High-Dose Statins Preserve Tendon-Bone Interface Healing Without Adversely Affecting Fatty Infiltration in a Rotator Cuff Repair Rat Model.

Excessive expression of proinflammatory cytokines after rotator cuff (RC) surgery impairs the quality of tendon-bone interface (TBI) healing. There is evidence that the asthma drug montelukast (MS) inhibits the expression of proinflammatory cytokines. This study was conducted to verify the effect of MS administration on TBI healing after RC repair. Thirteen rats in the MS group were intraperitoneally administered 10 mg/kg of the drug daily for 2 weeks after RC surgery, and 13 rats in the control group were administered only 0.9% saline. The healing effect of the TBI was assessed through histologic and biomechanical analysis 4 weeks after tendon repair. In the MS group, the expression of interleukin-1 beta (IL-1β; P<.01) and interleukin 6 (IL-6; P<.01) was significantly reduced compared with the control group. In the evaluation of supraspinatus fatty infiltration, the MS group showed significant inhibition of fatty infiltration compared with the control group (P<.001). Histologic analysis showed that the MS group had significant improvements in collagen density (P=.035) and alignment (P=.011). Biomechanical analysis after systemic administration of MS showed an increase in the cross-sectional area (P<.001) and elongation (P<.01) of the TBI. The use of MS improved tendon elasticity through suppressing fatty infiltration and improving TBI collagen density and arrangement. The mechanism is down-regulation of IL-1β and IL-6. These results strongly support the use of MS as an anti-inflammatory agent that does not impair tendon healing. [Orthopedics. 2025;48(2):e105-e112.].

Background: Multiple factors, such as muscle fatty infiltration (FI), tendon collagen content, and collagen arrangement, determine bone-tendon interface (BTI) healing after rotator cuff (RC) repair. Purpose: To evaluate the effects of systemic administration of ezetimibe-atorvastatin (EZE/ATZ) combination on muscle FI and tendon collagen density and arrangement in an RC repair rat model. Study design: Controlled laboratory study. Methods: A total of 26 male Sprague-Dawley rats were randomly divided equally into control and EZE/ATZ groups and subjected to RC tendon repair surgery. Postoperatively, the EZE/ATZ group rats received a combination of EZE (10 mg/kg/d) and ATZ (20 mg/kg/d) for 4 weeks, after which they were sacrificed. Oil Red O staining was used to assess FI in the supraspinatus muscle. The expression of biomarkers related to muscle atrophy and FI was measured using quantitative real-time polymerase chain reaction. For the qualitative and quantitative analysis of FI-related biomarkers, immunohistochemical staining was performed. Biomechanical and histological analyses were performed to evaluate the quality of BTI healing after RC repair. Results: The EZE/ATZ group showed significantly lower FI compared with the control group (P < .001) and significantly downregulated expression of gene markers related to muscle atrophy and FI. On histological analysis, the EZE/ATZ group exhibited increased collagen type I contents, consistent collagen arrangement (P = .005), and significantly higher collagen density (P = .003) compared with the control group. Biomechanical analysis of the BTI healing revealed that the EZE/ATZ group had significantly increased ultimate strength (P = .006) compared with the control group. Conclusion: Systemic EZE/ATZ administration suppressed supraspinatus FI by downregulating muscle atrophy–related and FI-related genes after RC repair. Additionally, EZE/ATZ use improved collagen biosynthesis, density, and arrangement at the BTI and significantly increased tensile strength. Clinical Relevance: The results of the current study strongly advocate the use of EZE/ATZ to improve shoulder function and tendon healing after RC repair.

