All surgical procedures performed on the rats were conducted under general anesthesia, ensuring the absence of pedal reflex. An intraperitoneal injection of 80 mg/kg ketamine hydrochloride (Narkamon 50 mg/mL, BIOVETA, Czechia) and 10 mg/kg xylazine (Xylazinbio 2% BIOVETA, Czechia) was administered for anesthesia induction. Furthermore, the rats were continuously monitored for pain sensation throughout the procedure, and additional anesthesia doses were administered as needed to maintain adequate pain control. Prior to incision, the surgical areas were shaved and cleansed with povidone-iodine (Batix^®, Denizpharma, Istanbul, Turkey), and all surgical procedures were carried out under sterile conditions.

A well-established tendon injury model, widely utilized in numerous studies in the literature (6, 41, 42), was employed in this study. Following the surgical preparation of the extremity, an incision was made along the posterior line of the ankle to expose the Achilles tendon and plantaris tendon. Tenotomy was performed approximately 0,5 cm proximal to the calcaneus insertion of the Achilles tendon using a No. 15 scalpel blade. The tendons were then repaired end-to-end using the modified Kessler method with 4/0 round polypropylene monofilament sutures (TıpKimSan Limited Company, Istanbul, Türkiye). Following irrigation of the wound area with saline, the incision was closed using 3/0 polypropylene monofilament sutures (TıpKimSan Limited Company, Istanbul, Türkiye) to achieve skin integrity. The surgical procedure is illustrated in Figure 1.

No bandages, dressings, or plasters were applied to the rats postoperatively. From the early postoperative period, all rats were allowed free joint movements and were permitted to load their lower extremities. Rats in the quercetin group received a daily dose of 100 mg/kg quercetin (Sigma-Aldrich, United States), administered as a suspension in corn oil via oral gavage at the same time each day. The control group received only corn oil via oral gavage. All rats were sacrificed on the 28th day following surgery. The quercetin dosage was adjusted based on previous studies (10, 43).

Following administration of high-dose anesthesia (ketamine 75 mg/kg and xylazine 10 mg/kg), rats were euthanized by cervical dislocation. The Achilles tendons of the euthanized rats were then carefully dissected, ensuring removal distally from the bone-tendon junction of the calcaneus and proximally from the bone-tendon junctions of the femur and tibia. Subsequently, one of the Achilles tendons from each rat underwent histopathological and immunohistochemical evaluation, while the other tendon was subjected to biomechanical analysis.

The samples were dissected and kept in 10% formalin fixative solution for 3 days. Then, the tissues were left under running water overnight, and the next day they were passed through a series of 60, 70, 80, and 100% alcohol, respectively, and then taken into xylene. Tissues were embedded in paraffin to obtain thin sections, and 3 μm sections were taken with a microtome. After the sections were kept in an oven at 60°C overnight and deparaffinized, routine Hematoxylin–Eosin (HE) and Masson Trichrome (MT) staining was performed as the first step of the morphological evaluation. After staining, the tissues were photographed with an Olympus CX41 microscope (Olympus Corp., Tokyo, Japan) at 20, 40, and 100 magnifications. The most common and established system for evaluating pathological changes in tendon tissue, particularly utilized in animal experiments, is the Bonar scoring system (44–46). Tenocytes, ground substance, collagen, and vascularization were assessed using the Bonar scoring system, each out of four points. The Bonar scoring system utilizes a four-point scale; ‘0’ represents a normal tendon appearance, ‘1’ indicates mild abnormal tendon appearance (10–20% of total tissue), ‘2’ represents moderately abnormal appearance (over 20% of total tissue), and ‘3’ indicates a markedly abnormal appearance. Total scores were scored from 0 (normal tendon, strong healing) to 12 (most severe detectable pathology, poor healing) and are shown in Figures 2A–D.

