Independent and combined effects of obesity and traumatic joint injury to the structure and composition of rat knee cartilage.

Osteoarthritis (OA) is a multifactorial joint disease characterized by articular cartilage degradation. Risk factors for OA include joint trauma, obesity, and inflammation, each of which can affect joint health independently, but their interaction and the associated consequences of such interaction were largely unexplored. Here, we studied compositional and structural alterations in knee joint cartilages of Sprague-Dawley rats exposed to two OA risk factors: joint injury and diet-induced obesity. Joint injury was imposed by surgical transection of anterior cruciate ligaments (ACLx), and obesity was induced by a high fat/high sucrose diet. Depth-dependent proteoglycan (PG) content and collagen structural network of cartilage were measured from histological sections collected previously in Collins et al.. (2015). We found that ACLx primarily affected the superficial cartilages. Compositionally, ACLx led to reduced PG content in lean animals, but increased PG content in obese rats. Structurally, ACLx caused disorganization of collagenous network in both lean and obese animals through increased collagen orientation in the superficial tissues and a change in the degree of fibrous alignment. However, the cartilage degradation attributed to joint injury and obesity was not necessarily additive when the two risk factors were present simultaneously, particularly for PG content and collagen orientation in the superficial tissues. Interestingly, sham surgeries caused a through-thickness disorganization of collagen network in lean and obese animals. We conclude that the interactions of multiple OA risk factors are complex and their combined effects cannot be understood by superposition principle. Further research is required to elucidate the interactive mechanism between OA subtypes.

A Comparative Analysis of Orthotopic and Subcutaneous Pancreatic Tumour Models: Tumour Microenvironment and Drug Delivery.

Pancreatic ductal adenocarcinoma (PDAC) remains a challenging malignancy, mainly due to its resistance to chemotherapy and its complex tumour microenvironment characterised by stromal desmoplasia. There is a need for new strategies to improve the delivery of drugs and therapeutic response. Relevant preclinical tumour models are needed to test potential treatments. This paper compared orthotopic and subcutaneous PDAC tumour models and their suitability for drug delivery studies. A novel aspect was the broad range of tumour properties that were studied, including tumour growth, histopathology, functional vasculature, perfusion, immune cell infiltration, biomechanical characteristics, and especially the extensive analysis of the structure and the orientation of the collagen fibres in the two tumour models. The study unveiled new insights into how these factors impact the uptake of a fluorescent model drug, the macromolecule called 800CW. While the orthotopic model offered a more clinically relevant microenvironment, the subcutaneous model offered advantages for drug delivery studies, primarily due to its reproducibility, and it was characterised by a more efficient drug uptake facilitated by its collagen organisation and well-perfused vasculature. The tumour uptake seemed to be influenced mainly by the structural organisation and the alignment of the collagen fibres and perfusion. Recognising the diverse characteristics of these models and their multifaceted impacts on drug delivery is crucial for designing clinically relevant experiments and improving our understanding of pancreatic cancer biology.

MATLAB-Based Algorithm and Software for Analysis of Wavy Collagen Fibers.

Knowledge of soft tissue fiber structure is necessary for accurate characterization and modeling of their mechanical response. Fiber configuration and structure informs both our understanding of healthy tissue physiology and of pathological processes resulting from diseased states. This study develops an automatic algorithm to simultaneously estimate fiber global orientation, abundance, and waviness in an investigated image. To our best knowledge, this is the first validated algorithm which can reliably separate fiber waviness from its global orientation for considerably wavy fibers. This is much needed feature for biological tissue characterization. The algorithm is based on incremental movement of local regions of interest (ROI) and analyzes two-dimensional images. Pixels belonging to the fiber are identified in the ROI, and ROI movement is determined according to local orientation of fiber within the ROI. The algorithm is validated with artificial images and ten images of porcine trachea containing wavy fibers. In each image, 80-120 fibers were tracked manually to serve as verification. The coefficient of determination R2 between curve lengths and histograms documenting the fiber waviness and global orientation were used as metrics for analysis. Verification-confirmed results were independent of image rotation and degree of fiber waviness, with curve length accuracy demonstrated to be below 1% of fiber curved length. Validation-confirmed median and interquartile range of R2, respectively, were 0.90 and 0.05 for curved length, 0.92 and 0.07 for waviness, and 0.96 and 0.04 for global orientation histograms. Software constructed from the proposed algorithm was able to track one fiber in about 1.1 s using a typical office computer. The proposed algorithm can reliably and accurately estimate fiber waviness, curve length, and global orientation simultaneously, moving beyond the limitations of prior methods.

