Tail Tendon Research Articles

Tendon Fascicles Exhibit a Linear Correlation Between Poisson's Ratio and Force During Uniaxial Stress Relaxation

The underlying mechanisms for the viscoelastic behavior of tendon and ligament tissue are poorly understood. It has been suggested that both a flow-dependent and flow-independent mechanism may contribute at different structural levels. We hypothesized that the stress relaxation response of a single tendon fascicle is consistent with the flow-dependent mechanism described by the biphasic theory (Armstrong et al., 1984, "An Analysis of the Unconfined Compression of Articular Cartilage," ASME J. Biomech. Eng., 106, pp. 165-173). To test this hypothesis, force, lateral strain, and Poisson's ratio were measured as a function of time during stress relaxation testing of six rat tail tendon fascicles from a Sprague Dawley rat. As predicted by biphasic theory, the lateral strain and Poisson's ratio were time dependent, a large estimated volume loss was seen at equilibrium and there was a linear correlation between the force and Poisson's ratio during stress relaxation. These results suggest that the fluid dependent mechanism described by biphasic theory may explain some or all of the apparent viscoelastic behavior of single tendon fascicles.

Advanced glycation end-products diminish tendon collagen fiber sliding

Connective tissue aging and diabetes related comorbidity are associated with compromised tissue function, increased susceptibility to injury, and reduced healing capacity. This has been partly attributed to collagen cross-linking by advanced glycation end-products (AGEs) that accumulate with both age and disease. While such cross-links are believed to alter the physical properties of collagen structures and tissue behavior, existing data relating AGEs to tendon mechanics is contradictory. In this study, we utilized a rat tail tendon model to quantify the micro-mechanical repercussion of AGEs at the collagen fiber-level. Individual tendon fascicles were incubated with methylglyoxal (MGO), a naturally occurring metabolite known to form AGEs. After incubation in MGO solution or buffer only, tendons were stretched on the stage of a multiphoton confocal microscope and individual collagen fiber stretch and relative fiber sliding were quantified. Treatment by MGO yielded increased fluorescence and elevated denaturation temperatures as found in normally aged tissue, confirming formation of AGEs and related cross-links. No apparent ultrastructural changes were noted in transmission electron micrographs of cross-linked fibrils. MGO treatment strongly reduced tissue stress relaxation (p<0.01), with concomitantly increased tissue yield stress (p<0.01) and ultimate failure stress (p=0.036). MGO did not affect tangential modulus in the linear part of the stress–strain curve (p=0.46). Microscopic analysis of collagen fiber kinematics yielded striking results, with MGO treatment drastically reducing fiber-sliding (p<0.01) with a compensatory increase in fiber-stretch (p<0.01). We thus conclude that the main mechanical effect of AGEs is a loss of tissue viscoelasticity driven by matrix-level loss of fiber–fiber sliding. This has potentially important implications to tissue damage accumulation, mechanically regulated cell signaling, and matrix remodeling. It further highlights the importance of assessing viscoelasticity – not only elastic response – when considering age-related changes in the tendon matrix and connective tissue in general.

Metabolic transit of Nε-carboxymethyl-lysine after consumption of AGEs from bread crust

Our aim was to investigate carboxymethyl-lysine (CML) intake and excretion after feeding rats with diets containing advanced glycation end-products (AGEs) from bread crust (BC) or its soluble or insoluble fractions, and to identify the factors responsible for the effects observed. CML in serum and different tissues was measured to detect possible accumulations. For 88 days, weanling rats were fed with either a control diet or one containing BC, or its soluble low molecular weight (LMW), soluble high molecular weight (HMW) or insoluble fractions. In the last week of the assay, faeces and urine were collected daily and stored as a 1 week pool. After sacrifice, blood was drawn to obtain serum and some organs were removed. CML analysis was performed by HPLC/MS/MS in diets, faeces, urines, serum and tissues. Faecal excretion of CML was strongly influenced by dietary CML levels and represents the major route of excretion (i.e. 33.2%). However, the urinary elimination of CML was probably limited or saturated, especially when more complex compounds were present in the diet. BC consumption increased CML in the cardiac tissue (170 ± 18 vs. 97 ± 3 μmol per mol lysine for BC and control groups), which correlated with the CML intake. The levels of this AGE in bone were unaffected by the dietary treatment, but in tail tendons CML was greatly increased in the animals that consumed the BC diet (102 ± 13 vs. 51 ± 8 μmol per mol lysine for BC and control groups, P = 0.006), which was associated with the intake of soluble LMW compounds present in BC. Despite the CML accumulation detected in different tissues, serum levels of protein-bound CML were unchanged, indicating the importance of measuring the free CML in this fluid as a real index of dietary CML.

