Aerobic Exercise and Celastrol: A Promising Combination for Achilles Tendon Regeneration

Background: An Achilles tendon rupture is a common orthopaedic injury in athletes and other people. Therefore, it is essential to investigate methods that may yield beneficial results for tendon healing by addressing the inflammatory and gene expression aspects of the healing process. Purpose: The objective of this study was to examine the effect of combining aerobic exercise and omega-3 supplementation on the process of Achilles tendon healing in male Wistar rats. Study Design: Controlled laboratory study. Methods: For the purpose of this experiment, 50 male Wistar rats were randomly assigned to 5 groups, with each group containing 10 rats. These groups were identified as a sham group (surgery without tendon damage), a negative control group (tendon damage), and 3 experimental groups: exercise group (tendon damage + aerobic exercise), omega-3 group (tendon damage + omega-3 supplement), and exercise + omega-3 group (tendon damage + aerobic exercise + omega-3 supplement). The Achilles tendon was cut and subsequently repaired through a surgical intervention. Beginning the day after the injury, the groups receiving omega-3 received a dose of 4.6 g/kg EPA [eicosapentaenoic acid] and 3.8 g/kg DHA [docosahexaenoic acid] orally each day, and those in the exercise groups ran on a treadmill 6 days a week. At 48 hours after completing the protocol, both tissue and blood samples were taken and immediately sent for biochemical (TNF-α, IL-1β, MDA, and SOD), gene expression (MMP-3, MMP-9, and SCX), histological (hematoxylin and eosin and Masson trichrome staining), and tensile analyses. Rats were also administered the Achilles functional index at various time points. Results: The histological results revealed a marked improvement in the arrangement of cells and collagen in the extracellular matrix of the exercise + omega-3 group, displaying a notably uniform and condensed alignment of collagen fibers compared with the other groups, which exhibited a lack of organization. Biochemical results showed that TNF-α, IL-1β, and MDA levels were significantly reduced in the exercise + omega-3 group (P < .05). On the other hand, a significant increase in the Achilles functional index score was shown in the exercise + omega-3 group at all time points (P < .05). In the same group, real-time polymerase chain reaction results demonstrated that the expression of MMP-3 and SCX genes increased and that the expression of the MMP-9 gene decreased (P < .05). Conclusion: Aerobic exercise with omega-3 supplementation was more beneficial for healing a surgically cut Achilles tendon in a rat model than aerobic exercise or omega-3 supplementation alone. Clinical Relevance: Our findings suggest that omega-3 supplementation combined with aerobic exercise after an Achilles tendon injury may be able to accelerate the process of Achilles tendon regeneration and healing.

