We utilized an in vivo animal model and an in vitro tenocyte model to evaluate whether repetitive loading would alter genes associated with tendinopathy in a load-dependent manner. Adult female Sprague-Dawley rats (N = 48; 390 ± 30 g) (Charles River Laboratories, Wilmington, MA, USA) were used in the in vivo model. Under isoflurane anesthesia, left patellae were surgically exposed and clamped to a 50 lb (222 N) load cell and actuator of a servohydraulic testing system (Instron 8841, Canton, MA, USA). All procedures were approved by the Institutional Animal Care and Use Committee. Tendons were fatigue loaded based on a damage accumulation loading protocol adapted from cortical bone fatigue damage studies [19
]. After preloading and initial length measured, tendons were cyclically loaded between 1 and 35 N (~40% maximum monotonic strength) at 1 Hz until reaching either 0.6% or 1.7% increases in elongation (relative to initial length) at peak cyclic load beyond baseline measurement. These loading endpoints were chosen because they produced different and repeatable levels of damage in our previous ex vivo tendon fatigue studies. Sham-operated animals, treated identically except for loading, and naïve control animals, were included in the study. Animals were allowed to resume normal cage activity for 1 or 3 days (n = 6/load group/time point) after loading. Animals were then sacrificed and hindlimbs dissected and frozen in liquid N2
prior to RNA and protein analysis. Animal numbers were chosen based on preliminary studies of gene expression using this loading protocol. For microstructural damage analysis, additional animals (n = 2/load group/time point) were used and sacrificed immediately after loading, and the hindlimbs fixed in 10% neutral buffered formalin and embedded in methacrylate [23
We utilized an in vitro tenocyte model to determine whether MMP-13 expression would be dependent on expression of IL-1β. A clonal cell line derived from rat patellar tendon was cultured in DMEM with 10% FBS, then starved in DMEM containing 1% FBS for 18 hours prior to intermittent hydrostatic pressure loading at 0, 2.5, 5.0, or 7.5 MPa at 1 Hz for 1 hour using a custom-made loading device. After loading, cells were lysed for isolation of total RNA and proteins. In some experiments, cells were either transfected for 24 hours prior to serum starvation with si-IL-1β RNA or a scrambled RNA control (Upstate Biologics, Lake Placid, NY, USA).
Total RNA isolated from tendons or cultured tenocytes (RNeasy Kit, Eppendorf, Westbury, NY, USA) was reverse transcribed with MMLV reverse transcriptase and an Oligo(dT)12–18 primer and expression of MMP-13 was quantitated by real time PCR (ABI Prism 7900HT Real-Time PCR System, Applied Biosystems, Framingham, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization.
Extracts from tendon tissue or cultured tenocytes containing approximately 10 μg of total protein were separated by electrophoresis on 10% SDS PAGE, transferred electrophoretically onto nitrocellulose membranes, blocked with a commercial blocking reagent (Amersham Biosciences, Piscataway, NJ, USA) and incubated overnight with antibody against MMP-13 (rabbit polyclonal IgG, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or IL-1β (goat polyclonal IgG, Santa Cruz Biotechnology) followed by peroxidase-conjugated secondary antibodies. Levels of MMP-13 or IL-1β proteins were detected using ECL Western blotting analysis system (Amersham Biosciences). To measure MMP-13 activity, extracts of tendon tissue or concentrates of tenocyte media were assayed using an MMP-13 assay kit (Sensolyte, Anaspec, Inc., San Jose, CA, USA) following the manufacturer’s instructions.
For microstructural analysis, 200 μm thick, sagittal sections were cut using a diamond wafering saw and mounted unstained. Tendon morphology was examined using a laser multiphoton microscope (LSM 510; Carl Zeiss, Jena, Germany) tuned to 840 nm. Backward second harmonic signals (generated by the collagen fibers) [47
] were collected using an external detector through a narrow bandpass (450/40 nm) filter. Area fraction of damage was quantitated in (400 × 400 μm) fields from at least 10 multiphoton micrographs per tendon.
We compared expression of MMP-13 and IL-1β at the mRNA and protein level for 0.6% and 1.7% cyclic strain in vivo and intermittent hydrostatic pressure loading at 0, 2.5, 5.0, or 7.5 MPa in vitro using ANOVA with Bonferroni post-hoc testing for multiple comparisons. We used Minitab v13 (Minitab, Inc., State Collage, PA, USA) for all analyses.