Substance P is a mechanoresponsive, autocrine regulator of human tenocyte proliferation

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell© technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10⁻⁷ M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

Gap junctions in IL-1β-mediated cell survival response to strain

Mechanical stimuli play important roles in proliferation and differentiation of connective tissue cells, and development and homeostatic maintenance of tissues. However, excessive mechanical loading to a tissue can injure cells and disrupt the matrix, as occurs in tendinopathy. Tendinopathy is a common clinical problem in athletes and in many occupational settings due to overuse of the tendon. Moreover, interleukin (IL)-1β is generally considered to be a "bad" cytokine, activating NF-κb and cell death and inducing matrix metalloproteinase (MMPs 1, 2, 3) expression and matrix destruction. However, activated NF-κB can also drive a cell survival pathway. We have reported that cyclic strain induced tenocyte death in three-dimensional (3D) cultures, and IL-1β could promote cell survival under strain. Therefore, it was hypothesized that 1) cyclic strain could induce cell death in tenocytes as observed in pathologic tendons in vivo; 2) a gene expression profile indicative of tendinopathy could be identified; and 3) low-dose IL-1β could protect cells from strain-induced, tendinopathy-like changes. Human tenocytes were cultured in 3D type I collagen hydrogels and subjected to 3.5% elongation at 1 Hz for 1 h/day for up to 5 days with or without IL-1β. Real-time RT-PCR data showed that cyclic strain regulated the expression of tendinopathy marker genes in a manner similar to that found in pathological tendons from patients and that addition of IL-1β reversed the gene expression changes to control levels. Results of further studies showed that IL-1β may modulate cell survival through upregulating the expression of connexin 43, which is involved in the modulation of cell death/survival in a variety of cells and tissues. The elucidation of the mechanisms underlying strain-induced cell death and recovery from strain injury will facilitate our understanding of the pathogenesis of tendinopathy and may lead to the discovery of new molecular targets for early diagnosis and treatment of tendinopathy.     

Gap junction permeability between tenocytes within tendon fascicles is suppressed by tensile loading

Gap junction communication is an essential component in the mechanosensitive response of tenocytes. However, little is known about direct mechanoregulation of gap junction turnover and permeability. The present study tests the hypothesis that mechanical loading alters gap junction communication between tenocyte within tendon fascicles. Viable tenocytes within rat tail tendon fasicles were labelled with calcein-AM and subjected to a fluorescent loss induced by photobleaching (FLIP) protocol. A designated target cell within a row of tenocytes was continuously photobleached at 100% laser power whilst recording the fluorescent intensity of neighbouring cells. A mathematical compartment model was developed to estimate the intercellular communication between tenocytes based upon the experimental FLIP data. This produced a permeability parameter, k, which quantifies the degree of functioning gap functions between cells as confirmed by the complete inhibition of FLIP by the inhibitor 18α-glycyrrhentic acid. The application of 1N static tensile load for 10?min had no effect on gap junction communication. However, when loading was increased to 1?h, there was a statistically significant reduction in gap junction permeability. This coincided with suppression of connexin 43 protein expression in loaded samples as determined by confocal immunofluorescence. However, there was an upregulation of connexin 43 mRNA. These findings demonstrate that tenocytes remodel their gap junctions in response to alterations in mechanical loading with a complex mechanosensitive mechanism of breakdown and remodelling. This is therefore the first study to show that tenocyte gap junctions are not only important in transmitting mechanically activated signals but that mechanical loading directly regulates gap junction permeability.     

Predicting tenocyte expression profiles and average molecular concentrations in Achilles tendon ECM from tissue strain and fiber damage

In this study, we propose a method for quantitative prediction of changes in concentrations of a number of key signaling, structural and effector molecules within the extracellular matrix of tendon. To achieve this, we introduce the notion of elementary cell responses (ECRs). An ECR defines a normal reference secretion profile of a molecule by a tenocyte in response to the tenocyte’s local strain. ECRs are then coupled with a model for mechanical damage of tendon collagen fibers at different straining conditions of tendon and then scaled up to the tendon tissue level for comparison with experimental observations. Specifically, our model predicts relative changes in ECM concentrations of transforming growth factor beta, interleukin 1 beta, collagen type I, glycosaminoglycan, matrix metalloproteinase 1 and a disintegrin and metalloproteinase with thrombospondin motifs 5, with respect to tendon straining conditions that are consistent with the observations in the literature. In good agreement with a number of in vivo and in vitro observations, the model provides a logical and parsimonious explanation for how excessive mechanical loading of tendon can lead to under-stimulation of tenocytes and a degenerative tissue profile, which may well have bearing on a better understanding of tendon homeostasis and the origin of some tendinopathies.     