BackgroudRotator cuff (RC) tears are a common shoulder disorder associated with pain, dysfunction, and progressive muscle degeneration. Postoperative muscle atrophy and fatty infiltration are key factors contributing to poor healing outcomes, increased rerupture rates, and long-term functional impairment. While acetaminophen (APAP) is widely used for pain management, its potential effects on muscle fatty infiltration and tendon-bone interface (TBI) healing remain unclear. This study aimed to investigate whether APAP influences fatty infiltration and TBI healing after RC surgery. We hypothesized that APAP would reduce fatty infiltration by downregulating peroxisome proliferator-activated receptor gamma (PPAR-γ) while preserving histological and biomechanical TBI integrity.MethodsA RC repair rat model was created using 12-week-old male Sprague-Dawley rats. Ten rats in the APAP group received an oral dose of 30 mg/kg APAP daily for 4 weeks after RC tendon transection, and 10 rats in the control group were administered only 0.9% saline. All rats were sacrificed 4 weeks after surgery. Fatty infiltration was analyzed qualitatively and quantitatively in the proximal part of the supraspinatus muscle. TBI was evaluated for RC healing effect through general tissue staining and biomechanical strength measurements.ResultsFour weeks after tendon repair, the APAP group showed significant prevention of supraspinatus fatty infiltration compared to the control group (p < 0.001). These results were confirmed through immunohistochemical results showing that PPAR-γ, a marker gene for fatty infiltration, was significantly decreased in the APAP group (p < 0.001). Histological healing assessments between the 2 groups were not significantly different (p = 0.634). Additionally, mechanical strength was not negatively affected.ConclusionsAPAP treatment was associated with prevention of supraspinatus fatty infiltration without adversely affecting TBI healing in an RC repair model.

Study of Rotator Cuff Repair Techniques: We Really Are Trying

Background: The tendon-bone interface (TBI) in the rotator cuff has a poor intrinsic capacity for healing, which increases the risk of retear after rotator cuff repair (RCR). However, facilitating regeneration of the TBI still remains a great clinical challenge. Herein, the authors established a novel strategy based on magnetic seeding to enhance the TBI regeneration. Hypothesis: Magnetic seeding bone marrow mesenchymal stem cells labeled with superparamagnetic iron oxide (SPIO-BMSCs) into a biphasic scaffold can promote tendon-bone healing after RCR. Study Design: Controlled laboratory study. Methods: BMSCs were labeled with SPIOs. Prussian blue staining, CCK-8 tests, Western blot, and quantitative reverse transcription polymerase chain reaction (PCR) were used to determine the optimal effect concentration of SPIOs on cell bioactivities and abilities. Then SPIO-BMSCs were magnetically seeded into a biphasic scaffold under a magnetic field. The seeding efficacy was assessed by a scanning electron microscope, and the potential mechanism in chondrogenic differentiation after seeding SPIO-BMSCs into the scaffold was evaluated by Western blot and PCR. Furthermore, the effect of SPIO-BMSC/biphasic scaffold on tendon-bone healing after RCR using a rat model was examined using histological analysis, enzyme-linked immunosorbent assay, and biomechanical evaluation. Results: BMSCs labeled with 100 μg/mL SPIO had no effect on cell bioactivities and the ability of chondrogenic differentiation. SPIO-BMSCs were magnetically seeded into a biphasic scaffold, which offered a high seeding efficacy to enhance chondrogenic differentiation of SPIO-BMSCs via the CDR1as/miR-7/FGF2 pathway for TBI formation in vitro. Furthermore, in vivo application of the biphasic scaffold with magnetically seeded SPIO-BMSCs showed their regenerative potential, indicating that they could significantly accelerate and promote TBI healing with superior biomechanical properties after RCR in a rat rotator cuff tear model. Conclusion: Magnetically seeding SPIO-BMSCs into a biphasic scaffold enhanced seeding efficacy to promote cell distribution and condensation. This construct enhanced the chondrogenesis process via the CDR1as/miR-7/FGF2 pathway and further promoted tendon-bone healing after RCR in a rat rotator cuff tear model. Clinical Relevance: This study provides an alternative strategy for improving TBI healing after RCR.

