Ageing-Related Structural and Cellular Alterations in the Mouse Muscle-Tendon Junction

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Thorpe, Nodoka Iwasaki This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8305364/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The muscle tendon junction (MTJ) is a specialised interface between muscle and tendon and transmits muscle-generated force to the tendon. The MTJ is particularly vulnerable to injuries compared to muscle and tendon and becomes more injury prone with age. Further, current treatments for MTJ injuries are insufficient as indicated by scar tissue formation and a high re-injury rate. Despite its clinical importance, the mechanisms driving MTJ ageing and age-related functional deterioration remain poorly understood. In this study, the first comprehensive three-dimensional characterisation of age-related structural and cellular changes at the mouse MTJ was performed using the high-resolution imaging techniques, micro-computed tomography (µCT) and confocal microscopy. µCT analysis revealed a 27% reduction in muscle fibre diameter with age, accompanied by a trend toward increased MTJ surface area and a 19% reduction in pennation angle, indicating diminished force generation capacity. Confocal imaging showed a 49% reduction in endothelial cell volume (VWF-labelled) in the old mouse muscle-tendon unit, suggesting a loss of vascularity. In situ hybridisation and immunofluorescence demonstrated increased expression of senescence markers p16 and p21 in endothelial and MTJ-specific cells, with MTJ-specific cells showing the greatest accumulation of p16 and p21 (270% and 310% increases, respectively) with age. These findings suggest that vascular and MTJ-specific cells are particularly susceptible to ageing and may collectively contribute to the age-related functional decline of the MTJ. Understanding these mechanisms may help to develop targeted therapeutic strategies to preserve or restore MTJ integrity and function in ageing populations. Muscle tendon junction ageing µCT immunolabelling endothelial cells senescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The muscle tendon junction (MTJ), also known as the myotendinous junction, is a specialised interface between muscle and tendon, and transmits the force generated by the muscle to its connecting tendon [ 1 ]. The MTJ is commonly associated with muscle strains and tears [ 2 ] and is particularly vulnerable to tensile failures compared to the neighbouring muscle and tendon [ 3 ], [ 4 ]. Injuries and failure at the MTJ increase the morbidity of patients and affect their quality of life [ 5 ], [ 6 ]. MTJ injuries are common, with 28% of injuries in the muscle-tendon-bone unit and 52% of acute hamstring injuries occurring at the MTJ [ 7 ], [ 8 ], [ 9 ]. In the non-athletic population, the average age for injuries involving the gastrocnemius-Achilles aponeurosis and myoaponeurotic junction is 48.7 ± 8.1 years [ 10 ], which is notably older than the average age reported for tendon injuries (approximately 40 years) [ 11 ], [ 12 ], suggesting that the MTJ becomes more vulnerable to injury with age. Moreover, 2D structural studies in ageing rodents have revealed that the length of the MTJ region approximately doubles with age [ 13 ], indicating that the MTJ undergoes degenerative changes with age and may contribute to an increased risk of injury. However, the specific effects of ageing on MTJ structure and function remain poorly understood [ 14 ]. This gap in knowledge limits our understanding of the mechanisms underlying age-related MTJ deterioration and hinders advances in the discovery of targeted therapeutic strategies. MTJ injuries are often treated with physical therapy, however, this conservative treatment often fails to achieve reduced recovery times without increasing the risk of reinjury and cannot be used to treat a complete MTJ tear [ 15 ]. Another common treatment is surgical suturing. This, however, fails to yield satisfactory long-term outcomes due to adhesions and reduced mechanical properties, which further increase the risk of re-rupture [ 16 ]. Traditionally, the autograft approach is the gold standard for tendon injuries, including MTJ, yet this is generally limited and can lead to higher donor site morbidity and infection risk compared to other grafts such as allografts [ 17 ]. Therefore, there is a need for more effective therapies to overcome the limitations of the current treatments. The extracellular matrix (ECM) of the MTJ contains MTJ-specific adhesion proteins, including collagen type XXII (Col22), paxillin, and talin [ 18 ], [ 19 ]. These proteins are critical for efficient force transmission between muscle and tendon and play key roles in maintaining MTJ structural and functional integrity [ 20 ]. Col22 is a well-characterised MTJ-specific protein, which is expressed by both muscle cells and tenocytes at the MTJ [ 21 ], [ 22 ], [ 23 ], and has been reported to maintain vascular integrity by regulating vascular permeability [ 24 ]. Ageing significantly affects the vasculature in the musculoskeletal system, and both aged muscle and tendon have been reported to exhibit reduced vascularisation and blood flow [ 25 ], [ 26 ], [ 27 ], [ 28 ], [ 29 ], [ 30 ], [ 31 ], which may contribute to the higher incidence of injury and impaired regenerative capacity observed with age. However, the effect of ageing on the MTJ vasculature has not been reported to date. Cellular senescence, a state of permanent cell cycle arrest triggered by various stressors, plays a critical role in the ageing process, with the proportion of senescent cells increasing across multiple tissues with age [ 32 ]. Cellular senescence has been reported to affect muscle and tendon health. For example, cells positive for the senescence marker p16 have been shown to contribute to the development of age-associated pathologies in mice, particularly within skeletal muscle [ 33 ], and inhibit tenogenic differentiation of tendon stem/progenitor cells [ 34 ]. Similarly, overexpression of p21, another key marker of senescence, has been shown to drive cellular senescence in skeletal muscle and is associated with muscle loss and functional decline [ 35 ]. While these findings indicate that MTJ-localised cells are likely to suffer from senescence, the precise effects of senescence on the ageing MTJ remain to be elucidated. The aim of this study was therefore to investigate age-related alterations in MTJ structure and vasculature using high resolution 3D imaging techniques, including micro-computed tomography (µCT) and confocal microscopy. In addition, cellular senescence in MTJ-specific cells (Col22-positive cells) and vascular endothelial cells was examined using in situ hybridisation and immunofluorescence. Understanding these structural and cellular alterations with age may provide insight into the mechanisms underlying MTJ degeneration and support the development of targeted therapeutic strategies to enhance MTJ repair and regeneration. Materials and methods 2.1. Sample acquisition Mouse hindlimbs were obtained as residual tissues from animals euthanised as part of an unrelated study (a kind gift from Dr. Linterman, Babraham Institute). In this unrelated study, male and female young (3-month-old, the life phase equivalent for humans ranges from 20–30 years) and old (23-month-old, the life phase equivalent for humans ranges from 56–69 years of age) C57BL/6 mice (n = 6 per age group) were immunised by intramuscular injection of 50 µg/mL LNP-mRNA vaccine into the right biceps femoris muscle; the mRNA encoded the spike protein of the ancestral SARS-CoV-2 strain. Ten days post-injection, mice were euthanised and tissues were collected. All procedures were approved by the Babraham Institute Animal Welfare and Ethical Review Body and conducted in accordance with European Union and UK Home Office regulations (Home Office Licence P4D4AF812). Achilles tendon and gastrocnemius muscle junctions (Achilles MTJs) were subsequently harvested from both hindlimbs of the immunised mice 3 hours after euthanasia at the Royal Veterinary College. The experiments were approved by the Royal Veterinary College Clinical Research Ethical Review Board (URN 2024-2336-A). 