Rat Tail Tendon Research Articles

Static magnetic field effects on the secondary structure and elasticity of collagen molecules; a possible biophysical approach to treat keratoconus

Type I collagen is among the major extracellular proteins that play a significant role in the maintenance of the cornea's structural integrity and is essential in cell adhesion, differentiation, growth, and integrity. Here, we investigated the effect of 300 mT Static Magnetic Field (300 mT SMF) on the structure and molecular properties of acid-solubilized collagens (ASC) isolated from the rat tail tendon. The SMF effects at molecular and atomic levels were investigated by various biophysical approaches like Circular Dichroism Spectropolarimetery (CD), Fourier Transform Infrared Spectroscopy (FTIR), Zetasizer light Scattering, and Rheological assay. Exposure of isolated type I collagen to 300 mT SMF retained its triple helix. The elasticity of collagen molecules and the keratoconus (KCN) cornea treated with SMF decreased significantly after 5 min and slightly after 10, 15, and 20 min of treatments. The exposure to 300 mT SMF shifted the Amid I bond random coil to antiparallel wave number from 1647 to 1631 cm−1. The pH of the 300 mT SMF treated collagen solution increased by about 25 %. The treatment of the KCN corneas with 300 mT SMF decreased their elasticity significantly. The promising results of the effects of 300 mT SMF on the collagen molecules and KCN cornea propose a novel biophysical approach capable of manipulating the collagen's elasticity, surface charges, electrostatic interactions, cross binding, network formation and fine structure. Therefore, SMF treatment may be considered as a novel non-invasive, direct, non-chemical and fast therapeutic and manipulative means to treat KCN cornea where the deviated physico-chemical status of collagen molecules cause deformation.

Neural network auto-segmentation of serial-block-face scanning electron microscopy images exhibit collagen fibril structural differences with tendon type and health.

A U-Net machine learning algorithm was adapted to automatically segment tendon collagen fibril cross-sections from serial block face scanning electron microscopy (SBF-SEM) and create three-dimensional (3D) renderings. We compared the performance of routine Otsu thresholding and U-Net for a positional tendon that has low fibril density (rat tail tendon), an energy-storing tendon that has high fibril density (rat plantaris tendon), and a high fibril density tendon hypothesized to have disorganized 3D ultrastructure (degenerated rat plantaris tendon). The area segmentation of the tail and healthy plantaris tendon had excellent accuracy for both the Otsu and U-Net, with an Intersection over Union (IoU) of 0.8. With degeneration, only the U-Net could accurately segment the area, whereas Otsu IoU was only 0.45. For boundary validation, the U-Net outperformed Otsu segmentation for all tendons. The fibril diameter from U-Net was within 10% of the manual segmentation, however, the Otsu underestimated the fibril diameter by 39% in healthy plantaris and by 84% in the degenerated plantaris. Fibril geometry was averaged across the entire image stack and compared across tendon types. The tail had a lower fibril area fraction (58%) and larger fibril diameter (0.31 µm) than the healthy plantaris (67% and 0.21 µm) and degenerated plantaris tendon (66% and 0.19 µm). This method can be applied to a large variety of tissues to quantify 3D collagen fibril structure.

Tendon extracellular-matrix-derived tissue engineering micro-tissue for Achilles tendon injury regeneration in rats

BackgroundAchilles tendon is vital in maintaining the stability and function of ankle joint. It is quite difficult to achieve the structural and functional repair of Achilles tendon in tissue engineering.MethodsA tissue-engineered tendon micro-tissue was prepared using rat tail tendon extracellular matrix (TECM) combined with rat adipose stem cells (ADSCs) to repair Achilles tendon injuries. The TECM was prepared by repeated freezing and thawing. The in vitro characteristics of TECM and its effect on ADSCs proliferation were detected. This tissue-engineered tendon micro-tissue for Achilles tendon repair in vivo was evaluated based on general characteristics, gait analysis, ultrasound findings, histological analysis, and biomechanical testing.ResultsThe results showed that the TECM scaffold had good biocompatibility for ADSCs. At 2 weeks post-surgery, collagen types I and III and tenomodulin expression were higher, and vascular endothelial growth factor expression was lower in the micro-tissue group than other groups. At 4 and 8 weeks post-surgery, the results of histological analysis and ultrasound findings showed that the repaired tendon tissue was smooth and lustrous, and was arranged regularly and evenly in the micro-tissue group. Gait analysis confirmed that better motor function recovery was noted in micro-tissue group than other groups. In addition, the mechanical properties of the repaired tendon tissue in micro-tissue group were better than other groups.ConclusionTissue-engineered tendon micro-tissue fabricated by TECM and ADSCs has good biocompatibility and can promote structural and functional repair of tendon in vivo. This composite biomaterial has broad application prospects in tissue engineering.