The role of an elevated critical shoulder angle (CSA) in rotator cuff healing following rotator cuff repair (RCR) remains a subject of clinical controversy. The present study aimed to investigate the effect of increased CSA on tendon-bone interface healing following RCR. A bilateral chronic rotator cuff tear model was established in 48 Sprague-Dawley rats. Acromion lateralization (Acr) surgery was performed unilaterally to increase CSA. After 4 weeks, bilateral RCR was performed. Micro-computed tomography was utilized to measure CSA. Tendon-bone interface healing was assessed at 3, 6, and 9 weeks post-RCR with use of magnetic resonance imaging (MRI), biomechanical testing, gait analysis, and histological evaluation. The mean CSA in the Acr group was significantly greater than that in the RCR-only group (37.2° ± 2.6° versus 29.7° ± 3.1°; p < 0.001). At 6 and 9 weeks postoperatively, the Acr group demonstrated significantly poorer outcomes on MRI (i.e., higher signal-to-noise quotient), biomechanical strength (i.e., lower ultimate failure load and stiffness), and gait parameters compared with the RCR-only group (p < 0.05). Histological analysis revealed inferior tendon-bone interface integration in the Acr group (p < 0.01), including reduced fibrocartilage formation, disorganized collagen fibers, and a lower collagen I/III ratio. Immunohistochemistry showed significantly higher Piezo1 expression in the Acr group (p < 0.001), suggesting a mechanobiological response to increased mechanical stress. An increased CSA impaired tendon-bone interface healing following RCR in a rat model. Although these findings were preclinical, they provide experimental evidence that an increased CSA may influence rotator cuff healing, supporting the potential role of CSA modification (e.g., with acromioplasty) in reducing the risk of retear. The present study provides experimental evidence to support the consideration of CSA reduction in selected high-risk patients undergoing RCR to promote rotator cuff healing and potentially reduce retear rates.

Isotretinoin improves tendon-bone interface healing and inhibits muscle-fatty infiltration through GATA6 activation in a rat model of rotator cuff repair.

Background: The results of rotator cuff (RC) repair surgery can be influenced by the presence of muscle atrophy and fatty infiltration. Three-dimensional (3D) quantitative data regarding those degenerative muscle changes after successful or failed RC repair are rare in the current literature. Hypothesis/Purpose: The purpose of this study was to analyze muscle volume and fatty infiltration of the supraspinatus muscle after successful and failed arthroscopic RC tendon repair, with a minimum follow-up of 5 years. It was hypothesized that RC muscle volume and fatty infiltration would improve after successful repair and only to a limited extent after failed repair. Study Design: Cohort study; Level of evidence 2. Methods: A total of 115 patients (mean age, 59 ± 8 years; 33% women) with RC repair for full-thickness supraspinatus tendon tear were prospectively followed at 3 and 12 months. Of them, 18 patients with unsuccessful RC repairs were matched to 21 patients with successful repairs and reevaluated at a minimum follow-up of 60 months. All patients underwent quantitative 2-point Dixon magnetic resonance imaging at all time points to evaluate full 3D muscle volume and 3D fatty infiltration. The clinical examination included the full Constant-Murley score (CS) and subjective shoulder value. Results: The relative changes in supraspinatus muscle volume were statistically significant between the 2 groups over time (P < .01). Successful repairs showed a mean volume increase of 18% (P < .001) and 23% (P < .001) from preoperatively and the 3-month follow-up, respectively, and failed repairs were remodeled by 3% (P = .586) and 12% (P = .001), respectively. However, a direct comparison of the volumes revealed comparable results at the latest follow-up with 43 cm3 (95% CI, 38-47 cm3) and 40 cm3 (95% CI, 33-46 cm3) for successful and failed repairs (P = .494), respectively. The supraspinatus 3D fatty infiltration also showed lower fat content for the successful repair preoperatively (6.9% [95% CI, 4.7%-9.2%] vs 9.1% [95% CI, 7.2%-11.1%]; P < .01), at 3 months (7.9% [95% CI, 5.5%-10.4%] vs 12.8% [95% CI, 9.1%-16.5%]; P < .01), at 12 months (7.5% [95% CI, 4.8%-10.1%] vs 11.6% [95% CI, 9.4%-13.8%]; P < .01), and at 60 months (7.4% [95% CI, 4.7%-10.2%] vs 15.5% [95% CI, 11%-20%]; P < .01) postoperatively. Fatty infiltration remained unchanged between preoperatively and from 3-month follow-up in the successful group. However, it increased by 70% (P < .01) from preoperative and by 21% (P = .065) from 3-months follow-up in the failed group. The clinical outcome was similar for successful and failed repairs with an absolute CS of 81 ± 6 versus 72 ± 15 (P = .069) and a relative CS of 94% ± 7% versus 85% ± 17% (P = .078). Conclusion: Successful RC repair was associated with relevant improvement of supraspinatus muscle mass and an unchanged fatty infiltration at a midterm follow-up of 5 years. However, failed repairs achieved only mild improvement of supraspinatus muscle volume and showed deterioration of fatty infiltration.