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.     

ATP modulates load-inducible IL-1beta,COX 2, and MMP-3 gene expression in human tendon cells

Tendon cells receive mechanical signals from the load bearing matrices. The response to mechanical stimulation is crucial for tendon function. However, overloading tendon cells may deteriorate extracellular matrix integrity by activating intrinsic factors such as matrix metalloproteinases (MMPs) that trigger matrix destruction. We hypothesized that mechanical loading might induce interleukin-1beta (IL-1beta) in tendon cells, which can induce MMPs, and that extracellular ATP might inhibit the load-inducible gene expression. Human tendon cells isolated from flexor digitorum profundus tendons (FDPs) of four patients were made quiescent and treated with ATP (10 or 100 microM) for 5 min, then stretched equibiaxially (1 Hz, 3.5% elongation) for 2 h followed by an 18-h-rest period. Stretching induced IL-1beta, cyclooxygenase 2 (COX 2), and MMP-3 genes but not MMP-1. ATP reduced the load-inducible gene expression but had no effect alone. A medium change caused tendon cells to secrete ATP into the medium, as did exogenous UTP. The data demonstrate that mechanical loading induces ATP release in tendon cells and stimulates expression of IL-1beta, COX 2, and MMP-3. Load-induced endogenous IL-1beta may trigger matrix remodeling or a more destructive pathway(s) involving IL-1beta, COX 2, and MMP-3. Concomitant autocrine and paracrine release of ATP may serve as a negative feedback mechanism to limit activation of such an injurious pathway. Attenuation or failure of this negative feedback mechanism may result in the progression to tendinosis.     

Expression of scleraxis and tenascin C in equine adipose and umbilical cord blood derived stem cells is dependent upon substrata and FGF supplementation

Recovery from tendon injury is based on long periods of rest, which results in sub-optimal repair, often replacing tendon with fibrocartilage scar tissue. Recently, the use of stem cells in equine tendon repair has been attempted with variable success. The objective of this work was to determine the expression of scleraxis (scx) and tenascin C (TnC), two markers of tenocytes, in adipose (AdMSC) and umbilical cord blood (UCB) stem cells during culture on various substrata and in response to fibroblast growth factor (FGF) treatment. Equine UCB and AdMSC were cultured on gelatin-coated plasticware, 30 % matrigel or collagen-coated Cytodex beads and treated with 10 ng/ml FGF2, FGF4 or FGF5 prior to measurement of proliferation, kinase activity and tenocyte gene expression. Supplementation with FGF2 or FGF5 activated the ERK1/2 signaling pathway in AdMSC and UCB; no effect of FGF4 was observed in UCB. FGF2 increased proliferation in AdMSC but not UCB. Conversely, FGF5 stimulated proliferation of UCB. Culture in matrigel increased scx expression in both cell populations and increased TnC in AdMSC. In AdMSC grown in matrigel, supplementation with FGF2 or FGF5 increased TnC expression. Thus, culture conditions (substrata and FGF supplementation) impact markers of tenocytes in AdMSC and UCB stem cells, indicating that careful consideration should be given to culture conditions prior to use of UCB or AdMSC as therapeutic aids. Optimal culture conditions may promote early differentiation of these cells, improving their ability to aid tendon regeneration and facilitating more efficient recovery from tendon injury.     