Objective: To investigate the effect of DEAD-box helicase (DDX) 21 on myocardial ischemia-reperfusion (I/R) injury and its potential mechanisms. Methods: In vivo, adult male Bama pigs and C57BL/6J mice were used to establish a myocardial I/R injury model by ligating the left anterior descending coronary artery, with sham-operated groups set as controls. The expression of DDX21 in myocardium after I/R injury was assessed by quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence staining. Following the establishment of the myocardial I/R injury model in mice, AAV9 vectors with cardiac-specific expression were injected in situ into the peri-infarct region (The I/R+DDX21 group, I/R+negative control (NC) group, I/R+sh-NC group and I/R+sh-DDX21 group were injected with AAV9:cTnT-DDX21, AAV9:cTnT-NC, AAV9:cTnT-sh-NC and AAV9:cTnT-sh-DDX21, respectively). Additionally, the I/R+A-485 group received intraperitoneal injections of the cAMP response element-binding protein (CREB) binding protein inhibitor A-485, while the I/R+PBS group was injected with an equivalent volume of phosphate-buffered saline (PBS) as the control. Echocardiography was performed on postoperative days 1 and 28 to evaluate cardiac function (left ventricular ejection fraction and fractional shortening). At 28 days post-surgery, mice were euthanized and heart tissues were harvested for histological sectioning. Myocardial fibrosis was evaluated using Masson's trichrome staining. In vitro, primary cardiomyocytes were isolated from neonatal day 1 C57BL/6J mice using enzymatic digestion method. Cardiomyocytes were transfected with plasmids or small interfering RNA (siRNA). The cardiomyocytes transfected with DDX21-siRNA were assigned to the siDDX21 group, those transfected with the DDX21 plasmid were assigned to the DDX21 group, and those transfected with the corresponding empty plasmid or siRNA were assigned to the NC group. Additionally, cardiomyocytes were treated with A-485 (A-485 group) or PBS (PBS group). An oxygen-glucose deprivation/reoxygenation (OGD/R) model was used to simulate cellular injury. Transcriptome sequencing was performed to identify downstream mechanisms of DDX21. Differential gene expression analysis was conducted using software such as DESeq2, and alternative splicing events in the mRNA transcriptome were analyzed using rMATS software. Mitochondrial superoxide, mitochondrial membrane potential, ATP content, and mitochondrial respiratory chain complex enzyme activity in cardiomyocytes were detected using immunofluorescence staining and commercial assay kits. The oxidative phosphorylation level of the cells was assessed by the Seahorse extracellular flux analyzer. Acetylated DDX21 levels were measured using co-immunoprecipitation and Western blot assays. Results: The expression levels of DDX21 in myocardium from the Bama pigs and mice in the I/R injury model were significantly higher than those in the sham group (all P<0.001). Echocardiographic results showed that at 28 days post-surgery, compared to the I/R+NC group, the I/R+DDX21 group exhibited higher left ventricular ejection fraction and fractional shortening, while the I/R+sh-DDX21 group showed lower values; Masson staining results demonstrated that, compared to the I/R+NC group, the myocardial fibrosis area in the I/R+DDX21 group was significantly reduced, whereas it was significantly increased in the I/R+sh-DDX21 group (all P<0.001). Transcriptomic sequencing results suggested that DDX21 may influence myocardial injury by regulating mitochondrial metabolic activity. In vitro, compared to the OGD/R+NC group, the OGD/R+DDX21 group exhibited lower mitochondrial superoxide levels, higher polymer/monomer ratio, maximal oxygen consumption, reserve capacity, and ATP content. In contrast, the OGD/R+siDDX21 group showed the opposite results, with reduced activity of mitochondrial respiratory chain complex V (all P<0.05). Mechanistically, rMATS software and other analyses indicated that knockdown of DDX21 affected the alternative 3' splicing sites of ATP5J precursor mRNA, inhibiting the splicing of certain exonic sequences. Overexpression of DDX21 upregulated both mRNA and protein levels of ATP5J. Co-immunoprecipitation experiments showed that, compared to the PBS group, acetylated DDX21 levels were reduced in the A-485 group. Further in vivo experiments showed that, compared to the I/R+PBS group, the I/R+A-485 group exhibited higher left ventricular ejection fraction and fractional shortening, and a lower proportion of left ventricular fibrosis (all P<0.001). Conclusions: DDX21 improves cardiomyocyte energy metabolism and alleviates I/R injury by regulating the alternative splicing of ATP5J. A-485 holds potential as a novel small molecule candidate for the treatment of myocardial injury.

