A novel and efficient murine model for investigating tendon-to-bone healing

This study received approval from the Animal Ethics Committee of our university (approval number: AMUWEC20210782). We obtained forty-four 9-week-old male C57/BL6 mice (Hunan SJA Laboratory), designating their left hindfoot to the control group and their right hindfoot to the model group. In the model group, we applied novel surgical techniques, while the control group was left untreated to serve as a comparative baseline. At two and four weeks post-operation, the mice were killed for histological staining, Micro-CT scanning (Kontich, Belgium), biomechanical testing (TA Instruments, USA), and real-time PCR analysis (Fig. 1).

The surgery was performed by a clinical doctor and a laboratory technician, under the supervision of a senior clinical doctor from the Sports Medicine Department. They refined and practiced the model techniques using cadaveric specimens collected from abolished mice (Refer to Figs. 2, 3, and Additional file 1: Video S1).

The mice were anesthetized with isoflurane and placed in a prone position on a foam surgical pad (Fig. 2A). After sterilization of the hindfoot, a 2 mm incision was made centrally, about 2 mm above the calcaneus (Fig. 2B). First, the tissue was dissected using blunt techniques to expose the Achilles tendon and calcaneus (Fig. 2C). Second, a transverse bone tunnel was created in the calcaneus with a 30 G insulin needle (Fig. 2D). Next, a 6-0 absorbable suture was threaded through the bone tunnel and the Achilles tendon in an oblique pattern from lower left to upper right, looped around the tendon toward the lower right, then passed obliquely from lower right to upper left, and secured (Fig. 2E and F). Third, the Achilles tendon was carefully incised near its insertion with a No.11 blade, and the cartilage at the tendon-to-bone interface (TBI) was gently excised (Fig. 2G). The suture ends were then knotted and tightened (Fig. 2H). Finally, the incision was closed with 1–2 interrupted sutures (Fig. 2I). Post-surgery, the mice had unrestricted movement in their cages, with free access to water and food throughout the experiment.

At 2 and 4 weeks post-surgery, five mice were killed for specimen collection. The skin was removed, and the calcaneus along with a portion of the Achilles tendon was harvested. These specimens were preserved in 4% paraformaldehyde for 24 h. Subsequently, they underwent decalcification in an EDTA solution for 72 h at 37 degrees Celsius in a thermostatic shaker. The specimens were then embedded in paraffin and sectioned sagittally. Hematoxylin and eosin (H&E), Sirius red, and safranin O fast green staining were conducted to examine the tendon-to-bone interface. H&E staining assessed the overall condition, including new tissue formation and cell morphology. Sirius red staining evaluated tendon maturation, and safranin O fast green staining detected glycosaminoglycan and cartilage formation. Additionally, we used a modified tendon maturity assessment system [16], comprising eight items for a total score of 32, to evaluate tendon maturity, with a higher score indicating more advanced tendon-to-bone healing.

Two and four weeks post-surgery, three mice were killed for specimen collection to study bone formation within the bone tunnel. The samples were analyzed using Micro-CT with the Bruker MicroCT Skyscan 1272 system (Kontich, Belgium), which featured an isotropic voxel size of 10.0 µm to image the entire calcaneus. Scans were performed in 4% paraformaldehyde, utilizing an X-ray tube potential of 60 kV, X-ray intensity of 166 µA, and an exposure time of 1700 ms. Image reconstruction was conducted using Nrecon software (Ver. 1.6.10), and three-dimensional (3D) images were generated from two-dimensional (2D) scans using CTvox software (Ver. 3.0.0), based on distance transformation of the grayscale original images.

Seven mice were killed, and only the entire feet and tendons were harvested at both the 2-week and 4-week postoperative time points. Ultimate tensile strength (MPa), stiffness (N/mm), and failure force were assessed using ElectroForce Mechanical Testing Instruments (TA Instruments, USA). To enhance fixation friction, the specimens were wrapped in gauze before being secured in the tensile testing machine. Following calibration, a preload of 0.05 N was applied for 1 min, and then, the samples were stretched at a rate of 0.03 mm/s. Data associated with terminal tendon slips and fractures not related to the tendon-to-bone insertion sites were excluded from our analysis.

At each postoperative time point, five mice were killed, and we collected samples from the tendon-to-bone interfaces, which included small portions of both the bone and tendon. These specimens were then sectioned, and total RNA was extracted using TRIzol (Thermo Fisher Scientific, USA). The concentration of the extracted RNA was determined by spectrophotometry and normalized against the expression of the housekeeping gene GAPDH. The primer sequences for genes ACAN, COL1a1, SOX9, MMP3, MMP13, MMP14, and GAPDH are provided below (See Table 1).

The average skin-to-skin time for beginners was approximately 10 min. However, this time decreased to about 6 min after they practiced on several cadaveric mice. Notably, there were no instances of significant bleeding during the surgical procedures.

After harvesting, we observed thickening of the Achilles tendon, structural disorder, and a loss of original luster and toughness compared to native tissue at both 2 and 4 weeks postoperatively. At 2 weeks, H&E staining revealed thickening of the Achilles tendon, disordered fibers, and incomplete reattachment to the calcaneus. This was coupled with a disruption of the original four layers and the formation of a fibrovascular scar at the tendon-to-bone interface. At 4 weeks, H&E staining indicated complete reattachment of the Achilles tendon to the calcaneus, yet the tendon and tendon-to-bone interface structure remained disordered. Chondrocytes in the tendon-to-bone interface were noted, but they lacked a smooth transition structure. Similarly, safranin O fast green staining showed glycosaminoglycan deposition at this site, but the original non-calcified and calcified structures were absent. Observations under a polarization microscope of Sirius red-stained slices revealed aligned tendons rich in type I collagen (red) in the control group, in contrast to the model group, where the tendon was disrupted and replaced by a fibrovascular scar (green). The average tendon maturity score in the model group was 14.67 ± 0.47 at two weeks postoperatively, increasing to 20 ± 0.82 at four weeks, suggesting an enhancement in the tendon-to-bone connection (Fig. 4).

3D image reconstruction allowed us to observe the persistence of the bone tunnel at two weeks postoperatively. By four weeks postoperatively, significant healing and closure of the bone tunnel were evident. Additionally, we noted an increase in bone mass at the tendon-bone interface over time, accompanied by a more complete structural formation (Fig. 5).

The failure force of the experimental group at 2 and 4 weeks post-operation was 8.08 ± 1.82 and 13.85 ± 5.32, respectively (P = 0.03), accounting for approximately 44.2% and 77.5% of that in intact tissues. The stiffness of the experimental group at 2 and 4 weeks post-operation was 12.37 ± 5.66 and 10.58 ± 3.7, respectively, with no significant difference between the two time points (P = 0.53). The ultimate tensile strength of the experimental group at 2 and 4 weeks post-operation was 2.02 ± 0.46 and 4.62 ± 1.78, respectively (P = 0.005). These biomechanical test results indicate that the biomechanical strength of the model group increases over time, correlating with the normal healing process (Table 2 and Fig. 6).

According to the data, key catabolic indicators, including MMP3 and MMP14, peaked at 2 weeks postoperatively. Although MMP13 exhibited a slight increase, there was no significant difference between the 2-week and 4-week time points. In contrast, anabolic indicators like SOX9 and Collagen I reached their peak in the fourth week. However, ACAN demonstrated a decreasing trend by the fourth week. These results suggest that most tissue inflammation occurs during the second week post-surgery, while tissue remodeling and maturation predominantly occur in the fourth week (Fig. 7).