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Response to external stimuli such as mechanical signals is critical for normal function of cells, especially when subjected to repetitive motion. Tenocytes receive mechanical stimuli from the load-bearing matrix as tension, compression, and shear stress during tendon gliding. Overloading a tendon by high strain, shear, or repetitive motion can cause matrix damage. Injury may induce cytokine expression, matrix metalloproteinase (MMP) expression and activation resulting in loss of biomechanical properties. These changes may result in tendinosis or tendinopathy. Alternatively, an immediate effector molecule may exist that acts in a signal-dampening pathway. Adenosine 5'-triphosphate (ATP) is a candidate signal blocker of mechanical stimuli. ATP suppresses load-inducible inflammatory genes in human tendon cells in vitro. ATP and other extracellular nucleotide signaling are regulated efficiently by two distinct mechanisms: purinoceptors via specific receptor-ligand binding and ecto-nucleotidases via the hydrolysis of specific nucleotide substrates. ATP is released from tendon cells by mechanical loading or by uridine 5'-triphosphate (UTP) stimulation. We hypothesized that mechanical loading might stimulate ecto-ATPase activity. Human tendon cells of surface epitenon (TSC) and internal compartment (TIF) were cyclically stretched (1 Hz, 0.035 strain, 2 h) with or without ATP. Aliquots of the supernatant fluids were collected at various time points, and ATP concentration (ATP) was determined by a luciferin-luciferase bioluminescence assay. Total RNA was isolated from TSC and TIF (three patients) and mRNA expression for ecto-nucleotidase was analyzed by RT-PCR. Human tendon cells secreted ATP in vitro (0.5-1 nM). Exogenous ATP was hydrolyzed within minutes. Mechanical load stimulated ATPase activity. ATP was hydrolyzed in mechanically loaded cultures at a significantly greater rate compared to no load controls. Tenocytes (TSC and TIF) expressed ecto-nucleotidase mRNA (ENTPD3 and ENPP1, ENPP2). These data suggest that motion may release ATP from tendon cells in vivo, where ecto-ATPase may also be activated to hydrolyze ATP quickly. Ecto-ATPase may act as a co-modulator in ATP load-signal modulation by regulating the half-life of extracellular purine nucleotides. The extracellular ATP/ATPase system may be important for tendon homeostasis by protecting tendon cells from responding to excessive load signals and activating injurious pathways.

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.

Background context: Whole body vibration is an important factor contributing to low back and radicular pain. Vibratory loading as a mechanical stimulus is transferred to connective tissues as energy from ground reaction forces, as well as a direct input from the use of motorized tools and vehicles. Extracellular matrix degradation parallels increased age and mechanical stimuli resulting in disc degeneration and eventual spinal deformity. Purpose: The objective of this study was to investigate the relationship between vibratory loading and extracellular matrix expression in cultured rabbit annulus cells. Study design/setting: An in vitro rabbit model using cultured annulus fibrosis cells isolated from normal intervertebral disc was used to study matrix and metalloproteinase expression in response to vibration. Methods: Annulus fibrosis cells were isolated by collagenase digestion from New Zealand White rabbits. Vibratory stimulation was applied to annulus cells in vitro, using an oscillating platform to deliver 0.1 × gravity load at 6 Hz for 2, 4, 6 or 8 hours. Gene expression was assessed by reverse transcriptase polymerase chain reaction. Results: Aggrecan, collagen Type III and matrix metalloproteinase–3 gene expression was suppressed by vibratory loading in rabbit annulus cells. Suppression of the aggrecan gene might lead to a decrease in proteoglycan synthesis. Conclusions: These data suggest that vibratory load may play an important role in extracellular matrix metabolism of intervertebral disc cells, especially in the gene expression pathway of proteoglycans. It has been proposed that vibratory loading increases production of matrix-degrading, proteolytic enzymes. We have demonstrated that gene expression for key matrix messages and matrix metalloproteinase is decreased by vibration. In conclusion, we believe that study of the roles between extracellular matrix gene suppression and mechanical stress may clarify the pathomechanism of disc degeneration, such as disc herniation or degeneration.

