Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system

Identification of functional programmable mechanical stimulation (PMS) on tendon not only provides the insight of the tendon homeostasis under physical/pathological condition, but also guides a better engineering strategy for tendon regeneration. The aims of the study are to design a bioreactor system with PMS to mimic the in vivo loading conditions, and to define the impact of different cyclic tensile strain on tendon. Rabbit Achilles tendons were loaded in the bioreactor with/without cyclic tensile loading (0.25 Hz for 8 h/day, 0–9% for 6 days). Tendons without loading lost its structure integrity as evidenced by disorientated collagen fiber, increased type III collagen expression, and increased cell apoptosis. Tendons with 3% of cyclic tensile loading had moderate matrix deterioration and elevated expression levels of MMP‐1, 3, and 12, whilst exceeded loading regime of 9% caused massive rupture of collagen bundle. However, 6% of cyclic tensile strain was able to maintain the structural integrity and cellular function. Our data indicated that an optimal PMS is required to maintain the tendon homeostasis and there is only a narrow range of tensile strain that can induce the anabolic action. The clinical impact of this study is that optimized eccentric training program is needed to achieve maximum beneficial effects on chronic tendinopathy management. Biotechnol. Bioeng. 2013; 110: 1495–1507. © 2012 Wiley Periodicals, Inc.     

Strain-related collagen gene expression in human osteoblast-like cells

The gene expression of cells in the musculoskeletal system, such as in bone, cartilage, ligament and tendon, is profoundly affected by mechanical loading. Previous studies have demonstrated that the expression of many genes, including collagen types I and III, are affected by mechanical strain in diverse cell types, such as human osteoblast-like SaOs-2 cells. However, whether the effect of mechanical loading on collagen gene expression is strain-related remains unclear. The goal of this study was to determine the relationship between mechanical strain and the gene expression of collagen types I and III in SaOs-2 cells. A Flexercell cellular mechanical loading system was used to subject SaOs-2 cells to equibiaxial cyclic tensile stress at a rate of 0.5 Hz with various strains of 5%, 7.5%, 10%, and 12.5% for 24 h. The relative amount of mRNA of both collagen I and collagen III increased at 5% strain compared with that of the control. As the strain increased, the relative amount of mRNA of collagen I remained stable at strain levels up to 12.5%. However, the mRNA for collagen III began to drop when the strain was greater than 5%, until a 10% strain was reached. From the application of a 10% strain through the maximum loading of a 12.5% strain, the relative amount of collagen III mRNA remained stable at amounts lower than that of the control. Thus, the gene expression of collagen types I and III responds differentially to mechanical strain at various magnitudes.     

Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.     

Aging enhances a mechanically-induced reduction in tendon strength by an active process involving matrix metalloproteinase activity

Age-associated and degenerative loss of functional integrity in soft tissues develops from effects of cumulative and subtle changes in their extracellular matrix (ECM). The highly ordered tendon ECM provides the tissue with its tensile strength during loading. As age and exercise collide in the high incidence of tendinopathies, we hypothesized that aged tendons fail due to cumulative damage resulting from a combination of diminished matrix repair and fragmentation of ECM proteins induced by prolonged cyclical loading, and that this is an active cell-mediated process. We developed an equine tendon explant model to examine the effect of age on the influence of prolonged cyclical loading at physiologically relevant strain rates (5% strain, 1 Hz for 24 h) on tissue mechanical properties, loss of ECM protein and matrix metalloproteinase (MMP) expression. We show significantly diminished mechanical strength of cyclically loaded tissue compared to controls (39.7 +/- 12%, P 相似文献   

The effect of step-wise increased stretching on rat calvarial osteoblast collagen production

