Supramolecular order following binding of the dichroic birefringent sulfonic dye Ponceau SS to collagen fibers

The optical anisotropies (linear dichroism or LD and birefringence) of crystalline aggregates of the sulfonic azo-dye Ponceau SS and of dye complexed with chicken tendon collagen fibers were investigated in order to assess their polarizing properties and similarity to liquid crystals. In some experiments, the staining was preceded by treatment with picric acid. Crystalline fibrous aggregates of the dye had a negative LD, and their electronic transitions were oriented perpendicular to the filamentary structures. The binding of Ponceau SS molecules to the collagen fibers altered the LD signal, with variations in the fiber orientation affecting the resulting dichroic ratios. The long axis of the rod-like dye molecule was assumed to be bound in register, parallel to the collagen fiber. Picric acid did not affect the oriented binding of the azo dye to collagen fibers. There were differences in the optical anisotropy of Ponceau SS-stained tendons from 21-day-old and 41-day-old chickens, indicating that Ponceau SS was able to distinguish between different ordered states of macromolecular aggregation in chicken tendon collagen fibers. In the presence of dichroic rod-like azo-dye molecules such as Ponceau SS, collagen also formed structures with a much higher degree of orientation. The presence of LD in the Ponceau SS-collagen complex even in unpolarized light indicated that this complex can act as a polarizer.     

Organization of collagen bundles during tendon healing in rats treated with L-NAME

The Achilles tendon can support high tension forces and may experience lesions. The damaged tissue does not regenerate completely, with the organization and mechanical properties of the repaired tendon being inferior to those of a healthy tendon. Nitric oxide (NO) plays an important role in wound repair. We have examined the structural reorganization and repair in Achilles tendon after injury in rats treated with the NO synthase inhibitor L-NAME. The right Achilles tendon of male Wistar rats was partially transected. One group of rats was treated with L-NAME (~300 mg/kg per day, given in drinking water) for 4 days prior to tendon sectioning and throughout the post-operative period. Control rats received water without L-NAME. The tendons were excised at 7, 14, and 21 days post-injury and used to quantify hydroxyproline and for mechanical tests. Tendons were also processed for histomorphological analysis by polarized light microscopy, which showed that the collagen fibers were disorganized by day 7 in non-treated and L-NAME-treated rats. In non-treated rats, the organization of the extracellular matrix was more homogeneous by days 14 and 21 compared with day 7, although this homogeneity was less than that in normal tendon. In contrast, in injured tendons from L-NAME-treated rats, the collagen fibers were still disorganized on day 21. Tendons from treated rats had more hydroxyproline but lower mechanical properties compared with those from non-treated rats. Thus, NO modulates tendon healing, with a reduction in NO biosynthesis delaying reorganization of the extracellular matrix, especially collagen. T.C.T. and W.R.N were supported by studentships from CAPES, and S.H. was supported by a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).     

Tendon crimps and peritendinous tissues responding to tensional forces

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.     

Regeneration of rabbit calcaneal tendon

Summary The regenerated tissue which fills the gap between the stumps of sectioned and unsutured rabbit calcaneal tendon was studied by immuno-fluorescence, light and electron microscopy from 2 days to 30 weeks after surgery. In the early stages, the newly formed tissue consisted of few connective tissue cells of variable shape dispersed in an abundant intercellular matrix. At 7 days after tenotomy most of the cells were spindle shaped and arranged along the major tendon axis. They showed a well developed rough endoplasmic reticulum, a prominent Golgi complex and bundles of thin and thick filaments. Moreover, they appeared intensely stained when treated with anti-actin and anti-myosin sera. The bulk of the intercellular matrix consisted of bundles of collagen fibers, mostly arranged parallel to the cells.In the subsequent stages the regenerating tissue became more compact, acquiring the morphological characteristics of tendon tissue. At 30 weeks after tenotomy, however, it did not show yet the typical texture of the normal adult tendon. The tenocytes were more numerous and less uniformly distributed, and contained a greater amount of ergastoplasm and contractile proteins. The collagen fibers were similar in size to those of the neonatal normal tendon and the elastic fibers appeared often immature.These findings suggest that, at least on the experimental conditions under which this study was performed, the regenerated tendon does not acquire the typical morphology of the normal adult tendon, possibly owing to the reduced mechanical stress acting on it.     