Exosomes derived from bone marrow mesenchymal stem cell preconditioned by low-intensity pulsed ultrasound stimulation promote bone–tendon interface fibrocartilage regeneration and ameliorate rotator cuff fatty infiltration

Background: Rotator cuff tendon heals by formation of an interposed zone of fibrovascular scar tissue. Recent studies demonstrate that transforming growth factor–beta 3 (TGF-β3) is associated with tissue regeneration and “scarless” healing, in contrast to scar-mediated healing that occurs with TGF-β1. Hypothesis: Delivery of TGF-β3 in an injectable calcium-phosphate matrix to the healing tendon-bone interface after rotator cuff repair will result in increased attachment strength secondary to improved bone formation and collagen organization and reduced scar formation of the healing enthesis. Study Design: Controlled laboratory study. Methods: Ninety-six male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon followed by acute repair using transosseous suture fixation. Animals were allocated into 1 of 3 groups: (1) repair alone (controls, n = 32), (2) repair augmented by application of an osteoconductive calcium-phosphate (Ca-P) matrix only (n = 32), or (3) repair augmented with Ca-P matrix + TGF-β3 (2.75 µg) at the tendon-bone interface (n = 32). Animals were euthanized at either 2 weeks or 4 weeks postoperatively. Biomechanical testing of the supraspinatus tendon-bone complex was performed at 2 and 4 weeks (n = 8 per group). Microcomputed tomography was utilized to quantitate bone microstructure at the repair site. The healing tendon-bone interface was evaluated with histomorphometry and immunohistochemical localization of collagen types I (COLI) and III (COLIII). Statistical analysis was performed using 2-way analysis of variance with significance set at P < .05. Results: There was significantly greater load to failure of the Ca-P matrix + TGF-β3 group compared with matrix alone or untreated controls at 4 weeks postoperatively (P = .04). At 2 weeks, microcomputed tomography revealed a larger volume of newly formed bone present at the healing enthesis in both experimental groups compared with the control group. By 4 weeks, this newly formed, woven bone had matured into calcified, lamellar bone. Histomorphometric analysis demonstrated significantly greater fibrocartilage and increased collagen organization at the healing tendon-bone insertion site in both experimental groups compared with the control group at 2 weeks (P = .04). Over time, TGF-β3 delivery led to greater COLI expression compared with COLIII at the healing enthesis, indicating a more favorable COLI to COLIII ratio with administration of TGF-β3. Conclusion: Augmentation with an osteoconductive Ca-P matrix at the tendon-bone repair site is associated with new bone formation, increased fibrocartilage, and improved collagen organization at the healing tendon-bone interface in the early postoperative period after rotator cuff repair. The addition of TGF-β3 significantly improved strength of the repair at 4 weeks postoperatively and resulted in a more favorable COLI/COLIII ratio. Clinical Relevance: The delivery of TGF-β3 with an injectable Ca-P matrix at the supraspinatus tendon footprint has promise to improve healing after soft tissue repair.