Spectroscopic analysis of the sum-frequency response of the carbon-hydrogen stretching modes in collagen type I.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility. Using the three-dimensional atomic positions derived from published crystallographic data of collagen type I, we simulated its Xijk2 elements for the methylene stretching vibration and compared the predicted response with the experimental results. Our analysis revealed that the carbon-hydrogen stretching range of the SFG spectrum is dominated by symmetric stretching modes of methylene bridge groups on the pyrrolidine rings of the proline and hydroxyproline residues, giving rise to a dominant peak near 2942cm-1 and a shoulder at 2917cm-1. Weak asymmetric stretches of the methylene bridge group of glycine are observed in the region near 2870cm-1, whereas asymmetric CH2-stretching modes on the pyrrolidine rings are found in the 2980 to 3030cm-1 range. These findings help predict the protein's nonlinear optical properties from its crystal structure, thus establishing a connection between the protein structure and SFG spectroscopic measurements.

Strain inhibition of bacterial collagenase is consistent with a collagen fibril uncrimping mechanism in rat tail tendons

Mechanical strain inhibits bacterial collagenase from cleaving collagen. Additionally, the toe region of a soft tissue’s force–elongation curve arises from sequentially engaging collagen fibrils as the tissue lengthens. Together, these phenomena suggest that mechanical strain may gradually inhibit collagenase activity through a soft tissue’s toe region. Therefore, this investigation sought to test this hypothesis.92 rat tail tendon fascicles from 3 female sentinel animals underwent preliminary stiffness tests, and their force–elongation curves were fit to a collagen distribution model. This distribution-based model calculated the force magnitude corresponding to p% of collagen fibril engagement. Specimens were separated into one of five levels of p, and that level of force was maintained for two hours while being exposed to 0.054 U/mL of bacterial collagenase from C. histolyticum. The specimens were strained to failure following the creep test, and the relative reduction in stiffness was quantified to estimate the fraction of digested fibrils.Every 10% additional collagen engagement corresponded to a 6.3% (97% highest density interval: 4.3 – 8.4%) retention of stiffness, which indicated collagenase inhibition.The results of this investigation were consistent with a strain-inhibition hypothesis along with the established uncrimping mechanism in the toe region. These results support an interaction between mechanical strain and collagenolysis, which may be valuable for disease prevention or treatment.

Biochemical and morpho-mechanical properties, and structural organization of rat tail tendon collagen in diet-induced obesity model

We have investigated the impact of obesity on the structural organization, morpho-mechanical properties of collagen fibers from rat tail tendon fascicles (RTTFs). Polarized Raman microspectroscopy showed that the collagen bands 855, 875, 938, and 960 cm−1 as well as those 1631 and 1660 cm−1 were affected by diet. Mechanical properties exhibited an increase in the yield strength from control (CTRL) to high fat (HF) diet (9.60 ± 1.71 and 13.09 ± 1.81 MPa) (p < 0.01) and ultimate tensile strength (13.12 ± 2.37 and 18.32 ± 2.83 MPa) (p < 0.05) with no significant change in the Young's Modulus. During mechanical, the band at 875 cm−1 exhibited the most relevant frequency shift (2 cm−1). The intensity of those at 855, 875, and 938 cm−1 in HF collagen displayed a comparable response to mechanical stress as compared to CTRL collagen with no significant diet-related changes in the Full Width at Half Maximum. Second harmonic generation technique revealed i) similar fiber straightness (0.963 ± 0.004 and 0.965 ± 0.003) and ii) significant changes in fibers diameter (1.48 ± 0.07 and 1.52 ± 0.08 μm) (p < 0.05) and length (22.06 ± 2.38 and 29.00 ± 3.76 μm) (p < 0.001) between CTRL and HF diet, respectively. The quantification of advanced glycation end products (AGEs) revealed an increase in both carboxymethyl-lysine and total fluorescence AGEs from CTRL to HF RTTFs.

Cell-Laden 3D Hydrogels of Type I Collagen Incorporating Bacterial Nanocellulose Fibers.