Preparation of Enzyme-Soluble Swim Bladder Collagen from Sea Eel (Muraenesox cinereus) and Evaluation Its Wound Healing Capacity.

In the present research, the enzyme-facilitated collagen from sea eel (Muraenesox cinereus) swim bladder was isolated, and the collagen characteristics were analyzed. Then, the collagen sponge was prepared and its potential mechanism in promoting skin wound healing in mice was further investigated. Collagen was obtained from the swim bladder of sea eels employing the pepsin extraction technique. Single-factor experiments served as the basis for the response surface method (RSM) to optimize pepsin concentration, solid-liquid ratio, and hydrolysis period. With a pepsin concentration of 2067 U/g, a solid-liquid ratio of 1:83 g/mL, and a hydrolysis period of 10 h, collagen extraction achieved a yield of 93.76%. The physicochemical analysis revealed that the extracted collagen belonged to type I collagen, and the collagen sponge displayed a fibrous structure under electron microscopy. Furthermore, in comparison to the control group, mice treated with collagen sponge dressing exhibited elevated activities of superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px), and decreased levels of malondialdehyde (MDA), interleukin (IL)-1ß, interleukin (IL)-6, and tumor necrosis factor (TNF)-α. The collagen sponge dressing effectively alleviated inflammation in the wound area, facilitating efficient repair and rapid healing of the skin tissue. During the initial phase of wound healing, the group treated with collagen sponge dressing exhibited an enhancement in the expressions of cluster of differentiation (CD)31, epidermal growth factor (EGF), transforming growth factor (TGF)-ß1, and type I collagen, leading to an accelerated rate of wound healing. In addition, this collagen sponge dressing could also downregulate the expressions of CD31, EGF, and type I collagen to prevent scar formation in the later stage. Moreover, this collagen treatment minimized oxidative damage and inflammation during skin wound healing and facilitated blood vessel formation in the wound. Consequently, it exhibits significant potential as an ideal material for the development of a skin wound dressing.

Maternal obesity driven changes in collagen linearity of breast extracellular matrix induces invasive mammary epithelial cell phenotype.

Obesity has been linked with numerous health issues as well as an increased risk of breast cancer. Although effects of direct obesity in patient outcomes is widely studied, effects of exposure to obesity-related systemic influences in utero have been overlooked. In this study, we investigated the effect of multigenerational obesity on epithelial cell migration and invasion using decellularized breast tissues explanted from normal female mouse pups from a diet induced multigenerational obesity mouse model. We first studied the effect of multigenerational diet on the mechanical properties, adipocyte size, and collagen structure of these mouse breast tissues, and then, examined the migration and invasion behavior of normal (KTB-21) and cancerous (MDA-MB-231) human mammary epithelial cells on the decellularized matrices from each diet group. Breast tissues of mice whose dams had been fed with high-fat diet exhibited larger adipocytes and thicker and curvier collagen fibers, but only slightly elevated elastic modulus and inflammatory cytokine levels. MDA-MB-231 cancer cell motility and invasion were significantly greater on the decellularized matrices from mice whose dams were fed with high-fat diet. A similar trend was observed with normal KTB-21 cells. Our results showed that the collagen curvature was the dominating factor on this enhanced motility and stretching the matrices to equalize the collagen fiber linearity of the matrices ameliorated the observed increase in cell migration and invasion in the mice that were exposed to a high-fat diet in utero. Previous studies indicated an increase in serum leptin concentration for those children born to an obese mother. We generated extracellular matrices using primary fibroblasts exposed to various concentrations of leptin. This produced curvier ECM and increased breast cancer cell motility for cells seeded on the decellularized ECM generated with increasing leptin concentration. Our study shows that exposure to obesity in utero is influential in determining the extracellular matrix structure, and that the resultant change in collagen curvature is a critical factor in regulating the migration and invasion of breast cancer cells.