Continuous cultivation of human hamstring tenocytes on microcarriers in a spinner flask bioreactor system

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering‐based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon‐derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 × 10⁶ cells/1000 cm² surface) on Cytodex? type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD‐PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead‐to‐bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:142–151, 2014     

In situ fibril stretch and sliding is location-dependent in mouse supraspinatus tendons

Tendons are able to transmit high loads efficiently due to their finely optimized hierarchical collagen structure. Two mechanisms by which tendons respond to load are collagen fibril sliding and deformation (stretch). Although many studies have demonstrated that regional variations in tendon structure, composition, and organization contribute to the full tendon׳s mechanical response, the location-dependent response to loading at the fibril level has not been investigated. In addition, the instantaneous response of fibrils to loading, which is clinically relevant for repetitive stretch or fatigue injuries, has also not been studied. Therefore, the purpose of this study was to quantify the instantaneous response of collagen fibrils throughout a mechanical loading protocol, both in the insertion site and in the midsubstance of the mouse supraspinatus tendon. Utilizing a novel atomic force microscopy-based imaging technique, tendons at various strain levels were directly visualized and analyzed for changes in fibril d-period with increasing tendon strain. At the insertion site, d-period significantly increased from 0% to 1% tendon strain, increased again from 3% to 5% strain, and decreased after 5% strain. At the midsubstance, d-period increased from 0% to 1% strain and then decreased after 7% strain. In addition, fibril d-period heterogeneity (fibril sliding) was present, primarily at 3% strain with a large majority occurring in the tendon midsubstance. This study builds upon previous work by adding information on the instantaneous and regional-dependent fibrillar response to mechanical loading and presents data proposing that collagen fibril sliding and stretch are directly related to tissue organization and function.     

Age-related effect of static and cyclic loadings on the strain-force curve of the vastus lateralis tendon and aponeurosis

The objective of the present study was to investigate the age-related effects of submaximal static and cyclic loading on the mechanical properties of the vastus lateralis (VL) tendon and aponeurosis in vivo. Fourteen old and 12 young male subjects performed maximal voluntary isometric knee extensions (MVC) on a dynamometer before and after (a) a sustained isometric contraction at 25% MVC and (b) isokinetic contractions at 50% isokinetic MVC, both until task failure. The elongation of the VL tendon and aponeurosis was examined using ultrasonography. To calculate the resultant knee joint moment, the kinematics of the leg were recorded with eight cameras (120 Hz). The old adults displayed significantly lower maximal moments but higher strain values at any given tendon force from 400 N and up in all tested conditions. Neither of the loading protocols influenced the strain-force relationship of the VL tendon and aponeurosis in either the old or young adults. Consequently, the capacity of the tendon and aponeurosis to resist force remained unaffected in both groups. It can be concluded that in vivo tendons are capable of resisting long-lasting static (~4.6 min) or cyclic (~18.5 min) mechanical loading at the attained strain levels (4-5%) without significantly altering their mechanical properties regardless of age. This implies that as the muscle becomes unable to generate the required force due to fatigue, the loading of the tendon is terminated prior to provoking any significant changes in tendon mechanical properties.     

Uniaxial mechanical tension promoted osteogenic differentiation of rat tendon-derived stem cells (rTDSCs) via the Wnt5a-RhoA pathway

Chronic tendinopathy is a tendon disorder that is common in athletes and individuals whose tendons are subjected to repetitive strain injuries. The presence of ossification worsened the clinical manifestation of the disorder. The change of tendon loading due to mechanical overload, compression, or disuse have been implicated as the possible etiologies, but the pathological mechanisms of tendinopathy remain unclear. In this study, we demonstrated that ossification in tendon tissue might be due to the osteogenesis of tendon‐derived stem cells (TDSCs) induced by uniaxial mechanical tension (UMT) which mimics the mechanical loading in tendon. Rat TDSCs (rTDSCs) could be induced to differentiate into osteogenic lineage after treatment with 2% elongation UMT for 3 days as shown by the increased expression Runx2 mRNA and protein, Alpl mRNA, collagen type 1 alpha 1 (Col1a1) mRNA, ALP activity, and ALP cytochemical staining. RhoA, an osteogenesis regulator, was activated in rTDSCs upon UMT stimulation. Blockage of RhoA activity in rTDSCs by C3 toxin or ROCK activity, a downstream target of RhoA, by Y‐27632 inhibited UMT‐induced osteogenesis in rTDSCs. UMT up‐regulated the mRNA expression of Wnt5a but not the other non‐canonical Wnts. The inhibition of Wnt5a expression by siRNA abolished UMT‐induced Runx2 mRNA expression and RhoA activation in rTDSCs and the inhibition of Runx2 expression could be rescued by addition of LPA, a RhoA activator. In conclusion, our results showed that UMT induced osteogenic differentiation of rTDSCs via the Wnt5a‐RhoA pathway, which might contribute to ectopic ossification in tendon tissue due to mechanical loading. J. Cell. Biochem. 113: 3133–3142, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