Effects of Water on the Tensile Properties of Collagen Fibrils

Fibrils were isolated from rat tail tendons and tensile tests of the fibrils were performed in the air (dry) condition or in the physiological saline solution (wet) condition. After the fibrils were immersed in the physiological saline solution for 3 and 30 minutes, the fibrils were stretched to failure. There were no significant differences in the tensile properties between 3 and 30 minutes. These results indicate that the fibrils were sufficiently hydrated by the immersion of 3 minutes. The fibrils immersed for 3 minutes showed the tensile strength of 111±31 MPa (Mean±S.E.), strain at failure of 32.1±4.5 %, and tangent modulus of 350±53 MPa. Next, the fibrils were stretched to failure in the air. The dry fibrils showed the tensile strength of 702±137 MPa, strain at failure of 30.4±8.5 %, and tangent modulus of 2720±840 MPa. The tensile strength and tangent modulus of the dry fibrils were significantly larger than those of the wet fibrils. There were no significant differences in the strain at failure between the dry and wet fibrils. These results point to the great importance of water for the tensile properties of collagen fibrils.

Age-Related Changes in the Cellular, Mechanical, and Contractile Properties of Rat Tail Tendons

Tendon laxity following injury, cyclic creep, or repair has been shown to alter the normal homeostasis of tendon cells, which can lead to degenerative changes in the extracellular matrix. While tendon cells have been shown to have an inherent contractile mechanism that gives them some ability to retighten lax tendons and reestablish a homeostatic cellular environment, the effect of age on this process is unknown. To determine the effect of aging on cell number, cell shape, and tensile modulus on tendons as well as the rate of cell-mediated contraction of lax tendons, tail tendon fascicles from 1-, 3-, and 12-month-old rats were analyzed. Aging results in a decrease (p < 0.001) in cell number per mm2: 1 m (981 ± 119), 3 m (570 ± 108), and 12 m (453 ± 23), a more flattened (p < 0.001) cell nuclei shape and a higher (p < 0.001) tensile modulus (MPa) of the tendons: 1 m (291 ± 2), 3 m (527 ± 38), and 12 m (640 ± 102). Both the extent and rate of contraction over 7 days decreased with age (p = 0.007). This decrease in contraction rate with age correlates to the observed changes seen in aging tendons [increased modulus (r2 = 0.95), decreased cell number (r2 = 0.89)]. The ability of tendons to regain normal tension following injury or exercise-induced laxity is a key factor in the recovery of tendon function. The decreased contraction rate as a function of age observed in the current study may limit the ability of tendon cells to retighten lax tendons in older individuals. This, in turn, may place these structures at further risk for injury or altered function.

Age-dependent effects of systemic administration of oxytetracycline on the viscoelastic properties of rat tail tendons as a mechanistic basis for pharmacological treatment of flexural limb deformities in foals

