Study design
This study was performed using a rat model for Achilles tendon healing and a 3D cell culturing model using primary human tendon cells. All evaluations followed a blinded approach (Table 3).
Table 3
Experimental design. In vivo experiment used a rat model for Achilles tendon healing with high or moderate in vivo loading. The Achilles tendon was transected at day 0 and animals were treated with saline or dexamethasone (0.1mg/kg) from day 7–11. All animals were euthanized at day 12 and tendons were used for qPCR, immunohistochemistry, and immunofluorescence (IHC/IF). In vitro experiment used a 3D cell culturing model with primary human tendon cells treated with or without different doses of dexamethasone (1-50nM). D means day of intervention.
| | Group | Interventions | Analyzes |
| In vivo – rat model | High loading + Saline (Control) | Tendon transection (D0) Saline injections (D7-11) Euthanization (D12) | qPCR (N = 6) |
| High loading + Dexamethasone 0.1mg/kg | Tendon transection (D0) Dexa injections (D7-11) Euthanization (D12) | qPCR (N = 6) |
| Moderate Loading + Saline (Control) | Botox injections (D-4) Tendon transection (D0) Saline injections (D7-11) Euthanization (D12) | qPCR (N = 6) IHC/IF (N = 6) |
| Moderate Loading + Dexamethasone 0.1mg/kg | Botox injections (D-4) Tendon transection (D0) Dexa injections (D7-11) Euthanization (D12) | qPCR (N = 6) IHC/IF (N = 6) |
| In vitro – human tendon cells | Control | Cell seeding (D0) Imaging (D4-14) Sample collection (D14) | qPCR (N = 5) Imaging (N = 5) |
| Dexamethasone 1, 5, 10 or 50 nM | Cell seeding (D0) Dexa treatment (D2-14) Imaging (D4-14) Sample collection (D14) | qPCR (N = 5) Imaging (N = 5) |
Animals and housing
A total of 36 female Sprague-Dawley rats, Specific Pathogen Free (Taconic Biosciences), weighing on average 207g (SD17) were randomly divided by lottery into four groups: 1) High loading + saline, 2) High loading + dexamethasone, 3) moderate loading + saline, 4) moderate loading + dexamethasone (Table 1). The group sizes are based on experience using this model. The Regional Ethics Committee for animal experiments in Linköping, Sweden, approved all procedures (Djurförsöksnämnden in Linköping Dnr. ID1424). All experiments were performed in accordance with relevant guidelines and regulations. The study is also reported in accordance with the ARRIVE guidelines. Rats were placed two and two and acclimated for two weeks before the experiment started. The room had a standard humidity (55%) and temperature (22°C), and a 12-hour light-dark cycle (light from 7 am to 7 pm). Water and food pellets were offered ad libitum.
Botox injections to reduce loading
Rats with moderate loading received Botulinum toxin injections (Botox, Allergan, Irvine, USA) in the calf muscles four days prior to surgery. Injections were performed under anesthesia with isoflurane gas (Forene, Abbot Scandinavia, Solna, Sweden). A total of 3U of Botox (0.06mL/animal) was injected in the right hind leg into the gastrocnemius lateralis, gastrocnemius medialis, and soleus (1U Botox/muscle). Botox effectiveness was confirmed in all animals before tendon transection by visual inspection.
Achilles tendon transection
The surgery day was counted as day 0, and rats were anesthetized with isoflurane gas. The right hind leg was shaved and cleaned with chlorhexidine ethanol. The tendon complex was exposed through a minor transverse skin incision lateral to the Achilles tendon. The plantaris tendon was completely removed to avoid interference during healing and sample collection. The Achilles tendon was transected in the mid tendon portion and left to heal unsutured, while the skin was closed. Antibiotic was given preoperatively (25mg/kg, oxytetracycline, Engemycin, Intervet, Netherlands) to avoid post-operative infection. Analgesic was given subcutaneously pre- and post-operatively every 8-12h for 48h (0.045mg/kg, buprenorphine, Temgesic, Indivior Europe Limited, Ireland). Rats received systemic subcutaneous injections of saline solution 0,9% as control or dexamethasone (0.1mg/kg, Dexaject; Dopharma Research B.V.) from day 7–11 at 3.30 pm. The healing tendons were collected 12 days post-operatively. The rats were anesthetized with isoflurane, and the mid part of the newly formed tendon was collected and snap frozen in liquid nitrogen. The rats were euthanized by a cardiac pentobarbital injection and the samples were stored at -80º until analysis. No animals had to be excluded from the study.
