A total of 50 male Sprague-Dawley rats [10 rats aged 2–3 weeks (50±5 g) and 40 rats aged 8 weeks (250±10 g)] were purchased from the Experimental Animal Research Center of Southern Medical University (Guangzhou, China) and housed in a specific pathogen-free animal facility with standard food and water under constant temperature (20–22°C), controlled humidity (45–55%) and a standard 12-h light/dark cycle. All animal experimental protocols were approved by the Animal Care and Use Committee of Southern Medical University and performed according to the Southern Medical University Guide for the Care and Use of Laboratory Animals.

The middle portions of tendons used for cell culture were obtained from 10 Sprague-Dawley rats (aged 2–3 weeks, 50±5 g). The rats were sacrificed and the middle portion of tendons was obtained by cutting the tendon sample 5 mm from the tendon-muscle junction to tendon-bone insertion. The tendons were minced into small pieces (1 mm³) and digested with 0.1% collagenase type I (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in 1 ml phosphate-buffered saline at 37°C for 1 h. After centrifugation at 1,000 × g at 37°C for 5 min and removal of the supernatant, a single-cell suspension was obtained, which was cultured in complete medium [CM; Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA] at 37°C with 5% CO₂. Passages 3 and 4 were used for experiments in this study. For osteogenic differentiation, 1×10⁵ cells were seeded into BioFlex six-well plates (Flexcell International Corporation, Burlington, NC, USA) in duplicate and incubated at 37°C in an atmosphere of 5% CO₂. After 24 h incubation, medium containing non-adherent cells was removed and replaced with osteogenic induction medium (OIM). OIM consisted of α-MEM (Gibco; Thermo Fisher Scientific, Inc.), 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 50 µM ascorbic acid (Sigma-Aldrich; Merck KGaA), 0.1 µM dexamethasone (Sigma-Aldrich; Merck KGaA) and 10 mM β-glycero-phosphate (Sigma-Aldrich; Merck KGaA).

Mechanical loading was applied using the Flexcell FX-5000 Tension System (FX5K; Flexcell International Corporation). When cultures reached 60–70% confluence, cells were serum-starved for 24 h to arrest cells at G₀. The cells were then subjected to mechanical stimulation, which was applied for 8 h daily for a week with the OIM replaced every 3rd day, using a sinusoidal waveform at a frequency of 0.5 Hz and a strain of 4 or 12%, with or without 10 nM rapamycin (Sigma-Aldrich; Merck KGaA). Control cultures were cultured under the same conditions without mechanical stimulation (NS).

Total RNA was extracted from cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) followed by cDNA synthesis performed with a StarScript II First-strand cDNA Synthesis kit (GenStar BioSolutions Co., Ltd., Beijing, China). The primers are listed in Table I. The RT-qPCR reactions were performed using RealStar Green Power Mixture with ROX (GenStar BioSolutions Co., Ltd) and an ABI Step One Plus PCR Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling parameters were as follows: Pre-denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 94°C for 20 sec, annealing at 65°C for 20 sec and extension at 72°C for 30 sec. All assays were performed in triplicate and samples were normalized to GAPDH using the delta delta Cq method (15).

Following the stretching procedure, cells were rinsed with cold PBS and lysed in cold RIPA buffer [1×PBS, 1% NP-40, 0.5% sodium dexoycholate, 0.1% SDS, protease inhibitor cocktail tablet (Roche Diagnostics, Basel, Switzerland) and 1 mM phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology)]. Lysates were incubated on ice for 20 min with frequent vortexing and cleared twice by centrifugation (11,000 × g, 10 min, 4°C). Protein was subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked for 60 min at room temperature in 5% non-fat milk/Tris-buffered saline/0.1% Tween-20 (TBST). Following washing with TBST, the membranes were incubated overnight at 4°C in primary antibodies, which were phospho-P70S6K (Thr389; diluted 1:1,000 in 5% BSA; #9234; Cell Signaling Technology Inc., Danvers, MA, USA), runt-related transcription factor 2 (RUNX2; 1:1,000; #8486; Cell Signaling Technology, Inc.), osterix (OSX; 1:1,000; ab22552; Abcam, Cambridge, UK) or GAPDH (1:1,000; #2118; Cell Signaling Technology, Inc.). Membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:2,000; #7047; Cell Signaling Technology, Inc.) at room temperature for 1 h, and subsequently washed. Image detection was performed according to the manufacturer's instructions using SignalFire enhanced chemiluminescence reagent (#6883; Cell Signaling Technology, Inc.). Data are representative of at least three experiments with similar results performed in triplicate. Images were analyzed using Image Pro-Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

A total of 40 male Sprague-Dawley rats aged 8 weeks and weighing 250±10 g were randomly distributed into 8 groups of 5 animals per group: Sham; Sham + rapamycin (Rapa); Control (Ctrl); Ctrl + Rapa; low elongation (LE); LE + Rapa; high elongation (HE); HE + Rapa. Rats were anesthetized with 10% chloral hydrate [300 mg/kg, intraperitoneally (i.p.)]. The rats in groups Ctrl, Ctrl + Rapa, LE, LE + Rapa, HE and HE + Rapa underwent midpoint Achilles tenotomy on the right leg through a posterior approach under aseptic conditions; rats in the Sham and Sham + Rapa groups received an incision only as a sham operation. Incisions were routinely closed with an interrupted 4-0 silk suture. Subsequently, the operated hind limbs of rats in Ctrl and Ctrl + Rapa groups and the non-operated hind limbs of the rats in the HE and HE + Rapa groups were fixed in plaster casts from the toes up to ~2.5 cm above the knee. The operated hind limbs of the rats in the LE and LE + Rapa groups were fixed in a plaster cast from ~1 cm below the ankle up to ~2.5 cm above the knee, allowing the toes to touch the ground directly. The ankle and knee were fixed at functional positions. The outer surface of the cast was coated with black pepper powder for protection (16). Thus, the animal models were established with three tendon stretch levels: Totally fixed (groups Ctrl and Ctrl + Rapa), low elongation stretch (groups LE and LE + Rapa) and high elongation stretch (groups HE and HE + Rapa). The rats in groups Sham + Rapa, Ctrl + Rapa, LE + Rapa and HE + Rapa received rapamycin (20 mg/kg/day, i.p.) every day for 6 weeks after operation (Table II).

