Determining the contribution of glycosaminoglycans to tendon mechanical properties with a modified shear-lag model

Tendon has a complex hierarchical structure composed of both a collagenous and a non-collagenous matrix. Despite several studies that have aimed to elucidate the mechanism of load transfer between matrix components, the roles of glycosaminoglycans (GAGs) remain controversial. Thus, this study investigated the elastic properties of tendon using a modified shear-lag model that accounts for the structure and non-linear mechanical response of the GAGs. Unlike prior shear-lag models that are solved either in two dimensions or in axially symmetric geometries, we present a closed-form analytical model for three-dimensional periodic lattices of fibrils linked by GAGs. Using this approach, we show that the non-linear mechanical response of the GAGs leads to a distinct toe region in the stress–strain response of the tendon. The critical strain of the toe region is shown to decrease inversely with fibril length. Furthermore, we identify a characteristic length scale, related to microstructural parameters (e.g. GAG spacing, stiffness, and geometry) over which load is transferred from the GAGs to the fibrils. We show that when the fibril lengths are significantly larger than this length scale, the mechanical properties of the tendon are relatively insensitive to deletion of GAGs. Our results provide a physical explanation for the insensitivity for the mechanical response of tendon to the deletion of GAGs in mature tendons, underscore the importance of fibril length in determining the elastic properties of the tendon, and are in excellent agreement with computationally intensive simulations.     

Hysteresis measurements in intact human tendon

Mechanical hysteresis in tendons has traditionally been quantified from tensile testing of isolated specimens. Limitations associated with tendon displacement measurement and clamping, and uncertainties as to whether in vitro material represents intact tendon function necessitate measuring hysteresis under in vivo conditions. In the present study such measurements were taken in the human tibialis anterior (TA) tendon. Having the foot fixed on a dynamometer footplate, the displacement of the TA tendon during stimulation and relaxation of the TA muscle was recorded by means of ultrasonography in six men. Combining moment data corresponding to 0, 20, 40, 60, 80 and 100% of maximum voltage moment and the respective tendon-displacement data, a hysteresis loop was obtained between the load–displacement curves during contraction and relaxation. Measurement of the hysteresis loop area yielded a value of 19%. This value agrees with results from in vitro tensile tests of low-stress tendons, suitable for tensile force transmission and joint displacement control. In fact, the human TA tendon has such functional characteristics. The methodology presented allows design of longitudinal and cross-sectional experimental protocols, and in vivo assessment of tendon function and propensity to overheat.     

Heparin--a unique stimulator of human colon cancer cells' growth

The cancer microenvironment and the interactions between cancer and surrounding tissue cells are thought to play a pivotal role in tumor development and progression. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are major constituents of the extracellular matrix, the composition of which may affect various cellular functions. In the present study, the effects of GAGs on the proliferation of HT29, SW1116, and HCT116 human colon cancer cell lines were examined using exogenously added GAGs, an inhibitor of endogenous GAG sulfation and specific glycosidase digestions. Our results demonstrate that colon cancer cell growth was exclusively stimulated by exogenously added heparin and insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related manner. Treatment of the tested cell lines with the FGF-2 neutralizing antibody showed that the stimulatory effect of heparin on the cells' growth was not FGF-2-dependent. Responsiveness of colon cancer cell lines to exogenous heparin/heparan sulfate may play a role in their growth and metastasis.     

Force and surface mechanomyogram frequency responses in cat gastrocnemius

Muscle surface displacement is a mechanical event taking place simultaneously with the tension generation at the tendon. The two phenomena can be studied by the surface mechanomyogram signal (MMG) (produced by a laser distance sensor) and the force signal (from a load cell). The aim of this paper was to provide data on the reliability of the laser detected MMG in muscle mechanics research. To this purpose it was verified if the laser detected MMG was suitable to estimate a frequency response in the cat medial gastrocnemius and its frequency response was compared with the one retrieved by the force signal at the tendon level. The force and MMG from the exposed medial gastrocnemius of four cats were analysed. The frequency response was investigated by sinusoidally changing the number of orderly recruited motor units, in different trials, in the 0.4-6 Hz range. It resulted that it was possible to model the force and MMG frequency response by a critically damped second-order system with two real double poles and a pure time delay. On the average, the poles were at 1.83 Hz (with 22.6 ms delay) and at 2.75 Hz (with 38 ms delay) for force and MMG, respectively. It can be concluded that MMG appears to be a reliable tool to investigate the muscle frequency response during stimulated isometric contraction. Even though not statistically significant. the differences in the second-order system parameters suggest that different components of the muscle mechanical model may specifically affect the force or MMG.     