There is a growing interest in developing natural hydrogel-based scaffolds to culture cells in a three-dimensional (3D) millieu that better mimics the in vivo cells' microenvironment. A promising approach is to use hydrogels from animal tissues, such as decellularized extracellular matrices; however, they usually exhibit suboptimal mechanical properties compared to native tissue and their composition with hundreds of different protein complicates to elucidate which stimulus triggers cell's responses. As simpler scaffolds, type I collagen hydrogels are used to study cell behavior in mechanobiology even though they are also softer than native tissues. In this work, type I collagen is mixed with bacterial nanocellulose fibers (BCf) to develop reinforced scaffolds with mechanical properties suitable for 3D cell culture. BCf were produced from blended pellicles biosynthesized from Komagataeibacter xylinus. Then, BCf were mixed with concentrated collagen from rat-tail tendons to form composite hydrogels. Confocal laser scanning microscopy and scanning electron microscopy images confirmed the homogeneous macro- and microdistribution of both natural polymers. Porosity analysis confirmed that BCf do not disrupt the scaffold structure. Tensile strength and rheology measurements demonstrated the reinforcement action of BCf (43% increased stiffness) compared to the collagen hydrogel while maintaining the same viscoelastic response. Additionally, this reinforcement of collagen hydrogels with BCf offers the possibility to mix cells before gelation and then proceed to the culture of the 3D cell scaffolds. We obtained scaffolds with human bone marrow-derived mesenchymal stromal cells or human fibroblasts within the composite hydrogels, allowing a homogeneous 3D viable culture for at least 7 days. A smaller surface shrinkage in the reinforced hydrogels compared to type I collagen hydrogels confirmed the strengthening of the composite hydrogels. These collagen hydrogels reinforced with BCf might emerge as a promising platform for 3D in vitro organ modeling, tissue-engineering applications, and suitable to conduct fundamental mechanobiology studies.

Electromechanical Coupling in Collagen Measured under Increasing Relative Humidity.

The functional role of collagen piezoelectricity has been under debate since the discovery of piezoelectricity in bone in 1957. The possibility that piezoelectricity plays a role in bone remodeling has generated interest in the investigation of this effect in relevant physiological conditions; however, there are conflicting reports as to whether collagen is piezoelectric in a humid environment. In macroscale measurements, the piezoelectricity in hydrated tendon has been shown to be insignificant compared to dehydrated tendon, whereas, at the nanoscale, the piezoelectric effect has been observed in both dry and wet bone using piezoresponse force microscopy (PFM). In this work, the electromechanical properties of type I collagen from a rat tail tendon have been investigated at the nanoscale as a function of humidity using lateral PFM (LPFM) for the first time. The relative humidity (RH) was varied from 10% to 70%, allowing the piezoelectric behavior to be studied dry, humid, as well as in the hydrated range for collagen in physiological bone (12% moisture content, corresponding to 40-50% RH). The results show that collagen piezoresponse can be measured across the humidity range studied, suggesting that piezoelectricity remains a property of collagen at a biologically relevant humidity.

Polarimetric second harmonic generation microscopy of partially oriented fibers II: Imaging study

Polarimetric second harmonic generation (SHG) microscopy imaging is employed to investigate the ultrastructural organization of biological and biomimetic partially oriented fibrillar structures. The linear polarization-in polarization-out SHG microscopy measurements are conducted with rat tail tendon, rabbit cornea, pig cartilage, and biomimetic meso-tetra(4-sulfonatophenyl)porphine (TPPS4) cylindrical aggregates, which represent different two- and three-dimensional (2D and 3D) configurations of C6 symmetry fibril structures in the focal volume (voxel) of the microscope. The polarization-in polarization-out imaging of rat tail tendon reveals that SHG intensity is affected by parallel/antiparallel arrangements of the fibers, and achiral (R) and chiral (C) susceptibility component ratio values change by tilting the tendon fibers out of image plane. The R ratio changes for the 2D crossing fibers observed in cornea tissue. The 3D crossing of fibers also affects R ratio in cartilage tissue. The distinctly different dependence of R on crossing and tilting of fibers is demonstrated in collagen and TPPS4 aggregates, due to the achiral molecular susceptibility ratio having values below and above 3, respectively. The polarimetric microscopy results correspond well with the analytical expressions of amplitude and R and C ratios dependence on the crossing angle of the fibers. The experimentally measured SHG intensity and R and C ratio maps are consistent with the computational modeling of various fiber configurations presented in the preceding article. The demonstrated SHG intensity and R and C ratio dependencies on fibril configurations provide the basis for interpreting polarimetric SHG microscopy images in terms of 3D ultrastructural organization of fibers in each voxel of the samples.