Following the operation, the tendon samples were immediately fixed in 4% buffered paraformaldehyde at 4°C for 24 h. The samples were then decalcified, embedded in paraffin, cut into 5 µm sections and placed on poly-L-lysine-coated slides for further procedures. For hematoxylin and eosin staining, the sections were stained with hematoxylin and eosin carried out according to the protocol in the product manual. For immunohistochemical staining, antigen retrieval of the sections was performed using the microwave method (17). Sections were incubated in 3% H₂O₂ at room temperature for 30 min to quench endogenous peroxidase activity, the sections were subsequently incubated with 10% normal goat serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 30 min to block nonspecific binding. The sections were then incubated with antibody against RUNX2 (diluted 1:100 in 5% BSA; ab23981; Abcam) and OSX (diluted 1:100; ab22552; Abcam), followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (diluted 1:100; #7074; Cell Signaling Technology, Inc.). The protein of interest was visualized using 3,3′-diaminobenzidine using a light microscope and the images were analyzed by applying Image Pro-Plus 6.0 software (Media Cybernetics, Inc.).

µCT analysis of the right Achilles tendons was performed using a high-resolution µCT (Skyscan 1172; Bruker Corporation, Ettlingen, Germany). Hind limb samples were collected and fixed overnight at 4°C in 4% paraformaldehyde, then scanned using the settings 60 kV, 150 µA with a mean 20 µm slice thickness. The reconstructed tendon images were selected for quantification of bone mass. Bone volume density [bone volume/total volume (BV/TV)%], trabecular number (Tb.N; l/mm) and trabecular thickness (Tb.Th; mm) were calculated.

Data were analyzed using one-way analysis of variance while homogeneity of variance tests were used to evaluate data homogeneity (SPSS 21.0 software; IBM SPSS, Armonk, NY, USA). If the variances were equal, least-significant difference tests were employed; otherwise, Dunnett's tests were used. Results are presented as the mean ± standard deviation unless otherwise indicated. P<0.05 was considered to indicate a statistically significant difference.

In order to evaluate the effects of mechanical loading on HO in the Achilles tenotomy rat model, µCT analysis was employed (Fig. 1A and B). Compared with the ctrl group, lower BT/VT, Tb.N and Tb.Th were observed in the LE group; whereas the HE group exhibited higher BT/VT, Tb.N and Tb.Th compared with the control group. Hematoxylin and eosin staining revealed the same phenomenon (Fig. 1C). Therefore, these data suggested that the HO in rats was attenuated by low elongation mechanical loading, but enhanced by high elongation mechanical loading.

Tendon cells in OIM were subjected to 4% elongation mechanical loading and protein and mRNA expression of RUNX2 and OSX were measured using western blot and RT-qPCR analyses, respectively. Protein and mRNA expression of RUNX2 and OSX were significantly decreased in the 4% stretch group compared with the NS group (P<0.05; Fig. 2A and B) while the 12% stretch group exhibited the opposite result (P<0.05; Fig. 2A and B). These data suggested that osteogenic differentiation in tendon cells was reduced by low elongation mechanical loading and enhanced by high elongation mechanical loading.

To examine the effects of mechanical loading on the mTORC1 signaling pathway in rat tendon cells, a downstream protein in the mTORC1 signaling pathway, p-P70S6K, was investigated using western blot analyses. Mechanical loading at 4% elongation decreased the protein expression of p-P70S6K, while mechanical loading at 12% elongation significantly increased p-P70S6K levels in rat tendon cells compared with the NS group (P<0.05; Fig. 3). These data suggested that low elongation mechanical loading suppressed the mTORC1 signaling pathway, while high elongation had the opposite effect on tendon cells.

In the in vitro tendon cells stretch model, the presence of rapamycin decreased protein expression of RUNX2 and OSX in the NS + Rapa group compared with the NS group (P<0.05; Fig. 4). Rapamycin-treated cells at 4% elongation mechanical loading decreased protein expression of RUNX2 and OSX compared with the untreated 4% group (P<0.05; Fig. 4). Additionally, the presence of rapamycin decreased 12% elongation mechanical loading-induced protein expression of RUNX2 compared with the untreated 12% group (P<0.05; Fig. 4). These data suggested that the osteogenic differentiation of tendon cells was mTORC1-dependent and that mechanical loading regulated the osteogenic differentiation of rat tendon cells through the mTORC1 signaling pathway.

In order to investigate the roles the mTORC1 signaling pathway plays in mechanical loading-induced HO of the tendon, the mTORC1 selective inhibitor rapamycin was employed. The presence of rapamycin decreased expression of RUNX2 and OSX in rats in the Ctrl + Rapa group compared with the Ctrl group (Fig. 5). Rapamycin treated rats in the LE + Rapa group had decreased expression of RUNX2 and OSX compared with the untreated LE group (Fig. 5). Additionally, rapamycin decreased the HE mechanical loading-induced expression of RUNX2 and OSX in the HE + Rapa group compared with the untreated HE group (Fig. 5). These data suggested that HO in tendon was mTORC1-dependent and that mechanical loading regulated HO in rat tendon through the mTORC1 signaling pathway.