Synergistic modification of collagen structure using ionic liquid and ultrasound to promote the production of DPP-IV inhibitory peptides.

BACKGROUND: Dual modification of collagen was performed using ionic liquid (IL) and ultrasound (US) to modulate the activity of collagen hydrolyzed peptides and reveal the production mechanism of cowhide-derived dipeptidyl peptidase (DPP-IV) inhibitory peptides. RESULTS: The results revealed that dual modification (IL + US) significantly improved the hydrolytic degree of collagen (P < 0.05). Meanwhile, IL and US tended to promote the break of hydrogen bonds, but inhibit the crosslinking between collagens. The double modification reduced the thermal stability and accelerated the exposure of tyrosine and phenylalanine of collagen, and improved the proportion of small molecular (< 1 kDa) peptides in collagen hydrolysates. Interestingly, the hydrophobic amino acid residues and DPP-IV inhibitory activity of collagen peptides with small molecular weight (< 1 kDa) was increased further under the combination of IL and US. CONCLUSION: Enhanced hypoglycemic activity of collagen peptides can be attained through the dual modification of IL and US. © 2023 Society of Chemical Industry.

Nanoscale characterization of collagen structural responses to in situ loading in rat Achilles tendons.

The specific viscoelastic mechanical properties of Achilles tendons are highly dependent on the structural characteristics of collagen at and between all hierarchical levels. Research has been conducted on the deformation mechanisms of positional tendons and single fibrils, but knowledge about the coupling between the whole tendon and nanoscale deformation mechanisms of more commonly injured energy-storing tendons, such as Achilles tendons, remains sparse. By exploiting the highly periodic arrangement of tendons at the nanoscale, in situ loading of rat Achilles tendons during small-angle X-ray scattering acquisition was used to investigate the collagen structural response during load to rupture, cyclic loading and stress relaxation. The fibril strain was substantially lower than the applied tissue strain. The fibrils strained linearly in the elastic region of the tissue, but also exhibited viscoelastic properties, such as an increased stretchability and recovery during cyclic loading and fibril strain relaxation during tissue stress relaxation. We demonstrate that the changes in the width of the collagen reflections could be attributed to strain heterogeneity and not changes in size of the coherently diffracting domains. Fibril strain heterogeneity increased with applied loads and after the toe region, fibrils also became increasingly disordered. Additionally, a thorough evaluation of radiation damage was performed. In conclusion, this study clearly displays the simultaneous structural response and adaption of the collagen fibrils to the applied tissue loads and provide novel information about the transition of loads between length scales in the Achilles tendon.

Rheological Method for Determining the Molecular Weight of Collagen Gels by Using a Machine Learning Technique.

This article presents, for the first time, the results of applying the rheological technique to measure the molecular weights (Mw) and their distributions (MwD) of highly hierarchical biomolecules, such as non-hydrolyzed collagen gels. Due to the high viscosity of the studied gels, the effect of the concentrations on the rheological tests was investigated. In addition, because these materials are highly sensitive to denaturation and degradation under mechanical stress and temperatures close to 40 °C, when frequency sweeps were applied, a mathematical adjustment of the data by machine learning techniques (artificial intelligence tools) was designed and implemented. Using the proposed method, collagen fibers of Mw close to 600 kDa were identified. To validate the proposed method, lower Mw species were obtained and characterized by both the proposed rheological method and traditional measurement techniques, such as chromatography and electrophoresis. The results of the tests confirmed the validity of the proposed method. It is a simple technique for obtaining more microstructural information on these biomolecules and, in turn, facilitating the design of new structural biomaterials with greater added value.