2.2. µCT imaging Phosphotungstic acid (PTA) was used to enhance the contrast of the MTJ, adapting a protocol from a previous study [ 3 ]. Young and old Achilles MTJs were immersed in an increasing ethanol concentration of 25, 50, and 70 % thanol for 90 min each followed by 1 % TA (79690, Sigma-Aldrich, Burlington, MA, USA) in 70 % thanol for 72 h (n = 4 per age group). Samples were then washed twice and immersed in Tris-buffered saline (TBS) for 30 mins prior to imaging. Samples were wrapped in clingfilm to avoid dehydration during imaging. A Skyscan 1172F (version 1.5, Skyscan, Kontich, Belgium) was used with an X-ray source at 50 kV tube voltage and 200 µA tube current with 2500 ms exposure time. The voxel size was 2 µm, and 180° scans were performed with 0.5 mm Aluminium filter, frame averaging at 2, and with a rotation step at 0.25°. Slice reconstruction was performed using NRecon (version 1.7.1.0). The reconstructed images were segmented to remove tendons and analysed using CTAn (version 1.17.7.1) to measure mean muscle fibre diameter. CTVox (version 3.3.0) was used to visualise the 3D reconstructed images. The images were also analysed using Avizo (Avizo 2021.1, ThermoFisher Scientific, MA, USA), and the images were cropped at 2000 x 2000 x 1000 voxels (32 mm 3 ) with the MTJ in the region of interest. Pennation angle was measured manually at each muscle–tendon sub-unit interface in Avizo and a mean value was calculated for each sample (Figure S1a). Tendon sub-unit diameter was also measured manually in Avizo. The images were then segmented to separate muscle and tendon, and the MTJ surface area was measured using the volume fraction function in Avizo and normalised to the whole tissue surface area (Figure S1b). 2.3. 3D immunolabelling MTJs from young and old mice were fixed in 4% paraformaldehyde (PFA) for 4 h for 3D immunolabelling (n = 4 per age group), using a protocol adapted from a previous study [ 36 ]. Permeabilization was performed using 50 % (/v) methanol:TBS, 80 % (/v) methanol:dH2O, and 100 % mthanol for 2 h, and 20 % (/v) dimethylsulphoxide (DMSO):methanol, 80 % (/v) methanol:dH2O, 50 % (/v) methanol:TBS for 30 min at 4°C, respectively, with gentle shaking. The samples were stored in TBS overnight at 4°C. Blocking was performed using blocking solution (0.2 % Titon X-100, 6 % dnkey serum, 6 % gat serum, 10 % DSO in TBS) for 72 h at 37°C with gentle shaking. The samples were then incubated with primary antibodies diluted in blocking solution for 72 h at 37°C with gentle shaking. The details of primary antibodies were as follows: Rabbit anti-von Willebrand factor (VWF, endothelial cell marker, 1:800, A0082, Dako, Ejby, Denmark) and rat anti-laminin alpha 2 (LAMA2, skeletal muscle marker, 1:1000, ab11576, Abcam, Cambridge, UK). The samples were then washed 5 times with 0.2 % Teen-20 in TBS for 1 h each at room temperature. The samples were incubated with secondary antibody diluted in blocking solution. The details of the secondary antibodies were as follows: Goat anti-rabbit IgG AF594 (1:800, A-11012, ThermoFisher Scientific, MA, USA) and Goat anti-rat IgG AF488 (1:800, ab150157, Abcam, Cambridge, UK) for 24 h at 37°C with gentle shaking, followed by 5 washes with 0.2 % Teen-20 in TBS for 1 h at room temperature. Samples were then incubated in DAPI solution (5 µg/mL in TBS) at 4°C overnight. Samples were dehydrated as described above with increasing concentrations of methanol. Two-step tissue clarification was performed by immersing samples in Visikol HISTO-1 (H1-30, Sigma-Aldrich, MA, USA) for 24 h, followed by immersion in HISTO-2 (H2-30, Sigma-Aldrich, MA, USA) for at least 48 h at room temperature with gentle shaking. 2.4. Confocal imaging The 3D immunolabelled samples were placed in a glass-bottom dish fitted with a polystyrene frame (220.220.042, IBL Baustoff + Labor GmbH, Austria) and a drop of Histo-2 was added to keep the sample hydrated. The samples were then imaged using a Leica TCS SP8 laser scanning confocal microscope (Leica Biosystems, Nussloch, Germany) with 10x objective, 512x512 pixel resolution with 2.27 µm pixel size and 2.27 µm z axis steps. The pinhole size was set to 1 Airy unit, frame average was set to 1, and line average was set to 2 using lasers emitting light at 405 nm (blue channel), 488 nm (green channel), and 561 nm (red channel). The images were visualised using Leica LAS X software (version 3.5.5) within the 3D module and reconstructed and analysed using Avizo. The reconstructed volume of immunolabeled vasculature was measured using the volume fraction function in Avizo. 2.5. In situ hybridisation In situ hybridisation was performed using OCT-embedded MTJ sections (n = 4 per age group), RNAscope Multiplex Fluorescent Reagent Kit v2 (323110, Bio-Techne Ltd, MN, USA) following the manufacturer’s protocol for fresh frozen tissues. The sections were fixed with 4% PFA for 2 h at 4°C and then dehydrated using an increasing ethanol concentration of 50, 70, and 100% ethanol for 5 min each at room temperature, followed by -20°C overnight incubation in 100% ethanol. The samples were dried for 5 minutes and encircled with a hydrophobic barrier pen, then incubated with hydrogen peroxide for 10 minutes and rinsed in distilled water. Custom pretreatment (300040, Bio-Techne Ltd, MN, USA) was added for 30 mins at 40°C, followed by two washes with distilled water. Probe solution was applied to the tissue sections for 2 h at 40°C. The details of the probes were as follows: Col22A1 (590911, C1, Bio-Techne Ltd, MN, USA), VWF (499111-C3, Bio-Techne Ltd, MN, USA), p16 (411011-C2, Bio-Techne Ltd, MN, USA) and p21 (408551-C2, Bio-Techne Ltd, MN, USA). The samples were stored overnight at room temperature in 5× saline-sodium citrate buffer (0.75 M sodium chloride, 75 mM trisodium citrate, pH 7.0). On the following day, signal amplification was performed according to the manufacturer’s instructions. All incubations were conducted at 40°C, followed by two 2-minute washes with RNAscope wash buffer. Amplifier incubations were carried out for 30 minutes for the first two amplifiers and 15 minutes for the third. Subsequently, slides were incubated with horseradish peroxidase (HRP) for 15 minutes, followed by a 30-minute incubation with a tyramide dye fluorophore (OPAL 520, FP1487001KT, Akoya Biosciences, MA, USA) diluted 1:1500 in RNAscope TSA dilution buffer, and a 30-minute incubation with HRP blocker. The HRP, fluorophore, and blocking steps were repeated using second and third tyramide dye fluorophores (OPAL 570 and OPAL 650, FP1488001KT and FP1496001KT, Akoya Biosciences, MA, USA). The sections were then incubated with DAPI for 30 seconds at room temperature, followed by mounting with ProLong™ Gold Antifade Mountant (P10144, ThermoFisher Scientific, MA, USA) and allowed to cure for 2–3 h before imaging using an Eclipse Ni-E upright microscope (Nikon Instruments Inc., Tokyo, Japan). Four images were obtained per sample, and they were analysed using ImageJ (National Instruments, Austin, USA) by manually counting cells expressing the positive signals. The percentages of p16 and p21 positive cells within Col22-positive MTJ-specific cell and VWF-positive endothelial cell populations were quantified and compared with their prevalence among all cells within the field of view. 2.6. 2D immunolabelling Mouse MTJs were snap frozen in hexane cooled on dry ice (n = 4 per age group), and then embedded using OCT (15212776, ThermoFisher Scientific, MA, USA). Longitudinal MTJ cryosections (10 µm thickness) were fixed in ice-cold methanol/acetone solution (1:1) for immunolabelling. Non-specific binding of antibodies was blocked by incubating samples with 5% goat serum (ab7481, Abcam, Cambridge, UK) in TBS for 45 mins at room temperature. The samples were incubated with primary antibodies in 5% goat serum for 2 h at room temperature. The details of primary antibodies were as follows: Guinea-pig anti‐collagen XXII (1:100, a kind gift from Manuel Koch, University of Cologne, Germany) and rabbit anti-p16 (1:50, 80772S, Cell Signaling Technology, MA, USA). After washing twice with TBS, the sections were incubated with secondary antibodies (A-11012 and SA5-10096, ThermoFisher Scientific) in 5% goat serum (1:400) for 1 h at room temperature. The sections were then incubated with DAPI (0.1 µg/mL) for 10 min at room temperature, followed by two washes in TBS. The sections were mounted with ProLong™ Gold Antifade Mountant (P10144, ThermoFisher Scientific, MA, USA) and allowed to cure for 2–3 h before imaging using an Eclipse Ni-E upright microscope (Nikon Instruments Inc., Tokyo, Japan). Four images were obtained per sample, and they were analysed using ImageJ by manually counting positively labelled cells. 2.7. Statistical analysis All data are expressed as the mean ± standard deviation (SD), and all experiments were conducted using 4 different animals from each age group. A D’Agostino and Pearson test was used to determine if the data followed a normal distribution. The Mann-Whitney test or two-way ANOVA was performed to calculate the differences (p < 0.05) between different sample groups using GraphPad Prism version 10.2.3 (La Jolla, CA, USA). Results 3.1. µCT analysis demonstrated age-related structural changes in mouse MTJs Quantitative analysis revealed a 27% reduction in muscle fibre diameter with age in the MTJ region (Fig. 1 b), whereas tendon sub-unit size was not significantly affected (Fig. 1 c). The MTJ surface area, normalised to total surface area, showed a trend towards an age-related increase (p = 0.0571; Fig. 1 d), suggesting enlargement of the MTJ with ageing. The pennation angle, an indicator of muscle force generation capacity, was significantly reduced by 19% in old mouse MTJs compared with those of young mice (Fig. 1 e). The muscle and tendon volume at the MTJ, normalised to the whole MTJ volume, showed no significant difference with age (Figure S2). 