Little is known about the factors that initiate and propagate tendon overuse injuries, but chronic inflammation and matrix destruction have been implicated. The purpose of this study was to evaluate the production of cyclooxygenase II (COX-2) and matrix metalloproteinases (MMPs) by tendon cells exposed to cyclic strain and inflammatory cytokines in vitro. Rabbit Achilles tendon cells were subjected to a stretching protocol with 5% elongation at 0.33 Hz for 6 h, or treated with 1000 pM interleukin-1beta (IL-1beta), or exposed to IL-1beta and stretching together. Gene expression was evaluated by RT-PCR and production of stromelysin was quantified with an ELISA. IL-1beta induced the expression of the collagenase-1 and stromelysin-1 genes. Production of stromelysin proenzyme by cells stimulated with IL-1beta was 17 times higher than production by control cells. Cells exposed to IL-1beta and stretching produced 20 times more stromelysin than control cells. Cells subjected to stretching alone did not produce more stromelysin than control cells. The synergistic effect of IL-1beta and stretching was observed at doses of IL-1beta ranging from 10 to 1000 pM. These data suggest that mechanical load and inflammatory cytokines can initiate a matrix destructive pathway in tendon that is more pronounced than with mechanical loading or inflammation alone.

Resident cells in the surface epitenon and internal compartment of flexor tendons are subjected to cyclic mechanical load as muscle contracts to move limbs or digits. Tendons are largely tensile load bearing tissues and are highly matrix intensive with nondividing cells providing maintenance functions. However, when an injury occurs, tendon cells are stimulated to divide by activated endogenous growth factors and those from platelets and plasma. We hypothesize that tendon cells detect mechanical load signals but do not interpret such signals as mitogenic unless an active growth factor is present. We have used an in vitro mechanical load model, application of cyclic strain to cells cultured on flexible bottomed culture plates, to test the hypothesis that tendon cells require platelet-derived growth factor (PDGF-BB) and insulin-like growth factor-I (IGF-I) in addition to mechanical load to stimulate DNA synthesis. In addition, we demonstrate that in avian tendon cells, load and growth factors stimulate phosphorylation of tyrosine residues in multiple proteins, including pp60src, a protein kinase that phosphorylates receptor protein tyrosine kinases. A lack of mitogenic responsiveness to mechanical load alone by tendon cells may be a characteristic of a regulatory pathway that modulates cell division.

Outer synovial tissues were separated from the remainder of avian flexor tendon and the collagens characterized biochemically and compared with those of the internal portion of tendon and sheath. The collagen content of tendon synovium was 23%, whereas that of tendon and sheath were 78% and 73%, respectively, based on dry weight. Four genetic types of collagen were found in the pepsin solubilized matrices: in the synovium, types I (78%) and III (19%) predominated; types V and possibly VI were present as minor collagens. Purified synovial type V collagen was a heterotrimer, with chain composition [alpha 1(V)]2 alpha 2(V). In contrast, the internal portion of tendon and sheath were comprised of only type I collagen. There was a large amount (41%) of ethanol extractable, noncollagenous material present in synovium, a part of which was proteoglycans. In addition, collagen cross-links of these tissues were quantified: the internal tendon had an abundant concentration of pyridinoline; synovium exhibited high amounts of labile, reducible cross-links, particularly dihydroxylysinonorleucine. In the case of sheath, lysine aldehyde-derived cross-links appeared to be predominant. These results indicate that each tissue has its own collagen type distribution as well as cross-linking pattern reflecting their maturational and functional differences.

Understanding the mechanisms that regulate mechanosensitivity in osteoblasts is important for controlling bone homeostasis and the development of new drugs to combat bone loss. It is believed that prestress or force generation (the tensile stress within the cell body) plays an important role in regulating cellular mechanosensitivity. In the present study, a three-dimensional (3D) collagen culture was used to monitor the change in prestress of the osteoblast-like cells. Collagen hydrogel compaction has been used as an indicator of the change in the degree of cell prestress. Previous results in this model demonstrated that extracellular ATP reduced the mechanosensitivity of osteoblasts by reducing cellular prestress. To elucidate the potential mechanisms involved in this process, the signaling pathways downstream of P2 purinoceptors involved in regulating the compaction of type I collagen gels were investigated. By using specific inhibitors to these signaling pathways, we found that ATP-induced reduction in collagen gel compaction rate is dependent on mitogen-activated protein kinase (MAKP) and NF-κB pathways. However, blocking protein kinase C with GF109203X did not change the compaction kinetics in the presence of ATPγS. Moreover, blocking cyclic AMP (cAMP), phosphatidylinositol-3 kinase (PI3K), calmodulin (CaM) or L-type voltage sensitive calcium channels did not affect ATP's ability to reduce collagen gel compaction. The results from the present and previous studies indicate that extracellular ATP may act as a negative feedback modulator in the mechanotransduction system since mechanical stimuli increase ATP release from stimulated cells.