Minimising chemical crosslinking for stabilising collagen in acellular bovine pericardium: Mechanistic insights via structural characterisations.

Chemically crosslinked acellular bovine pericardium (ABP) has been widely used in clinical practice as bioprostheses. To ensure its consistency and durability, crosslinkers are used in excess, with stability guided by indicators including the hydrothermal denaturation temperature, the enzymatic resistance and the degree of crosslinking. Yet, understanding of the intermolecular structure in collagen fibrils which imparts the intrinsic stability of the ABPs is lacking, and the discrepancies in the stability criteria in varied conditions are poorly explained. In this study, synchrotron small-angle X-ray scattering (SAXS) in combination with thermal and colorimetric methods are employed to investigate the changes in the structure and the stability of ABPs during crosslinking using glutaraldehyde (GA) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at different concentrations. Based on the findings, a mechanism is proposed to explicate the crosslinking effects on collagen structure and the relationship between the structure and each stability indicator. At low crosslinker concentrations, the telopeptidyl-helical linkages are preferred, which cause rearrangements in the intermolecular structure of collagen, and efficiently contribute to its stabilities. Excess crosslinking is revealed by a revert trend in structural changes and the plateauing of the stabilities, assigning to the helical-helical linkages and monovalent bindings. The former would improve thermal stability but not collagenase resistance, whereas the latter have negligible effects. Overall, this study provides a mechanistic understanding of the chemical crosslinking of ABPs, which will contribute to the future development of more efficient and economically viable strategies to produce bioprostheses. STATEMENT OF SIGNIFICANCE: Chemical crosslinking imparts suitable properties to acellular bovine pericardium (ABP) for clinical applications, yet the understanding is lacking on the structure-stability relationship especially under different crosslinking conditions. Structural evidence in this study differentiates the binding sites during crosslinking in collagen fibrils at different crosslinker concentrations, highlighting the excess usage in the conventional crosslinking treatments. The mechanism based on the structure of collagen also successfully explains the dissimilarity in hydrothermal and enzymatic stabilities with varied crosslinking conditions. Future researches focusing on developing biomaterials via chemical crosslinking of ABPs would benefit from this study, for its contribution to the better understanding of the relationship of collagen structure and functions.

A hyperelastic model to capture the mechanical behaviour and histological aspects of the soft tissues.

It is well established that the soft connective tissues show a nonlinear elastic response that comes from their microstructural arrangement. Tissues' microstructure alters with various physiological conditions and may affect their mechanical responses. Therefore, the accurate prediction of tissue's mechanical response is crucial for clinical diagnosis and treatments. Thus, a physically motivated and mathematically simplified model is required for the accurate prediction of tissues' mechanical and structural responses. This study explored the 'Exp-Ln' hyperelastic model (Khajehsaeid et al., 2013) to capture soft tissues' mechanical and histological behaviour. In this work, uniaxial tensile test data for the belly and back pig skin were extracted from the experiments performed in our laboratory, whereas uniaxial test data for other soft tissues (human skin, tendon, ligament, and aorta) were extracted from the literature. The 'Exp-Ln; and other hyperelastic models (e.g. Money Rivlin, Ogden, Yeoh, and Gent models) were fitted with these experimental data, and obtained results were compared between the models. These results show that the 'Exp-Ln' model could capture the mechanical behaviour of soft tissues more accurately than other hyperelastic models. This model was also found numerically stable for all modes and ranges of deformation. This study also investigated the link between 'Exp-Ln' material parameters and tissue's histological parameters. The histological parameters such as collagen content, fibre free length, crosslink density, and collagen arrangement were measured using staining and ATR-FTIR techniques. The material parameters were found statistically correlated with the histological parameters. Further, 'Exp-Ln' model was implemented in ABAQUS through the VUMAT subroutine, where the mechanical behaviour of various soft tissues was simulated for different modes of deformation. The finite element analysis results obtained using the 'Exp-Ln' model agreed with the experiments and were more accurate than other hyperelastic models. Overall, these results demonstrate the capability of 'Exp-Ln' model to predict the mechanical and structural responses of the soft tissues.