Mechanical forces regulate the function of bone cells. In this paper, the effects of cyclic stretching on osteoblasts derived from rat calvaria were studied at a magnitude occurring in physiological loaded bone tissue. A four-point bending apparatus was used to apply cyclic stretching on osteoblasts. Stretching at 500 microepsilon for 2-24 h resulted in an increase in matrix synthesis(P<0.01). In contrast, the cyclic stretching at 1000 and 1500 microepsilon for 2-24 h inhibited osteoblast collagen production (P<0.01). We also described our new loading method to increase strain magnitude step-by-step. The strain magnitude increased by 500 microepsilon increments from 500 to 1500 microepsilon every 2 or 12 h, respectively. Results showed that osteoblasts could absorb large amount of proline for collagen synthesis when stretched at 500 microepsilon. However, not all the absorbed proline was used to synthesize collagen. Some of it was stored in cells. When the suitable signal (500 microepsilon) was changed to an inhibiting signal (1000 microepsilon), cells responded to it accordingly and released proline to medium. These results demonstrate that the response of osteoblasts is dependent on the magnitude of the strain applied and cells can adjust their bio-chemical response to adapt to the changing environmental stimulation.     

In situ cell–matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell–matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell–matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (～2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell–matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.     

Involvement of stretch-activated ion channels in strain-regulated glycosaminoglycan synthesis in mesenchymal stem cell-seeded 3D scaffolds

The objective of this study was to determine if cyclic tensile strain would regulate the rate of glycosaminoglycan synthesis via stretch-activated ion channels in adult mesenchymal stem cells seeded in a collagen type I-glycosaminoglycan scaffold and treated with TGF-beta1. The application of 10% cyclic tensile loading at 1Hz for 7 days significantly increased the rate of glycosaminoglycan synthesis, as assessed using [(35)S] sulphate incorporation. This increase was attenuated in the presence of a stretch-activated ion channel inhibitor (10microM gadolinium chloride) demonstrating the involvement, in part, of these ion channels in the mechanotransduction pathway that couples cyclic tensile loading to matrix synthesis.     

Up-regulated α-actin expression is associated with cell adhesion ability in 3-D cultured myocytes subjected to mechanical stimulation

This study was aimed to investigate the alteration of α-actin in three-dimensionally (3-D) cultured myocytes under cyclic tensile stress loading. Myocytes were collected from neonatal SD rat’s lateral pterygoid muscle for primary cell culture. The third-passage cells were implanted and 3-D cultured in poly lactic-co-glycolic acid (PLGA) scaffold, and then subjected to cyclic tensile stress (0.5 Hz, 2,000 μstrain) for 0, 2, 4, 8, 12, and 24 h through a four-point bending strain system. The α-actin mRNA was investigated by semi-quantitative RT–PCR. The α-actin protein expression was examined by immunofluorescent cytochemistry, laser confocal scanning microscopy (LCSM), and image analysis technology. The dynamic adhesion of myocytes to PLGA scaffolds was investigated by fluorescence microscope and the viability of the myocytes was measured by MTT assay. After mechanical loading, the α-actin mRNA increased at 2 h and then declined. The α-actin protein expression kept increased until peaked at 12 h, but declined at 24 h. The time course changing of α-actin protein expression parallelled with that of cell adhesion ability. It is concluded that α-actin expression is probably associated with cell adhesion ability in myocytes subjected to mechanical stimulation.     

Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation.