Background: The muscle quality of the rotator cuff (RC), measured by atrophy and fatty infiltration (FI), is a key determinant of outcomes in RC injury and repair. The ability to regenerate muscle after repair has been shown to be limited. Purpose: To determine if there is a source of resident endogenous stem cells, fibroadipogenic progenitor cells (FAPs), within RC injury patients, and if these cells are capable of adipogenic, fibrogenic, and pro-myogenic differentiation. Study Design: Controlled laboratory study. Methods: A total of 20 patients between the ages of 40 and 75 years with partial- or full-thickness RC tears of the supraspinatus and evidence of atrophy and FI Goutallier grade 1, 2, or 3 were selected from 2 surgeons at an orthopaedic center. During the surgical repair procedure, supraspinatus muscle biopsy specimens were obtained for analysis as were deltoid muscle biopsy specimens to serve as the control. FAPs and satellite cells were quantified using fluorescence-activated cell sorting. Muscle FI and fibrosis was quantified using Oil Red O and Masson trichrome staining. FAP differentiation and gene expression profiles were compared across tear sizes after culture in adipogenic, fibrogenic, and beta-3 agonist (amibegron) conditions. Analysis of variance was used for statistical comparisons between groups, with P < .05 as statistically significant. Results: Histologic analysis confirmed the presence of fat in biopsy specimens from patients with full-thickness tears. There were more FAPs in the full-thickness tear group compared with the partial-thickness tear group (9.43% ± 4.25% vs 3.84% ± 2.54%; P < .01). Full-thickness tears were divided by tear size, with patients with larger tears having significantly more FAPs than those with smaller tears. FAPs from muscles with full-thickness tendon tears had more adipogenic and fibrogenic potential than those with partial tears. Induction of a beige adipose tissue (BAT) phenotype in FAPs was possible, as demonstrated by increased expression of BAT markers and pro-myogenic genes including insulin-like growth factor 1 and follistatin. Conclusion: Endogenous FAPs are present within the RC and likely are the source of FI. These FAPs were increased in muscles with in larger tears but are capable of adopting a pro-myogenic BAT phenotype that could be utilized to improve muscle quality and patient function after RC repair.

Extracellular Vesicles Delivered by Injectable Collagen Promote Bone–Tendon Interface Healing and Prevent Fatty Degeneration of Rotator Cuff Muscle

Background: Magnetic resonance imaging (MRI) is the most commonly used imaging modality to assess the rotator cuff. Currently, there are a limited number of studies assessing the interobserver and intraobserver reliability of MRI after rotator cuff repair. Hypothesis: Fellowship-trained orthopaedic shoulder surgeons will have good inter- and intraobserver agreement with regard to features of the repaired rotator cuff (repair integrity, fat content, muscle volume, number of tendons involved, tear size, and retract) on MRI. Study Design: Cohort study (diagnosis); Level of evidence, 3. Methods: Seven fellowship-trained orthopaedic shoulder surgeons reviewed 31 MRI scans from 31 shoulders from patients who had previous rotator cuff repair. The scans were evaluated for the following characteristics: rotator cuff repair status (full-thickness retear vs intact repair), tear location, tendon thickness, fatty infiltration, atrophy, number of tendons involved in retear, tendon retraction, status of the long head of the biceps tendon, and bone marrow edema in the humeral head. Surgeons were asked to review images at 2 separate time points approximately 9 months apart and complete an evaluation form for each scan at each time point. Multirater kappa (κ) statistics were used to assess inter- and intraobserver reliability. Results: The interobserver agreement was highest (80%, κ = 0.60) for identifying full-thickness retears, tendon retear retraction (64%, κ = 0.45), and cysts in the greater tuberosity (72%, κ = 0.43). All other variables were found to have fair to poor agreement. The worst interobserver agreement was associated with identifying rotator cuff footprint coverage (47%, κ = −0.21) and tendon signal intensity (29%, κ = −0.01). The mean intraobserver reproducibility was also highest (77%-90%, κ = 0.71) for full-thickness retears, quality of the supraspinatus (47%-83%, κ = 0.52), tears of the long head of the biceps tendon (58%-94%, κ = 0.49), presence of bone marrow edema in the humeral head (63%-87%, κ = 0.48), cysts in the greater tuberosity (70%-83%, κ = 0.47), signal in the long head of the biceps tendon (60%-80%, κ = 0.43), and quality of the infraspinatus (37-90%, κ = 0.43). The worst intraobserver reproducibility was found in identification of the location of bone marrow edema (22%-83%, κ = −0.03). Conclusion: The results of this study indicate that there is substantial variability when evaluating MRI scans after rotator cuff repair. Intact rotator cuff repairs or full-thickness retears can be identified with moderate reliability. These findings indicate that additional imaging modalities may be needed for accurate assessment of the repaired rotator cuff.