3.2. Three-dimensional immunolabelling demonstrated a significant reduction in VWF labelled endothelial cell volume with age. Whole-tissue immunolabelling was performed to visualise endothelial and muscle cell populations within the muscle-tendon unit including the MTJ. VWF was used to label endothelial cells and LAMA2 was used to label muscle cells, and DAPI staining was applied to identify the overall tissue volume. The images showed that the vasculature exists not only in the muscle, as indicated by LAMA2 labelling, but also within the tendon and across their interfaces, demonstrating the presence of vasculature throughout the MTJ region (Fig. 2 a). Quantitative analysis of VWF- and LAMA2-labelled volumes revealed a significant reduction of 49% in VWF-labelled volume in old mice (Fig. 2 b), whereas LAMA2-labelled volume showed no significant change with age (Fig. 2 c). This finding indicated an age-associated decline in vascularity within the muscle-tendon unit, without a corresponding reduction in muscle volume. 3.3 Collagen type 22 positive MTJ-specific cells and VWF-positive endothelial cells exhibit higher expression of senescence markers at the MTJ region In situ hybridisation, a technique used to visualise RNA expression within tissue sections, was employed to detect senescence markers at the MTJ. In old MTJs, senescence markers p16 and p21 were predominantly co-localised with the MTJ marker Col22 and endothelial cell marker VWF (Fig. 3 a and b). Col22-positive MTJ-specific cells exhibited significantly higher percentages of p16- and p21-positive cells with age (270% and 310% increases, respectively), while VWF-positive endothelial cells showed a 780% increase in p16-positive cells with age (Fig. 3 c and d). While p16 expression was significantly higher in Col22-positive MTJ-specific cells (52%) and VWF-positive endothelial cells (19%) compared with all cells in the field of view (3.2%) in the old MTJ, p16 expression was also significantly higher in Col22-positive MTJ-specific cells (19%) than VWF-positive endothelial cells (2.5%) and all cells (1.6%) in the young MTJ. Similarly, p21 expression was significantly higher in Col22-positive MTJ-specific cells (45%) than in all cells, whereas no significant difference was observed between VWF-positive endothelial cells and the overall cell population. However, in young MTJ, there was no significant difference in p21 expression among different cell types. Expression of Col22 and p16 was assessed at the protein level using 2D immunofluorescent labelling. In old MTJ, expression of p16 was predominantly co-localised with Col22 expression (Fig. 4 a), which was not observed in young MTJ. Quantitative analysis revealed a 270% increase in the proportion of p16-positive cells within the Col22-positive MTJ-specific cell population with age (Fig. 4 b), consistent with the increase observed in the RNA expression analysis shown in Fig. 3 . Discussion This study provides the first comprehensive investigation of structural and cellular alterations with age in the mouse MTJ, demonstrating that age-related structural changes occur within the MTJ, which are accompanied by reduced vascularity and increased cell senescence in this region. One of the limitations of this study was using immunised mice against the ancestral SARS-CoV-2 strain. However, both young and old mice were immunised simultaneously and underwent identical treatments after the immunisation. Although some individuals may experience musculoskeletal pain as a result of SARS-CoV-2 infection [ 37 ], it has been reported that SARS-CoV-2 infection has no association with musculoskeletal function in humans [ 38 ]. In addition, the vaccine was administered into the right biceps femoris muscle, and the tissues obtained in this study were anatomically distinct from the injection site. Therefore, it is unlikely that the immunisation affected the MTJ structure, vasculature, or cellular senescence. Age-related structural changes in the MTJ have traditionally been investigated using 2D imaging approaches. In this study, µCT imaging was employed to explore structural changes in the MTJ with age in 3D, demonstrating a significant decrease in muscle fibre diameter. Similar findings have been reported by several previous studies using 2D imaging, with the reduction in size depending on the types of muscle fibres; type II muscle fibre size decreases more than type I muscle fibres with age [ 39 ], [ 40 ], [ 41 ], [ 42 ], [ 43 ], [ 44 ], [ 45 ]. In the current study, the mean muscle fibre diameter reduction with age was 27 %, which is similar to the mean muscle ibre reduction reported previously in human quadriceps muscle (~ 20 %) [ 45 ]. In addition, pennation angle, whic is the angle between the muscle fibres and tendon long axis, significantly decreased with age. A larger pennation angle results in increased force generation capacity in muscle [ 46 ], [ 47 ] and therefore the decrease in pennation angle, combined with the reduction in muscle fibre diameter, strongly indicates a reduction in muscle force generation capacity in the MTJ region [ 46 ], [ 47 ], [ 48 ] and aligns with a previous report of age-related decreases in muscle force production observed in skeletal muscle [ 49 ]. Further analysis of µCT images showed a trend towards an age-related increase in MTJ surface area, which has also been reported in 2D image analysis of mouse soleus MTJ [ 13 ]. The structural alterations observed with age in this study indicate that mouse Achilles MTJ undergoes functional deterioration and loss of force generation capacity with age. However, age-related alterations in MTJ mechanical properties and force generating capacity were not directly measured in the current study and therefore remain an important area for future investigation. Three-dimensional visualisation of muscle-tendon unit vascularity was achieved by confocal imaging of whole-tissue immunolabelled for VWF to target endothelial cell populations, and LAMA2 labelling for muscle cell populations. Imaging revealed the presence of vasculature within both muscle and tendon tissues, as well as across their interface, the MTJ. Quantitative analysis showed a significant reduction in VWF-labelled endothelial cell volume normalised by the whole tissue volume, but no significant difference in LAMA2-labelled muscle volume with age, indicating an age-associated decline in vascularisation at the muscle-tendon unit without a corresponding reduction in muscle volume, which supports the findings from the µCT image analysis. Together with previous reports of vascular decline in aged muscle and tendon [ 25 ], [ 27 ], [ 29 ], these findings indicated that reduced vascularisation is a shared feature of musculoskeletal ageing across tissues, which may contribute to functional deterioration at the MTJ through limited delivery of oxygen, amino acids, nutrients and hormones [ 50 ]. In situ hybridisation was employed to investigate the expression of senescence markers, p16 and p21, in different cell populations in the MTJ. Both markers were predominantly localised to Col22-positive MTJ-specific cells and, to a lesser extent, VWF-positive endothelial cells. These results suggest that these cell types are particularly susceptible to senescence with age compared to other cell populations in the MTJ, especially Col22-positive MTJ-specific cells. Additionally, in Col22-positive MTJ-specific cells, elevated expression of p16 was observed in young MTJ compared to the other cell types, suggesting that Col22-positive MTJ-specific cells may experience senescence in early age. The age-related accumulation of p16 in Col22-positive MTJ-specific cells was further validated at the protein level using immunolabelling. The marked increases in p16 and p21 expression within Col22-positive MTJ-specific cells indicated that MTJ-specific cells are prone to senescence, which may impair junctional integrity and contribute to age-related disorders and functional decline of the MTJ. Supporting this notion, the development of age-related pathologies and tissue dysfunction induced by upregulation of p16 and p21 has previously been observed in muscle [ 33 ], [ 35 ]. Similarly, the elevated p16 expression observed in VWF-positive endothelial cells suggests induction of vascular cell senescence, which may result in vascular dysfunction, disrupted vascular ECM formation and fibrosis [ 51 ], [ 52 ], further exacerbating MTJ degeneration. While senescence in Col22-positive MTJ-specific cells and VWF-positive endothelial cells was demonstrated in this study, and Col22 plays an important role