Overcoming the limited ability of articular cartilage to self-repair may be possible through tissue engineering. However, bioengineered cartilage formed using current methods does not match the physical properties of native cartilage. In previous studies we demonstrated that mechanical stimulation improved cartilage tissue formation. This study examines the mechanisms by which this occurs. Application of uniaxial, cyclic compression (1 kPa, 1 Hz, 30 min) significantly increased matrix metalloprotease (MMP)-3 and MMP-13 gene expression at 2 h compared to unstimulated cells. These returned to constitutive levels by 6 h. Increased MMP-13 protein levels, both pro- and active forms, were detected at 6 h and these decreased by 24 h. This was associated with tissue degradation as more proteoglycans and collagen had been released into the culture media at 6 h when compared to the unstimulated cells. This catabolic change was followed by a significant increase in type II collagen and aggrecan gene expression at 12 h post-stimulation and increased synthesis and accumulation of these matrix molecules at 24 h. Mechanical stimulation activated the MAP kinase pathway as there was increased phosphorylation of ERK1/2 and JNK as well as increased AP-1 binding. Mechanical stimulation in the presence of the JNK inhibitor, SP600125, blocked AP-1 binding preventing the increased gene expression of MMP-3 and -13 at 2 h and type II collagen and aggrecan at 12 h as well as the increased matrix synthesis and accumulation. Given the sequence of changes, cyclic compressive loading appears to initiate a remodelling effect involving MAPK and AP-1 signalling resulting in improved in vitro formation of cartilage.     

Understanding strain-induced collagen matrix development in engineered cardiovascular tissues from gene expression profiles

Mechanical conditioning is often used to enhance collagen synthesis, remodeling and maturation and, hence, the structural and mechanical properties of engineered cardiovascular tissues. Intermittent straining, i.e., alternating periods of cyclic and static strain, has previously been shown to result in more mature tissue compared with continuous cyclic straining. Nevertheless, the underlying mechanism is unknown. We have determined the short-term effects of continuous cyclic strain and of cyclic strain followed by static strain at the gene expression level to improve insight into the mechano-regulatory mechanism of intermittent conditioning on collagen synthesis, remodeling and maturation. Tissue-engineered constructs, consisting of human vascular-derived cells seeded onto rapidly degrading PGA/P4HB scaffolds, were conditioned with 4% strain at 1 Hz for 3 h in order to study the immediate effects of cyclic strain (n=18). Next, the constructs were either subjected to ongoing cyclic strain (4% at 1 Hz; n=9) or to static strain (n=9). Expression levels of genes involved in collagen synthesis, remodeling and maturation were studied at various time points up to 24 h within each straining protocol. The results indicate that a period of static strain following cyclic strain favors collagen synthesis and remodeling, whereas ongoing cyclic strain shifts this balance toward collagen remodeling and maturation. The data suggest that, with prolonged culture, the conditioning protocol should be changed from intermittent straining to continuous cyclic straining to improve collagen maturation after its synthesis and, hence, the tissue (mechanical) properties.     

Mechanical response of individual collagen fibrils in loaded tendon as measured by atomic force microscopy

A precise analysis of the mechanical response of collagen fibrils in tendon tissue is critical to understanding the ultrastructural mechanisms that underlie collagen fibril interactions (load transfer), and ultimately tendon structure–function. This study reports a novel experimental approach combining macroscopic mechanical loading of tendon with a morphometric ultrascale assessment of longitudinal and cross-sectional collagen fibril deformations. An atomic force microscope was used to characterize diameters and periodic banding (D-period) of individual type-I collagen fibrils within murine Achilles tendons that were loaded to 0%, 5%, or 10% macroscopic nominal strain, respectively. D-period banding of the collagen fibrils increased with increasing tendon strain (2.1% increase at 10% applied tendon strain, p < 0.05), while fibril diameter decreased (8% reduction, p < 0.05). No statistically significant differences between 0% and 5% applied strain were observed, indicating that the onset of fibril (D-period) straining lagged macroscopically applied tendon strains by at least 5%. This confirms previous reports of delayed onset of collagen fibril stretching and the role of collagen fibril kinematics in supporting physiological tendon loads. Fibril strains within the tissue were relatively tightly distributed in unloaded and highly strained tendons, but were more broadly distributed at 5% applied strain, indicating progressive recruitment of collagen fibrils. Using these techniques we also confirmed that collagen fibrils thin appreciably at higher levels of macroscopic tendon strain. Finally, in contrast to prevalent tendon structure–function concepts data revealed that loading of the collagen network is fairly homogenous, with no apparent predisposition for loading of collagen fibrils according to their diameter.     