Sympathetic efferent nerves are present in tendons, but their function within tendon is unknown. α1-Adrenoceptors are expressed by a variety of cell types. In the presence of norepinephrine (NE), adrenoceptors activate Gq/11signaling pathways that subsequently increase intracellular Ca2+concentration ([Ca2+]ic). It was hypothesized that avian tendon cells express functional adrenoceptors that respond to NE by increasing [Ca2+]ic. Avian tendon cells were analyzed for mRNA expression of α1-adrenoceptors by RT-PCR. Avian tendons expressed the α1A- and α1B-adrenoceptor subtypes. Furthermore, both tendon surface epitenon cells and internal fibroblasts infused with a Ca2+-sensitive dye, fura 2, and stimulated with NE responded by increasing [Ca2+]ic. KMD-3213, an α1A-adrenoceptor antagonist, significantly reduced the Ca2+response. Other adrenoceptor antagonists had no effect on the Ca2+response. The absence of extracellular Ca2+also significantly reduced the response to NE, indicating that Ca2+influx contributed to the rise in [Ca2+]ic. This study provides the first evidence that tendon cells express adrenoceptors and that the NE-induced Ca2+response is coupled to the α1A-adrenoceptor subtype.

Mechanical forces regulate the developmental path and phenotype of a variety of tissues and cultured cells. Vibratory loading as a mechanical stimulus occurs in connective tissues due to energy returned from ground reaction forces, as well as a mechanical input from use of motorized tools and vehicles. Structures in the spine may be particularly at risk when exposed to destructive vibratory stimuli. Cells from many tissues respond to mechanical stimuli, such as fluid flow, by increasing intracellular calcium concentration ([Ca 2 + ] i c ) and releasing adenosine 5'-triphosphate (ATP), extracellularly, as a mediator to activate signaling pathways. Therefore, we examined whether ATP is released from rabbit (rAN) and human (hAN) intervertebral disc annulus cells in response to vibratory loading. ATP release from annulus cells by vibratory stimulation as well as in control cells was quantitated using a firefly luciferin-luciferase assay. Cultured hAN and rAN cells had a basal level of extracellular ATP ([ATP] e c ) in the range of 1-1.5 nM. Vibratory loading of hAN cells stimulated ATP release, reaching a net maximum [ATP] within 10 min of continuous vibration, and shortly thereafter, [ATP] declined and returned to below baseline level. [ATP] in the supernatant fluid of hAN cells was significantly reduced compared to the control level when the cells received vibration for longer than 15 min. In rAN cells, [ATP] was increased in response to vibratory loading, attaining a level significantly greater than that of the control after 30 min of continuous vibration. Results of the current study show that resting annulus cells secrete ATP and maintain a basal [ATP] e c . Annulus cells may use this nucleotide as a signaling messenger in an autocrine/paracrine fashion in response to vibratory loading. Rapid degradation of ATP to ADP may alternatively modulate cellular responses. It is hypothesized that exposure to repetitive, complex vibration regimens may activate signaling pathways that regulate matrix destruction in the disc. As in tendon cells, ATP may block subsequent responses to load and modulate the vibration response. Rabbit annulus cells were used as a readily obtainable source of cells in development of an animal model for testing effects of vibration on the disc. Human cells obtained from discarded surgical specimens were used to correlate responses of animal to human cells.

Overuse injuries and trauma in tendon often involve acute or chronic pain and eventual matrix destruction. Anti-inflammatory drugs have been used as a treatment, however, the cellular and molecular mechanisms of the destructive processes in tendon are not clearly understood. It is thought that an inflammatory event may be involved as an initiating factor. Mediators of the inflammatory response include cytokines released from macrophages and monocytes. Interleukin-1 beta (IL-1 beta) is a candidate proinflammatory cytokine that is active in connective tissues such as bone and cartilage. We hypothesized that tendon cells would express receptors and respond to IL-1 beta in an initial "molecular inflammation" cascade, that is, connective tissue cell expression of cytokines that induce matrix destructive enzymes. This cascade results in expression of matrix metalloproteinases (MMPs) and aggrecanases that may lead to matrix destruction. Normal human tendon cells from six patients were isolated, grown to quiescence and treated with human recombinant IL-1 beta in serum-free medium for 16 h. Total RNA was isolated and mRNA expression assessed by semiquantitative RT-PCR. IL-1 beta (1 nM) induced mRNAs for cyclooxygenase 2 (COX2), MMP-1, -3, -13 and aggrecanase-1 as well as IL-1 beta and IL-6, whereas mRNAs for COX1 and MMP-2 were expressed constitutively. The IL-1 beta-treated tendon cells released prostaglandin E(2) (PGE(2)) in the medium, suggesting that the inducible COX2 catalyzed this synthesis. Induction of PGE(2) was detectable at 10 pM IL-1 beta. IL-1 beta also stimulated MMP-1 and -3 protein secretion. Induction of MMP-1 and -3 was detectable at 10 pM IL-1 beta. Post-injury or after some other inciting events, exogenous IL-1 beta released upon bleeding or as leakage of local capillaries may drive a proinflammatory response at the connective tissue cell level. The resulting induction of COX2, MMP-1 and -3 may underscore a potential for nonlymphocyte-mediated cytokine production of MMPs that causes matrix destruction and a loss of tendon biomechanical properties. Endogenous IL-1 beta might contribute to the process through a positive feedback loop by stimulating expression and accumulation of MMPs in the tendon matrix.