in vascular integrity [ 24 ], it remains unclear whether these two cell types influence each other during ageing. Future studies should investigate the relationship between MTJ-specific cells and vascular endothelial cells in the context of ageing, potentially using in vitro MTJ co-culture models. Despite the high incidence of MTJ injuries, current treatments remain insufficient [ 15 ], [ 16 ]. MTJ regeneration, and the effects of ageing, are poorly understood at cellular and ultrastructure level [ 53 ], and it is essential to understand the regeneration mechanisms in the MTJ to develop more effective treatments. The results in this study revealed that Col22-positive MTJ-specific cells undergo senescence earlier and more extensively than other cell types, suggesting that these cells could be a new promising therapeutic target to preserve MTJ integrity and function during ageing. Conclusion This study provides the first comprehensive characterisation of age-related structural and cellular changes within the mouse MTJ. High-resolution µCT and confocal imaging revealed that ageing leads to a reduction in muscle fibre diameter and vascular volume at the muscle-tendon unit, alongside an expansion of the MTJ interface area, indicating altered force generation capacity and compromised tissue integrity. Cellular analysis demonstrated that both MTJ-specific Col22-positive cells and VWF-positive endothelial cells exhibit increased expression of senescence markers with age, suggesting that these cell types are particularly vulnerable to age-associated dysfunction. Together, these findings highlight that ageing drives structural remodelling and cell-type-specific senescence within the MTJ, which may contribute to impaired tissue functionality and repair capacity in older individuals. These findings give a crucial insight into understand mechanisms of age-related degeneration in the MTJ and to develop therapeutic approaches for preserving MTJ integrity and function during ageing. Declarations Acknowledgements The authors acknowledge Dr Michelle Linterman and Dr Theresa Pankhurst (Babraham Institute) for providing the mouse tissues for this project, and Prof. Manuel Koch (University of Cologne) for providing the Col22 antibody. Author contributions CT contributed to research design, interpretation of results, writing/revision of the original manuscript, and funding acquisition. NI contributed to all aspects of this study, including research design, sample collection and processing, data acquisition and analysis, writing/revision of the original manuscript, and funding acquisition. Funding This research was funded by the Gill Malone Memorial Award from the Royal Veterinary College. Competing interests The authors declare no competing interests. Ethics and Consent to Participate All procedures carried out at Babraham Institute were approved by the Babraham Institute Animal Welfare and Ethical Review Body and conducted in accordance with European Union and UK Home Office regulations (Home Office Licence P4D4AF812). All experiments performed at the RVC were approved by the Royal Veterinary College Clinical Research Ethical Review Board (URN 2024-2336-A). 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Int J Numer Method Biomed Eng 36(1). 10.1002/cnm.3295 Landers-Ramos RQ, Prior SJ (2018) The Microvasculature and Skeletal Muscle Health in Aging, Exerc Sport Sci Rev , vol. 46, no. 3, pp. 172–179, Jul. 10.1249/JES.0000000000000151 Graves SI, Meyer CF, Jeganathan KB, Baker DJ (2025) p16-expressing microglia and endothelial cells promote tauopathy and neurovascular abnormalities in PS19 mice, Neuron , vol. 113, no. 14, pp. 2251–2264.e4, Jul. 10.1016/j.neuron.2025.04.020 Grosse L et al (Jul. 2020) Defined p16High Senescent Cell Types Are Indispensable for Mouse Healthspan. Cell Metab 32(1):87–99. 10.1016/j.cmet.2020.05.002 . .e6 Mackey AL (2024) The Myotendinous Junction—Form and Function, Cold Spring Harb Perspect Biol , p. a041500, Aug. 10.1101/cshperspect.a041500 Additional Declarations The authors declare no competing interests. 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15:36:29","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119170,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/14a376aeb90498ade39a8ac9.html"},{"id":97896634,"identity":"4a7631c2-6cd8-46f0-927a-31651502f152","added_by":"auto","created_at":"2025-12-10 15:36:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170868,"visible":true,"origin":"","legend":"\u003cp\u003eµCT image analysis shows significant decreases in muscle fibre diameter and pennation angle at the MTJ with age. (a) Representative 3D reconstructed µCT images of young and old MTJ in longitudinal view. Scale bar is 500 µm. Quantitative analysis of µCT images showing (b) muscle fibre diameter, (c) tendon sub-unit diameter, (d) MTJ surface area normalised to total surface area, and (e) pennation angle at the MTJ. Data are presented as mean ± SD from analysis of 4 young and 4 old mice. Mann-Whitney test was used to calculate the significance between young and old MTJs. *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/6eb04a1c2549a4ebc85359d7.png"},{"id":97767006,"identity":"a3b7445d-09f1-4c15-bc86-8d0f1b1b8bf5","added_by":"auto","created_at":"2025-12-09 07:21:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":240307,"visible":true,"origin":"","legend":"\u003cp\u003eImmunolabeled 3D image analysis shows a significant decrease in muscle-tendon unit vascularity with age. (a) Representative reconstructed 3D immunolabelled images of young and old mouse MTJ with surrounding muscle and tendon (n = 4 per age group) with endothelial cell marker, VWF and muscle marker, LAMA2 antibodies. Scale bar is 1 mm. Immunolabelled volume of (b) VWF (endothelial cell marker) and (c) LAMA2 (skeletal muscle marker) in young and old MTJ normalised using DAPI labelled volume. Data are presented as mean ± SD (n = 4). A Mann-Whitney test was used to calculate the significance between young and old MTJs. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/e445fe54282643bec57b82a7.png"},{"id":97896362,"identity":"ef4ac50d-ee00-4cc0-849f-341c74587c5f","added_by":"auto","created_at":"2025-12-10 15:36:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":266053,"visible":true,"origin":"","legend":"\u003cp\u003eSenescence was detected more frequently in MTJ-specific and vascular endothelial cells compared to the overall cell population. Representative in situ hybridisation images of young and old mouse MTJ (n = 4 per age group) showing senescence markers (a) p16 and (b) p21.Red: Col22 (MTJ marker); yellow: (a) p16 and (b) p21; cyan: VWF (endothelial cell marker); grey: DAPI. Blue DAPI image on the right shows the interface between the muscle and tendon indicated by orange dashed lines. Scale bar is 50 µm. Percentage of (c) p16 and (d) p21 positive cells were measured in Col22-positive MTJ-specific cells, VWF-positive endothelial cells, and all cells in the field of view. Y: young; O: old. Two-way ANOVA followed by Tukey's multiple comparisons test was used to calculate the significance between the age groups and cell populations. Data are presented as mean ± SD: *p\u0026lt;0.05 **p\u0026lt;0.01 ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/a8a21571c6a957c6c2c82ab9.png"},{"id":97896566,"identity":"93b379dd-7716-40d4-b012-fbbf863da9c0","added_by":"auto","created_at":"2025-12-10 15:36:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":163932,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression analysis showed an increase in p16-positive cell proportion within Col22-positive MTJ-specific cells with age. (a) Representative immunofluorescent images of young and old MTJ (n = 4 per age group). Red: Col22; green: p16; grey: DAPI. Blue DAPI image on the right shows the interface between the muscle and tendon indicated by orange dashed lines. Scale bar is 50 µm. (b) Percentage of p16 positive cells in Col22-positive MTJ-specific cell population. Data are presented as mean ± SD (n = 4). A Mann-Whitney test was used to calculate the significance between young and old tendons. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/511100205f27e0fb463642ea.png"},{"id":97903125,"identity":"10245d7d-58cd-45c8-9337-35d78e02b57d","added_by":"auto","created_at":"2025-12-10 15:54:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1453620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/7bbf7557-bd96-4712-8781-474153deea72.pdf"},{"id":97897075,"identity":"9a0bcf56-891f-4ee1-a202-a87464d02b70","added_by":"auto","created_at":"2025-12-10 15:37:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":307932,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaldata1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8305364/v1/d272fc4a89cc026e63e22151.