Exogenous addition of a C-xylopyranoside derivative stimulates keratinocyte dermatan sulfate synthesis and promotes migration

As C-Xyloside has been suggested to be an initiator of glycosaminoglycan (GAG) synthesis, and GAGs such as Dermatan sulfate (DS) are potent enhancers of fibroblast growth factor (FGF)--10 action, we investigated if a C-Xylopyranoside derivative, (C-β-D-xylopyranoside-2-hydroxy-propane, C-Xyloside), could promote DS production by cultured normal human keratinocytes, how this occurs and if C-Xyloside could also stimulate FGF-dependent cell migration and proliferation. C-Xyloside-treated keratinocytes greatly increased secretion of total sulfated GAGs. Majority of the induced GAG was chondroitin sulfate/dermatan sulfate (CS/DS) of which the major secreted GAG was DS. Cells lacking xylosyltransferase enzymatic activity demonstrated that C-Xyloside was able to stimulate GAG synthesis without addition to core proteins. Consistent with the observed increase in DS, keratinocytes treated with C-Xyloside showed enhanced migration in response to FGF-10 and secreted into their culture media GAGs that promoted FGF-10-dependent cellular proliferation. These results indicate that C-Xyloside may enhance epithelial repair by serving as an initiator of DS synthesis.     

Tenocytic extract and mechanical stimulation in a tissue‐engineered tendon construct increases cellular proliferation and ECM deposition

Chemical and mechanical stimulation, when properly utilized, positively influence both the differentiation of in vitro cultured stem cells and the quality of the deposited extracellular matrix (ECM). This study aimed to find if cell‐free extract from primary tenocytes can positively affect the development of a tissue‐engineered tendon construct, consisting of a human umbilical vein (HUV) seeded with mesenchymal stem cells (MSCs) subjected to cyclical mechanical stimulation. The tenocytic cell‐free extract possesses biological material from tendon cells that could potentially influence MSC tenocytic differentiation and construct development. We demonstrate that the addition of tenocytic extract in statically cultured tendon constructs increases ECM deposition and tendon‐related gene expression of MSCs. The incorporation of mechanical stimulation (2% strain for 30 min/day at 0.5 cycles/min) with tenocytic extract further improved the MSC seeded HUV constructs by increasing cellularity of the construct by 37% and the ultimate tensile strength by 33% compared to the constructs with only mechanical stimulation after 14 days. Furthermore, the addition of mechanical stimulation to the extract supplementation produced longitudinal ECM fibril alignment along with dense connective tissue, reminiscent of natural tendon.     

The tensile and stress relaxation responses of human patellar tendon varies with specimen cross-sectional area.

In order to provide insight into the mechanical response of the collagen fascicle structures in tendon, a series of constant strain rate and constant displacement, stress relaxation mechanical tests were performed on sequentially sectioned human patellar tendon specimens (protocol 1) and specimens with both small (approximately 1 mm2) and large (approximately 20 mm2) cross-sectional areas (protocol 2). These data described the stress relaxation and constant strain rate tensile responses as a function of cross-sectional area and water content. The experimental data suggested that small portions of tendon exhibit a higher tensile modulus, a slower rate of relaxation and a lower amount of relaxation in comparison to larger specimens from the same location in the same tendon. The decrease in relaxation response and the increase in tensile modulus with decreasing cross-sectional area was nonlinear. These data suggest that there may be structures other than the subfascicle, such as the epitenon and other connective tissue components, which influence the tensile and stress relaxation responses in tendon.     

Mechanotransduction of stem cells for tendon repair

Tendon is a mechanosensitive tissue that transmits force from muscle to bone. Physiological loading contributes to maintaining the homeostasis and adaptation of tendon, but aberrant loading may lead to injury or failed repair. It is shown that stem cells respond to mechanical loading and play an essential role in both acute and chronic injuries, as well as in tendon repair. In the process of mechanotransduction, mechanical loading is detected by mechanosensors that regulate cell differentiation and proliferation via several signaling pathways. In order to better understand the stem-cell response to mechanical stimulation and the potential mechanism of the tendon repair process, in this review, we summarize the source and role of endogenous and exogenous stem cells active in tendon repair, describe the mechanical response of stem cells, and finally, highlight the mechanotransduction process and underlying signaling pathways.     

Endogenous heparan sulfate and heparin modulate bone morphogenetic protein-4 signaling and activity

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses.     