Cell extraction
Human tendon fibroblasts were isolated by collagenase digestion from semitendinosus tendons from patients (n = 5) undergoing anterior cruciate ligament reconstructive surgery as previously described [26]. The experiments were approved by the regional ethics review board in Linköping, Sweden (2015/408 − 31) and patients gave written informed consent to participation. The investigation has been conducted according to the Declaration of Helsinki and in accordance with relevant guidelines and regulations. Cells were seeded in flasks and cultured to confluence in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.
Construct formation
Tendon constructs were assembled as previously described [26]. Six-well plates were coated with silicon (SYLGARD, Dow-Chemicals) and two silk sutures (0.5 cm, Ethicon) were pinned downed as anchor points (15 mm apart). 250 000 cells (passage 2–5) were mixed into a fibrin gel and quickly spread in each well. The fibrin was left to set before it was covered with cell culture supernatant (DMEM/F12 supplemented with 10% FBS, 0.2 mM L-ascorbic acid 2-phosphate, 0.05 mM L-Proline and 1% Penicillin-Streptomycin). Cell culture supernatant was replaced every second to third day and adhesions to the side of the well were detached 2 days post-seeding using a fine pipette tip to allow gel contraction. Cells were treated with dexamethasone from day 2–14 post-seeding. Four different concentrations were used; 1, 5, 10, or 50 nM. The drug was diluted in cell culture supernatant and controls were treated with 1 mM of ethanol. DEXAJECT® includes ethanol and 1 mM corresponds to the levels in the 50 nM group. All samples were harvested 14 days post-seeding.
Image acquisition and analysis
Fibrin gel contraction was measured on images taken day 4–14 post-seeding by a mobile phone on a standardized distance (11 cm) above the 6-well plate. The vertical diameter of the fibrin gel was measured at its widest point and normalized to the vertical diameter of the well in ImageJ (1.51k National Institutes of Health, USA). Results are presented as a percentage of contraction of the well.
RNA extraction
Tissue: Samples (< 100 mg) were pulverized one by one by a tungsten ball using in a nitrogen cooled container using a Mixer Mill (Retsch, Germany) at 2,600 rpm for 45 seconds. The process was repeated if samples were not fully homogenized. 1000 µl of TRIzol (Life Technologies, Gibco BRL) was added to each sample and thawed at room temperature. RNA was separated by adding chloroform, followed by centrifugation (12000xg for 15min at 4ºC). The aqueous phase was collected in new tubes, and 70% ethanol was added. A RNeasy Total RNA mini kit (Qiagen, Germany) was used for further RNA extraction according to instructions.
Tendon constructs: Samples were rinsed with PBS and collected in tubes containing 1 ml TRIzol, 5 stainless steel beads (2.3 mm in diameter), and 5 silicon-carbide sharp particles (1 mm in diameter) for mechanical disruption (BioSpec Products Inc, USA). Samples were snap frozen and stored in -80°C until analysis. After thawing samples on ice, they were homogenized at 30 Hz, for 15 seconds using TissueLyser (Qiagen). Disruption was repeated 2–4 times with 2 minutes on ice in between the repetitions. RNA was separated by adding 100 µl bromo-chloropropane (Molecular Research Centre, USA) followed by centrifugation (12000xg for 15min at 4ºC). The aqueous phase was collected and mixed with isopropanol (equal parts) and 4 µl of glycogen (20 µg/µl, Invitrogen, USA). The pellet was washed repeatedly with ethanol and dissolved in RNAse-free water. RNA concentration, purity, and quality were verified with Nanodrop ND-1000 (NanoDrop Technologies, USA) and RNA 6000 Nano kit (Agilent Bioanalyzer Technologies, Germany). All samples had an RIN value above 5 and were used for further analysis.