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAgeing-Related Structural and Cellular Alterations in the Mouse Muscle-Tendon Junction\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe muscle tendon junction (MTJ), also known as the myotendinous junction, is a specialised interface between muscle and tendon, and transmits the force generated by the muscle to its connecting tendon [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The MTJ is commonly associated with muscle strains and tears [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and is particularly vulnerable to tensile failures compared to the neighbouring muscle and tendon [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Injuries and failure at the MTJ increase the morbidity of patients and affect their quality of life [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMTJ injuries are common, with 28% of injuries in the muscle-tendon-bone unit and 52% of acute hamstring injuries occurring at the MTJ [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the non-athletic population, the average age for injuries involving the gastrocnemius-Achilles aponeurosis and myoaponeurotic junction is 48.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 years [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], which is notably older than the average age reported for tendon injuries (approximately 40 years) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], suggesting that the MTJ becomes more vulnerable to injury with age. Moreover, 2D structural studies in ageing rodents have revealed that the length of the MTJ region approximately doubles with age [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], indicating that the MTJ undergoes degenerative changes with age and may contribute to an increased risk of injury. However, the specific effects of ageing on MTJ structure and function remain poorly understood [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This gap in knowledge limits our understanding of the mechanisms underlying age-related MTJ deterioration and hinders advances in the discovery of targeted therapeutic strategies.\u003c/p\u003e\u003cp\u003eMTJ injuries are often treated with physical therapy, however, this conservative treatment often fails to achieve reduced recovery times without increasing the risk of reinjury and cannot be used to treat a complete MTJ tear [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Another common treatment is surgical suturing. This, however, fails to yield satisfactory long-term outcomes due to adhesions and reduced mechanical properties, which further increase the risk of re-rupture [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Traditionally, the autograft approach is the gold standard for tendon injuries, including MTJ, yet this is generally limited and can lead to higher donor site morbidity and infection risk compared to other grafts such as allografts [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, there is a need for more effective therapies to overcome the limitations of the current treatments.\u003c/p\u003e\u003cp\u003eThe extracellular matrix (ECM) of the MTJ contains MTJ-specific adhesion proteins, including collagen type XXII (Col22), paxillin, and talin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These proteins are critical for efficient force transmission between muscle and tendon and play key roles in maintaining MTJ structural and functional integrity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Col22 is a well-characterised MTJ-specific protein, which is expressed by both muscle cells and tenocytes at the MTJ [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and has been reported to maintain vascular integrity by regulating vascular permeability [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Ageing significantly affects the vasculature in the musculoskeletal system, and both aged muscle and tendon have been reported to exhibit reduced vascularisation and blood flow [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which may contribute to the higher incidence of injury and impaired regenerative capacity observed with age. However, the effect of ageing on the MTJ vasculature has not been reported to date.\u003c/p\u003e\u003cp\u003eCellular senescence, a state of permanent cell cycle arrest triggered by various stressors, plays a critical role in the ageing process, with the proportion of senescent cells increasing across multiple tissues with age [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Cellular senescence has been reported to affect muscle and tendon health. For example, cells positive for the senescence marker p16 have been shown to contribute to the development of age-associated pathologies in mice, particularly within skeletal muscle [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and inhibit tenogenic differentiation of tendon stem/progenitor cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Similarly, overexpression of p21, another key marker of senescence, has been shown to drive cellular senescence in skeletal muscle and is associated with muscle loss and functional decline [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. While these findings indicate that MTJ-localised cells are likely to suffer from senescence, the precise effects of senescence on the ageing MTJ remain to be elucidated.\u003c/p\u003e\u003cp\u003eThe aim of this study was therefore to investigate age-related alterations in MTJ structure and vasculature using high resolution 3D imaging techniques, including micro-computed tomography (\u0026micro;CT) and confocal microscopy. In addition, cellular senescence in MTJ-specific cells (Col22-positive cells) and vascular endothelial cells was examined using \u003cem\u003ein situ\u003c/em\u003e hybridisation and immunofluorescence. Understanding these structural and cellular alterations with age may provide insight into the mechanisms underlying MTJ degeneration and support the development of targeted therapeutic strategies to enhance MTJ repair and regeneration.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Sample acquisition\u003c/h2\u003e\u003cp\u003eMouse hindlimbs were obtained as residual tissues from animals euthanised as part of an unrelated study (a kind gift from Dr. Linterman, Babraham Institute). In this unrelated study, male and female young (3-month-old, the life phase equivalent for humans ranges from 20\u0026ndash;30 years) and old (23-month-old, the life phase equivalent for humans ranges from 56\u0026ndash;69 years of age) C57BL/6 mice (n\u0026thinsp;=\u0026thinsp;6 per age group) were immunised by intramuscular injection of 50 \u0026micro;g/mL LNP-mRNA vaccine into the right biceps femoris muscle; the mRNA encoded the spike protein of the ancestral SARS-CoV-2 strain. Ten days post-injection, mice were euthanised and tissues were collected. All procedures were approved by the Babraham Institute Animal Welfare and Ethical Review Body and conducted in accordance with European Union and UK Home Office regulations (Home Office Licence P4D4AF812).\u003c/p\u003e\u003cp\u003eAchilles tendon and gastrocnemius muscle junctions (Achilles MTJs) were subsequently harvested from both hindlimbs of the immunised mice 3 hours after euthanasia at the Royal Veterinary College. The experiments were approved by the Royal Veterinary College Clinical Research Ethical Review Board (URN 2024-2336-A).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. \u0026micro;CT imaging\u003c/h2\u003e\u003cp\u003ePhosphotungstic acid (PTA) was used to enhance the contrast of the MTJ, adapting a protocol from a previous study [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Young and old Achilles MTJs were immersed in an increasing ethanol concentration of 25, 50, and 70 % thanol for 90 min each followed by 1 % TA (79690, Sigma-Aldrich, Burlington, MA, USA) in 70 % thanol for 72 h (n\u0026thinsp;=\u0026thinsp;4 per age group). Samples were then washed twice and immersed in Tris-buffered saline (TBS) for 30 mins prior to imaging. Samples were wrapped in clingfilm to avoid dehydration during imaging. A Skyscan 1172F (version 1.5, Skyscan, Kontich, Belgium) was used with an X-ray source at 50 kV tube voltage and 200 \u0026micro;A tube current with 2500 ms exposure time. The voxel size was 2 \u0026micro;m, and 180\u0026deg; scans were performed with 0.5 mm Aluminium filter, frame averaging at 2, and with a rotation step at 0.25\u0026deg;. Slice reconstruction was performed using NRecon (version 1.7.1.0). The reconstructed images were segmented to remove tendons and analysed using CTAn (version 1.17.7.1) to measure mean muscle fibre diameter. CTVox (version 3.3.0) was used to visualise the 3D reconstructed images. The images were also analysed using Avizo (Avizo 2021.1, ThermoFisher Scientific, MA, USA), and the images were cropped at 2000 x 2000 x 1000 voxels (32 mm\u003csup\u003e3\u003c/sup\u003e) with the MTJ in the region of interest. Pennation angle was measured manually at each muscle\u0026ndash;tendon sub-unit interface in Avizo and a mean value was calculated for each sample (Figure S1a). Tendon sub-unit diameter was also measured manually in Avizo. The images were then segmented to separate muscle and tendon, and the MTJ surface area was measured using the volume fraction function in Avizo and normalised to the whole tissue surface area (Figure S1b).