To describe the effect of systemically administered oxytetracycline on the viscoelastic properties of rat tail tendon fascicles (TTfs) to provide a mechanistic rationale for pharmacological treatment of flexural limb deformities in foals. TTfs from ten 1-month-old and ten 6-month-old male Sprague-Dawley rats. 5 rats in each age group were administered oxytetracycline (50 mg/kg, IP, q 24 h) for 4 days. The remaining 5 rats in each age group served as untreated controls. Five days after initiation of oxytetracycline treatment, TTfs were collected and their viscoelastic properties were evaluated via a stress-relaxation protocol. Maximum modulus and equilibrium modulus were compared via a 2-way ANOVA. Collagen fibril size, density, and orientation in TTfs were compared between treated and control rats. Viscoelastic properties were significantly decreased in TTfs from 1-month-old oxytetracycline-treated rats, compared with those in TTfs from 1-month-old control rats. Oxytetracycline had no effect on the viscoelastic properties of TTfs from 6-month-old rats. Collagen fibril size, density, and orientation in TTfs from 1-month-old rats did not differ between oxytetracycline-treated and control rats. Results confirmed that systemically administered oxytetracycline decreased the viscoelastic properties of TTfs from 1-month-old rats but not those of TTfs from 6-month-old rats. The decrease in viscoelastic properties associated with oxytetracycline treatment does not appear to be caused by altered collagen fibril diameter or organization. The age-dependent effect of oxytetracycline on the viscoelastic properties of tendons may be related to its effect on the maturation of the extracellular matrix of developing tendons.

Analysis of collagen fiber domain organization by Fourier second harmonic generation microscopy

We present an automated and systematic two-dimensional discrete Fourier transform (2D-FFT) approach to analyze collagen fiber organization through the use of second harmonic generation (SHG) microscopy. Average orientations of individual domains and Ising-like order parameters introduced to characterize the correlation between orientations of adjacent domains may be used to quantitatively characterize fibrous tissues. Our approach was applied to analyze tissues including rat tail tendon, mouse skin, bovine corneas, and human corneas. We also show that collagen fiber organization in normal and keratokonus human corneas may be distinguished. The current approach may be used for the quantitative differentiation of SHG collagen fiber morphology in different tissues and may be applied for diagnostic purposes.

Hierarchical Model of Fibrillar Collagen Organization for Interpreting the Second-Order Susceptibility Tensors in Biological Tissue

The second-order nonlinear polarization properties of fibrillar collagen in various rat tissues (vertebrae, tibia, tail tendon, dermis, and cornea) are investigated with polarization-dependent second-harmonic generation (P-SHG) microscopy. Three parameters are extracted: the second-order susceptibility ratio, R =χZZZ(2)′/χZXX(2)′; a measure of the fibril distribution asymmetry, |A|; and the weighted-average fibril orientation, 〈δ〉. A hierarchical organizational model of fibrillar collagen is developed to interpret the second-harmonic generation polarization properties. Highlights of the model include: collagen type (e.g., type-I, type-II), fibril internal structure (e.g., straight, constant-tilt), and fibril architecture (e.g., parallel fibers, intertwined, lamellae). Quantifiable differences in internal structure and architecture of the fibrils are observed. Occurrence histograms of R and |A| distinguished parallel from nonparallel fibril distributions. Parallel distributions possessed low parameter values and variability, whereas nonparallel distributions displayed an increase in values and variability. From the P-SHG parameters of vertebrae tissue, a three-dimensional reconstruction of lamellae of intervertebral disk is presented.

Structural Origins of Chiral Second-Order Optical Nonlinearity in Collagen: Amide I Band

The molecular basis of nonlinear optical (NLO) chiral effects in the amide I region of type I collagen was investigated using sum-frequency generation vibrational spectroscopy; chiral and achiral tensor elements were separated using different input/output beam polarization conditions. Spectra were obtained from native rat tail tendon (RTT) collagen and from cholesteric liquid crystal-like (LC) type I collagen films. Although RTT and LC collagen both possess long-range order, LC collagen lacks the complex hierarchical organization of RTT collagen. Their spectra were compared to assess the role of such organization in NLO chirality. No significant differences were observed between RTT and LC with respect to chiral or achiral spectra. These findings suggest that amide I NLO chiral effects in type I collagen assemblies arise predominantly from the chiral organization of amide chromophores within individual collagen molecules, rather than from supramolecular structures. The study suggests that sum-frequency generation vibrational spectroscopy may be uniquely valuable in exploring fundamental aspects of chiral nonlinearity in complex macromolecular structures.