Achilles tendon rupture is a common and debilitating injury that significantly impacts mobility and quality of life. Effective treatment options that promote faster and more complete healing are needed. Fibroblast growth factor-2 (FGF-2) has shown potential in enhancing tendon repair. This study aims to investigate the efficacy of FGF-2 in promoting tendon healing in a rat model of Achilles tendon rupture, providing insights into its potential as a therapeutic option. Forty-eight rat hind legs with complete Achilles tendon ruptures were divided into four equal groups: the Sham (S) group (tendon repair only), the Polymer (P) group (tendon repair with scaffold wrapping), the Produced FGF-2 (PF) group (scaffold coated with lab-produced FGF-2), and the Commercial FGF-2 (CF) group (scaffold coated with commercially sourced FGF-2). Histological analyses at two and four weeks post-surgery evaluated healing based on nuclear morphology, vascularity, fibril organization, inflammation, and adipogenesis. At the end of the second week, no macroscopic healing was observed in one rat each from the S and P groups. By the end of the fourth week, macroscopic healing was observed in all groups. The S and P groups exhibited similarly severe fibril disorganization, pathological adipogenesis, and sustained inflammation, particularly at the fourth week. In contrast, the CF group demonstrated improved tendon healing with increased vascularity and extracellular matrix, lower inflammatory cell infiltration, and better fibril organization. Pathological adipogenesis was absent in the CF group, especially at the fourth week. The PF group showed comparable improvements at the second week but experienced a relapse by the 4th week, with increased inflammation and adipogenesis. FGF-2 coated scaffolds significantly enhanced tendon healing in a rat Achilles tendon rupture model by improving fibril organization, increasing vascularity, and reducing inflammation and pathological adipogenesis. These findings suggest that FGF-2 could be a promising therapeutic option for accelerating tendon repair. Future perspectives on tendon repair will focus on enhancing FGF-2 delivery using innovative scaffolds, paving the way for more effective therapies and improved patient outcomes.

Achilles tendon ruptures are recognized as one of the most widespread musculoskeletal injuries. Tendon injuries are a notable clinical challenge, primarily due to the restricted regenerative capacity of the tissue and the associated risks of fibrosis and incomplete functional recovery. Recent studies suggest that cell-free therapies, including stem cell-derived secretomes, may facilitate tendon regeneration. Additionally, mechanical stimulation through exercise can enhance tissue remodeling. This study aimed to investigate the combined effects of tendon-derived stem cell (TDSC) secretome and treadmill-based rehabilitation on Achilles tendon regeneration in a rat model. TDSCs were isolated from rat Achilles tendon and characterized using morphology, cytochemical staining, and flow cytometry. In vitro scratch assays were performed to assess cell migration and wound healing in a laboratory setting. The secretome was collected from fourth-passage TDSCs and incorporated into a collagen-based, injectable hydrogel. A total of 42 adult female Wistar rats were categorized into eight distinct experimental groups, including injury-only, treadmill-only, secretome-only, and treadmill + secretome groups. A surgical procedure was performed to induce a partial rupture of the Achilles tendon, followed by the injection of a secretome-loaded hydrogel at the site of injury. Subsequently, a structured treadmill training program was initiated post-surgery. Regenerative outcomes were evaluated using footprint analysis, histological staining (hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome), and biomechanical testing. In vitro scratch assays demonstrated that TDSCs treated with secretome exhibited enhanced migratory capabilities. Flow cytometry confirmed the identity of these mesenchymal stem cells (MSCs). In vivo studies showed that the combination therapy group (secretome-loaded hydrogel and treadmill training) achieved superior histological recovery. This group exhibited organized collagen bundles, aligned spindle-shaped tenocytes, minimal inflammation, and restored extracellular matrix integrity. PAS staining indicated reduced glycosaminoglycan degradation, while Masson's trichrome staining revealed partial collagen maturation. Additionally, footprint analysis showed improved functional performance, with the combination group achieving significantly higher Achilles functional index scores. Biomechanical testing confirmed enhanced tensile strength and elastic modulus, approaching values comparable to those of intact tendon healing. The synergistic application of TDSC-derived secretome along with treadmill training significantly improved tendon regeneration, matrix remodeling, and functional recovery. This cell-free, bioactive approach offers a promising therapeutic alternative for enhancing tendon recovery.

Connecting Art and Science: How Far Have We Come?