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. 3D immunolabelling\u003c/h2\u003e\u003cp\u003eMTJs from young and old mice were fixed in 4% paraformaldehyde (PFA) for 4 h for 3D immunolabelling (n\u0026thinsp;=\u0026thinsp;4 per age group), using a protocol adapted from a previous study [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Permeabilization was performed using 50 % (/v) methanol:TBS, 80 % (/v) methanol:dH2O, and 100 % mthanol for 2 h, and 20 % (/v) dimethylsulphoxide (DMSO):methanol, 80 % (/v) methanol:dH2O, 50 % (/v) methanol:TBS for 30 min at 4\u0026deg;C, respectively, with gentle shaking. The samples were stored in TBS overnight at 4\u0026deg;C. Blocking was performed using blocking solution (0.2 % Titon X-100, 6 % dnkey serum, 6 % gat serum, 10 % DSO in TBS) for 72 h at 37\u0026deg;C with gentle shaking. The samples were then incubated with primary antibodies diluted in blocking solution for 72 h at 37\u0026deg;C with gentle shaking. The details of primary antibodies were as follows: Rabbit anti-von Willebrand factor (VWF, endothelial cell marker, 1:800, A0082, Dako, Ejby, Denmark) and rat anti-laminin alpha 2 (LAMA2, skeletal muscle marker, 1:1000, ab11576, Abcam, Cambridge, UK). The samples were then washed 5 times with 0.2 % Teen-20 in TBS for 1 h each at room temperature. The samples were incubated with secondary antibody diluted in blocking solution. The details of the secondary antibodies were as follows: Goat anti-rabbit IgG AF594 (1:800, A-11012, ThermoFisher Scientific, MA, USA) and Goat anti-rat IgG AF488 (1:800, ab150157, Abcam, Cambridge, UK) for 24 h at 37\u0026deg;C with gentle shaking, followed by 5 washes with 0.2 % Teen-20 in TBS for 1 h at room temperature. Samples were then incubated in DAPI solution (5 \u0026micro;g/mL in TBS) at 4\u0026deg;C overnight. Samples were dehydrated as described above with increasing concentrations of methanol. Two-step tissue clarification was performed by immersing samples in Visikol HISTO-1 (H1-30, Sigma-Aldrich, MA, USA) for 24 h, followed by immersion in HISTO-2 (H2-30, Sigma-Aldrich, MA, USA) for at least 48 h at room temperature with gentle shaking.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Confocal imaging\u003c/h2\u003e\u003cp\u003eThe 3D immunolabelled samples were placed in a glass-bottom dish fitted with a polystyrene frame (220.220.042, IBL Baustoff\u0026thinsp;+\u0026thinsp;Labor GmbH, Austria) and a drop of Histo-2 was added to keep the sample hydrated. The samples were then imaged using a Leica TCS SP8 laser scanning confocal microscope (Leica Biosystems, Nussloch, Germany) with 10x objective, 512x512 pixel resolution with 2.27 \u0026micro;m pixel size and 2.27 \u0026micro;m z axis steps. The pinhole size was set to 1 Airy unit, frame average was set to 1, and line average was set to 2 using lasers emitting light at 405 nm (blue channel), 488 nm (green channel), and 561 nm (red channel). The images were visualised using Leica LAS X software (version 3.5.5) within the 3D module and reconstructed and analysed using Avizo. The reconstructed volume of immunolabeled vasculature was measured using the volume fraction function in Avizo.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. \u003cem\u003eIn situ\u003c/em\u003e hybridisation\u003c/h2\u003e\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridisation was performed using OCT-embedded MTJ sections (n\u0026thinsp;=\u0026thinsp;4 per age group), RNAscope Multiplex Fluorescent Reagent Kit v2 (323110, Bio-Techne Ltd, MN, USA) following the manufacturer\u0026rsquo;s protocol for fresh frozen tissues. The sections were fixed with 4% PFA for 2 h at 4\u0026deg;C and then dehydrated using an increasing ethanol concentration of 50, 70, and 100% ethanol for 5 min each at room temperature, followed by -20\u0026deg;C overnight incubation in 100% ethanol. The samples were dried for 5 minutes and encircled with a hydrophobic barrier pen, then incubated with hydrogen peroxide for 10 minutes and rinsed in distilled water. Custom pretreatment (300040, Bio-Techne Ltd, MN, USA) was added for 30 mins at 40\u0026deg;C, followed by two washes with distilled water. Probe solution was applied to the tissue sections for 2 h at 40\u0026deg;C. The details of the probes were as follows: Col22A1 (590911, C1, Bio-Techne Ltd, MN, USA), VWF (499111-C3, Bio-Techne Ltd, MN, USA), p16 (411011-C2, Bio-Techne Ltd, MN, USA) and p21 (408551-C2, Bio-Techne Ltd, MN, USA). The samples were stored overnight at room temperature in 5\u0026times; saline-sodium citrate buffer (0.75 M sodium chloride, 75 mM trisodium citrate, pH 7.0). On the following day, signal amplification was performed according to the manufacturer\u0026rsquo;s instructions. All incubations were conducted at 40\u0026deg;C, followed by two 2-minute washes with RNAscope wash buffer. Amplifier incubations were carried out for 30 minutes for the first two amplifiers and 15 minutes for the third. Subsequently, slides were incubated with horseradish peroxidase (HRP) for 15 minutes, followed by a 30-minute incubation with a tyramide dye fluorophore (OPAL 520, FP1487001KT, Akoya Biosciences, MA, USA) diluted 1:1500 in RNAscope TSA dilution buffer, and a 30-minute incubation with HRP blocker. The HRP, fluorophore, and blocking steps were repeated using second and third tyramide dye fluorophores (OPAL 570 and OPAL 650, FP1488001KT and FP1496001KT, Akoya Biosciences, MA, USA). The sections were then incubated with DAPI for 30 seconds at room temperature, followed by mounting with ProLong\u0026trade; Gold Antifade Mountant (P10144, ThermoFisher Scientific, MA, USA) and allowed to cure for 2\u0026ndash;3 h before imaging using an Eclipse Ni-E upright microscope (Nikon Instruments Inc., Tokyo, Japan). Four images were obtained per sample, and they were analysed using ImageJ (National Instruments, Austin, USA) by manually counting cells expressing the positive signals. The percentages of p16 and p21 positive cells within Col22-positive MTJ-specific cell and VWF-positive endothelial cell populations were quantified and compared with their prevalence among all cells within the field of view.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. 2D immunolabelling\u003c/h2\u003e\u003cp\u003eMouse MTJs were snap frozen in hexane cooled on dry ice (n\u0026thinsp;=\u0026thinsp;4 per age group), and then embedded using OCT (15212776, ThermoFisher Scientific, MA, USA). Longitudinal MTJ cryosections (10 \u0026micro;m thickness) were fixed in ice-cold methanol/acetone solution (1:1) for immunolabelling. Non-specific binding of antibodies was blocked by incubating samples with 5% goat serum (ab7481, Abcam, Cambridge, UK) in TBS for 45 mins at room temperature. The samples were incubated with primary antibodies in 5% goat serum for 2 h at room temperature. The details of primary antibodies were as follows: Guinea-pig anti‐collagen XXII (1:100, a kind gift from Manuel Koch, University of Cologne, Germany) and rabbit anti-p16 (1:50, 80772S, Cell Signaling Technology, MA, USA). After washing twice with TBS, the sections were incubated with secondary antibodies (A-11012 and SA5-10096, ThermoFisher Scientific) in 5% goat serum (1:400) for 1 h at room temperature. The sections were then incubated with DAPI (0.1 \u0026micro;g/mL) for 10 min at room temperature, followed by two washes in TBS. The sections were mounted with ProLong\u0026trade; Gold Antifade Mountant (P10144, ThermoFisher Scientific, MA, USA) and allowed to cure for 2\u0026ndash;3 h before imaging using an Eclipse Ni-E upright microscope (Nikon Instruments Inc., Tokyo, Japan). Four images were obtained per sample, and they were analysed using ImageJ by manually counting positively labelled cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll data are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), and all experiments were conducted using 4 different animals from each age group. A D\u0026rsquo;Agostino and Pearson test was used to determine if the data followed a normal distribution. The Mann-Whitney test or two-way ANOVA was performed to calculate the differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between different sample groups using GraphPad Prism version 10.2.3 (La Jolla, CA, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1. \u0026micro;CT analysis demonstrated age-related structural changes in mouse MTJs\u003c/h2\u003e\u003cp\u003eQuantitative analysis revealed a 27% reduction in muscle fibre diameter with age in the MTJ region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), whereas tendon sub-unit size was not significantly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The MTJ surface area, normalised to total surface area, showed a trend towards an age-related increase (p\u0026thinsp;=\u0026thinsp;0.0571; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), suggesting enlargement of the MTJ with ageing. The pennation angle, an indicator of muscle force generation capacity, was significantly reduced by 19% in old mouse MTJs compared with those of young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The muscle and tendon volume at the MTJ, normalised to the whole MTJ volume, showed no significant difference with age (Figure S2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Three-dimensional immunolabelling demonstrated a significant reduction in VWF labelled endothelial cell volume with age.