Fractal analysis of rat dermal tissue in the different injury states.

Scar formation and chronic ulcers can develop following a skin injury. They are the result of the over- or underproduction of collagen. It is very important to evaluate the quality and quantity of the collagen that is produced during wound healing, especially with respect to its structure, as these factors are very important to a complicated outcome. However, there is no standard way to quantitatively analyse dermal collagen. As prior work characterised some potentially fractal properties of collagen, it was hypothesised that collagen structure could be evaluated with fractal dimension analysis. Small-angle X-ray scattering technology (SAXS) was used to evaluate the dermis of rats exposed to graft harvest, burn, and diabetic pathologic states. It was found that almost all collagen structures could be quantitatively measured with fractal dimension analysis. Further, there were significant differences in the three-dimensional (3-D) structure of normal collagen versus that measured in pathologic tissues. There was a significant difference in the 3-D structure of collagen at different stages of healing. The findings of this work suggest that fractal analysis is a good tool for wound healing analysis, and that quantitative collagen analysis is very useful for assessing the structure of dermal collagen.

Efficient Decellularization by Application of Moderate High Hydrostatic Pressure with Supercooling Pretreatment.

Decellularized tissues are considered superior scaffolds for cell cultures, preserving the microstructure of native tissues and delivering many kinds of cytokines. High hydrostatic pressure (HHP) treatment could remove cells physically from biological tissues rather than chemical methods. However, there are some risks of inducing destruction or denaturation of extracellular matrices (ECMs) at an ultrahigh level of HHP. Therefore, efficient decellularization using moderate HHP is required to remove almost all cells simultaneously to suppress tissue damage. In this study, we proposed a novel decellularization method using a moderate HHP with supercooling pretreatment. To validate the decellularization method, a supercooling device was developed to incubate human dermal fibroblasts or collagen gels in a supercooled state. The cell suspension and collagen gels were subjected to 100, 150, and 200 MPa of HHP after supercooling pretreatment, respectively. After applying HHP, the viability and morphology of the cells and the collagen network structure of the gels were evaluated. The viability of cells decreased dramatically after HHP application with supercooling pretreatment, whereas the microstructures of collagen gels were preserved and cell adhesivity was retained after HHP application. In conclusion, it was revealed that supercooling pretreatment promoted the denaturation of the cell membrane to improve the efficacy of decellularization using static application of moderate HHP. Furthermore, it was demonstrated that the HHP with supercooling pretreatment did not degenerate and damage the microstructure in collagen gels.

Skin Substitute Preparation Method Induces Immunomodulatory Changes in Co-Incubated Cells through Collagen Modification.

