Research Article

Micro-Injury of the Tendon-Bone Junction Caused by Acute Exhaustive Exercise in Rats: Ultrastructural Changes and Mechanism

DOI:

10.3791/69184

⸱

November 14th, 2025

 ,  ,  ,  , 
1Department of Rehabilitation, Sichuan college of traditional Chinese medicine, 2Department of Rehabilitation, Mian-yang Orthopedic hospital, 3Sports Medicine Center, West China Hospital, Sichuan University, 4Department of Orthopedics and Orthopedic Research Institute, West China Hospital, 5Department of Geriatric Medicine, Mian-yang hospital of traditional Chinese medicine, 6Department of Rehabilitation Medicine, Sichuan Provincial Orthopedic Hospital

In This Article

Summary

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This study aimed to explore the ultrastructural changes and mechanisms at the tendon-bone junction due to acute exhaustive exercise. Acute exhaustive exercise induces micro-injury at the tendon-bone junction, transiently activating ER stress and its mediated apoptotic signaling pathways. Nevertheless, these micro-injuries and ER stress can be progressively restored post-exercise.

Abstract

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Exercise-induced micro-injuries at the tendon-bone junction (enthesis) can lead to tendinopathy. Investigating the ultrastructural changes and mechanisms within the enthesis following acute exhaustive exercise can improve our understanding of tendinopathy and guide clinical treatments. In this study, thirty-six male Sprague-Dawley rats were randomly assigned to two groups: control and exhaustive exercise. The exhaustive exercise group was further subdivided into five subgroups based on the time post-exercise: 0 h, 6 h, 12 h, 24 h, and 48 h. Hematoxylin-eosin staining and electron microscopy were employed to examine changes in the calcaneal tendon-bone junction. The expression of endoplasmic reticulum stress (ERS)-related proteins GRP78, CHOP, and Caspase-12 at the tendon-bone junction was quantified using immunohistochemistry (IHC). Following acute exhaustive exercise, the tendon-bone junctions of rats that underwent exhaustive exercise displayed significant structural alterations. Chondrocytes and collagen fibers exhibited notable ultrastructural changes indicative of ERS. ERS protein levels increased immediately after exercise, peaking at 6 h. These protein levels began to decline at 12 h post-exercise and returned to baseline by 48 h. In conclusion, acute exhaustive exercise induces micro-damage and endoplasmic reticulum stress at the tendon-bone junction in rats, but no irreversible damage ensues. Timely treatment and repair of these micro-injuries can prevent excessive damage and the development of tendinopathy.

Introduction

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Achilles tendinopathy is a common clinical condition caused by frequent ankle overuse injuries. This disorder is characterized by tendon swelling, pain, and dysfunction. It is more prevalent in older individuals and athletes, particularly runners and jumpers. Achilles tendinopathy is categorized into non-insertional and insertional tendinopathy. Non-insertional tendinopathy affects the tendon 2 to 6 cm above its bone insertion, while insertional tendinopathy affects the tendon-bone junction1. Moreover, non-insertional tendinopathy tends to occur in overweight, older, and less active persons, whereas insertional tendinopathy tends to occur in more active persons1.

The tendon-bone junction, where the tendon inserts into the bone, involves a gradual transition from tendon tissue to bone tissue, including uncalcified fibrocartilage and calcified fibrocartilage2. This change provides a transition from a soft and flexible tendon tissue to hard bone. In engineering terms, this region serves as an anchor point, transmitting muscle strength to facilitate joint movement3,4. Therefore, this region is a site of stress concentration and is commonly subjected to overuse injuries in sports.

Clinical research has primarily focused on non-insertional tendinopathy, with less attention given to insertional tendinopathy at the tendon-bone junction2. Research indicates that overuse loading promotes disorganization and fragmentation of the collagen, adipose tissue infiltration, and neovascularization in the tendon. It also leads to fibrochondrocyte hyperplasia, endochondral ossification, and bone spurs in both the uncalcified and calcified fibrocartilage regions5. However, recent studies suggest that muscle contraction patterns may play a more significant role than the amount of exercise in insertional tendinopathy6,7. Therefore, further research is needed to comprehend the impact of overuse on tendon-bone junctions. Previous studies have explored the effects of overuse, such as long-term jumping or running, on these junctions. However, few have investigated the impact of acute exhaustive exercise. A recent study suggests that acute exercise induces endoplasmic reticulum stress in the kidneys of rats, indicating an upregulation of IL-6 signaling and heat shock proteins (HSPs), and a decrease in mitochondrial respiratory complex mRNA8. Whether such changes occur in tendon-bone junctions has not been reported. This study focuses on the tendon-bone junction (enthesis) to elucidate the changes and underlying mechanisms induced by acute exhaustive exercise. The findings aim to contribute to the development of preventive strategies for insertional tendinopathy.