Tenogenic differentiation of equine adipose-tissue-derived stem cells under the influence of tensile strain, growth differentiation factors and various oxygen tensions

Mesenchymal stem cells have become extremely interesting for regenerative medicine and tissue engineering in the horse. Stem cell therapy has been proven to be a powerful and successful instrument, in particular for the healing of tendon lesions. We pre-differentiated equine adipose-tissue-derived stem cells (ASCs) in a collagen I gel scaffold by applying tensile strain, growth differentiation factors (GDFs) and various oxygen tensions in order to determine the optimal conditions for in vitro differentiation toward the tenogenic lineage. We compared the influence of 3% versus 21% oxygen tension, the use of GDF 5, GDF 6 and GDF 7 and the application of uniaxial tensile strain versus no mechanical stimulation on differentiation results as evaluated by cell morphology and by the expression of the tendon-relevant genes collagen I, collagen III, cartilage oligomeric matrix protein and scleraxis. The best results were obtained with an oxygen tension of 21%, tensile stimulation and supplementation with GDF 5 or GDF 7. This approach raises the hope that the in vivo application of pre-differentiated stem cells will improve healing and recovery time in comparison with treatment involving undifferentiated stem cells.     

Viscoelastic properties of collagen: synchrotron radiation investigations and structural model

Collagen type I is the most abundant structural protein in tendon, skin and bone, and largely determines the mechanical behaviour of these connective tissues. To obtain a better understanding of the relationship between structure and mechanical properties, tensile tests and synchrotron X-ray scattering have been carried out simultaneously, correlating the mechanical behaviour with changes in the microstructure. Because intermolecular cross-links are thought to have a great influence on the mechanical behaviour of collagen, we also carried out experiments using cross-link-deficient tail-tendon collagen from rats fed with beta-APN, in addition to normal controls. The load-elongation curve of tendon collagen has a characteristic shape with, initially, an increasing slope, corresponding to an increasing stiffness, followed by yielding and then fracture. Cross-link-deficient collagen produces a quite different curve with a marked plateau appearing in some cases, where the length of the tendon increases at constant stress. With the use of in situ X-ray diffraction, it was possible to measure simultaneously the elongation of the collagen fibrils inside the tendon and of the tendon as a whole. The overall strain of the tendon was always larger than the strain in the individual fibrils, which demonstrates that some deformation is taking place in the matrix between fibrils. Moreover, the ratio of fibril strain to tendon strain was dependent on the applied strain rate. When the speed of deformation was increased, this ratio increased in normal collagen but generally decreased in cross-link-deficient collagen, correlating to the appearance of a plateau in the force-elongation curve indicating creep. We proposed a simple structural model, which describes the tendon at a hierarchical level, where fibrils and interfibrillar matrix act as coupled viscoelastic systems. All qualitative features of the strain-rate dependence of both normal and cross-link-deficient collagen can be reproduced within this model. This complements earlier models that considered the next smallest level of hierarchy, describing the deformation of collagen fibrils in terms of changes in their molecular packing.     