A quantitative collagen fibers orientation assessment using birefringence measurements: calibration and application to human osteons

Even though mechanical properties depend strongly on the arrangement of collagen fibers in mineralized tissues, it is not yet well resolved. Only a few semi-quantitative evaluations of the fiber arrangement in bone, like spectroscopic techniques or circularly polarized light microscopy methods are available. In this study the out-of-plane collagen arrangement angle was calibrated to the linear birefringence of a longitudinally fibered mineralized turkey leg tendon cut at variety of angles to the main axis. The calibration curve was applied to human cortical bone osteons to quantify the out-of-plane collagen fibers arrangement. The proposed calibration curve is normalized to sample thickness and wavelength of the probing light to enable a universally applicable quantitative assessment. This approach may improve our understanding of the fibrillar structure of bone and its implications on mechanical properties.     

Fiberoptic measurement of tendon forces is influenced by skin movement artifact

Fiberoptic cables have previously been used for tendon force measurements in vivo. To measure forces in the Achilles tendon, a cable is passed mediolaterally through the skin and tendon, transverse to the loading axis. As the tendon is loaded, its fibers compress the cable and modulate the intensity of transmitted light, which can be related to tendon force by an in situ calibration. The relative movement between skin and tendon at the cable entry and exit sites may cause error by bending the cable and thus altering transducer output. Cadaver simulations of walking were conducted to compare fiberoptic measurements of Achilles tendon forces to known loads applied to the tendon by actuators attached in series. Force measurement errors, which were high when the skin was intact (RMS errors 24-81% peak forces), decreased considerably after skin removal (RMS errors 10-33% peak forces). The fiberoptic transducer is a useful tool for measurement of tendon forces in situ under natural loading conditions when skin can be removed, but caution should be exercised during in vivo use of this technique or under circumstances where skin is in contact with the fiberoptic cable at the insertion and exit sites.     

Biaxial tensile testing and constitutive modeling of human supraspinatus tendon

The heterogeneous composition and mechanical properties of the supraspinatus tendon offer an opportunity for studying the structure-function relationships of fibrous musculoskeletal connective tissues. Previous uniaxial testing has demonstrated a correlation between the collagen fiber angle distribution and tendon mechanics in response to tensile loading both parallel and transverse to the tendon longitudinal axis. However, the planar mechanics of the supraspinatus tendon may be more appropriately characterized through biaxial tensile testing, which avoids the limitation of nonphysiologic traction-free boundary conditions present during uniaxial testing. Combined with a structural constitutive model, biaxial testing can help identify the specific structural mechanisms underlying the tendon's two-dimensional mechanical behavior. Therefore, the objective of this study was to evaluate the contribution of collagen fiber organization to the planar tensile mechanics of the human supraspinatus tendon by fitting biaxial tensile data with a structural constitutive model that incorporates a sample-specific angular distribution of nonlinear fibers. Regional samples were tested under several biaxial boundary conditions while simultaneously measuring the collagen fiber orientations via polarized light imaging. The histograms of fiber angles were fit with a von Mises probability distribution and input into a hyperelastic constitutive model incorporating the contributions of the uncrimped fibers. Samples with a wide fiber angle distribution produced greater transverse stresses than more highly aligned samples. The structural model fit the longitudinal stresses well (median R(2) ≥ 0.96) and was validated by successfully predicting the stress response to a mechanical protocol not used for parameter estimation. The transverse stresses were fit less well with greater errors observed for less aligned samples. Sensitivity analyses and relatively affine fiber kinematics suggest that these errors are not due to inaccuracies in measuring the collagen fiber organization. More likely, additional strain energy terms representing fiber-fiber interactions are necessary to provide a closer approximation of the transverse stresses. Nevertheless, this approach demonstrated that the longitudinal tensile mechanics of the supraspinatus tendon are primarily dependent on the moduli, crimp, and angular distribution of its collagen fibers. These results add to the existing knowledge of structure-function relationships in fibrous musculoskeletal tissue, which is valuable for understanding the etiology of degenerative disease, developing effective tissue engineering design strategies, and predicting outcomes of tissue repair.     