\u003c/h2\u003e\u003cp\u003eWhole-tissue immunolabelling was performed to visualise endothelial and muscle cell populations within the muscle-tendon unit including the MTJ. VWF was used to label endothelial cells and LAMA2 was used to label muscle cells, and DAPI staining was applied to identify the overall tissue volume. The images showed that the vasculature exists not only in the muscle, as indicated by LAMA2 labelling, but also within the tendon and across their interfaces, demonstrating the presence of vasculature throughout the MTJ region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eQuantitative analysis of VWF- and LAMA2-labelled volumes revealed a significant reduction of 49% in VWF-labelled volume in old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), whereas LAMA2-labelled volume showed no significant change with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). This finding indicated an age-associated decline in vascularity within the muscle-tendon unit, without a corresponding reduction in muscle volume.\u003c/p\u003e\u003cp\u003e\u003cem\u003e3.3 Collagen type 22 positive MTJ-specific cells and VWF-positive endothelial cells exhibit higher expression of senescence markers at the MTJ region\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridisation, a technique used to visualise RNA expression within tissue sections, was employed to detect senescence markers at the MTJ. In old MTJs, senescence markers p16 and p21 were predominantly co-localised with the MTJ marker Col22 and endothelial cell marker VWF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and b). Col22-positive MTJ-specific cells exhibited significantly higher percentages of p16- and p21-positive cells with age (270% and 310% increases, respectively), while VWF-positive endothelial cells showed a 780% increase in p16-positive cells with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and d).\u003c/p\u003e\u003cp\u003eWhile p16 expression was significantly higher in Col22-positive MTJ-specific cells (52%) and VWF-positive endothelial cells (19%) compared with all cells in the field of view (3.2%) in the old MTJ, p16 expression was also significantly higher in Col22-positive MTJ-specific cells (19%) than VWF-positive endothelial cells (2.5%) and all cells (1.6%) in the young MTJ. Similarly, p21 expression was significantly higher in Col22-positive MTJ-specific cells (45%) than in all cells, whereas no significant difference was observed between VWF-positive endothelial cells and the overall cell population. However, in young MTJ, there was no significant difference in p21 expression among different cell types.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eExpression of Col22 and p16 was assessed at the protein level using 2D immunofluorescent labelling. In old MTJ, expression of p16 was predominantly co-localised with Col22 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), which was not observed in young MTJ. Quantitative analysis revealed a 270% increase in the proportion of p16-positive cells within the Col22-positive MTJ-specific cell population with age (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), consistent with the increase observed in the RNA expression analysis shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides the first comprehensive investigation of structural and cellular alterations with age in the mouse MTJ, demonstrating that age-related structural changes occur within the MTJ, which are accompanied by reduced vascularity and increased cell senescence in this region.\u003c/p\u003e\u003cp\u003eOne of the limitations of this study was using immunised mice against the ancestral SARS-CoV-2 strain. However, both young and old mice were immunised simultaneously and underwent identical treatments after the immunisation. Although some individuals may experience musculoskeletal pain as a result of SARS-CoV-2 infection [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], it has been reported that SARS-CoV-2 infection has no association with musculoskeletal function in humans [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In addition, the vaccine was administered into the right biceps femoris muscle, and the tissues obtained in this study were anatomically distinct from the injection site. Therefore, it is unlikely that the immunisation affected the MTJ structure, vasculature, or cellular senescence.\u003c/p\u003e\u003cp\u003eAge-related structural changes in the MTJ have traditionally been investigated using 2D imaging approaches. In this study, \u0026micro;CT imaging was employed to explore structural changes in the MTJ with age in 3D, demonstrating a significant decrease in muscle fibre diameter. Similar findings have been reported by several previous studies using 2D imaging, with the reduction in size depending on the types of muscle fibres; type II muscle fibre size decreases more than type I muscle fibres with age [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the current study, the mean muscle fibre diameter reduction with age was 27 %, which is similar to the mean muscle ibre reduction reported previously in human quadriceps muscle (~\u0026thinsp;20 %) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, pennation angle, whic is the angle between the muscle fibres and tendon long axis, significantly decreased with age. A larger pennation angle results in increased force generation capacity in muscle [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and therefore the decrease in pennation angle, combined with the reduction in muscle fibre diameter, strongly indicates a reduction in muscle force generation capacity in the MTJ region [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and aligns with a previous report of age-related decreases in muscle force production observed in skeletal muscle [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Further analysis of \u0026micro;CT images showed a trend towards an age-related increase in MTJ surface area, which has also been reported in 2D image analysis of mouse soleus MTJ [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The structural alterations observed with age in this study indicate that mouse Achilles MTJ undergoes functional deterioration and loss of force generation capacity with age. However, age-related alterations in MTJ mechanical properties and force generating capacity were not directly measured in the current study and therefore remain an important area for future investigation.\u003c/p\u003e\u003cp\u003eThree-dimensional visualisation of muscle-tendon unit vascularity was achieved by confocal imaging of whole-tissue immunolabelled for VWF to target endothelial cell populations, and LAMA2 labelling for muscle cell populations. Imaging revealed the presence of vasculature within both muscle and tendon tissues, as well as across their interface, the MTJ. Quantitative analysis showed a significant reduction in VWF-labelled endothelial cell volume normalised by the whole tissue volume, but no significant difference in LAMA2-labelled muscle volume with age, indicating an age-associated decline in vascularisation at the muscle-tendon unit without a corresponding reduction in muscle volume, which supports the findings from the \u0026micro;CT image analysis. Together with previous reports of vascular decline in aged muscle and tendon [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], these findings indicated that reduced vascularisation is a shared feature of musculoskeletal ageing across tissues, which may contribute to functional deterioration at the MTJ through limited delivery of oxygen, amino acids, nutrients and hormones [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn situ\u003c/em\u003e hybridisation was employed to investigate the expression of senescence markers, p16 and p21, in different cell populations in the MTJ. Both markers were predominantly localised to Col22-positive MTJ-specific cells and, to a lesser extent, VWF-positive endothelial cells. These results suggest that these cell types are particularly susceptible to senescence with age compared to other cell populations in the MTJ, especially Col22-positive MTJ-specific cells. Additionally, in Col22-positive MTJ-specific cells, elevated expression of p16 was observed in young MTJ compared to the other cell types, suggesting that Col22-positive MTJ-specific cells may experience senescence in early age. The age-related accumulation of p16 in Col22-positive MTJ-specific cells was further validated at the protein level using immunolabelling. The marked increases in p16 and p21 expression within Col22-positive MTJ-specific cells indicated that MTJ-specific cells are prone to senescence, which may impair junctional integrity and contribute to age-related disorders and functional decline of the MTJ. Supporting this notion, the development of age-related pathologies and tissue dysfunction induced by upregulation of p16 and p21 has previously been observed in muscle [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Similarly, the elevated p16 expression observed in VWF-positive endothelial cells suggests induction of vascular cell senescence, which may result in vascular dysfunction, disrupted vascular ECM formation and fibrosis [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], further exacerbating MTJ degeneration.