Perspective into the regulation of cell-generated forces toward stem cell migration and differentiation

Stem cells are promising candidates for cell-based therapies in diverse conditions including regenerating damaged tissues, treating inflammation in virtue of sepsis, acute renal failure, and cardiovascular disease. Advancement of these therapies relies on the ability to guide stem cells to migrate directly and differentiate towards specific cell phenotypes. During the past decade, many researchers have demonstrated that exogenous applied forces could significantly affect the migration and lineage differentiation of stem cells. Besides, recent advances have highlighted the critical role of internal forces due to cell-matrix interaction in the function of stem cells. Stem cells can generate contractile forces to sense the mechanical properties of cell-generated force microenvironment, and thereby perceive mechanical information that directs broad aspects of stem cell functions, including migration and lineage commitment. In the review, we recount the cell-generated force microenvironment of stem cells and discuss the interactions between cell-generated forces with migration and differentiation of stem cells. We also summarize key experimental evidence of a tight linkage between migration and lineage differentiation of stem cells and pose important unanswered questions in this field.     

Basic characteristics between mechanomyogram and muscle force during twitch and tetanic contractions in rat skeletal muscles

The mechanomyogram (MMG) is a signal measured by various vibration sensors for slight vibrations induced by muscle contraction, and it reflects the muscle force during electrically induced-contraction or until 60%–70% maximum voluntary contraction, so the MMG is considered an alternative and novel measurement tool for muscle strength. We simultaneously measured the MMG and muscle force in the gastrocnemius (GC), vastus intermedius (VI), and soleus (SOL) muscles of rats. The muscle force was measured by attaching a hook to the tendon using a load cell, and the MMG was measured using a charged-coupled device-type displacement sensor at the middle of the target muscle. The MMG-twitch waveform was very similar to that of the muscle force; however, the half relaxation time and relaxation time (10%), which are relaxation parameters, were prolonged compared to those of the muscle force. The MMG amplitude correlated with the muscle force. Since stimulation frequencies that are necessary to evoke tetanic progression have a significant correlation with the twitch parameter, there is a close relationship between twitch and tetanus in the MMG signal. Therefore, we suggest that the MMG, which is electrically induced and detected by a laser displacement sensor, may be an alternative tool for measuring muscle strength.     

Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells.

Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface-expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2-terminal part as well as in the C-repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC-chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein-mediated adhesion of S. pyogenes.     

Conformation and sequence evidence for two-fold symmetry in left-handed beta-helix fold

The glycosaminoglycan (GAG) side-chains of small leucine-rich proteoglycans have been postulated to mechanically cross-link adjacent collagen fibrils and contribute to tendon mechanics. Enzymatic depletion of tendon GAGs (chondroitin and dermatan sulfate) has emerged as a preferred method to experimentally assess this role. However, GAG removal is typically incomplete and the possibility remains that extant GAGs may remain mechanically functional. The current study specifically investigated the potential mechanical effect of the remaining GAGs after partial enzymatic digestion.A three-dimensional finite element model of tendon was created based upon the concept of proteoglycan mediated inter-fibril load sharing. Approximately 250 interacting, discontinuous collagen fibrils were modeled as having a length of 400 μm, being composed of rod elements of length 67 nm and E-modulus 1 GPa connected in series. Spatial distribution and diameters of these idealized fibrils were derived from a representative cross-sectional electron micrograph of tendon. Rod element lengths corresponded to the collagen fibril D-Period, widely accepted to act as a binding site for decorin and biglycan, the most abundant proteoglycans in tendon. Each element node was connected to nodes of any neighboring fibrils within a radius of 100 nm, the slack length of unstretched chondroitin sulfate. These GAG cross-links were the sole mechanism for lateral load sharing among the discontinuous fibrils, and were modeled as bilinear spring elements. Simulation of tensile testing of tendon with complete cross-linking closely reproduced corresponding experiments on rat tail tendons. Random reduction of 80% of GAG cross-links (matched to a conservative estimate of enzymatic depletion efficacy) predicted a drop of 14% in tendon modulus. Corresponding mechanical properties derived from experiments on rat tail tendons treated in buffer with and without chondroitinase ABC were apparently unaffected, regardless of GAG depletion. Further tests for equivalence, conservatively based on effect size limits predicted by the model, confirmed equivalent stiffness between enzymatically depleted tendons and their native controls.Although the model predicts that relatively small quantities of GAGs acting as primary collagen cross-linking elements could provide mechanical integrity to the tendon, partial enzymatic depletion of GAGs should result in mechanical changes that are not reflected in analogous experimental testing. We thus conclude that GAG side chains of small leucine-rich proteoglycans are not a primary determinant of tensile mechanical behavior in mature rat tail tendons.     

Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.     

Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells

Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation.     

