ABSTRACT: Background:The attempted healing of tendon after acute injury (overloading, partial tear or complete rupture) proceeds via the normal wound healing cascade involving hemostasis, inflammation, matrix synthesis and matrix remodeling. Depending on the degree of trauma and the nature of the post-injury milieu, a variable degree of healing and recovery of function occurs. Post-injury analgesia is often achieved with NSAIDs such as Ibuprofen, however there is increasing evidence that NSAID usage may interfere with the healing process. This study aimed to investigate the cellular mechanism by which IBU therapy might lead to a worsening of tendon pathology. Methods:We have examined the effect of oral Ibuprofen, on Achilles tendon healing in a TGFb1-induced murine tendinopathy model. Dosing was started 3 days after initial injury (acute cellular response phase) and continued for 22 days or started at 9 days after injury (transition to matrix regeneration phase) and given for 16 days. Cellular changes in tendon and surrounding peritenon were assessed using Hematoxylin/Eosin, chondroid accumulation with Safranin O and anti-aggrecan immunohistochemistry, and neo-vessel formation with GSI Lectin histochemistry. Markers of inflammation included histochemical localization of hyaluronan, immunohistochemistry of heavy chain 1 and TNF?-stimulated glycoprotein-6 (TSG6). Cell responses were further examined by RT-qPCR of 84 NF?B target genes and 84 wound healing genes. Biomechanical properties of tendons were evaluated by tensile testing. Results:At a clinically-relevant dosage, Ibuprofen prevented the process of remodeling/removal of the inflammatory matrix components, hyaluronan, HC1 and TSG6. Furthermore, the aberrant matrix remodeling was accompanied by activation at day 28 of genes (Col1a2, Col5a3, Plat, Ccl12, Itga4, Stat3, Vegfa, Mif, Col4a1, Rhoa, Relb, F8, Cxcl9, Lta, Ltb, Ccl12, Cdkn1a, Ccl22, Sele, Cd80), which were not activated at any time without the drug, and so appear most likely to be involved in the pathology. Of these, Vegfa, Col4a1, F8, Cxcl9 and Sele, have been shown to play a role in vascular remodeling, consistent with the appearance at 25 days of vasculogenic cell groups in the peritenon and fat pad stroma surrounding the Achilles of the drug-dosed mice. Tensile stiffness (p = 0.004) and elastic modulus (p = 0.012) were both decreased (relative to age-matched uninjured and non-dosed mice) in mice dosed with Ibuprofen from day 3 to day 25, whether injured or not. Conclusion:We conclude that the use of Ibuprofen for pain relief during inflammatory phases of tendinopathy, might interfere with the normal processes of extracellular matrix remodeling and cellular control of expression of inflammatory and wound healing genes. It is proposed that the known COX2-mediated anti-inflammatory effect of ibuprofen has detrimental effects on the turnover of a pro-inflammatory HA matrix produced in response to soft-tissue injury, thus preventing the switch to cellular responses associated with functional matrix remodeling and eventual healing.
