer Riboflavin and 370 nm UVA Light on Human Corneoscleral Tissues

This study quantitatively investigated the immediate effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin (RF) and 370 nm UVA light in in vitro human corneoscleral collagen fibrils using histology, thickness, scanning electron microscopy, and atomic force microscopy analyses. Twenty 8 x 2 mm corneoscleral strips were dissected sagittally from donor tissue using a scalpel. Four parameters were investigated, including the density, thickness, adhesion force, and stiffness of corneoscleral tissues before and after the collagen cross-linking treatment. The RFUVA-catalyzed collagen cross-linking treatment led to an increase in the density of both corneal (8%) and scleral (23%) stromal collagens. However, there was no difference in corneoscleral thickness. Furthermore, RFUVA-catalyzed collagen cross-linking treatment led to an increased biomechanical response of corneosclera: 25 and 8% increases in corneoscleral stiffness, and 24 and 22% increases in corneoscleral adhesion force. The collagen cross-linking treatment through RF-sensitized photoreaction may cause structural and biomechanical changes in the collagen fibril network of the cornea and the sclera. This is due to narrowing of the interfibrillar spacing and the stromal edema.

Quantitative analysis of type I collagen fibril regulation by lumican and decorin using AFM

Lumican and decorin, two members of the small leucine-rich repeat proteoglycan (SLRP) family, have been implicated as regulators of collagen I fibril structure in different tissues. Both proteoglycans consist of a core protein and a glycosaminoglycan (GAG) chain, but quantitative information regarding the precise role of the protein and GAG moieties in regulating collagen structure is still limited. In this study, we used AFM imaging and a model system of aligned collagen I nanofibrils to investigate the role of lumican and decorin on collagen I fibril structure with high resolution. When co-assembled with collagen I, recombinant lumican or decorin proteins lacking the GAG chains decreased collagen fibril width to values below <100nm and increased interfibrillar spacing in a dose-dependent manner. At lower concentrations, lumican appeared to have a stabilizing effect on newly-formed collagen fibrils, while at higher concentrations both lumican and decorin inhibited collagen fibrillogenesis. GAG-containing decorin also increased interfibrillar spacing, decreased fibril width and ultimately inhibited fibrillogenesis, but these effects required lower concentrations compared to recombinant decorin, indicating that the decorin core protein alone cannot compensate for the full regulatory and structural contribution of the GAG chain during collagen I fibrillogenesis. Using a 2D autocorrelation approach, we furthermore analyzed and compared the effects of recombinant and glycosylated decorin on collagen ultrastructure, providing a quantitative measure for the observed structural differences. AFM analysis of ordered fibrillar collagen arrays in combination with quantitative autocorrelation image analysis thus provides a useful tool for investigating SLRP-dependent nanoscale effects on collagen fibril structure.

Bioengineering Organized, Multilamellar Human Corneal Stromal Tissue by Growth Factor Supplementation on Highly Aligned Synthetic Substrates

Recapitulating the microstructure of the native human corneal stromal tissue is believed to be a key feature in successfully engineering the corneal tissue. The stratified multilayered collagen fibril lamellae with orthogonal orientation determine the robust biomechanical properties of this tissue, and the uniform collagen fibril size and interfibrillar spacing are critical to its optical transparency. The objective of this investigation was to develop a highly organized collagen-fibril construct secreted by human corneal stromal stem cells (hCSSCs) to mimic the human corneal stromal tissue. In culture on a highly aligned fibrous substrate made from poly(ester urethane) urea, the fibroblast growth factor-2 (FGF-2, 10 ng/mL) and transforming growth factor-beta 3 (TGF-β3, 0.1 ng/mL) impacted the organization and abundance of the secreted collagen fibril matrix. hCSSCs differentiated into keratocytes with significant upregulation of the typical gene markers, including KERA, B3GnT7, and CHST6. FGF-2 treatment stimulated hCSSCs to secrete collagen fibrils strongly aligned in a single direction, whereas TGF-β3 induced collagenous layers with orthogonal fibril orientation. The combination of FGF-2 and TGF-β3 induced multilayered lamellae with orthogonally oriented collagen fibrils, in a pattern mimicking the human corneal stromal tissue. The constructs were 60-70 μm thick and had an increased content of cornea-specific extracellular matrix components, including keratan sulfate, lumican, and keratocan. The approach of combining substrate cues with growth factor augmentation offers a new means to engineer well-organized, collagen-based constructs with an appropriate nanoscale structure for corneal repair and regeneration.

The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

PurposeTo examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour.MethodsOne hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm2) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm2). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques.ResultsCorneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion.ConclusionsIt seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.