Visualizing molecular polar order in tissues via electromechanical coupling.

Electron microscopy (EM) and atomic force microscopy (AFM) techniques have long been used to characterize collagen fibril ordering and alignment in connective tissues. These techniques, however, are unable to map collagen fibril polarity, i.e., the polar orientation that is directed from the amine to the carboxyl termini. Using a voltage modulated AFM-based technique called piezoresponse force microscopy (PFM), we show it is possible to visualize both the alignment of collagen fibrils within a tissue and the polar orientation of the fibrils with minimal sample preparation. We demonstrate the technique on rat tail tendon and porcine eye tissues in ambient conditions. In each sample, fibrils are arranged into domains whereby neighboring domains exhibit opposite polarizations, which in some cases extend to the individual fibrillar level. Uniform polarity has not been observed in any of the tissues studied. Evidence of anti-parallel ordering of the amine to carboxyl polarity in bundles of fibrils or in individual fibrils is found in all tissues, which has relevance for understanding mechanical and biofunctional properties and the formation of connective tissues. The technique can be applied to any biological material containing piezoelectric biopolymers or polysaccharides.

A novel in vitro loading system for high frequency loading of cultured tendon fascicles

Tendons are known to adapt to their mechanical environment, however high frequency low magnitude (HFLM) loading regimes (10–50Hz), which are effective in promoting bone anabolic effects, have not been investigated in controlled conditions in tendon. In vitro loading systems (IVLS) enable precise characterisation of the link between their controlled mechanical environment and cultured tissue biological response.We report a novel IVLS design using an applied magnetic field to produce time varying loading in cultured rat tail tendon fascicles (RTTF). The design was validated through magnetic flux, load cell and viability measurements, and we report the results of preliminary experiments testing the hypothesis that an HFLM loading regime will maintain the biochemical and mechanical properties of fresh RTTF in culture over 7 days.Tissue viability was maintained for 7 days under all loading conditions, and the average peak load applied to RTTFs using the IVLS at 20Hz was 0.125N. RTTFs cultured for 7 days with HFLM loading showed a trend for a higher tangent modulus than fresh tissue, and significantly higher modulus than unloaded RTTFs. GAG content of HFLM cultured RTTFs was not significantly changed from that of fresh RTTFs.This novel, validated IVLS will provide new knowledge of tendon mechanobiology and has already shown the potential of clinically relevant HFLM loading for influencing tendon biology.

Smad3 Modulates Tendon Gene Expression and Interacts with Key Tendon Transcription Factors: Not a clinical study

METHODS Gene Expression qPCR Analyses: Hindlimb and tail tendons were isolated from 6-week old mice. RNA was extracted using the Purelink RNA Mini Kit (Invitrogen) and cDNA was reverse transcribed using the iScript cDNA synthesis kit (BioRad). Expression was assessed by SYBR green fluorescence during qRT-PCR with established primers. Data was analyzed using a two-tailed t-test assuming unequal variances.

Bimodal collagen fibril diameter distributions direct age-related variations in tendon resilience and resistance to rupture

Scaling relationships have been formulated to investigate the influence of collagen fibril diameter (D) on age-related variations in the strain energy density of tendon. Transmission electron microscopy was used to quantify D in tail tendon from 1.7- to 35.3-mo-old (C57BL/6) male mice. Frequency histograms of D for all age groups were modeled as two normally distributed subpopulations with smaller (D(D1)) and larger (D(D2)) mean Ds, respectively. Both D(D1) and D(D2) increase from 1.6 to 4.0 mo but decrease thereafter. From tensile tests to rupture, two strain energy densities were calculated: 1) u(E) [from initial loading until the yield stress (σ(Y))], which contributes primarily to tendon resilience, and 2) u(F) [from σ(Y) through the maximum stress (σ(U)) until rupture], which relates primarily to resistance of the tendons to rupture. As measured by the normalized strain energy densities u(E)/σ(Y) and u(F)/σ(U), both the resilience and resistance to rupture increase with increasing age and peak at 23.0 and 4.0 mo, respectively, before decreasing thereafter. Multiple regression analysis reveals that increases in u(E)/σ(Y) (resilience energy) are associated with decreases in D(D1) and increases in D(D2), whereas u(F)/σ(U) (rupture energy) is associated with increases in D(D1) alone. These findings support a model where age-related variations in tendon resilience and resistance to rupture can be directed by subtle changes in the bimodal distribution of Ds.