Chronic inflammation and altered mechanical loading are implicated as contributors to intervertebral disc degeneration. Biomechanical and biochemical factors play a role in disc degeneration but have received limited study. Mechanically, intervertebral discs are sheared during bending or twisting of the trunk. Biochemically, IL-1beta, detected in degenerative discs, promotes metalloproteinase expression. We hypothesized that disc cells might respond to shear stress and IL-1beta in a calcium signaling response. We measured the effect of single and combined stimuli on intracellular calcium concentration ([Ca2+]ic) and signaling. Cells were isolated from annulus tissue, cultured to quiescence, plated on collagen-bonded Culture Slips and incubated with Fura-2AM. Cells then were incubated in IL-1beta. Cell response to the effects of fluid flow was tested using FlexFlo, a laminar flow device. Human annulus (hAN) cells responded to laminar fluid flow with a one to three-fold increase in [Ca2+]ic. IL-1beta alone produced a small, transient stimulation. hAN cells pretreated with IL-1beta responded to shear with a more dramatic and sustained increase in [Ca2+]ic, six to ten-fold over basal level, when compared to shear then IL-1beta or shear and IL-1beta alone (P

Cells in normal tendon are in a resting G0 state, performing maintenance functions. However, traumatic injury introduces growth factors such as platelet-derived growth factor and insulin-like growth factor from blood as well as activates endogenous growth factors. These factors stimulate migration and proliferation of tendon cells at the wound area. Tendon cells require growth-promoting factors to transit the cell cycle. To evaluate the contribution of endogenous growth factors in tendon, extracts of the epitenon and internal compartment of avian flexor tendon as well as medium of cultured cells from the epitenon (tendon surface cells) and internal tendon (tendon internal fibroblasts) were collected to assess their ability to stimulate DNA synthesis. Acid-ethanol extracts of tissues and medium were chromatographed on a P-30 molecular sieve column and assayed for mitogenic activity by quantitating [3H]thymidine incorporation into tendon cell DNA. The extract from the internal tendon compartment was more stimulatory for DNA synthesis than that from the epitenon, particularly when tested on tendon internal fibroblasts. However, conditioned medium fractions from surface epitenon cells stimulated DNA synthesis to a high degree on both tendon surface cells and tendon internal fibroblasts. Conditioned medium from tendon internal fibroblasts was also stimulatory. An anti-insulin-like growth factor-I antibody ablated most of the mitogenic activity present in both tissues and conditioned medium. The levels of acid-extractable insulin-like growth factor-I in tendon were determined by competitive radioimmunoassay as 1.48 ± 0.05 ng/g tissue for the epitenon and 3.83 ± 0.03 ng/g tissue for the internal compartment. Results of Western immunoblots of conditioned medium revealed insulin-like growth factor-I at the 7.5 kDa position. Cultured tendon surface cells and tendon internal fibroblasts as well as cells in intact flexor tendon expressed insulin-like growth factor-I mRNA detected by reverse transcriptase-polymerase chain reaction. In situ hybridization histochemistry positively identified insulin-like growth factor-I mRNA in tendons from 52-day-old chickens. Platelet-derived growth factor was not detected at the protein or message levels. Furthermore, tendon surface cells and tendon internal fibroblasts both expressed receptors for insulin-like growth factor-I detected by flow cytometry. These data suggest that tendon cells express insulin-like growth factor-I mRNA and synthesize insulin-like growth factor-I in both the epitenon and the internal compartment of tendon, which is present in an inactive from, most likely bound to insulin-like growth factor-binding proteins.