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Protocol

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All animal experiments were conducted in accordance with the ethical standards for experimental animals established by the Science and Technology Department of Sichuan Higher Institute of Traditional Chinese Medicine (Approval No: 2023DS01). The reagents and the equipment used are listed in the Table of Materials.

1. Experimental design and procedure

  1. Animals
    Thirty-six male, 12-week-old CD-Sprague-Dawley rats were housed and provided with ad libitum feeding throughout the experimental period. The environment was maintained at a temperature of 20-25 °C and a humidity level of 50%-60%, with a 12-h light/dark cycle.
  2. Group design
    After a week of adaptive feeding, the rats were randomly assigned to either a control group (Con, n = 6) or an exhaustive exercise group (EE, n = 30). The rats in the EE group underwent adaptation training by running at a speed of 10 m/min for 15 min on an uphill treadmill with a 10-degree incline for three days. Following the adaptive training, the EE group was further divided into five time-subgroups: 0 h, 6 h, 12 h, 24 h, and 48 h after exhaustive exercise.
  3. Intervention procedure
    Rats in the control group did not exercise, whereas those in the EE group underwent exhaustive running on an uphill treadmill at a 10-degree incline (Figure 1A). Following the Bedford protocol9, the exercise load was divided into three levels: Level 1 at 10 m/min for 15 min, Level 2 at 15 m/min for 15 min, and Level 3 at 20 m/min until exhaustion (Figure 1B). Exhaustion was determined when a rat could no longer run and remained stuck on the track three times within a period of less than 1 min10, despite repeated low-intensity electrical (150 V, 0.5 mA alternating current) and sound stimulation (70 dB).

The rats were sacrificed via excessive intraperitoneal injection of 3% sodium pentobarbital (200 mg/kg) at the specified times (0 h, 6 h, 12 h, 24 h, and 48 h post-exhaustive exercise). The skin and subcutaneous tissues of the calcaneus were carefully dissected, and the Achilles tendon was cut 2 cm above the calcaneus with ophthalmic scissors. The right calcaneus (including the attached Achilles tendon), was preserved in 4% paraformaldehyde for histopathological examination and immunohistochemical testing. Additionally, three random samples from the left side were stored in 2% paraformaldehyde-2.5% glutaraldehyde solution for electron microscopy to study ultrastructural changes ( Figure 1C).

2. Tendon-bone junction histology

The samples were fixed in 4% paraformaldehyde for 48 h and decalcified with 15% EDTA for 4 weeks at room temperature (20-25 °C), with the solution changed daily. The decalcification process concluded when the bone could be easily pierced by a needle without applying any force. Following decalcification, the samples underwent dehydration through an automated process involving a graded series of ethanol and xylene, followed by three washes with PBS. The samples were embedded in paraffin sagittally and sectioned at 5 µm. Hematoxylin-eosin staining was performed according to standard procedures, and images were captured with a Panthera digital triocular microcamera system at 20× and 40× magnification.

3. Electron Microscopy scan

The left samples were prefixed with 2% paraformaldehyde-2.5% glutaraldehyde for 48 h, then dehydrated in a series of acetone solutions, infiltrated in epoxy curing analyzer, and embedded sagittally. Semithin sections were stained using methylene blue to identify the tendon-bone junction region. Ultrathin sections, approximately 60-90 nm thick, were cut using a diamond knife. These sections were stained with uranyl acetate for 10-15 min, followed by lead citrate for 1-2 min. The sections were examined under a Transmission Electron Microscope at magnifications of 6000×,12000×, and 25000×.

4. Immunohistochemical assay

The tendon-bone junction was routinely decalcified and sectioned as previously described. After incubation with a citrate buffer antigen retrieval solution for 10 min, the samples were treated with 3% H2O2 for 10 min to block endogenous peroxidase activity, followed by blocking with normal goat serum for 20 min at 25 °C.The sections were then incubated overnight at 4 °C with primary antibodies, including GRP78 (1:100), CHOP (1:100 ), and Caspase-12 (1:100). Subsequently, the samples were incubated with secondary antibodies for 2 h at 25 °C. Color development was achieved using DAB for 8 min at 25 °C, resulting in a brown-yellow positive signal. The nuclei were restained with Hematoxylin for 3 min. Image-Pro Plus 6.0 software was used to analyze mean density. Three regions of interest of the tendon-bone junction region (tendon, fibrocartilage, and mineralized fibrocartilage) were selected for analysis, and the average value was calculated.