Insight into the synthesis of biocompatible polymer for collagen stabilization and leather process

Collagen-based biomaterials are highly effective owing to their high biocompatibility and non-immunogenicity, but their biological application is still constrained because of poor thermal stability. The greener crosslinkers devoid of aldehydes and toxic precursors have recently evoked considerable interest. In this work, biocompatible and multifunctional carboxyl polymer (MCP) has been synthesized by a simple one-step carboxylation reaction by modifying the hydroxyl group in the monomer, followed by polymerization in aqueous conditions. The synthesized polymer has been used for both collagen stabilization and leather processing. The circular dichroism studies revealed a significant change in the secondary structure of the collagen with increasing concentration of the MCP, and thermal stability of the rat tail tendon is enhanced up to 70 °C. The application of MCP as a crosslinker for collagen in the leather-making process along with metal salts increased the leather's thermal stability and improved uptake of the metal salts. The interaction mechanism of MCP and the metals during the tanning process is through complexation with carboxyl pendants available on the MCP backbone, as confirmed by UV–Visible and IR spectroscopy. The experimental leathers showed a uniform penetration of tanning agents and excellent mechanical properties compared to the control leathers. Further, biocompatibility studies using 3 T3 fibroblast cells revealed that the MCP did not induce any significant cytotoxicity with cell viability of 96%, confirming the non-toxic nature of the synthesized polymer. Thus, the current study opens a new path for sustainable alternative polymeric crosslinkers for collagen stabilization and its potential application in biomaterial and leather-making fields.

Single collagen fibrils isolated from high stress and low stress tendons show differing susceptibility to enzymatic degradation by the interstitial collagenase matrix metalloproteinase-1 (MMP-1)

Bovine forelimb flexor and extensor tendons serve as a model for examining high stress, energy storing and low stress, positional tendons, respectively. Previous research has shown structural differences between the collagen fibrils of these tissues. The nanoscale collagen fibrils of flexor tendons are smaller in size, more heavily crosslinked, and respond differently to mechanical loading. Meanwhile, energy storing tendons undergo less collagen turnover compared to positional tendons and are more commonly injured. These observations raise the question of whether collagen fibril structure influences the collagen degradation processes necessary for remodelling. Atomic force microscopy was used to image dry collagen fibrils before and after 5-hour exposure to matrix metalloproteinase-1 (MMP-1) to detect changes in fibril size. Collagen fibrils from three tissue types were studied: bovine superficial digital flexor tendons, matched-pair bovine lateral digital extensor tendons, and rat tail tendons. Compared to control fibrils exposed only to buffer, a significant decrease in fibril cross-sectional area (CSA) following MMP-1 exposure was observed for bovine extensor and rat tail fibrils, with larger fibrils experiencing a greater magnitude of CSA decrease in both fibril types. Fibrils from bovine flexor tendons, on the other hand, showed no decrease in CSA when exposed to MMP-1. The result did not appear to be linked to the small size of flexor fibrils, as equivalently sized extensor fibrils were readily degraded by the enzyme. Increased proteolytic resistance of collagen fibrils from high stress tendons may help to explain the longevity of collagen within these tissues in vivo.

Fabrication of nanofibrous mat surrounded hydrogel scaffold as an encapsulation device for encapsulating pancreas β cells

The main barriers to cells or organ transplantation such as pancreatic β-cells are the need for lifelong immune suppression and the shortage of donors. It may be overcome via cell encapsulation and transplantation techniques. Hydrogels provide a suitable ECM-like microenvironment for cells to adhere, survive, and function, while weakly performing as an immune barrier. In this study, we aimed to macro-encapsulate islet cells in a dual encapsulation device with collagen hydrogel and PCL nanofiber to provide an immune-isolated environment for cells to function more efficiently, where immune cells are not allowed to enter but oxygen, insulin, and nutrients can pass through. PCL thin mats with the pores diameter of 500 nm were synthesized by electrospinning and characterized by scanning electron microscope, porosity measurement, tensile strength test, and contact angle measurement. Collagen hydrogel was fabricated by extracting collagen fibers from rat tail tendons and solving them in acetic acid. β-cells (CRI-D2 cell line) encapsulated after neutralizing collagen solution (pH ≈ 7.4). Cell-collagen gel complex was poured into the nanofibrous mat packets to fabricate the whole device. Histology evaluation, cell viability, and cell function tests were done in 10 days. Live/dead assay of Cri-D2 cells encapsulated within the device showed that cells have diffuse distribution at the core of the hydrogel and the device. Also, cluster formation was seen and shows these cells can live in groups. To identify cells’ function within the device in these 10 days samples’ supernatant insulin level was measured by chemiluminescent immunoassay. It just showed a positive result for existing insulin within the medium. Based on our results, this device presents adequate features to be a good immune-isolation device for cell transplanting.