Five micrometer thick sections taken from paraffin blocks with the help of a microtome were then placed on polylysine slides (Sigma, St Louis, MO, United States). After the dehydration stage, microwave application was made in citrate solution with a pH of 6 (Carlo Erba, 368,057). Sections were kept in 3% hydrogen peroxide (Dako, Glastrup, Denmark) to prevent endogenous peroxidase activity. After blocking with normal goat serum, the sections were incubated with primary antibodies, which are type I collagen (1:100, Sigma, United States), type III collagen (1:100, Sigma, United States), alpha smooth muscle actin (SMA) (1:100, RTU, Leica, BK), and Galectin-3 (1:100, RTU, Cell Marque, CA, United States). After the biotin-labeled secondary antibody (Abcam, United Kingdom) step, the tissues treated with the streptavidin-HRP kit (Santa Cruz, USA) were finally treated with 0.05% diaminobenzidine (Zymed Histostain Plus CA, United States). All washes during the staining process were performed using 1X phosphate-buffered saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, pH 7.4), purchased from Sigma-Aldrich (St. Louis, MO, United States). Immunohistochemical staining results were evaluated with H-score. Staining ratio was graded semiquantitatively (0 = staining in less than 1% of cells, 1+ = staining in 1–10% of cells, 2+ = staining in 11–50% of cells, 3+ = staining in 51–80% of cells, 4+ = staining in more than 80% of cells). Staining intensity was also determined by the blind method (0 = no staining, 1 = pale, 2 = moderate, 3 = intense). Then, the total score was calculated with the formula “(1 + staining intensity/3) x staining ratio.” The highest marked immunohistochemical images for H-score are shown in Figures 2E–L.

The transverse diameters of the rat’s Achilles tendons were measured using a digital caliper (TESA Unimaster, Renens, Switzerland) (47). It was assumed that the transverse area of the Achilles tendons followed circular geometry. To ensure tissue integrity, the tendons were kept moistened with gauze soaked in a physiological solution during preparation and mounting in the materials testing equipment. The proximal tendon was delicately separated from the muscle for clamping. The length of the tendon between the grips was estimated using a caliper. A tensile test was conducted on both the experimental and control groups’ Achilles tendons (total number of samples 32). Biomechanical tests were performed using the ELISTA, TST 2500 mxe brand axial tensile testing device. The tensile tests were executed under identical conditions for each Achilles tendon sample. Figure 3 illustrates the tensile tests, with the Achilles tendons secured between the jaws of the device, one end attached to the calcaneus and the other to the musculotendinous junction. A 5 kN load cell was employed to conduct the biomechanical tests on the specimens, fixed vertically in the machine using a fixture, at a crosshead speed of 1 mm/min at room temperature. Force (N) versus elongation (mm) was recorded during the tensile test. Parameters such as failure load (N), displacement (mm), total energy (J), ultimate stress (MPa), and strain (%) were derived from the force (N)-elongation (mm) graphs at the conclusion of the biomechanical tests.

In this study, double-blinding and randomization strategies were implemented to reduce potential biases and enhance the reliability of the results. These double-blinding and randomization strategies were employed to enhance the internal validity of the study and enable the evaluation of results independently of external factors. The reliability of the obtained data and the scientific robustness of the study were supported by the integration of these methodological approaches.

The allocation of animals to groups was performed randomly using computer-based randomization methods. This helped balance potential differences among animals, initially while reducing the likelihood of systematic errors between experimental and control groups. In double-blinding, during the execution of the experiment, both the observers applying interventions to the animals and the individuals evaluating the data (microscopic preparations) were kept unaware of group assignments. This approach helped prevent potential biases in subjective assessments and data analysis.

SPSS 23 (Statistical Package for the Social Sciences) program was used for data analysis (Graph Pad Software, Inc., La Jolla, CA). Data were expressed as mean ± standard deviation (SD) or median (min–max). The skewness and kurtosis values of the data were observed to adhere to a normal distribution. Accordingly, an independent two-sample t-test (unpaired t-test) was utilized. A p-value of <0.05 was considered statistically significant.