Influence of a single loading episode on gene expression in healing rat Achilles tendons

Mechanical loading stimulates tendon healing via mechanisms that are largely unknown. Genes will be differently regulated in loaded healing tendons, compared with unloaded, just because of the fact that healing processes have been changed. To avoid such secondary effects and study the effect of loading per se, we therefore studied the gene expression response shortly after a single loading episode in otherwise unloaded healing tendons. The Achilles tendon was transected in 30 tail-suspended rats. The animals were let down from the suspension to load their tendons on a treadmill for 30 min once, 5 days after tendon transection. Gene expression was studied by Affymetrix microarray before and 3, 12, 24, and 48 h after loading. The strongest response in gene expression was seen 3 h after loading, when 150 genes were up- or downregulated (fold change ≥2, P ≤ 0.05). Twelve hours after loading, only three genes were upregulated, whereas 38 were downregulated. Fewer than seven genes were regulated after 24 and 48 h. Genes involved in the inflammatory response were strongly regulated at 3 and 12 h after loading; this included upregulation of iNOS, PGE synthase, and IL-1β. Also genes involved in wound healing/coagulation, angiogenesis, and production of reactive oxygen species were strongly regulated by loading. Microarray results were confirmed for 16 selected genes in a repeat experiment (N = 30 rats) using real-time PCR. It was also confirmed that a single loading episode on day 5 increased the strength of the healing tendon on day 12. In conclusion, the fact that there were hardly any regulated genes 24 h after loading suggests that optimal stimulation of healing requires a mechanical loading stimulus every day.     

Development of a technique to determine strains in tendons using the cell nuclei

Tenocytes detect mechanical stimuli in vivo, and respond through mechanotransduction pathways to initiate matrix remodelling in tendons. Due to the crimped nature of tendon fascicles, the strain field throughout is non-homogeneous. The present study has developed a means to quantify the local strain fields within a fascicle by monitoring the relative movement and deformation of fluorescently labelled tenocyte nuclei. A stage mounted test rig was designed to apply tensile strain to fascicles. Rat tail and bovine extensor tendons were harvested for analysis, and the cell nuclei stained and visualised using an inverted confocal microscope. As the fascicles were subjected to gross strains of up to 5%, the movement of selected tenocyte nuclei were recorded. Results from a series of cell nuclei from both tendon sources revealed that local strains were significantly less than the applied strain. The nuclei length to width ratio, an indicator of cell deformation, also increased with applied strain, most significantly between 2 and 3% applied strain.     

Connexin 32 and 43 gap junctions differentially modulate tenocyte response to cyclic mechanical load

Gap junctions allow rapid exchange of ions and small metabolites between cells. They can occur between connective tissue cells, and in tendons there are two prominent types, composed of connexin 32 or 43. These form distinct networks - tenocyte rows are linked by both longitudinally, but only by connexin 43 laterally. We hypothesised that the junctions had different roles in cell response to mechanical loading, and measured the effects of inhibitors of gap junction function on secretion of collagen by tenocyte cultures exposed to mechanical strain. Chicken tendon fibroblasts were exposed to cyclic tensile loading in the presence or absence of general gap junction inhibitors (halothane or the biomimetic peptide gap27), or antisense oligonucleotides to chicken connexin 32 or 43. Untreated cultures increased collagen secretion by around 25% under load. Halothane eliminated this response but caused cell damage. Gap27 peptide reduced secretion but maintained loading effects - strained cultures secreting more collagen than unstrained. Antisense downregulation showed major differences between connexins: antisense 32 reduced, and antisense 43 increased, collagen secretion. In both cases loading effects were maintained. This shows that (i) gap junctional integration of signals is important in load response of tenocyte populations - mechanotransduction occurs in individual cells but integration of signals markedly enhances it and (ii) communication via connexin 32 and 43 have differential effects on the load response, with connexin 32 being stimulatory and connexin 43 being inhibitory. Cells coordinate and control their response to mechanical signals at least in part by differential actions of these two types of gap junction.