Study design
Total of 120 mature (12 weeks) male C57BL/6 mice were used in this study, and a rotator cuff repair model were established in the left forelimb, following Scott A. Rodeo’s method [14]. The operated mice were randomly allocated to 1 of 3 groups: control (n=40), rhBMP-4 (n=40), and noggin (n=40), intervened with fibrin sealant, fibrin sealant containing rhBMP-4 (Bio-tool, Beijing), and fibrin sealant containing noggin (Huabio, Hangzhou), respectively. Due to the ineffectivity of fibrin sealant to rotator cuff repair in a rodent model [15], the natural healing group was cancelled in this study. Animals were housed in cages and allowed unrestrained cage activity after surgery. At postoperative 2 and 4 weeks, mice were sacrificed and bilateral supraspinatus-supraspinatus tendon-humerus (SSTH) complexes were harvested for subsequent detections (Figure 1).
Fibrin sealants preparation and in vitro drug sustained-release test
Fibrinogen solution (Sigma-Aldrich) and thrombin solution (Sigma-Aldrich) were prepared following the manufacturer’s manual. RhBMP-4 and noggin were added to the thrombin solution, respectively. 10 μL fibrinogen solution and 2 μL thrombin solution (containing 0.5μg rhBMP-4 or 0.2 ng noggin, the dosage used in prior studies [16, 17]) were utilized to make each fibrin sealant, with a 0.22 μm syringe filter (Millex, Sigma). Similarly, fibrin sealant in control group was also prepared, but containing nothing.
To measure BMP-4 and noggin release kinetics from the fibrin sealant, which was comprised of 250μL fibrinogen solution and 50 μL thrombin solution, and 12.5μg rhBMP-4 or 5 ng noggin. The gels were incubated 0.5 mL phosphate buffered saline (PBS) with 10% fetal bovine serum at 37 ℃. The PBS solution was replaced and collected five times over the first 24 hours, and then was changed and collected daily. ELISA for BMP-4 and noggin were performed on the washed solutions, respectively. The release experiment was carried 3 times and cumulative percentages were presented in the results.
Surgical technique
Briefly, mice were placed in a right lateral decubitus position after being anesthetized with an intraperitoneal injection of 0.3% pentobarbital sodium. An incision was made on the left shoulder, and the deltoid muscle was revealed, which was dissected minimally to expose the supraspinatus tendon. Using a customized retractor to elevate the acromion, the supraspinatus-supraspinatus tendon (SST) was isolated. Then, the SST was sutured with a 6-0 prolene with double needles (Ethicon) in an “8” figure fashion, and sharply detached from its footprint on the humeral head. Any soft tissues and fibrocartilaginous tissues remaining on the bone were gently removed with a scalpel. One transosseous bone tunnel was then created with a 30-G needle in the humeral head under the footprint, and the two sutures limbs were crossed through the tunnel, one from lateral to the interior, and the other from interior to lateral. After tightening the two sutures limbs, the SST was reattached to the footprint. Before tying the knot, fibrin sealant, fibrin sealant containing rhBMP-4, or fibrin sealant containing noggin was placed on the tendon-to-bone interface. Finally, the wounds were layered sutured, and buprenorphine (0.05mg/kg) was administrated subcutaneously for analgesic postoperatively. All animal surgeries were operated by two sports medicine doctors under surgical microscopy.
Micro-computed tomography (micro-CT) evaluation
Micro-CT (viva CT 80; Scanco Medical, Switzerland) was performed in this study to assess the new bone formation at the tendon attachment site. After fixing in 10% paraformaldehyde in 4 °C for 24 hours, each specimen was kept in a tube surrounded by formalin and scanned with 10.4 μm voxel size, at 55 KVp, 0.36° rotation step (180° angular range) and a 400 ms exposure per view. Then data of each specimen were processed with a 3-dimensional Gaussian filter and a global threshold, to identify the bone voxels from the surrounded saline in bone marrow and soft tissues. After thresholding, the micro-parameters of the osteogenesis at TBA were calculated, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular spacing (Tb.Sp). The region of interest (ROI) was selected randomly under the footprint, at the center of the two suture bone holes in the image. In the analysis, the ROI was defined as a cube with a length of 1 mm, from the bone surface and extending distally, so as to avoid involvement of adjacent cortical bone.
Histomorphometric analysis
Once dissection, the fresh SSTH structures were fixed in 10% neutral buffered formalin for 48 hours. After 7 days decalcification in 10% EDTA, the specimens were dehydrated in grade ethanol, and embedded in paraffin. The paraffin blocks were sectioned with 5µm thickness in the coronal plane, through the supraspinatus tendon and the greater tuberosity.
The sections were stained with Hematoxylin and eosin (H&E, Solarbio Co. Ltd, Beijing, China) and Toluidine blue-fast green (Sigma-Aldrich, St. Louis, MO). H&E stained sections were used for the histological description of healing of BTI, and semi-quantitative analysis for cell density around the tendon to bone interface [15]. Toluidine blue O/fast green (TB) stained sections were used for semi-quantitative analysis for the fibrocartilage zone thickness using the ImageJ software (National Institutes of Health, Bethesda, Maryland) [18]. All sections were observed using a transmitted light microscopy (Olympus CX31; Olympus Inc) under a same condition.
Immunoflorescence analysis
Immunoflorescence was used to detect SOX 9 expressing cells in the interface zone of tendon to bone. After fixation, decalcification, dehydration and embedded in OCT, the SSTHC specimens were longitudinally sectioned with 10 µm. For immunofluorescence staining, the sections were washed with PBS, permeabilized with 0.1% TritonX‐100, and blocked with 5% bovine serum albumin (BSA; Sigma‐Aldrich, St. Louis, MO). Then, primary antibody against mouse SOX 9 (ab76997, abcam) was applied at 4℃ overnight, subsequently incubated with anti-rabbit secondary antibody (ab150116, abcam) at room temperature for 1 hour, and finally incubated with DAPI (ab228549, abcam). All the images were observed and captured with using a Zeiss Axio Imager (Zeiss, Solms, Germany). To quantitate the positive signals of SOX 9, a region of interest (ROI) was defined at the SST attachment site according to aprevious similar study[19]. The SOX 9 positive cells areas were counted for three times, and the mean number per square millimeter for each sample was reported. The assessor was blinded to the groups during image analysis.
Biomechanical testing
For biomechanical testing, each SSTHC was carefully dissected from surrounding tissues under a surgical microscope, and stored at -80 ℃. At the time of conduct of testing, the specimen was thawed at room temperature, and the supraspinatus muscle and all suture were carefully removed. Each SSTHC was dipped in 0.9% saline solution water bath at 37 ℃ before biomechanical testing. The humerus was mounted and the supraspinatus tendon was fixed in custom grip with sandpaper and ethyl cyanoacrylate. A microcomputer controlled electronic material testing system (WD-T, Shanghai Zhuoji Instrument Equipment Co., Ltd.) was used to test the failure load of TBA. The SSTHC was placed in the testing system machine and allowing a uniaxial tensile following approximate the anatomic position of the supraspinatus tendon. After pre-tensioning for 3 times under a preload between 0.0-0.5 N, the SSTHC was loaded to failure at a rate of 1mm/min, and the ultimate failure load was recorded. The stiffness was calculated from the curve of load to deformation.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism (Version 6.0.1; San Diego, CA, USA). All quantitative data were presented as mean ± SD (standard deviation) and analyzed statistically using 2-way analysis of variance (ANOVA) with the Bonferroni post hoc test to detect differences among groups. The significant level was sat to P < 0.05.