Tendon tissue microdamage and the limits of intrinsic repair

The transmission of mechanical muscle force to bone for musculoskeletal stability and movement is one of the most important functions of tendon. The load-bearing tendon core is composed of highly aligned collagen-rich fascicles interspersed with stromal cells (tenocytes). Despite being built to bear very high mechanical stresses, supra-physiological/repetitive mechanical overloading leads to tendon microdamage in fascicles, and potentially to tendon disease and rupture. To date, it is unclear to what extent intrinsic healing mechanisms of the tendon core compartment can repair microdamage. In the present study, we investigated the healing capacity of the tendon core compartment in an ex vivo tissue explant model. To do so, we isolated rat tail tendon fascicles, damaged them by applying a single stretch to various degrees of sub-rupture damage and longitudinally assessed downstream functional and structural changes over a period of several days. Functional damage was assessed by changes in the elastic modulus of the material stress-strain curves, and biological viability of the resident tenocytes. Structural damage was quantified using a fluorescent collagen hybridizing peptide (CHP) to label mechanically disrupted collagen structures. While we observed functional mechanical damage for strains above 2% of the initial fascicle length, structural collagen damage was only detectable for 6% strain and beyond. Minimally loaded/damaged fascicles (2–4% strain) progressively lost elastic modulus over the course of tissue culture, despite their collagen structures remaining intact with high degree of maintained cell viability. In contrast, more severely overloaded fascicles (6–8% strain) with damage at the molecular/collagen level showed no further loss of the elastic modulus but markedly decreased cell viability. Surprisingly, in these heavily damaged fascicles the elastic modulus partially recovered, an effect also seen in further experiments on devitalized fascicles, implying the possibility of a non-cellular but matrix-driven mechanism of molecular repair. Overall, our findings indicate that the tendon core has very little capacity for self-repair of microdamage. We conclude that stromal tenocytes likely do not play a major role in anabolic repair of tendon matrix microdamage, but rather mediate catabolic matrix breakdown and communication with extrinsic cells that are able to effect tissue repair.     

Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions,Changes Matrix Architecture,and Induces an Inflammatory Phenotype

Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α₁₁ mRNA and protein expression, and an increase in α₂ integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced.     

Role of TGF-beta1 in relation to exercise-induced type I collagen synthesis in human tendinous tissue.

Mechanical loading of tissue is known to influence local collagen synthesis, and microdialysis studies indicate that mechanical loading of human tendon during exercise elevates tendinous type I collagen production. Transforming growth factor-beta1 (TGF-beta1), a potent stimulator of type I collagen synthesis, is released from cultured tendon fibroblasts in response to mechanical loading. Thus TGF-beta1 could link mechanical loading and collagen synthesis in tendon tissue in vivo. Tissue levels of TGF-beta1 and type I collagen metabolism markers [procollagen I COOH-terminal propeptide (PICP) and COOH-terminal telopeptide of type I collagen (ICTP)] were measured by microdialysis in peritendinous tissue of the Achilles' tendon in six male volunteers before and after treadmill running (1 h, 12 km/h, 3% uphill). In addition, blood levels of TGF-beta1, PICP, and ICTP were obtained. PICP levels increased 68 h after exercise (P < 0.05). Dialysate levels of TGF-beta1 changed from 303 +/- 46 pg/ml (at rest) to 423 +/- 86 pg/ml 3 h postexercise. This change was nonsignificant, but the decay of tissue TGF-beta1 after catheter insertion was markedly delayed by exercise compared with the decay seen in resting subjects. Plasma concentrations of TGF-beta1 rose 30% in response to exercise (P < 0.05 vs. pre). Our observations indicate an increased local production of type I collagen in human peritendinous tissue in response to uphill running. Although not conclusive, changes in circulating and local TGF-beta1, in response to exercise, suggest a role for TGF-beta1 in mechanical regulation of local collagen type I synthesis in tendon-related connective tissue in vivo.     

The dynamics of collagen uncrimping and lateral contraction in tendon and the effect of ionic concentration

