Flexor tendons are designed to transmit the force of muscle contraction to bone to effect limb movement. The matrix is the major load-bearing component of tendon; however the cells are passively loaded. The tendon surface is subjected to shear stress during gliding, while the whole tendon receives cyclic tension. Two major cell populations exist in flexor tendon; the surface epitenon cells (TSC) residing in a pulse dampening milieu of half collagen and proteoglycan and half lipid, and the internal fibroblasts (TIF) of the tendon interior nestled in linear arrays, that appear optimal for junctional connectivity, amidst aligned collagen fibers. The applicants hypothesize that tendon cells can receive and interpret mechanical signals by intercommunicating with junctionally competent neighboring cells. Intercellular communication occurs after a target cell releases intracellular calcium stores whose signal is propagated to neighboring cells through gap junctions by an IP3-dependent mechanism. Treatment of target cells with heparin prevents signal propagation by blocking IP3 receptors and treatment with halothane also blocks the signal by interfering at gap junctions. Gap junctions are comprised of hemichannel connexons assembled from 6 identical connexin subunits. Avian cells synthesize several connexins, of which connexin 43 is prominent. The CXN-43 phosphorylation forms may regulate channel gating to the open/closed states. The investigators have found that avian tendon cells have connexin 43 and that it is phosphorylated in internal fibroblasts, but not surface synovial cells. Moreover, cultured tendon cells can require time (up to days) after plating to reestablish gap junction connections and the ability to signal each other after a mechanical stimulus. Therefore, reestablishing gap junction competency may require new synthesis and alteration in the phosphorylation state. In a healing tendon, days may be required before migrating cells populating a wound can reestablish their ability to intercommunicate. The applicants hypothesize that cyclic mechanical load will increase the number of gap junction connections in tendon resulting in improved intercellular signalling with time. Likewise, immobilization will decrease intercellular communication. The investigator have designed experiments to test these hypotheses in tendon cells in both in vivo and in vitro models of wounding and mechanical perturbation with the following specific aims: (1) to test the ability of cells in normal and wounded tendon to mount a release of [Ca2+}i and propagate the signal in response to a mechanical stimulus to a single cell. (2) to test the same response in a separate or mixed culture of TSC and TIF in freshly isolated log growth or quiescent cells +/- cyclic mechanical load applied, to stimulate dynamic vs resting phases of healing, +/- motion; and (3) to quantitate the amount and synthetic rates of gap junction mRNA and connexin 43 protein in quiescent or log phase cells +/- mechanical load and serum stimulation, and quantitate and correlate the connexin 43 phosphorylation state and junctional competency. Results of these studies should elucidate how cyclic mechanical loading applied to tendon or its isolated cells affects the ability of a single target cell to respond to a single mechanical stimulation and intercommunicate with neighboring cells during healing. An important clinical aspect is that the mechanism underlying the beneficial effects of passive progressive motion to injured connective tissues during convalescence may be identified.
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| Wall, Michelle E; Weinhold, Paul S; Siu, Tung et al. (2007) Comparison of cellular strain with applied substrate strain in vitro. J Biomech 40:173-81 |
| Qi, Jie; Chi, Liqun; Faber, James et al. (2007) ATP reduces gel compaction in osteoblast-populated collagen gels. J Appl Physiol 102:1152-60 |
| Qi, Jie; Chi, Liqun; Maloney, Melissa et al. (2006) Interleukin-1beta increases elasticity of human bioartificial tendons. Tissue Eng 12:2913-25 |
| Qi, Jie; Fox, Ann Marie; Alexopoulos, Leonidas G et al. (2006) IL-1beta decreases the elastic modulus of human tenocytes. J Appl Physiol 101:189-95 |
| Jones, Bertina F; Wall, Michelle E; Carroll, R Lloyd et al. (2005) Ligament cells stretch-adapted on a microgrooved substrate increase intercellular communication in response to a mechanical stimulus. J Biomech 38:1653-64 |
| Tsuzaki, M; Bynum, D; Almekinders, L et al. (2005) Mechanical loading stimulates ecto-ATPase activity in human tendon cells. J Cell Biochem 96:117-25 |
| Wall, Michelle E; Faber, James E; Yang, Xi et al. (2004) Norepinephrine-induced calcium signaling and expression of adrenoceptors in avian tendon cells. Am J Physiol Cell Physiol 287:C912-8 |
| Tsuzaki, M; Bynum, D; Almekinders, L et al. (2003) ATP modulates load-inducible IL-1beta, COX 2, and MMP-3 gene expression in human tendon cells. J Cell Biochem 89:556-62 |
| Yamazaki, Satoru; Weinhold, Paul S; Graff, Ronald D et al. (2003) Annulus cells release ATP in response to vibratory loading in vitro. J Cell Biochem 90:812-8 |
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