A Possible Anti-Inflammatory Role of Angiotensin II Type 2 Receptor in Immune-Mediated Glomerulonephritis during Type 1 Receptor Blockade

Tendon healing is characterized mostly by slow rehabilitation and, as in tendinopathy, aberrant, protracted sensory nerve ingrowth. This study investigated whether administration of the sensory neuropeptide substance P (SP) could enhance healing and modulate sensory nerve plasticity after Achilles tendon rupture. Fifty-four male Sprague-Dawley rats were allocated to three groups, all receiving six daily injections post-rupture of; (1) SP (10(-6) mol/kg body weight)+endopeptidase inhibitors captopril and thiorphan, (2) captopril/thiorphan only and (3) saline control. At 1, 3 and 6 weeks post-rupture tendon healing was evaluated by assessments of fibroblast proliferation, collagen III-LI (like) occurrence, diameter of newly organized collagen and sensory nerve fiber ingrowth. At 1 week, the SP-treated group exhibited increased occurrence of collagen III-LI (P=0.03) and of organized collagen (P=0.04) compared with control. At 3 weeks, the SP group notably displayed reduced SP-nerve fiber ingrowth (P=0.02), and higher fibroblast density (P=0.004). Both the SP and captopril/thiorphan groups demonstrated increase in collagen fiber organization compared with control (P=0.02 and 0.004, respectively). At 6 weeks, no significant differences were observed between the groups. SP supply in tendon repair promotes early tissue proliferation and regulation of endogenous sensory nerve ingrowth, suggesting implications for novel treatment in tendinopathy.

The effect of manufactured nanoparticles on the expression of proinflammatory cytokine genes was examined. THP-1 cells differentiated into macrophage cells were exposed to TiO2and NiO medium dispersions. After 2, 6, 12, or 24 hours exposure, the expression of IL-1β, IL-6, IL-8, TNF-α and HO-1 genes was determined by real-time PCR. TiO2nanoparticles did not affect cytokine production. In addition, TiO2nanoparticles did not dissolve in the dispersion. On the other hand, NiO nanoparticles enhanced the expression of all the genes tested. NiO dispersions were composed of 58.3 μg/mL of NiO nanoparticles and 45.8 μg/mL of Ni2+. The release of metal ions from the nanoparticles is associated with their cytotoxicity. Therefore, the effect of an NiCl2solution containing 45.8 μg/mL of Ni2+on the expression of cytokine genes was also examined. The effects of NiCl2were similar to those of the NiO nanoparticles. Furthermore, the effect of ZnO, SiO2-coated ZnO, Sb2O3, and Cr2O3nanoparticles on the expression of IL-1β, IL-8 and TNF-α genes was examined. Soluble nanoparticles, such as ZnO, SiO2-coated ZnO, and Cr2O3enhanced the gene expression of cytokines. Sb2O3nanoparticles showed poor solubility and did not affect the expression of cytokine genes. In conclusion, these results suggest that nanoparticle solubility plays an important role in regulating the expression of proinflammatory cytokines.

BackgroundIn rheumatoid arthritis (RA) the combination of glucocorticoids (GC) and the fusion protein CTLA4-Ig (abatacept) allows to obtain better clinical improvement than CTLA4-Ig monotherapy. Our recent data showed that CTLA4-Ig...

The Selective A3AR Antagonist LJ-1888 Ameliorates UUO-Induced Tubulointerstitial Fibrosis

Primary infection with Epstein-Barr virus (EBV) is asymptomatic in children with immature immune systems but may manifest as infectious mononucleosis, a vigorous immune activation, in adolescents or adults with mature immune systems. Infectious mononucleosis and chronic immune activation are linked to increased risk for EBV-associated lymphoma. Here we show that EBV initiates progressive lytic infection by expression of BZLF-1 and the late lytic genes gp85 and gp350/220 in cord blood mononuclear cells (CBMC) but not in peripheral blood mononuclear cells (PBMC) from EBV-naive adults after EBV infection ex vivo. Lower levels of proinflammatory cytokines in CBMC, used to model a state of minimal immune activation and immature immunity, than in PBMC were associated with lytic EBV infection. Triggering the innate immunity specifically via Toll-like receptor-9 of B cells substantially suppressed BZLF-1 mRNA expression in acute EBV infection ex vivo and in anti-IgG-stimulated chronically latently EBV-infected Akata Burkitt lymphoma cells. This was mediated in part by IL-12 and IFN-gamma. These results identify immune activation as critical factor for the suppression of initiation of lytic EBV infection. We hypothesize that immune activation contributes to EBV-associated lymphomagenesis by suppressing lytic EBV and in turn promotes latent EBV with transformation potential.