Under tensile loading, tendon undergoes a number of unique structural changes that govern its mechanical response. For example, stretching a tendon is known to induce both the progressive “uncrimping” of wavy collagen fibrils and extensive lateral contraction mediated by fluid flow out of the tissue. However, it is not known whether these processes are interdependent. Moreover, the rate-dependence of collagen uncrimping and its contribution to tendon's viscoelastic mechanical properties are unknown. Therefore, the objective of this study was to (a) develop a methodology allowing for simultaneous measurement of crimp, stress, axial strain and lateral contraction in tendon under dynamic loading; (b) determine the interdependence of collagen uncrimping and lateral contraction by testing tendons in different swelling conditions; and (c) assess how the process of collagen uncrimping depends on loading rate. Murine flexor carpi ulnaris (FCU) tendons in varying ionic environments were dynamically stretched to a set strain level and imaged through a plane polariscope with the polarizer and analyzer at a fixed angle. Analysis of the resulting images allowed for direct measurement of the crimp frequency and indirect measurement of the tendon thickness. Our findings demonstrate that collagen uncrimping and lateral contraction can occur independently and interstitial fluid impacts tendon mechanics directly. Furthermore, tensile stress, transverse contraction and degree of collagen uncrimping were all rate-dependent, suggesting that collagen uncrimping plays a role in tendon's dynamic mechanical response. This study is the first to characterize the time-dependence of collagen uncrimping in tendon, and establishes structure–function relationships for healthy tendons that can be used to better understand and assess changes in tendon mechanics after disease or injury.     

Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves

This study addressed the following questions: 1) Does cyclic tensile strain induce protein expression patterns consistent with myxomatous degeneration in mitral valves? 2) Does cyclic strain induce local serotonin synthesis in mitral valves? 3) Are cyclic strain-induced myxomatous protein expression patterns in mitral valves dependent on local serotonin? Cultured sheep mitral valve leaflets were subjected to 0, 10, 20, and 30% cyclic strain for 24 and 72 h. Protein levels of activated myofibroblast phenotype markers, α-smooth muscle actin (α-SMA) and nonmuscle embryonic myosin (SMemb); matrix catabolic enzymes, matrix metalloprotease (MMP) 1 and 13, and cathepsin K; and sulfated glycosaminoglycan (GAG) content in mitral valves increased with increased cyclic strain. Serotonin was present in the serum-free media of cultured mitral valves and concentrations increased with cyclic strain. Expression of the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1) increased in strained mitral valves. Pharmacologic inhibition of the serotonin 2B/2C receptor or TPH1 diminished expression of phenotype markers (α-SMA and SMemb) and matrix catabolic enzyme (MMP1, MMP13, and cathepsin K) expression in 10- and 30%-strained mitral valves. These results provide first evidence that mitral valves synthesize serotonin locally. The results further demonstrate that tensile loading modulates local serotonin synthesis, expression of effector proteins associated with mitral valve degeneration, and GAG synthesis. Inhibition of serotonin diminishes strain-mediated protein expression patterns. These findings implicate serotonin and tensile loading in mitral degeneration, functionally link the pathogeneses of serotoninergic (carcinoid, drug-induced) and degenerative mitral valve disease, and have therapeutic implications.     

An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery

Tendons attach muscles to bone and thereby transmit tensile forces during joint movement. However, a detailed understanding of the mechanisms that establish the mechanical properties of tendon has remained elusive because of the practical difficulties of studying tissue mechanics in vivo. Here we have performed a study of tendon-like constructs made by culturing embryonic tendon cells in fixed-length fibrin gels. The constructs display mechanical properties (toe–linear–fail stress–strain curve, stiffness, ultimate tensile strength, and failure strain) as well as collagen fibril volume fraction and extracellular matrix (ECM)/cell ratio that are statistically similar to those of embryonic chick metatarsal tendons. The development of mechanical properties during time in culture was abolished when the constructs were treated separately with Triton X-100 (to solubilise membranes), cytochalasin (to disassemble the actin cytoskeleton) and blebbistatin (a small molecule inhibitor of non-muscle myosin II). Importantly, these treatments had no effect on the mechanical properties of the constructs that existed prior to treatment. Live-cell imaging and ¹⁴C-proline metabolic labeling showed that blebbistatin inhibited the contraction of the constructs without affecting cell viability, procollagen synthesis, or conversion of procollagen to collagen. In conclusion, the mechanical properties per se of the tendon constructs are attributable to the ECM generated by the cells but the improvement of mechanical properties during time in culture was dependent on non-muscle myosin II-derived forces.