NMClab, a model to assess the contributions of muscle visco-elasticity and afferent feedback to joint dynamics

The dynamic behavior of a neuromusculoskeletal system results from the complex mechanical interaction between muscle visco-elasticity resulting from (co-)contraction and afferent feedback from muscle spindles and Golgi tendon organs. As a result of the multiple interactions the individual effect of each of the structures to the overall dynamics is hard to recognize, if not impossible. Here a neuromuscular control (NMC) model is developed to analyze the functional contribution of the various physiological structures on the mechanical behavior of a limb. The dynamics of a joint are presented in admittances, i.e. the dynamic relation between input force (or torque) and the output displacement, which can be represented by either frequency or impulse response functions. With the model it can be shown that afferent feedback reduces, while muscle visco-elasticity increases, the stability margins. This implicates that there is a delicate balance between muscle co-contraction and afferent feedback, which depends on the joint specific physiological properties. The main application of the model is educational; it is implemented in a graphical user interface allowing users to explore the role of the various physiological structures on joint dynamics. Other applications of the model are more experimental, e.g. to elucidate experimentally measured admittances and to compare the quantified parameter values with the theoretically optimal ones. It is concluded that the NMC model is a useful and intuitive tool to investigate human motor control, in a theoretical as well as an experimental way.     

Analysis of the gliding pattern of the canine flexor digitorum profundus tendon through the A2 pulley

Friction between a tendon and its pulley was first quantified using the concept of the arc of contact. Studies of human tendons conformed closely to a theoretical nylon cable/nylon rod model. However, we observed differences in measured friction that depended on the direction of motion in the canine model. We hypothesized that fibrocartilaginous nodules in the tendon affected the measurements and attempted to develop a theoretical model to explain the observations we made. Two force transducers were connected to each end of the canine flexor digitorum profundus tendon and the forces were recorded when it was moved through the A2 pulley toward a direction of flexion by an actuator and then reversed a direction toward extension. The changes of a force as a function of tendon excursion were evaluated in 20 canine paws. A bead cable/rod model was developed to simulate the canine tendon-pulley complex. To interpret the results, a free-body diagram was developed. The two prominent fibrocartilaginous nodules in the tendon were found to be responsible for deviation from a theoretical nylon cable gliding around the rod model, in a fashion analogous to the effect of the patella on the quadriceps mechanism. A bead cable/rod model qualitatively reproduced the findings observed in the canine tendon-pulley complex. Frictional coefficient of the canine flexor tendon-pulley was 0.016+/-0.005. After accounting for the effect created by the geometry of two fibrocartilaginous nodules within the tendon, calculation of frictional force in the canine tendon was possible.     

Prefabrication of 3D Cartilage Contructs: Towards a Tissue Engineered Auricle – A Model Tested in Rabbits

The reconstruction of an auricle for congenital deformity or following trauma remains one of the greatest challenges in reconstructive surgery. Tissue-engineered (TE) three-dimensional (3D) cartilage constructs have proven to be a promising option, but problems remain with regard to cell vitality in large cell constructs. The supply of nutrients and oxygen is limited because cultured cartilage is not vascular integrated due to missing perichondrium. The consequence is necrosis and thus a loss of form stability. The micro-surgical implantation of an arteriovenous loop represents a reliable technology for neovascularization, and thus vascular integration, of three-dimensional (3D) cultivated cell constructs. Auricular cartilage biopsies were obtained from 15 rabbits and seeded in 3D scaffolds made from polycaprolactone-based polyurethane in the shape and size of a human auricle. These cartilage cell constructs were implanted subcutaneously into a skin flap (15×8 cm) and neovascularized by means of vascular loops implanted micro-surgically. They were then totally enhanced as 3D tissue and freely re-implanted in-situ through microsurgery. Neovascularization in the prefabricated flap and cultured cartilage construct was analyzed by microangiography. After explantation, the specimens were examined by histological and immunohistochemical methods. Cultivated 3D cartilage cell constructs with implanted vascular pedicle promoted the formation of engineered cartilaginous tissue within the scaffold in vivo. The auricles contained cartilage-specific extracellular matrix (ECM) components, such as GAGs and collagen even in the center oft the constructs. In contrast, in cultivated 3D cartilage cell constructs without vascular pedicle, ECM distribution was only detectable on the surface compared to constructs with vascular pedicle. We demonstrated, that the 3D flaps could be freely transplanted. On a microangiographic level it was evident that all the skin flaps and the implanted cultivated constructs were well neovascularized. The presented method is suggested as a promising alternative towards clinical application of engineered cartilaginous tissue for plastic and reconstructive surgery.