MP47-09 HKLK1 IMPROVES THE ERECTILE FUNCTION IN AGED RATS BY ENHANCING ENOS AND INOS EXPRESSON AND ANTI-TISSUE FIBROSIS EFFECT

Outcomes after intrasynovial tendon repair are highly variable. An intense inflammatory cascade followed by a delayed healing response can cause adhesion formation and repair-site failure that severely impair the function of repaired digits. No effective remedies exist to fully address these issues. Cell- and growth factor-based therapies have been shown to modulate inflammation and improve cell proliferation and matrix synthesis and therefore are promising treatment approaches for intrasynovial tendon repair. (1) Can autologous adipose-derived mesenchymal stromal cells (ASCs) and recombinant bone morphogenetic protein-12 (rBMP-12) be effectively delivered to an intrasynovial flexor tendon repair without adverse effects? (2) Do autologous ASCs modulate the inflammatory response after intrasynovial tendon injury and repair? (3) Does the combined application of autologous ASCs and rBMP-12 modulate the proliferative and remodeling responses after intrasynovial tendon injury and repair? Sixteen 1- to 2-year-old female canines were used in this study. Autologous ASC sheets, with and without rBMP-12, were applied to the surface of sutured flexor tendons. Fourteen days after repair, the effects of treatment were determined using quantitative PCR (six per group) for the expression of genes related to macrophage phenotype or inflammation (IL-4, CD163, VEGF, NOS2, IL-1B, and IFNG), cell proliferation (CCND1), and tendon formation (SCX, TNMD, COL1A1 and COL3A1). Proteomics analysis (four per group) was performed to examine changes in tendon protein abundances. CD146 immunostaining and hematoxylin and eosin staining (four per group) were used to detect tendon stem or progenitor cells and to semiquantitatively evaluate cellularity at the tendon repair; analyses were done blinded to group. Gross inspection and cell tracing showed that autologous ASCs and rBMP-12 were delivered to the flexor tendon repair site without the deleterious effects of adhesion and repair-site gap formation. Quantitative assessment of gene and protein expression showed effects of treatment: ASC-sheet treatment modulated the postrepair inflammatory response and facilitated healing by increasing regenerative M2 macrophages (M2 marker CD204, twofold of normal, p = 0.030), inflammatory inhibitor (prostaglandin reductase 1 [PTRG1], 1.6-fold of normal, p = 0.026), and proteins involved in tendon formation (periostin [POSTN], 1.9-fold of normal, p = 0.035). Consistently, semiquantitative and qualitative evaluations of repaired tissue showed that ASC-sheet treatment reduced mononuclear cell infiltration (12% less than nontreated tendons, p = 0.021) and introduced CD146+ stem or progenitor cells to the repair site. The combined administration of ASCs and rBMP-12 further stimulated M2 macrophages by increasing IL-4 (116-fold of normal, p = 0.002) and led to the increase of M2 effector matrix metalloproteinase-12 involved in matrix remodeling (twofold of normal, p = 0.016) and reduction of a negative regulator of angiogenesis and cell migration (StAR-related lipid transfer domain protein13 [STARD13]; 84% of normal, p = 0.000), thus facilitating the proliferative stage of tendon repair. ASCs and BMP-12 accelerated the progression of healing in the proliferative stage of tendon repair. The effects of ASCs and BMP-12 on tendon functional recovery should be evaluated in future studies. The cell sheet approach is an effective, biocompatible, and surgeon-friendly approach for cell and growth factor delivery during tendon repair. Combined application of ASCs and BMP-12 may accelerate intrasynovial tendon healing while suppressing the adverse inflammatory response.