Chronic ulcerative and hard-healing wounds are a growing global concern. Skin substitutes, including acellular dermal matrices (ADMs), have shown beneficial effects in healing processes. Presently, the vast majority of currently available ADMs are processed from xenobiotic or cadaveric skin. Here we propose a novel strategy for ADM preparation from human abdominoplasty-derived skin. Skin was processed using three different methods of decellularization involving the use of ionic detergent (sodium dodecyl sulfate; SDS, in hADM 1), non-ionic detergent (Triton X-100 in hADM 2), and a combination of recombinant trypsin and Triton X-100 (in hADM 3). We next evaluated the immunogenicity and immunomodulatory properties of this novel hADM by using an in vitro model of peripheral blood mononuclear cell culture, flow cytometry, and cytokine assays. We found that similarly sourced but differentially processed hADMs possess distinct immunogenicity. hADM 1 showed no immunogenic effects as evidenced by low T cell proliferation and no significant change in cytokine profile. In contrast, hADMs 2 and 3 showed relatively higher immunogenicity. Moreover, our novel hADMs exerted no effect on T cell composition after three-day of coincubation. However, we observed significant changes in the composition of monocytes, indicating their maturation toward a phenotype possessing anti-inflammatory and pro-angiogenic properties. Taken together, we showed here that abdominoplasty skin is suitable for hADM manufacturing. More importantly, the use of SDS-based protocols for the purposes of dermal matrix decellularization allows for the preparation of non-immunogenic scaffolds with high therapeutic potential. Despite these encouraging results, further studies are needed to evaluate the beneficial effects of our hADM 1 on deep and hard-healing wounds.

Can fractal dimension analysis be used in quantitating collagen structure?

It is well known that collagen tissue, especially the collagen structure, plays an important role in wound healing. However, most research on collagen has been qualitative and morphological, based on sections, and cannot be used for real-time monitoring and clinical prediction. There are no standardized methods of quantitative analysis based on the whole skin sample in three dimensions (3-D). In order to explore a 3-D quantitative analysis, we developed a method that was derived from that of material science and physics, combined with our previous technique, X-ray scattering (SAXS). We hypothesized that the dermis might be analysed by fractal dimensions. To test this hypothesis, we performed the analysis in different pathological conditions, such as scar tissue, different time points after wounding, skin in different degrees of burns and skin in diabetes. The results showed that fractal dimension analysis could detect differences in different locations of the scar tissue, at different time points after wounding, and at a different extent of the severity of skin in diabetes. The research demonstrated that fractal dimension analysis can describe the 3-D structure of the collagen tissue of the skin, which will be beneficial for studying wound healing and finding new clinical treatments.

The effect of universal adhesives on dentine collagen.

OBJECTIVES: The purpose of the study was to evaluate the integrity of dentine type I collagen after self-etching (SE) treatments with strong and mild universal adhesives. METHODS: Coronal dentine specimens (n=10/product) were imaged by optical microscopy and analyzed by ATR-FTIR spectroscopy before and after treatment with 32% phosphoric acid gel (PA-negative control), 17% neutral EDTA (ED-positive control) conditioners and Adhese Universal (AD), Clearfil Universal Bond Quick (CQ), G-Premio Bond (GP), Prelude One (PR) and Scotchbond Universal (SB) adhesives. From the spectroscopic analysis the following parameters were determined: a) Extent of dentine demineralization (DM%) and b) percentage area of the Amide I curve-fitted components of ß-turns, 310-helix/ß-turns, α-helix, random coils, ß-sheets and collagen maturation (R) index. Statistical analysis was performed by one-way ANOVA (DM%), paired t-test/Wilcoxon test (Amide I components) and Spearman correlation coefficient (DM% vs Amide I components) at an a=0.05 level. RESULTS: PA, ED and GP removed the smear-layer and opened tubule orifices, whereas all other treatments removed only the intratubular smear-layer fraction. The ranking of the statistically significant differences in DM% was PA>GP>ED>AD, SB, CQ, PR, with AD being significantly different from PR. Regarding the Amide I components, PA demonstrated a significant reduction in ß-turns, α-helices and an increase in ß-sheets, GP a reduction in ß-turns, AD an increase in ß-turns and random coils, and CQ an increase in ß-turns. PR, SB and ED showed insignificant differences in all the Amide I components. Significant correlations were found between DM%-random coils and DM%-R. SIGNIFICANCE: The universal adhesives used in the SE mode induced none to minimal changes in dentine collagen structure, without evidence of the destabilization pattern observed after conventional phosphoric acid treatments.