Photoreactive Hydrogels Based on Type I Collagen Extracted from Different Sources as Scaffolds for Tissue Engineering Applications: A Comparative Study

Collagen-based hydrogels are promising constructs used frequently as scaffolds in various tissue engineering applications. The source of collagen extraction can influence the physiochemical properties, mechanical strength, as well as cellular behaviour of the tissue-engineered hydrogels. In the present study, type I collagen was isolated from three different animal sources, namely bovine Achilles tendon, bighead carp fish skin, and rat tail tendons. After characterizing the extracted collagens, their structures were modified via covalent functionalization of the free amines with methacrylate groups. Methacrylated collagens were combined with PEG-diacrylate to act as three-dimensional photocrosslinked scaffolds for culturing human corneal stromal cells. Physicochemical properties, mechanical strength, morphological features, and cellular behaviour were different depending on the collagen sources. Human corneal stromal cells more easily penetrated within hydrogels containing rat tail type I collagen, which also had greater pore sizes and porosity. However, the expressions of type I collagen and lumican were observed to be a greater extent in fish skin collagen-based hydrogels. Such observations highlight the significant effect of collagen extraction source on cellular behaviour and the importance of having a proper selection in accordance with the application of the tissue-engineered scaffold.

Mechanical Properties of Mineralized Collagen Type I Rat-Tail Tendon Fascicles

Type I collagen fibers transmit forces, dissipate energy, and prevent mechanical failure in normal tissues. Mineralization of collagen fibers is proposed to strengthen these structural proteins as a possible therapeutic option for small tears in soft tissues such as cemental tears in periodontal ligaments. In the present study, collagen fascicles extracted from the tail tendons of male Sprague-Dawley rats were tested in a MiniMat tensile tester at different strain rates. The fascicles were previously immersed in SBF to induce mineralization and were maintained in a moist condition during mechanical testing. Mineralization of the fascicles resulted in an increase in the modulus of elasticity and tensile strength with minimal change in percentage elongation to failure. From SEM/EDS, calcium phosphate deposits formed on the fascicles indicating a high probability of intrafibrillar mineralization resulting in the observed improvement in mechanical properties.

Effect of copper and zinc ions on type I collagen self-assembly

Owing to their central roles in cellular signaling, construction, and biochemistry, collagen-collagen interactions, and collagen self-assembly have become a major focus of molecular design and synthetic biology. The well-ordered assembly of collagen molecules on mica has attracted extensive attention for its essential applications and studies on the self-assembly process as a model system. Although collagen assembly mediated by various ions on mica has been reported over these years, specific effects of transition metal ions in this field have never been deeply surveyed and discussed till now. In this work, type I collagen extracted from rat tail tendon by pepsin solubilization method was selected to explore the kinetic process and self-assembly characteristics. In the presence of Cu2+ and Zn2+ sulfates, the turbidity method was used to directly measure the kinetic process of collagen self-assembly and atomic force microscopy (AFM) was applied to observe the patterns of collagen self-assembly on mica. The results showed that the favorable properties of transition metal coordination (bonding strength, directionality, and reversibility) were helpful for increasing the collagen fibrillogenesis rate, and at different ion concentrations, Cu2+ and Zn2+ ions affected the interactions between collagen molecules and mica surface by destroying the hydration membranes on the surface of collagen molecules and the hydrogen bonds between the amino acid residues of collagen molecules. Circular dichroism (CD) analysis demonstrated that collagen had complete triple-strand conformation in the presence of Cu2+ and Zn2+. These results and analysis would be helpful for directing the patterns of collagen self-assembly on a solid surface and provide theoretical guidance for creating collagen-based high-performance materials and/or adding functionality in materials for various applications.