Objective: To clarify the effect and the mechanism of G protein-coupled receptor class C group 5 member A (GPRC5A) on lipopolysaccharide (LPS)-induced inflammatory response in human gingival fibroblasts (GFs), thus to provide a foundation for delving into the role of G protein coupled receptor (GPCR) in periodontitis. Methods: Gingival tissue samples were collected from 3 individuals periodontally healthy (health group) and 3 patients with periodontitis (periodontitis group) in Shandong Stomatological Hospital from December 2022 to February 2023. The expressions of GPRC5A of the two groups were detected by immunohistochemistry staining. GFs used in this study were isolated from a portion of gingiva for the extraction of impacted teeth in School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University from December 2022 to February 2023. GFs were isolated with enzymic digestion and transfected with 30, 50 and 80 μmol/L small interfering RNA-GPRC5A (siGPRC5A) or small interfering RNA-negative control (siNC), regarded as the experimental group and the negative control one, respectively. The silencing efficiency of siGPRC5A was evaluated by real-time fluorescence quantitative PCR (RT-qPCR). Experiments were then conducted using these cells which were divided into four groups of negative control (NC), LPS, siGPRC5A+LPS and siGPRC5A. The mRNA and protein levels of GPRC5A in GFs under 1 mg/L LPS-induced GFs inflammatory state were evaluated by RT-qPCR and Western blotting analysis after GPRC5A knockdown. RT-qPCR was used to detect the gene expression levels of the inflammatory cytokines in GFs induced by LPS, namely, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, prostaglandin endoperoxide synthase 2 (PTGS2) after GPRC5A knockdown. Western blotting analysis and immunofluorescence staining were used to further investigate the activation of nuclear factor-kappa B (NF-κB) signaling pathway. Results: Immunohistochemistry staining showed that the expression of GPRC5A in gingival tissues of periodontitis group (0.132±0.006) increased compared with that in periodontally healthy group (0.036±0.019) (t=8.24, P=0.001). Meanwhile, RT-qPCR results showed that the gene expression levels of GPRC5A at different time point (2, 6, 12, 24 h) in LPS-induced GFs (0.026±0.002, 0.042±0.005, 0.004±0.000, 0.016±0.000) were upregulated compared with those in the NC group (0.004±0.000, 0.004±0.000, 0.002±0.000, 0.007±0.000) (all P<0.001), respectively, and peaked at 6 h. The 50 μmol/L group displayed the most significant decrease in siGPRC5A expression (31.16±3.29) compared with that of the siNC group (100.00±4.88) (F=297.98, P<0.001). The results of RT-qPCR and Western blotting analysis showed that siGPRC5A (0.27±0.03, 0.71±0.00) suppressed the expressions of GPRC5A at both gene and protein levels, while LPS (1.30±0.10, 1.43±0.03) was able to promote the expressions of GPRC5A compared with those of the NC group (1.00±0.01, 1.00±0.00)(all P<0.001). The siGPRC5A+LPS group (0.39±0.03, 1.06±0.16) also inhibited the increase of GPRC5A at both gene and protein levels induced by LPS (1.30±0.10, 1.43±0.03) (F=208.38, P<0.001; F=42.04, P<0.001). RT-qPCR results showed that the expressions of IL-8, IL-1β, IL-6, TNF-α, and PTGS2 at the gene level in LPS group were highly increased compared with those in the NC group (all P<0.001). siGPRC5A significantly suppressed LPS-induced expressions of these inflammatory cytokines in GFs (all P<0.001). Western blotting analysis showed that the levels of p65 and IκBα protein phosphorylation in the LPS group were highly increased compared with those in the NC group, and siGPRC5A could effectively suppressed LPS-induced protein phosphorylation (all P<0.01). Furthermore, immunofluorescence staining showed that NF-κB p65 in the control group was mainly concentrated in the cytoplasm, and partially translocated to the nucleus under the stimulation of LPS. siGPRC5A was able to inhibit LPS-induced intranuclear translocation of p65 to a certain extent. Conclusions: GPRC5A expression was upregulated in periodontitis, and GPRC5A knockdown inhibited LPS-induced inflammation. Moreover, GPRC5A played a role in inflammation regulation by interacting with NF-κB signaling pathway.