Variation of Passive Biomechanical Properties of the Small Intestine along Its Length: Microstructure-Based Characterization.

Multiaxial testing of the small intestinal wall is critical for understanding its biomechanical properties and defining material models, but limited data and material models are available. The aim of the present study was to develop a microstructure-based material model for the small intestine and test whether there was a significant variation in the passive biomechanical properties along the length of the organ. Rat tissue was cut into eight segments that underwent inflation/extension testing, and their nonlinearly hyper-elastic and anisotropic response was characterized by a fiber-reinforced model. Extensive parametric analysis showed a non-significant contribution to the model of the isotropic matrix and circumferential-fiber family, leading also to severe over-parameterization. Such issues were not apparent with the reduced neo-Hookean and (axial and diagonal)-fiber family model, that provided equally accurate fitting results. Absence from the model of either the axial or diagonal-fiber families led to ill representations of the force- and pressure-diameter data, respectively. The primary direction of anisotropy, designated by the estimated orientation angle of diagonal-fiber families, was about 35° to the axial direction, corroborating prior microscopic observations of submucosal collagen-fiber orientation. The estimated model parameters varied across and within the duodenum, jejunum, and ileum, corroborating histologically assessed segmental differences in layer thicknesses.

Depth- and direction-dependent changes in solute transport following cross-linking with riboflavin and UVA light in ex vivo porcine cornea.

Diffusion is an important mechanism of transport for nutrients and drugs throughout the avascular corneal stroma. The purpose of this study was to investigate the depth- and direction-dependent changes in stromal transport properties and their relationship to changes in collagen structure following ultraviolet A (UVA)-riboflavin induced corneal collagen cross-linking (CXL). After cross-linking in ex vivo porcine eyes, fluorescence recovery after photobleaching (FRAP) was performed to measure fluorescein diffusion in the nasal-temporal (NT) and anterior-posterior (AP) directions at corneal depths of 100, 200, and 300 µm. Second harmonic generation (SHG) imaging was also performed at these three corneal depths to quantify fiber alignment. For additional confirmation, an electrical conductivity method was employed to quantify ion permeability in the AP direction in corneal buttons and immunohistochemistry (IHC) was used to image collagen structure. Cross-linked corneas were compared to a control treatment that received the riboflavin solution without UVA light (SHAM). The results of FRAP revealed that fluorescein diffusivity decreased from 23.39 ± 11.60 µm2/s in the SHAM group to 19.87 ± 10.10 µm2/s in the CXL group. This change was dependent on depth and direction: the decrease was more pronounced in the 100 µm depth (P = 0.0005) and AP direction (P = 0.001) when compared to the effect in deeper locations and in the NT direction, respectively. Conductivity experiments confirmed a decrease in solute transport in the AP direction (P < 0.0001). FRAP also detected diffusional anisotropy in the porcine cornea: the fluorescein diffusivity in the NT direction was higher than the diffusivity in the AP direction. This anisotropy was increased following CXL treatment. Both SHG and IHC revealed a qualitative decrease in collagen crimping following CXL. Analysis of SHG images revealed an increase in coherency in the anterior 200 µm of CXL treated corneas when compared to SHAM treated corneas (P < 0.01). In conclusion, CXL results in a decrease in stromal solute transport, and this decrease is concentrated in the most anterior region and AP direction. Solute transport in the porcine cornea is anisotropic, and an increase in anisotropy with CXL may be explained by a decrease in collagen crimping.

Model studies of advanced glycation end product modification of heterograft biomaterials: The effects of in vitro glucose, glyoxal, and serum albumin on collagen structure and mechanical properties.