Local NO synthase inhibition produces histological and functional recovery in Achilles tendon of rats after tenotomy

Repair of injured tendon is a very slow process and involves the release of many molecules, including nitric oxide. We investigate the influence of local nitrergic inhibition in histological and functional recovery of injured Achilles tendon. A standard murine model of tendon injury by rupture was used. The animals were divided into three experimental groups: control, injury + vehicle (normal saline) and injury + Nω-nitro-L-arginine methyl ester (L-NAME). The products were injected into the paratendinous region every 2 days and body weight gain and Achilles functional index (AFI) were evaluated on days 0, 7, 14 and 21 after tendon injury. On day 21 post-injury, the animals were killed to evaluate nitric oxide production and tissue organization. We observed that tendon surgical division led to increased tissue nitrite levels, which were reduced in L-NAME-treated rats. The AFI revealed functional recovery of L-NAME-treated animals on day 21 post-injury, which was not observed in the saline-treated group. Microscopic analysis of hematoxylin-eosin staining and collagen autofluorescence showed that L-NAME-treated rats had more aligned areas of collagen fibers and that the diameter of newly organized collagen in this group was also greater than that in the vehicle-treated one. We demonstrate that local treatment with L-NAME significantly improves the functional parameters and accelerates histomorphological recovery.     

The absence of oestrogen receptor beta disturbs collagen I type deposition during Achilles tendon healing by regulating the IRF5‐CCL3 axis

Achilles tendon healing (ATH) remains an unanswered question in the field of sports medicine because it does not produce tissue with homology to the previously uninjured tissue. Oestrogen receptor β (ERβ) is involved in the injury and repair processes of tendons. Our previous study confirmed that ERβ plays a role in the early stage of ATH by affecting adipogenesis, but its role in extracellular matrix (ECM) remodelling is unknown. We established a 4‐week Achilles tendon repair model to investigate the mechanism through which ERβ affects ATH at the very beginning of ECM remodelling phase. In vitro studies were performed using tendon‐derived stem cells (TDSCs) due to their promising role in tendon healing. Behavioural and biomechanical tests revealed that ERβ‐deficient mice exhibit weaker mobility and inferior biomechanical properties, and immunofluorescence staining and qRT‐PCR showed that these mice exhibited an erroneous ECM composition, as mainly characterized by decreased collagen type I (Col I) deposition. The changes in gene expression profiles between ERβ‐knockout and WT mice at 1 week were analysed by RNA sequencing to identify factors affecting Col I deposition. The results highlighted the IRF5‐CCL3 axis, and this finding was verified with CCL3‐treated TDSCs. These findings revealed that ERβ regulates Col I deposition during ATH via the IRF5‐CCL3 axis.     

Changes induced by ozone and ultraviolet light in type I collagen. Bovine Achilles tendon collagen versus rat tail tendon collagen

High-molecular-mass aggregates were made soluble from insoluble collagens of bovine Achilles tendon and rat tail tendon by limited thermal hydrolysis. These polymeric collagen aggregates were cross-linked by 390-nm-fluorescent 3-hydroxy-pyridinium residues (excited at 325 nm) in the former tendon and by unknown non-fluorescent residues in the latter. With the solubilized insoluble-collagens from both tendons, as well as with acid-soluble collagen from rat tail tendon, other 350-385-nm fluorescence intensities (excited at 300 nm) were found to be higher in monomeric chains than in dimeric and polymeric chains. Low levels of ozone inhibited fibril formation of acid-soluble collagen particularly from young rat tail tendon, reacting with tyrosine residues and the 350-385-nm fluorophores. Aldehyde groups, involved in cross-linking, were not effectively modified by ozone. beta-Components (alpha-chain dimers) were not efficiently dissociated even by higher doses of ozone compared to gamma-components (alpha-chain trimers). Polymeric chain aggregates from bovine Achilles tendon collagen, whose 3-hydroxy-pyridinium cross-links are cleaved by ozone, were more readily dissociated by ozone than those from rat tail tendon collagen. Ultraviolet (300-nm) light, which destroyed the 350-385-nm fluorophores, inhibited fibril formation less effectively than ultraviolet (275-nm) light, which is absorbed by tyrosine residues, and did not dissociate collagen polymers from rat tail tendon. On the other hand, ultraviolet (320-nm) light, absorbed by 3-hydroxy-pyridinium cross-links which were rapidly photolyzed, partially dissociated polymeric collagen aggregates from bovine Achilles tendon after subsequent heating.     