Development of a continuum damage model to predict accumulation of sub-failure damage in tendons

Many painful and physically debilitating conditions involve sub-failure mechanical damage to seemingly intact connective tissues such as tendons and ligaments. We found that the amount of denatured collagen in rat tail tendon (RTT) fascicles increased over experiments of cyclic loading to a constant load level (creep cyclic fatigue) with fluorescently tagged collagen hybridizing peptides (CHPs) that bind to denatured collagen. To better understand tendon sub-failure damage progression, computational modeling of tendon materials via finite element analysis in FEBio has been conducted. The objective of this project was to develop, implement, and test the ability of a new continuum damage mechanics (CDM) model in FEBio to represent the sub-failure damage behavior seen in our RTT fascicle creep cyclic fatigue experimental data.There appeared to be two distinct mechanisms responsible for the creep cyclic fatigue softening behavior of RTT fascicles over the number of cycles to failure: the preconditioning effect and overall collagen damage. In our finite element (FE) models, the RTT fascicle undamaged elastic constitutive material was composed of a matrix and fibers described by the Coupled Veronda-Westmann and exponential-linear materials. This undamaged elastic material was convolved with a modified CDM model adapted from Balzani et al., in 2012.The novelty of the Balzani damage model is the inclusion of two interrelated mechanisms described as continuous and discontinuous damage. The continuous damage formulation calculates damage accumulation during the loading and reloading of each new cycle, while the discontinuous damage approach accumulates damage from the maximum strain over the loading history to the current time. We modified the Balzani damage model formulations to represent exponential and sigmoidal increases in damage marked by the preconditioning effect and collagen damage in RTT fascicles as functions of continuous and discontinuous damage. The original Balzani damage model was first verified, then the modified CDM model was implemented into FEBio and used to reproduce the sample specific experimental creep cyclic fatigue stress-strain data as well as predict incremental cyclic fatigue. The resulting model will be useful for future experimental and computational studies of damage mechanics to understand tendon pathologies.

VEGF-D-mediated signaling in tendon cells is involved in degenerative processes.

Vascular endothelial growth factor (VEGF) signaling is crucial for a large variety of cellular processes, not only related to angiogenesis but also in nonvascular cell types. We have previously shown that controlling angiogenesis by reducing VEGF-A signaling positively affects tendon healing. We now hypothesize that VEGF signaling in non-endothelial cells may contribute to tendon pathologies. By immunohistochemistry we show that VEGFR1, VEGFR2, and VEGFR3 are expressed in murine and human tendon cells in vivo. In a rat Achilles tendon defect model we show that VEGFR1, VEGFR3, and VEGF-D expression are increased after injury. On cultured rat tendon cells we show that VEGF-D stimulates cell proliferation in a dose-dependent manner; the specific VEGFR3 inhibitor SAR131675 reduces cell proliferation and cell migration. Furthermore, activation of VEGFR2 and -3 in tendon-derived cells affects the expression of mRNAs encoding extracellular matrix and matrix remodeling proteins. Using explant model systems, we provide evidence, that VEGFR3 inhibition prevents biomechanical deterioration in rat tail tendon fascicles cultured without load and attenuates matrix damage if exposed to dynamic overload in a bioreactor system. Together, these results suggest a strong role of tendon cell VEGF signaling in mediation of degenerative processes. These findings give novel insight into tendon cell biology and may pave the way for novel treatment options for degenerative tendon diseases.

Inhibition of Advanced Glycation End Product Formation in Rat Tail Tendons by Polydatin and p-Coumaric acid: an In Vitro Study.

