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 p) 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 b 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-lP in serum-free medium for 16 h. Total RNA was isolated and mRNA expression assessed by semiquantitative RT-PCR. IL-lP (1 nM) induced mRNAs for cyclooxygenase 2 (COX2), MMP-I, -3, -13 and aggrecanase-1 as well as IL-1 j3 and IL-6, whereas mRNAs for COX1 and MMP-2 were expressed constitutively. The IL-lj3-treated tendon cells released prostaglandin E2 (PGE2) in the medium, suggesting that the inducible COX2 catalyzed this synthesis. Induction of PGE2 was detectable at 10 pM IL-1 j3. IL-10 also stimulated MMP-1 and -3 protein secretion. Induction of MMP-1 and -3 was detectable at 10 pM IL-1 fi. Post-injury or after some other inciting events, exogenous IL-1 P 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 b might contribute to the process through a positive feedback loop by stimulating expression and accumulation of MMPs in the tendon matrix.

Cells cultured in three-dimensional collagen gels express a more native state phenotype because they form a syncytial network that can be mechanically loaded. Moreover, cells remodel their matrix by eliminating water, and by reorganizing and aligning the collagen fibrils. Last, the ability to subject cells to mechanical loading in a native matrix is desirable because cells, in tissues as well as the matrix, bear strains and alter their expression profile consistent with either immobilization, moderate activity, or repetitive loading. This is the first report of a model bioreactor system to fabricate and culture tendon cell-populated, linear, tethered matrix constructs that can be mechanically loaded by a computer-driven, pressure-controlled system. Bioartificial tissues (BATs) as tendon constructs were molded in a novel, rubber bottom Tissue Train culture plate bearing nonwoven nylon mesh anchors at the east and west poles of each culture well. Mechanical loading was achieved by placing an Arctangle loading post (an Arctangle is a rectangle with curved short ends) beneath each well of the six-well culture plate and using vacuum to displace the flexible membrane downward, resulting in uniaxial strain on the BAT. BATs populated with avian flexor tendon cells expressed collagen genes I, III, and XII as well as aggrecan, fibronectin, prolyl hydroxylase, and tenascin, consistent with expression levels of cells grown on collagen-bonded two-dimensional surfaces or in native, whole, avian flexor tendon. Likewise, cells in BATs established a morphology of linearly arranged cells aligned with the principal strain direction as in fasicles of whole tendons. Last, BATs that were mechanically loaded had an ultimate tensile strength that was nearly 3-fold greater than that of nonloaded BATs in the first week of culture. Taken together, these results indicate that tendon cells fabricated in a mechanically loaded, linear collagen gel construct assume a phenotype that is similar to that of a native tendon in terms of appearance and expression and are stronger than nonexercised counterparts yet far weaker than native adult tendons. This technique represents a novel approach to culturing cells in a mechanically active, three-dimensional culture environment that can be readily used for the fabrication of tissue simulates for drug testing or tissue engineering.

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.

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 11 (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 interleukin1b (IL-lB), or exposed to IL-I 0 and stretching together. Gene expression was evaluated by RT-PCR and production of stromelysin was quantified with an ELISA. IL-I@ induced the expression of the collagenase-1 and stromelysin-I genes. Production of stromelysin proenzyme by cells stimulated with IL-ID was 17 times higher than production by control cells. Cells exposed to IL-lp 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-1 p and stretching was observed at doses of IL-lp 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.