\u003c/p\u003e\u003cp\u003eWhile senescence in Col22-positive MTJ-specific cells and VWF-positive endothelial cells was demonstrated in this study, and Col22 plays an important role in vascular integrity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], it remains unclear whether these two cell types influence each other during ageing. Future studies should investigate the relationship between MTJ-specific cells and vascular endothelial cells in the context of ageing, potentially using \u003cem\u003ein vitro\u003c/em\u003e MTJ co-culture models.\u003c/p\u003e\u003cp\u003eDespite the high incidence of MTJ injuries, current treatments remain insufficient [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. MTJ regeneration, and the effects of ageing, are poorly understood at cellular and ultrastructure level [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and it is essential to understand the regeneration mechanisms in the MTJ to develop more effective treatments. The results in this study revealed that Col22-positive MTJ-specific cells undergo senescence earlier and more extensively than other cell types, suggesting that these cells could be a new promising therapeutic target to preserve MTJ integrity and function during ageing.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first comprehensive characterisation of age-related structural and cellular changes within the mouse MTJ. High-resolution \u0026micro;CT and confocal imaging revealed that ageing leads to a reduction in muscle fibre diameter and vascular volume at the muscle-tendon unit, alongside an expansion of the MTJ interface area, indicating altered force generation capacity and compromised tissue integrity. Cellular analysis demonstrated that both MTJ-specific Col22-positive cells and VWF-positive endothelial cells exhibit increased expression of senescence markers with age, suggesting that these cell types are particularly vulnerable to age-associated dysfunction. Together, these findings highlight that ageing drives structural remodelling and cell-type-specific senescence within the MTJ, which may contribute to impaired tissue functionality and repair capacity in older individuals. These findings give a crucial insight into understand mechanisms of age-related degeneration in the MTJ and to develop therapeutic approaches for preserving MTJ integrity and function during ageing.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge Dr Michelle Linterman and Dr Theresa Pankhurst (Babraham Institute) for providing the mouse tissues for this project, and Prof. Manuel Koch (University of Cologne) for providing the Col22 antibody.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT contributed to research design, interpretation of results, writing/revision of the original manuscript, and funding acquisition. NI contributed to all aspects of this study, including research design, sample collection and processing, data acquisition and analysis, writing/revision of the original manuscript, and funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Gill Malone Memorial Award from the Royal Veterinary College.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics and Consent to Participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures carried out at Babraham Institute were approved by the Babraham Institute Animal Welfare and Ethical Review Body and conducted in accordance with European Union and UK Home Office regulations (Home Office Licence P4D4AF812). All experiments performed at the RVC were approved by the Royal Veterinary College Clinical Research Ethical Review Board (URN 2024-2336-A).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the outcomes of this study are available from the paper and supporting material. Upon request, raw data are available from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTidball JG (1983) The geometry of actin filament-membrane associations can modify adhesive strength of the myotendinous junction. 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Cell Metab 32(1):87\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cmet.2020.05.002\u003c/span\u003e\u003cspan address=\"10.1016/j.cmet.2020.05.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. .e6\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMackey AL (2024) The Myotendinous Junction\u0026mdash;Form and Function, \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e, p. a041500, Aug. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/cshperspect.a041500\u003c/span\u003e\u003cspan address=\"10.1101/cshperspect.a041500\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"60307796-80ee-426b-b39c-0a71500244b6","identifier":"10.13039/100015226","name":"Royal Veterinary College","awardNumber":"Gill Malone Memorial Award","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Royal Veterinary College","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Muscle tendon junction, ageing, µCT, immunolabelling, endothelial cells, senescence","lastPublishedDoi":"10.21203/rs.3.rs-8305364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8305364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe muscle tendon junction (MTJ) is a specialised interface between muscle and tendon and transmits muscle-generated force to the tendon. The MTJ is particularly vulnerable to injuries compared to muscle and tendon and becomes more injury prone with age. Further, current treatments for MTJ injuries are insufficient as indicated by scar tissue formation and a high re-injury rate. Despite its clinical importance, the mechanisms driving MTJ ageing and age-related functional deterioration remain poorly understood. In this study, the first comprehensive three-dimensional characterisation of age-related structural and cellular changes at the mouse MTJ was performed using the high-resolution imaging techniques, micro-computed tomography (\u0026micro;CT) and confocal microscopy. \u0026micro;CT analysis revealed a 27% reduction in muscle fibre diameter with age, accompanied by a trend toward increased MTJ surface area and a 19% reduction in pennation angle, indicating diminished force generation capacity. Confocal imaging showed a 49% reduction in endothelial cell volume (VWF-labelled) in the old mouse muscle-tendon unit, suggesting a loss of vascularity. \u003cem\u003eIn situ\u003c/em\u003e hybridisation and immunofluorescence demonstrated increased expression of senescence markers p16 and p21 in endothelial and MTJ-specific cells, with MTJ-specific cells showing the greatest accumulation of p16 and p21 (270% and 310% increases, respectively) with age. These findings suggest that vascular and MTJ-specific cells are particularly susceptible to ageing and may collectively contribute to the age-related functional decline of the MTJ. Understanding these mechanisms may help to develop targeted therapeutic strategies to preserve or restore MTJ integrity and function in ageing populations.\u003c/p\u003e","manuscriptTitle":"Ageing-Related Structural and Cellular Alterations in the Mouse Muscle-Tendon Junction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-09 07:21:16","doi":"10.21203/rs.3.rs-8305364/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be9c3a2a-7b21-4973-a665-83c6daf6c8b7","owner":[],"postedDate":"December 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-09T07:21:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-09 07:21:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8305364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8305364","identity":"rs-8305364","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}