cDNA synthesis and Quantitative real-time PCR
Total RNA (1.5 µg) was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, UK). Primers for genes related to extracellular matrix, resolution, and tendon cell markers were bought from Applied Biosystems (Supplementary table 1). CYPA, RPLPO, and UBC were tested as internal controls for the rat tissue samples and each sample was normalized to a geometric mean of these three genes. GUSB, RPLP0, and YWHAZ were tested as internal controls for the tendon constructs samples and YWHAZ was chosen as it was stably expressed between the groups.
Fast PCR Master Mix (Life Technologies) was used for amplification (15µL/reaction). Quantification was calculated using a standard curve from universal rat reference gene (C.N. #QS0641, Life Technologies) or universal human reference gene (C.N. #QS0639). Reactions with no reverse transcription and no template were added as negative controls. Samples were analyzed with the 7.500 software, version 2.3 (Life Technologies).
Immunohistochemistry
Tendons were embedded in OCT, snap-frozen, and sectioned longitudinally (7 µm) using a Cryostat Microtome (Leica, CM1950, Heidelberg, Germany). Samples were quickly stored at -80 ºC for further immunostaining. Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (catalogue #CTS005, R&D Systems, USA) was used for blocking, secondary antibody and chromogenic staining according to instructions [33,34].
The primary antibodies used were: rabbit monoclonal [EPR5368] to alpha smooth muscle Actin (αSMA, 1:500, C.N #ab124964, Abcam, UK), and rabbit polyclonal to Fibronectin (FN1, 1:100, C.N #ab2413), Collagen type I (1:100, C.N#34710), Collagen type III (1:100,C.N#7778), scleraxis (1:100, C.N # ab58655), and Mannose Receptor (1:250, C.N # ab64693), and goat polyclonal to S100A4 (1:100; C.N #ab58597). The primary antibodies were incubated overnight at 4 ºC. Washing steps and incubations with primary antibody were performed in PBS with 0.1% saponin. Tissue sections were blocked with 5% normal goat serum after the primary antibody and followed by a biotinylated anti- mouse secondary antibody (R&D Systems, USA). Tissue sections were developed by 3,3′-Diaminobenzidine (DAB) and counterstained with Mayer’s Hematoxylin (Sigma Aldrich, USA) before image visualization, using a standard light microscope.
Immunofluorescence
Staining was performed as described [33,34] with some modification. Briefly, after the primary antibody incubation (as described above) and washing in PBS (2×5 min). A mixture of biotinylated secondary antibodies including anti-rabbit conjugated with Alexa Fluor 594, and anti-goat conjugated with Alexa Fluor 488 (1:500 dilution in PBS-0.1% saponin) was incubated for 60 minutes. DAPI (4,6-diamidino-2-phenylindole, Invitrogen, USA) staining was performed for nuclei visualization.
Image acquisition and analysis
Digital images were captured by a confocal and/or a super resolution microscope (Upright Zeiss LSM700 or LSM710, Carl Zeiss, Germany) and analyzed with ZEN 2009 software (LSM 710; Carl Zeiss). All pictures were taken at original magnifications of 200x, 400x or 630x with oil objective and analyzed with ImageJ software (Fiji ImageJ 1.52i, USA).
Statistics
Results were analyzed using SPSS software version 21, and graphs were created using GraphPad Prism version 9. Gene expression data were log-transformed to obtain similar variances between the groups. Independent t-tests were used in the animal experiment to compare dexamethasone treated and saline group (controls) within each loading condition. Protein quantification data was analyzed with Mann-Whitney U tests. The in vitro study used a repeated measured ANOVA with Holm-Šídák multiple comparisons test. Results were considered significant when p < 0.05. Potential confounders were minimized by random locations of the different groups in the animal facility, a random order of surgery and euthanization from the different groups. The surgeon was kept blinded from group allocation during surgery and euthanization and analyzes were performed blinded from group allocation until statistical analysis.