Advanced glycation end products (AGEs) formed through non-enzymatic glycosylation between a protein and sugar molecule are highly harmful to the human body. In hyperglycemic patients, AGE formation is more due to high glucose circulating in the blood, causing inter and intra molecular cross-linking of collagen leading to reduction of collagen elasticity. This cross-linked collagen develops resistance to matrix metalloproteinases leading to impaired collagen turnover. The aim of this work is to determine the anti-glycation effects of polydatin and p-coumaric acid in preventing collagen cross-linking by incubating rat tail tendons (RTTs) as collagen source in high glucose concentration (50mM) for a week. The RTTs were then characterized for tensile strength, cross-linking efficiency, circular dichroism spectrometry, collagen, glucose, and aldehyde contents. Electrophoresis was carried out to evaluate the level of cross-linking in collagen and the results confirmed the ability of the drugs in preventing complex intermolecular cross-link formation induced by non-enzymatic glycosylation. CD data showed alteration in the secondary structure of collagen where AGE formation had occurred. More collagen was extracted by pepsin from RTTs treated with glucose alone (6.88mg/10mg tendon) when compared with drug-treated groups (4.25, 2.56mg/10mg tendon for polydatin and p-coumaric acid, respectively). Tensile strength (20.66% and 18.95%), cross-linking percentage (32.5% and 29.84%), and glucose content (2.3 and 1.8mg/100mg) of drug-treated groups were similar to the positive control (19.07%, 30.13%, and 2.61mg/100mg) thus proving the anti-glycation potential of the drugs. Hence, both polydatin and p-coumaric acid could play a pivotal role in preventing AGE formation.

Effect of Solvent on the Hydrodynamic Properties of Collagen.

In this study, the effect of solvent on the hydrodynamic properties of collagen extracted from tail tendons of young rats was researched. Collagen was dissolved in various aqueous carboxylic acid solutions, including acetic acid (AA), acetic acid with the addition of sodium chloride (AA/NaCl), formic acid (FA), lactic acid (LA), citric acid (CA), and also citrate buffer at pH = 3.7 (CB). The properties of collagen solutions at a concentration of 0.45 mg/mL were characterized based on the viscometric method. The reduced viscosity, intrinsic viscosity, and Huggins coefficient of collagen solutions and effect of solvent, temperature, and UV irradiation on these properties were investigated. Collagen solutions in acetic acid, acetic acid/NaCl, and citrate buffer were irradiated with UV light up to 1 h, and the viscosity of collagen solutions was measured. It was found that the organic acids used as solvent affected viscosity behavior, denaturation temperature, and stability of collagen solutions. The lowest values of studied parameters were obtained for the collagen solutions in acetic acid with the addition of sodium chloride. Thus, the effect of various aqueous carboxylic acid solutions on collagen solutions properties and denaturation temperature can also be affected by the sodium chloride addition. The results of this research can be crucial for the preparation of collagen solutions for both cosmetic and biomedical applications.

An integrated approach to investigate age-related modifications of morphological, mechanical and structural properties of type I collagen

The main propose of this study is to characterize the impact of chronological aging on mechanical, structural, biochemical, and morphological properties of type I collagen. We have developed an original approach combining a stress-strain measurement device with a portable Raman spectrometer to enable simultaneous measurement of Raman spectra during stress vs strain responses of young adult, adult and old rat tail tendon fascicles (RTTFs). Our data showed an increase in all mechanical properties such as Young's modulus, yield strength, and ultimate tensile strength with aging. At the molecular level, Raman data revealed that the most relevant frequency shift was observed at 938 cm−1 in Old RTTFs, which is assigned to the C–C. This suggested a long axis deformation of the peptide chains in Old RTTFs during tensile stress. In addition, the intensity of the band at 872 cm−1, corresponding to hydroxyproline decreased for young adult RTTFs and increased for the adult ones, while it remained unchanged for Old RTTFs during tensile stress. The amide III band (1242 and 1265 cm−1) as well as the band ratios I1631/ I1663 and I1645 / I1663 responses to tensile stress were depending on mechanical phases (toe, elastic and plastic). The quantification of advanced glycation end-products by LC-MS/MS and spectrofluorometry showed an increase in their content with aging. This suggested that the accumulation of such products was correlated to the alterations observed in the mechanical and molecular properties of RTTFs. Analysis of the morphological properties of RTTFs by SHG combined with CT-FIRE software revealed an increase in length and straightness of collagen fibers, whereas their width and wavy fraction decreased. Our integrated study model could be useful to provide additional translational information to monitor progression of diseases related to collagen remodeling in musculoskeletal disorders. Statement of significanceType I collagen is the major component of the extracellular matrix. Its architectural and structural organization plays an important role in the mechanical properties of many tissues at the physiological and pathological levels. The objective of this work is to develop an integrated approach to bring a new insight on the impact of chronological aging on the structural organization and mechanical properties of type I collagen. We combined a portable Raman spectrometer with a mechanical tensile testing device in order to monitor in real time the changes in the Raman fingerprint of type I collagen fibers during the mechanical stress. Raman spectroscopy allowed the identification of the type I collagen bonds that were affected by mechanical stress in a differential manner with aging.