Glutaraldehyde cross-linked heterograft tissues, bovine pericardium (BP) or porcine aortic valves, are the leaflet materials in bioprosthetic heart valves (BHV) used in cardiac surgery for heart valve disease. BHV fail due to structural valve degeneration (SVD), often with calcification. Advanced glycation end products (AGE) are post-translational, non-enzymatic reaction products from sugars reducing proteins. AGE are present in SVD-BHV clinical explants and are not detectable in un-implanted BHV. Prior studies modeled BP-AGE formation in vitro with glyoxal, a glucose breakdown product, and serum albumin. However, glucose is the most abundant AGE precursor. Thus, the present studies investigated the hypothesis that BHV susceptibility to glucose related AGE, together with serum proteins, results in deterioration of collagen structure and mechanical properties. In vitro experiments studied AGE formation in BP and porcine collagen sponges (CS) comparing 14C-glucose and 14C-glyoxal with and without bovine serum albumin (BSA). Glucose incorporation occurred at a significantly lower level than glyoxal (p<0.02). BSA co-incubations demonstrated reduced glyoxal and glucose uptake by both BP and CS. BSA incubation caused a significant increase in BP mass, enhanced by glyoxal co-incubation. Two-photon microscopy of BP showed BSA induced disruption of collagen structure that was more severe with glucose or glyoxal co-incubation. Uniaxial testing of CS demonstrated that glucose or glyoxal together with BSA compared to controls, caused accelerated deterioration of viscoelastic relaxation, and increased stiffness over a 28-day time course. In conclusion, glucose, glyoxal and BSA uniquely contribute to AGE-mediated disruption of heterograft collagen structure and deterioration of mechanical properties.

Constitutive modeling using structural information on collagen fiber direction and dispersion in human superficial femoral artery specimens of different ages.

Arterial mechanics plays an important role in vascular pathophysiology and repair, and advanced imaging can inform constitutive models of vascular behavior. We have measured the mechanical properties of 14 human superficial femoral arteries (SFAs) (age 12-70, mean 48±19 years) using planar biaxial extension, and determined the preferred collagen fiber direction and dispersion using multiphoton microscopy. The collagen fiber direction and dispersion were evaluated using second-harmonic generation imaging and modeled using bivariate von Mises distributions. The microstructures of elastin and collagen were assessed using two-photon fluorescence imaging and conventional bidirectional histology. The mechanical and structural data were used to describe the SFA mechanical behavior using two- and four-fiber family invariant-based constitutive models. Older SFAs were stiffer and mechanically more nonlinear than younger specimens. In the adventitia, collagen fibers were undulated and diagonally-oriented, while in the media, they were straight and circumferentially-oriented. The media was rich in collagen that surrounded the circumferentially-oriented smooth muscle cells, and the elastin was present primarily in the internal and external elastic laminae. Older SFAs had a more circumferential collagen fiber alignment, a decreased circumferential-radial fiber dispersion, but the same circumferential-longitudinal fiber dispersion as younger specimens. Both the two- and the four-fiber family constitutive models were able to capture the experimental data, and the fits were better for the four-fiber family formulation. Our data provide additional details on the SFA intramural structure and inform structurally-based constitutive models.

Anion-regulated binding selectivity of Cr(III) in collagen.

We present a mechanism for the selectivity of covalent/electrostatic binding of the Cr(III) ion to collagen, mediated by the kosmotropicity of the anions. Although a change in the long-range ordered structure of collagen is observed after covalent binding (Cr(III)-OOC) in the presence of SO4 2- at pH 4.5, the νsym (COO- ) band remains intense, suggesting a relatively lower propensity for the Cr(III) to bind covalently instead of electrostatically through Cr(H2 O)6 3+ . Replacing SO4 2- with Cl- reduces the kosmotropic effect which further favors the electrostatic binding of Cr(III) to collagen. Our findings allow a greater understanding of mechanism-specific metal binding in the collagen molecule. We also report for the first time, surface-enhanced Raman spectroscopy to analyze binding mechanisms in collagen, suggesting a novel way to study chemical modifications in collagen-based biomaterials.