Breast epithelial tissue morphology is affected in 3D cultures by species‐specific collagen‐based extracellular matrix

Collagen-based gels have been widely used to determine the factors that regulate branching morphogenesis in the mammary gland. The patterns of biomechanical gradients and collagen reorganization influence the shape and orientation of epithelial structures in three-dimensional (3D) conditions. We explored in greater detail whether collagen type I fibers with distinct biomechanical and fiber-assembling properties, isolated from either bovine or rat tail tendon, differentially affected the epithelial phenotype in a tissue culture model of the human breast. Rat tail collagen fibers were densely packed into significantly longer and thicker bundles compared to those of the bovine type (average fascicle length 7.35 and 2.29 μm, respectively; p = 0.0001), indicating increased fiber alignment and biomechanical enablement in the former. MCF10A epithelial cells formed elaborated branched tubular structures in bovine but only nonbranched ducts and acini in rat tail collagen matrices. Ductal branching in bovine collagen was associated with interactions between neighboring structures mediated through packed collagen fibers; these fiber-mediated interactions were absent in rat tail collagen gels. Normal breast fibroblasts increased the final size and number of ducts only in rat tail collagen gels while not affecting branching. Our results suggest that the species of origin of collagen used in organotypic cultures may influence epithelial differentiation into alveolar or ductal structures and the patterns of epithelial branching. These observations underscore the importance of considering the species of origin and fiber alignment properties of collagen when engineering branching organs in 3D matrices and interpreting their role in the tissue phenotype.

Matrix Disruptions, Growth, and Degradation of Cartilage with Impaired Sulfation

Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in the SLC26A2 transporter responsible for sulfate uptake by chondrocytes. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally. To understand underlying mechanisms, we studied newborn dtd mice. We developed, verified and used high-definition infrared hyperspectral imaging of cartilage sections at physiological conditions, to quantify collagen and its orientation, noncollagenous proteins, and chondroitin chains, and their sulfation with 6-μm spatial resolution and without labeling. We found that chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with the chondroitin synthesis rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth. The results provide clues to in vivo sulfation, DTD treatment, and cartilage growth.

Designed to Fail: A Novel Mode of Collagen Fibril Disruption and Its Relevance to Tissue Toughness

Collagen fibrils are nanostructured biological cables essential to the structural integrity of many of our tissues. Consequently, understanding the structural basis of their robust mechanical properties is of great interest. Here we present what to our knowledge is a novel mode of collagen fibril disruption that provides new insights into both the structure and mechanics of native collagen fibrils. Using enzyme probes for denatured collagen and scanning electron microscopy, we show that mechanically overloading collagen fibrils from bovine tail tendons causes them to undergo a sequential, two-stage, selective molecular failure process. Denatured collagen molecules—meaning molecules with a reduced degree of time-averaged helicity compared to those packed in undamaged fibrils—were first created within kinks that developed at discrete, repeating locations along the length of fibrils. There, collagen denaturation within the kinks was concentrated within certain subfibrils. Additional denatured molecules were then created along the surface of some disrupted fibrils. The heterogeneity of the disruption within fibrils suggests that either mechanical load is not carried equally by a fibril's subcomponents or that the subcomponents do not possess homogenous mechanical properties. Meanwhile, the creation of denatured collagen molecules, which necessarily involves the energy intensive breaking of intramolecular hydrogen bonds, provides a physical basis for the toughness of collagen fibrils.