Background: The current nature of tendon-bone healing after rotator cuff (RC) repair is still the formation of granulation tissue at the tendon-bone interface rather than the formation of fibrocartilage, which is the crucial structure in native tendon insertion and can be observed after knee ligament reconstruction. The interposition of calcium phosphate materials has been found to be able to enhance tendon-bone healing in knee ligament reconstruction. However, whether the interposition of these kinds of materials can enhance tendon-bone healing or even change the current nature of tendon-bone healing after RC repair still needs to be explored. Hypothesis: The interposition of calcium phosphate materials during RC repair would enhance tendon-bone healing or change its current nature of granulation tissue formation into a more favorable process. Study Design: Controlled laboratory study. Methods: A total of 144 male Sprague-Dawley rats underwent unilateral detachment of the supraspinatus tendon, followed by delayed repair after 3 weeks. The animals were allocated into 1 of 3 groups: (1) repair alone, (2) repair with Ca5(PO4)2SiO4 (CPS) bioceramic interposition, or (3) repair with hydroxyapatite (HA) bioceramic interposition at the tendon-bone interface. Animals were sacrificed at 2, 4, or 8 weeks postoperatively, and microcomputed tomography (micro-CT) was used to quantify the new bone formation at the repair site. New fibrocartilage formation and collagen organization at the tendon-bone interface was evaluated by histomorphometric analysis. Biomechanical testing of the supraspinatus tendon-bone complex was performed. Statistical analysis was performed using 1-way analysis of variance. Significance was set at P < .05. Results: The micro-CT analysis demonstrated remarkable osteogenic activity and osteoconductivity to promote new bone formation and ingrowth of CPS and HA bioceramic, with CPS bioceramic showing better results than HA. Histological observations indicated that CPS bioceramic had excellent biocompatibility and biodegradability. At early time points after the RC repair, CPS bioceramic significantly increased the area of fibrocartilage at the tendon-bone interface compared with the control and HA groups. Moreover, CPS and HA bioceramics had significantly improved collagen organization. Biomechanical tests indicated that the CPS and HA groups have greater ultimate load to failure and stiffness than the control group at 4 and 8 weeks, and the CPS specimens exhibited the maximum ultimate load to failure, stiffness, and stress of the healing enthesis. Conclusion: Both CPS and HA bioceramics aid in cell attachment and proliferation and accelerate new bone formation, and CPS bioceramic has a more prominent effect on tendon-to-bone healing. Clinical Relevance: Local application of CPS and HA bioceramic at the tendon-bone interface shows promise in improving healing after rotator cuff tear repair.