Objective: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strain in vitro than in vivo . Design: We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF) in vitro that were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loading in vivo and in vitro . Methods: We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1h 45min with the balance of time at rest to 6h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1Hz, 5% average elongation for 6h, ±PDGF-BB, IGF-I, TGF-β1, PTH, estrogen, PGE 2 , or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis. Conclusions: Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cells in vivo and in vitro will lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.

Cells from diverse tissues detect mechanical load signals by similar mechanisms but respond differently. The diversity of responses reflects the genotype of the cell and the mechanical demands of the resident tissue. We hypothesize that cells maintain a basal equilibrium stress state that is a function of the number and quality of focal adhesions, the polymerization state of the cytoskeleton, and the amount of extrinsic, applied mechanical deformation. A load stimulus detected by a mechano-electrochemical sensory system, including mechanically sensitive ion channels, integrin–cytoskeleton machinery, and (or) a load-conformation sensitive receptor or nonreceptor tyrosine kinase, may activate G proteins, induce second messengers, and activate an RPTK or JAK/STAT kinase cascade to elicit a response. We propose the terms autobaric to describe a self-loading process, whereby a cell increases its stress state by contracting and applying a mechanical load to itself, and parabaric, whereby a cell applies a load to an adjacent cell by direct contact or through the matrix. We predict that the setpoint for maintaining this basal stress state is affected by continuity of incoming mechanical signals as deformations that activate signalling pathways. A displacement of the cytoskeletal machinery may result in a conformational change in a kinase that results in autophosphorylation and cascade initiation. pp60Src is such a kinase and is part of a mechanosensory protein complex linking integrins with the cytoskeleton. Cyclic mechanical load induces rapid Src phosphorylation. Regulation of the extent of kinase activation in the pathway(s) may be controlled by modulators such as G proteins, kinase phosphorylation and activation, and kinase inhibitors or phosphatases. Intervention at the point of ras–raf interaction may be particularly important as a restriction point.Key words: mechanoreception, cells, in vitro, load deformation.

Cross-linking is believed to be one of the major factors that characterize the calcifiability of dentin and bone collagens. Dehydro-dihydroxylysinonorleucine and pyridinoline which constitute the principal cross-links of dentin collagen have so far been located only in the carboxy terminal telopeptide of the molecules [alpha 1(I)-chain 87 x alpha 1(I)-chain 16C]. This situation suggested that the amino terminal telopeptide portion might be "open" without intermolecular cross-linking in hard tissue collagen fibrils. However, the present study provided evidence that pyridinoline is also located in amino-terminal telopeptides (alpha 1-chain 9N or alpha 2-chain 5N) and alpha 1-chain 930. Bovine dentin collagen was digested with trypsin followed by heating at 60 degrees C before and after the digestion. This method gave complete trypsin peptides of dentin collagen. Fluorescent pyridinoline peptides with a smaller molecular size were isolated by Sephadex G-50 superfine, DEAE-cellulose and reverse-phase HPLC. Automatic Edman analysis of several isolated peptides revealed the five-residue sequence, Gly-Ile-X-Gly-His-Arg, the only assignment of which was alpha 1-chain 928-933. The above evidence together with the amino acid compositions of the peptides led to the conclusion that pyridinoline is located not only in the carboxy-terminal but also in the amino-terminal telopeptide in dentin collagen.

Nachweis, dass bei Entwicklungsstadien von Mausesauglingen, die mit Mitomycin C injiziert wurden, Hyperplasien mit Melanin-Ablagerungen auftraten, was mit «Dysdifferenzierungen» von Zellen der Neuralleisten zusammenhangen durfte.

[3H]NaBH4 reduced bovine dentin collagen was denatured at 60°C for 1 hr and then digested with trypsin. The digest was still substantially insoluble suspension, but it was found that 99% of dentin collagen can be solubilized if the digest was heated again at 60°C for 15 min. Two cross-linked tryptic peptides were isolated from this digest by sequential chromatographies on Sephadex G50, phosphocellulose and DEAE-cellulose column. One isolated peptide was characterized as a 59 residue cross-linked peptide including one residue of dihydroxylysinonorleucine and the other was 103 residue including one residue of pyridinoline. The amino acid compositions were consistent with the identification of the 59 residue peptide as the sequence in α1-CB4-5 (76–90) linked to the sequence in α1-CB6 (990-23c), and the 103 residue peptide as the sequence 76–90 linked to two of the sequence 990-23c. These results strongly support the previously proposed precursor-product relationship between dihydroxylysinonorleucine and pyridinoline.