Introduction The role of inflammation in tendon injury is uncertain and is a topic of current interest.1 Injury is likely the result of damage accumulation during cyclic loading for extended periods of time. However, the role of inflammation in damage accumulation and the initiation of tendinopathy is not easily investigated in human subjects, because the affected tendons are normally only assessed when advanced tendinopathic changes have occurred. Therefore, in vitro studies of the inflammatory response to induced tendon overload damage can serve as a valuable source of information on the early stages of tendinopathy.
Methods 12 samples of fascicle bundles were dissected from three bovine extensor tendons. Samples were divided into three groups: 1. statically loaded to 2% strain for 24hrs; 2. Cyclically loaded for 300 cycles (2–12% strain; 1Hz) followed by 2% static strain for 24hrs; 3. Cyclically loaded for 1800 cycles (2–12% strain;1
Hz) followed by 2% static strain for 24hrs. Tests were performed in the same custom-made loading chambers, with samples maintained in sterile DMEM at 37 degrees.
After testing, samples were snap frozen in OCT embedding medium and then three 20 μm cryo-sections cut from each sample resulting in 108 sections for immunostaining. Dual immunostaining for inflammatory markers COX-2 and IL-6 or matrix degradation markers MMP-1 and MMP-3, MMP-13 and C1,2C was performed. Sections were imaged with a confocal microscope (Leica TCS SP2) and a qualitative assessment made.
Results Cyclic loading of samples resulted in visible matrix damage, with disrupted collagen fibres and fibre kinks. Damage was especially concentrated in the interfascicular matrix. Inflammatory markers were only expressed in the cyclically loaded samples, and were more highly expressed in the samples loaded for 1800 than 300 cycles. COX-2 staining appeared stronger than IL-6 staining and was localised predominantly in the interfascicular matrix, see Figure 1.
Matrix degradation markers MMP-1 and C1,2C were present in statically strained control samples. Staining become increasingly intense with increasing levels of cyclic loading. In the loaded samples MMP-1 and C1,2C were co-localised in many areas, but MMP-1 staining was more intense and more diffuse than C1,2C. These stains were mainly interfascicular matrix related, but with the high loading regime, C1,2C and MMP-1 were also expressed within fascicles and associated with tenocytes.
Very little MMP-3 or MMP-13 was evident in control sections. However, in loaded samples, some sections shown intensive staining of these markers, again localised to interfascicular matrix regions.
Discussion This study suggests that inflammatory markers may be expressed rapidly and early after tendon overload exercise, indicating that inflammation may be an early occurrence in tendinopathy. Interestingly, both inflammation and damage-induced matrix remodelling seem to be concentrated in the interfascicular matrix.
Reference 1 Dakin, et al. Vet immunol immunop. 2014;158:121–127
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