Is the tendon embryogenesis process resurrected during tendon healing?

The process of embryonic tendon development, including the nature and purpose of collagen fibril segments, is reviewed. It is proposed that tendon fibrillogenesis of repair is related to the fibrillogenesis of tendon embryonic development. The assembly of collagen fibril segment units into longer fibers occurs on the surface of tendon fibroblasts in embryonic tendon development. The biochemist's view of tendon healing, whereby the spontaneous polymerization of tropocollagen monomers regenerates lost tendon collagen fibers, needs to be reconsidered. Furthermore, the importance of direct fibroblast involvement in collagen fiber reassembly during tendon healing needs to be studied in tendon intrinsic regenerative repair.     

Polarization microscopy and microspectrophotometry of Sirius Red, Picrosirius and Chlorantine Fast Red aggregates and of their complexes with collagen

Summary A detailed quantitative analysis of the anisotropic properties of Sirius Red F3B, Picrosirius, and Chlorantine Fast Red crystals, and of their complexes with a macromolecularly oriented protein either in a pure form or as part of a tissue structure was carried out. Collagen I was used as the protein model. Linear dichroism and dispersion of birefringence were investigated in dye aggregates, in stained filaments of collagen I and in collagen bundles in sections of tendon. A positive linear dichroism, the characteristics of which varied as a function of the dye type used, was demonstrated for the dye aggregates and stained substrates. However, even thin regions of the stained tendon collagen bundles showed very high absorbances, differing from the pattern reported previously, for collagen stained with another sulphonated azo dye, Xylidine Ponceau. Consequently, not all these dyes enable protein concentration and orientation to be determined in collagen-containing structures. From the linear dichroism patterns it is assumed that the long axis of the molecules of these azo dye is mostly parallel to that of filaments of pure collagen I and statistically parallel to the long axis of collagen bundles of tendon sections. The dye aggregates and, stained pure collagen I and tendon collagen bundles exhibited birefringent images with interference colours that varied as a function of thickness and packing state of the preparations, which is in agreement with reports in the literature. The optical retardations of the collagen bundles increased by a factor of 5–6 times after staining with Picrosirius. From data on form dichroism it is concluded that when studying the macromolecular orientation of collagen preparations stained with azo dyes, the choice of the mounting medium deserves consideration.     

Absorption spectral curves of dichroism on collagen bundles

Summary The dichroism on tendons stained with Toluidine Blue was investigated under conditions which reveal metachromasy. The spectral curves of the dichroism were determined by histophotometry and showed maxima of light absorption at different wavelengths, according to the fiber direction with respect to the plan of polarized light.The results were discussed in function of the occurrence of hybrids of resonance which would have their balance changed by inducing electronic transitions.     

Denervation does not change the ratio of collagen I and collagen III mRNA in the extracellular matrix of muscle

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle and the mechanics of tendon. It has been proposed that a shift in the collagen of the extracellular matrix (ECM) of the muscle, increasing type III and decreasing type I collagen, may be partially responsible for the observed changes. We directly investigated this hypothesis using quantitative real-time PCR on muscles and tendons that had been denervated for 5 wk. Five weeks of denervation resulted in a 2.91-fold increase in collagen concentration but no change in the content of collagen in the muscle, whereas in the tendon there was no change in either the concentration or content of collagen. The expression of collagen I, collagen III, and lysyl oxidase mRNA in the ECM of muscle decreased (76 +/- 1.6%, 73 +/- 2.3%, and 83 +/- 3.2%, respectively) after 5 wk of denervation. Staining with picrosirius red confirmed the earlier observation of a change in staining color from red to green. Taken with the observed equivalent decreases in collagen I and III mRNA, this suggests that there was a change in orientation of the ECM of muscle becoming more aligned with the axis of the muscle fibers and no change in collagen type. The change in collagen orientation may serve to protect the smaller muscle fibers from damage by increasing the stiffness of the ECM and may partly explain why the region of the tendon closest to the muscle becomes stiffer after inactivity.     

Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils.

Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.     

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.     

Functional Grading of Mineral and Collagen in the Attachment of Tendon to Bone

Attachment of dissimilar materials is a major challenge because high levels of localized stress may develop at their interfaces. An effective biologic solution to this problem exists at one of nature's most extreme interfaces: the attachment of tendon (a compliant, structural “soft tissue”) to bone (a stiff, structural “hard tissue”). The goal of our study was to develop biomechanical models to describe how the tendon-to-bone insertion derives its mechanical properties. We examined the tendon-to-bone insertion and found two factors that give the tendon-to-bone transition a unique grading in mechanical properties: 1), a gradation in mineral concentration, measured by Raman spectroscopy; and 2), a gradation in collagen fiber orientation, measured by polarized light microscopy. Our measurements motivate a new physiological picture of the tissue that achieves this transition, the tendon-to-bone insertion, as a continuous, functionally graded material. Our biomechanical model suggests that the experimentally observed increase in mineral accumulation within collagen fibers can provide significant stiffening of the partially mineralized fibers, but only for concentrations of mineral above a “percolation threshold” corresponding to formation of a mechanically continuous mineral network within each collagen fiber (e.g., the case of mineral connectivity extending from one end of the fiber to the other). Increasing dispersion in the orientation distribution of collagen fibers from tendon to bone is a second major determinant of tissue stiffness. The combination of these two factors may explain the nonmonotonic variation of stiffness over the length of the tendon-to-bone insertion reported previously. Our models explain how tendon-to-bone attachment is achieved through a functionally graded material composition, and provide targets for tissue engineered surgical interventions and biomimetic material interfaces.     

Pleochroism in tendon and its bearing to acid mucopolysaccharides

Summary and conclusions The pleochroism found in the histological sections dyed with toluidine blue buffered at pH 4 is caused by the selective absorption of polarized light by the molecule of the dye. This fact makes it possible to calculate the position of the molecules of acid mucopolysaccharides (AMP) by indirect method.The molecules of AMP, of linear or bastonet shape are placed parallel to the long axis of the collagen fibers, with their negative polar groups directed toward the collagen.This structural relation between the molecules of the AMP and the collagen not only facilitate a cementing and stabilizing action but contribute to the resistance to traction as well.The author would like to thank Prof. Lucien Lison for his help and encouragement.     

Histological staining as a measure of stress in collagen fibers

It has been reported previously that collagen fibers will stain either red or green by Masson's and other trichrome methods depending on whether they have been respectively stressed or relaxed prior to fixation. This was shown in skin [1, 2, 3] tendon [4, 5] bone [6] and films of collagen [7]. If this stain-stress dependence is of a unique quantitative nature, then staining could be used as a tension probe for collagen fibers. Relaxed and stressed collagen bundles of rat tail tendon and rat Achilles tendon have been stained using various staining periods, and results indicate that the change in staining may be associated with denser packing of the fibers in the bundle under stress rather than directly due to the stress itself. Denser packing may reduce the rate of penetration of the counterstain thus causing the staining differences. Since this rate of penetration is dependent on a number of other variables (unrelated to stress), it is concluded that collagen staining is not a reliable tension probe.     

Measuring collagen fiber orientation: a two-dimensional quantitative macroscopic technique.

This paper describes the design, evaluation, and application of a new system for quantifying two-dimensional collagen fiber orientation in soft tissue. Series of transmitted polarized light images were collected using a custom-designed macroscope. Combined analysis of pixel brightness, and hue from images collected with a compensator plate, permitted the assignment of each pixel into the appropriate orientation band. Experiments were performed to quantify the linearity and noise of the system. Validation was performed on a specimen composed of strain-birefringent plastic strips at various orientations. Preliminary collagen fiber orientation data is presented from a tendon specimen. This study demonstrates the utility of this approach for studying collagen fiber orientation across large areas.