5. Statistical analysis

GraphPad Prism 8 was used to analyze the data and generate charts. All data are expressed as mean ± standard deviation. Comparisons between different groups were made using one-way ANOVA, and the Least Significant Difference (LSD) test was used for pairwise comparisons. A P-value of < 0.05 was considered statistically significant.

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Results

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As shown in Figure 2A, in the control group, the chondrocytes and collagen fibrocytes in the tendon-bone junction are regularly arranged, and there is a smooth blue line between the fibrocartilage and mineralized fibrocartilage, known as the tidemark (yellow arrows). In the exhaustive exercise groups, ultrastructural changes occurred at the tendon-bone junction. The changes involve an increase in chondrocytes (green circles), thinning and rupture of collagen ...

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Discussion

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The tendon-bone junction, also known as entheses or insertion sites, allows the insertion of tendons into bone and is a site of stress concentration where ligaments and tendons attach to bone4. As an anchor point, it facilitates ankle movement, transmits the force of calf muscle contraction to the foot ligaments and tendons, and participates in ankle and foot movement2. The tendon-bone junction comprises four consecutive regions: tendon, fibrocartilage, mineralized fibrocar...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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The authors would like to express their sincere gratitude for the support received from the Natural Science Foundation (Grant No. 23ZRYB03), Research and Innovation Team Project (TD-2022-04), the Clinical Medicine Science and Education Innovation Group and of Sichuan College of Traditional Chinese Medicine, as well as the 2025 research project (25MSZX329) funded by the Sichuan Provincial Administration of Traditional Chinese Medicine.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
2% Paraformaldehyde-2.5% glutaraldehyde solutionMKBio,ChinaMM1515, Electron microscopy Scan
Anti-Caspase12AntibodyHangzhou Huaan Biotechnology Co., Ltd.HA500144Immunohistochemical assay
Anti-CHOP AntibodyHangzhou Huaan Biotechnology Co., Ltd.ET1703-05Immunohistochemical assay
Anti-GRP78 AntibodyHangzhou Huaan Biotechnology Co., Ltd.ER1706-50Immunohistochemical assay
BiomicroscopyMotic Industrial Group Co., Ltd.CHINABA210DigitalBiomicroscopy for Tendon-bone junction histology Immunohistochemical assay
CD-Sprague-Dawley ratsBeijing Vital River Laboratory Animal Technology Co., Ltd.SCXK2021-0007 CD
Citrate buffer antigen retrieval solutionZSGB-Bio,ChinaZLI-9065, Immunohistochemical assay
DAB  ZSGB-Bio,ChinaZLI-9018 Immunohistochemical assay
EDTASinopharm Chimical Reagant Co., Ltd.10009717Hematoxylin-eosin staining 
Epox 812Sinopharm Chimical Reagant Co., Ltd.XW00000020Electron microscopy Scan
Ethanol Sinopharm Chimical Reagant Co., Ltd.10009218Hematoxylin-eosin staining 
GraphPad Prism 8 GraphPad Software, Inc. GraphPad Prism 8 Statistical analysis
H2O2 Sinopharm Chimical Reagant Co., Ltd.011092708Immunohistochemical assay
Image-Pro Plus 6.0Media Cybernetics Co., Ltd. USAImage-Pro Plus 6.0Immunohistochemical assay
Paraformaldehyde Sinopharm Chimical Reagant Co., Ltd.20230408Hematoxylin-eosin staining  Immunohistochemical assay
PBSZSGB-Bio,ChinaZLI-9062Electron microscopy Scan
Transmission electron microscopeJAPAN ELECTRON OPTICS LABORATORY CO., LTDJEM-1400Flash, JapanElectron microscopy Scan
Treadmill for Rat & MouseBioMed Easy Technologies Co., Ltd  CHINASA101Performed exhaustive exercise on the treadmill
xyleneSinopharm Chimical Reagant Co., Ltd.10023418Hematoxylin-eosin staining 

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Tags

Tendon Bone JunctionAcute Exhaustive ExerciseUltrastructural ChangesEndoplasmic Reticulum StressRat ModelHematoxylin Eosin StainingElectron MicroscopyImmunohistochemistryChondrocyte UltrastructureCollagen Fiber Changes

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