A Nonlinear Constitutive Model for Stress Relaxation in Ligaments and Tendons

A novel constitutive model that describes stress relaxation in transversely isotropic soft collagenous tissues such as ligaments and tendons is presented. The model is formulated within the nonlinear integral representation framework proposed by Pipkin and Rogers (J. Mech. Phys. Solids. 16:59-72, 1968). It represents a departure from existing models in biomechanics since it describes not only the strain dependent stress relaxation behavior of collagenous tissues but also their finite strains and transverse isotropy. Axial stress-stretch data and stress relaxation data at different axial stretches are collected on rat tail tendon fascicles in order to compute the model parameters. Toward this end, the rat tail tendon fascicles are assumed to be incompressible and undergo an isochoric axisymmetric deformation. A comparison with the experimental data proves that, unlike the quasi-linear viscoelastic model (Fung, Biomechanics: Mechanics of Living Tissues. Springer, New York, 1993) the constitutive law can capture the observed nonlinearities in the stress relaxation response of rat tail tendon fascicles.

Re‐establishment of cytoskeletal tensional homeostasis in lax tendons occurs through an actin‐mediated cellular contraction of the extracellular matrix

Cytoskeletal tensional homeostasis is known to be an important factor in controlling catabolic gene expression in tendon cells. Loss of cell tension in lax rat tail tendon fascicles (RTTfs) has been associated with an upregulation of MMP-13 gene expression and protein synthesis. To determine the role of the actin cytoskeleton in re-establishing tensional homeostasis in lax tendons, RTTfs were allowed to freely contract in vitro for 8 days. The cultured RTTfs contracted rapidly, reaching 50% of their initial length by 3 days. This contraction was associated with the presence of α-smooth muscle actin positive cells within the tendon. Disruption of the actin network by cytochalasian D caused an immediate and significant elongation of the contracted RTTfs. Subsequent removal of the cytochalasian D re-initiated the contraction process. When lax RTTfs were allowed to contract between fixed clamps in culture and become taut, they demonstrated a marked decrease in MMP-13 staining intensity when compared to freely contracting RTTfs. The ability of native tendon cells to contract lax tendons and re-establish their homeostatic "set point" with respect to collagenase production may be an important mechanism in the recovery of tendons elongated by injury, surgical positioning, or cyclic, viscoelastic creep secondary to repetitive exercise.

Electromechanical properties of dried tendon and isoelectrically focused collagen hydrogels

Assembling artificial collagenous tissues with structural, functional, and mechanical properties which mimic natural tissues is of vital importance for many tissue engineering applications. While the electro-mechanical properties of collagen are thought to play a role in, for example, bone formation and remodeling, this functional property has not been adequately addressed in engineered tissues. Here the electro-mechanical properties of rat tail tendon are compared with those of dried isoelectrically focused collagen hydrogels using piezoresponse force microscopy under ambient conditions. In both the natural tissue and the engineered hydrogel D-periodic type I collagen fibrils are observed, which exhibit shear piezoelectricity. While both tissues also exhibit fibrils with parallel orientations, Fourier transform analysis has revealed that the degree of parallel alignment of the fibrils in the tendon is three times that of the dried hydrogel. The results obtained demonstrate that isoelectrically focused collagen has similar structural and electro-mechanical properties to that of tendon, which is relevant for tissue engineering applications.

Effects of Hydration on Nanoscale Structural Morphology and Mechanics of Individual Type I Collagen Fibrils

ABSTRACTType I collagen is one of the most vital proteins in our bodies and serves a number of structural roles. Despite collagen’s importance, little is known about its nanoscale morphology in tissues and how morphology relates to mechanical function. This study directly probes nanoscale structure and mechanics in collagen as a function of hydration utilizing atomic force microscopy investigations of the mouse tail tendon. We demonstrate that collagen morphology and mechanical properties at the nanoscale change with dehydration, indicating that hydration is a factor which must be considered when performing studies at any length scale in collagen-based tissues. Studies are underway to further investigate this phenomenon and to determine how these properties change with disease in tendon and other Type I collagen-based tissues.