Objectives: Recent studies have been using the anterior cruciate ligament reconstruction (ACLR) mouse model to study the tendon-bone healing process and the development of post-traumatic osteoarthritis. While it is known that mechanical load has an important effect on ACL graft healing, a limited number of studies have investigated the impact of treadmill exercise. No study has yet examined the effects of different exercise regimens following an ACLR in mice. The purpose of this study is to explore the effect of different mechanical loading stimuli on the tendon-bone interface (TBI) healing process following ACLR in mice. We hypothesized that moderate loading would positively affect TBI healing after ACLR. Methods: The study protocol was approved by our Institutional Animal Care and Use Committee (IACUC 2020-0024). A total of 58 C57BL/6 (12 weeks old) male mice underwent to an ACL reconstruction in the right knee as previously described. The first 30 mice were assigned into 5 different exercise groups (n=6 per group) and used to evaluate different exercise regimens (Study Arm 1- Fig. 1A). After identifying the optimal exercise regimen, an additional 28 mice were included in the study (Study arm 2 - Fig. 1B). All mice were acclimated on the treadmill following a 5-day acclimation protocol prior to surgery. Starting 7 days post-surgery, the treadmill exercise was performed 5 days/week for a total of 3 weeks. All animals were euthanized at 4 weeks post-surgery. Micro-CT imaging was performed using a Scanco mCT 45 system (Scanco Medical). The bone tunnel was identified, a region of interest (ROI) was defined as the bone tunnel diameter (0.64mm), and bone volume fraction (bone volume/total volume), bone mineral density, and trabecular number were evaluated in the tibial tunnel. Biomechanical Testing was performed in a materials-testing machine (EnduraTEC ELF 3200) (Fig. 2). The knee joints were carefully dissected to isolate the ACL graft. Custom fixtures were designed to ensure that the ACL was aligned in the direction of loading. The specimens were subjected to load until failure at a rate of 5.0 mm/min and the load-to-failure (N) data were recorded. The tendon-to-bone tunnel healing (TBTH) was further evaluated using a well-established histological scoring system [4]. Paraffin-embedded samples were cut into sagittal and axial planes. Hematoxylin and eosin as well as Picrosirius red staining were performed, followed by examination and photography using both brightfield and polarized light microscopy. Statistical analysis was performed by a Wilcoxon signed-rank test in GraphPad Prism 6 software. Statistical significance was defined as a p-value of less than 0.05 (P&lt;0.05). Results: In study arm 1 no significant difference was observed in micro-CT analysis and histological assessment (sagittal slices of the entire tibia) among the groups. Although the sample size was limited, the most relevant trend was observed in the 10m/min, 50min treadmill group compared to the free cage group. Further analysis of micro-CT and biomechanical testing in study arm 2 revealed no significant differences between the groups. However, histological evaluation (axial slices of the femoral/tibial intra-articular/extra-articular parts; Fig. 3) demonstrated significantly higher TBTH scores in the treadmill group compared to the free cage activity group. This difference was most pronounced in the extra-articular region of the femoral tunnel (4.1 vs. 8.4, *P=0.02), as well as in the intra-articular part of the tibial tunnel (6.9 vs. 18.0, **P&lt;0.01). Furthermore, significantly higher TBTH scores were observed in the extra-articular part of the tibia compared to those of the femur, both in the free cage (4.1 vs. 17.3, **P&lt;0.01) and the treadmill group (8.4 vs. 19.3, **P&lt;0.01). Additionally, within the treadmill group, the extra-articular part of the tibia exhibited a significantly higher TBTH score compared to that of the femur (6.6 vs. 18.0, **P&lt;0.01). Conclusions: The most important finding of this study is that TBI healing exhibited better outcomes in the extra-articular part of the femur and the intra-articular part of the tibia among the treadmill group compared to those in the free cage group. These findings suggest that moderate treadmill exercise following ACLR has a positive impact on TBI healing in both the femoral and tibial tunnels, confirming our hypothesis. The lack of a significant difference in the biomechanical test between the groups could potentially be attributed to incomplete healing of the graft in the femoral tunnel across all groups. Failure during biomechanical testing was most often observed on the femoral side. Further studies are warranted to achieve a more robust sample size, investigate alternative loading regimen such as periods of immobilization followed by remobilization, and higher intensity treadmill running (20m/min) to evaluate TBI healing following ACLR. A moderate load rehabilitation protocol might potentially result in improved healing of the tendon graft-to-bone interface following ACLR.