During the phases of tendon healing, structures typically observed microscopically, such as tenoblasts, inflammatory cell infiltration, collagen fibrils, and extracellular matrix, were identified in both the experimental and control groups. It was observed that tenoblasts lost their elongated appearance and alignment, while the density of extracellular matrix increased, and tenoblasts decreased in size. Inflammatory cells, newly formed collagen bundles, mature collagen bundles, and tenocytes starting to line up in wavy patterns were observed and depicted as shown in Figures 4A,B. According to the Bonar scoring, tenocytes were 2.31 ± 0.75 in the control group and 1.69 ± 0.73 in the quercetin group, indicating a significant difference between them (p = 0.0232). Ground substance was 2.56 ± 0.66 in the control group and 1.93 ± 0.68 in the quercetin group, showing a significant difference between them (p = 0.0128). Collagen was 2.44 ± 0.68 in the control group and 1.81 ± 0.83 in the quercetin group, indicating a significant difference between them (p = 0.0272). Vascularization was 2.44 ± 0.68 in the control group and 1.81 ± 0.83 in the quercetin group, showing a significant difference between them (p = 0.0307). As shown in Table 1, morphometric scoring revealed significant differences between the control and experimental groups in terms of parameters.

In immunohistochemical analyses, tendon healing and ground substance were evaluated using type I collagen, type III collagen, and SMA. To evaluate the relationship between Galectin-3 and fibrosis, Galectin-3 was applied using immunohistochemical methods. The evaluation of Galectin-3 involved counting stained fibroblastic cells under a light microscope at X400 magnification. Following staining with Galectin-3, fibroblasts were differentiated from monocytes, macrophages, and neutrophil cells, and the morphological characteristics of fibroblasts were analyzed in detail. In this analysis, the number of fibroblasts exposed to Galectin-3 was determined, and morphological features associated with fibrosis were identified. As shown in Figure 4A, the region with the highest number of fibroblasts was selected as the focal point of this evaluation.

As shown in Figure 4A, areas with the highest cell count were preferred. In H-score evaluations, Collagen-1 was 78.03 ± 15.17 in the control group and 91.15 ± 14.59 in the quercetin group, indicating a significant difference between them (p = 0.0166). Collagen-3 was 150.54 ± 9.53 in the control group and 162.13 ± 11.17 in the quercetin group, showing a significant difference between them (p = 0.0166). SMA was 19.36 ± 9.78 in the control group and 29.38 ± 14.99 in the quercetin group, indicating a significant difference between them (p = 0.0323). Galectin-3 was 8.99 ± 4.90 in the control group and 13.29 ± 5.71 in the quercetin group, showing a significant difference between them (p = 0.0295). All immunohistochemical parameters are provided in Table 2 and Figures 4C,D.

The biomechanical data regarding the resistance against applied loads of Achilles tendons utilized in this study are presented in Table 3. Biomechanical test outcomes of Achilles tendons for both the quercetin and control groups are depicted in Figure 5. Analysis of failure load (N), displacement (mm), total energy (J), ultimate stress (MPa), and strain (%) data derived from the biomechanical tests for both quercetin and control group samples was conducted in detail. Initially, upon examining the average failure load, the quercetin group’s results (36.5 N) demonstrated mechanically superior outcomes compared to the control group (32.06 N) by approximately 12% (Figure 5A). Displacement and strain values exhibited greater elongation in the quercetin group, suggesting that Achilles tendons exhibit higher elongation under mechanical load in this group (Figures 5B,I). In terms of total energy, the calculation was automatically derived from the program of the testing device, representing the area under the force-strain graph. The total energy (J) and maximum energy (J) values were found to be approximately 18 and 27% higher, respectively, in the quercetin group test results compared to the control group (Figures 5C,D). One of the most critical mechanical parameters in biomechanical tests is ultimate stress. The results of this study indicated that the average ultimate stress values of the quercetin group samples were approximately 7% higher than those of the control group (Figure 5H). Stiffness refers to the resistance of a specimen against deformation when subjected to a mechanical force. It is determined by the gradient of the force-strain curve within the ‘linear’ zone. In the present study, stiffness value was calculated to be approximately 20% higher in the quercetin group compared to the control group (Figure 5G). This outcome suggests a positive healing effect of quercetin in the experimental group.