Category:Basic Sciences/Biologics; SportsIntroduction/Purpose:Achilles tendon ruptures cause long-term functional deficits in nearly 2 out of 3 patients. Our group has established a complex muscle-tendon pathology following isolated Achilles tendon ruptures. Our long-term goal is to determine how rehabilitation loads can simultaneously accomplish 2 competing objectives: (1) prevent muscle remodeling while (2) not overloading the healing tendon that causes permanent tendon elongation - unloading the muscle and stimulating muscle remodeling. To do this, we developed a novel experimental framework to test the potential therapeutic effectiveness of different loading paradigms on the healing muscle-tendon following surgically induced Achilles tendon ruptures in an established rat model. We hypothesized that overloading the healing tendon would lead to worse functional outcomes compared to unloaded immobilization.Methods:Achilles tendon rupture, repair, and immobilization. Male Sprague-Dawley rats underwent a surgically induced Achilles tendon rupture by blunt dissection followed by a modified Kessler repair in this IACUC approved study. The repaired limb was immobilized in either unloaded plantar flexion (n=7) to simulate the clinical standard of rehabilitative care or loaded dorsiflexion (n=5) to simulate clinically damaging rehabilitation. Structural analyses. Following 2 weeks of immobilization and 2 weeks of cage recovery, we sacrificed our study animals and measured tendon elongation and serial sarcomere count. Plantar flexor functional assessment. We developed an ankle dynamometer to quantify plantar flexor function that mirrored our clinical assessments in patients. To demonstrate the clinical- relevance of this device, we tested 1 animal that was immobilized in unloaded plantar flexion for 1 week and 1 animal that was immobilized in loaded dorsiflexion for 1 week - both then completed 2 weeks of unrestricted cage activity.Results:Muscle-tendon structural changes. Achilles tendon ruptures caused the tendon to heal in an elongated position with fewer sarcomeres in series (Fig. A). Tendon length increased by 73% following 2-weeks of loaded immobilization in dorsiflexion and 23% in unloaded immobilization in plantar flexion (p<.001). We measured sarcomere length in a subset of these animals but found similar trends in serial sarcomere reductions, where unloaded immobilization in plantar flexion caused 14% reduced serial sarcomeres (p<.01, n=4) and loaded immobilization in dorsiflexion caused 32% reduced serial sarcomeres (n=2). Plantar flexor function. Plantar flexor work was adversely impacted by the Achilles tendon ruptures (Fig. B). We found that unloaded immobilization in plantar flexion caused a 13% decrease in plantar flexor work with the detectable deficits occurring in ankle plantar flexion. Loaded immobilization in dorsiflexion caused greater plantar flexor work deficits (28%) with greater torque deficits in dorsiflexion and continuing through plantar flexion angles.Conclusion:Our exciting experimental findings are the first of their kind to directly link muscle-tendon structural deficits with plantar flexor functional deficits in an animal model of Achilles tendon rupture using a clinically relevant ankle dynamometer. We established preliminary evidence that supports our overarching hypothesis that joint immobilization, whether unloaded or loaded, causes detrimental muscle-tendon structural changes that impose functional constraints on plantar flexor work. Our preliminary findings suggest that rehabilitation loading is critical to structural and functional outcomes following surgically repairing the Achilles tendon. Too little loading promotes muscle remodeling while too much loading elongates the healing tendon.