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Advanced glycation end products impair the repair of injured tendon: a study in rats

BMC Musculoskeletal Disorders volume 25, Article number: 700 (2024) Cite this article

Abstract

Background

The AGEs levels in tissues of diabetics and elderly tend to be higher than in normal individuals. This study aims to determine the effects of AGEs on Achilles tendon repair.

Materials and methods

Thirty-six male eight-week-old Sprague Dawley rats were selected in this study. The rats were randomly divided into two experimental groups and a control group after the transection of the Achilles tendon. During the tendon repair, the experimental groups were injected around the Achilles tendon with 350mmol/L (low dose group) and 1000mmol/L (high dose group) D-ribose 0.2 ml respectively to increase the AGEs level, while in the control group were given the same amount of PBS. The injections were given twice a week for six weeks. Collagen-I, TNF-α, and IL-6 expression in the healed Achilles tendon was assessed. Additionally, macroscopic, pathological, and biomechanical evaluations of Achilles tendon repair were conducted.

Results

The repaired Achilles tendons in the high dose group showed severe swelling and distinctive adhesions. The histological score went up with the increase of the AGEs in the Achilles tendon (p<0.001). TNF- α and IL-6 in the Achilles tendon increased (p<0.001, p<0.001), and the production of collagen-I decreased with the accumulation of AGEs in the repaired Achilles tendon (p<0.001). The tensile strength of Achilles tendon in the high dose group was impaired significantly.

Conclusion

In current study, the compromised tendon repair model induced by AGEs was successfully established in rat. The study demonstrated that AGEs significantly impair Achilles tendon repair.

Peer Review reports

Introduction

Diabetic patients are almost four times more likely to develop tendon injury than non-diabetic patients, especially in the lower extremities [1]. Achilles tendon repair is a long-term process that generally takes several months to restore the continuity of the tendon fibers and mechanical strength [2,3,4]. It has previously been observed that this process is usually abnormal when patients have diabetes mellitus [5, 6]. As reported, it could lead to a compromised tendon repair and increase peritendinous adhesion, which eventually causes pain, disability, mobility impairment, and reduced quality of life for patients [1, 7, 8]. Compromised tendon healing often indicates insufficient collagen fiber generation at the healing site and inadequate restoration of biomechanical properties, leading to suboptimal treatment outcomes. Therefore, it is essential to investigate the mechanisms responsible for impaired Achilles tendon repair due to diabetes, thus providing direction for clinical interventions.

Advanced glycation end products (AGEs) are a heterogeneous compound slowly formed in the environment of hyperglycemia [9, 10]. It is well known that diabetes can cause overproduction of AGEs and damage to physiological functions in tissues [11, 12]. Thus far, several studies have shown that the continuous progress of the course of diabetic patients will cause a large amount of AGEs accumulated in their Achilles tendon [13, 14]. However, it is still not known whether AGEs prolong the repair process, interfere with the recovery of biomechanical properties and lead to a higher rate of re-rupture during the repair stage of the Achilles tendon.

As reported, AGEs can exacerbate the degradation of physiological functions in the course of diabetes by increasing oxidative stress and inflammation [15,16,17]. The combination of AGEs and the specific receptor of AGEs (RAGE) could trigger the oxidative responses and cytokine cascade through the phosphorylation of mitogen-activated protein kinase p38 and the activation of NF-κB [18,19,20]. This intracellular signal conduction may cause cell apoptosis and disorder the metabolism [15]. Tendon fibroblast plays a leading role in tendon development, adaptation, and repair [21, 22]. To participate in tendon repair, they need to migrate into the injury, proliferate and form new extracellular matrix [23, 24]. Although the role of AGEs in tendon repair has not been well described, a large number of studies have shown that it seriously affects the proliferation of tendon fibroblasts [25, 26]. Furthermore, Li et al. [27] have previously revealed that AGEs also induce apoptosis of periodontal ligament fibroblasts. Patel et al. [28, 29] have found that AGEs accumulation limits fibroblast migration and has a dramatic impact on tenocyte mitochondrial respiration. Therefore, we could speculate that AGEs accumulation is a possible reason contributing to compromised Achilles tendon repair in patients with diabetes.

It is necessary to know whether the AGEs interfere with the normal tendon repair process and lead to the damage of biomechanical properties of the Achilles tendon and the increase of peritendinous adhesion. In clinical practice, the study results may have potential guiding value for doctors to determine the treatment and intervention measures. In order to avoid the interaction of various factors in the course of diabetes, a rat model of compromised tendon repair induced by AGEs with non-diabetic conditions is developed in this study. The study aims to determine the effects of AGEs on biomechanics and histology of Achilles tendon repair based on the model.

Materials and methods

Animals

This study was approved by the local ethics committee (D2022-118). A total of 36 adult male Sprague-Dawley rats [mean weight (g), 315 ± 10.6 g; age (months), eight months] were used. The rats were purchased from Laboratory Animal Center of Lanzhou University. The rats were exposed to standard 12 h light and 12 h dark cycle, and the room temperature (℃) was maintained to 22 ℃. Animals have access to water ad libitum and are fed a standard laboratory chow. The rats were randomly divided into two experimental groups and a control group.

Surgical technique

By referring previous study [30], the rats were anesthetized with an intraperitoneal injection of 0.3% sodium pentobarbital (50 mg/kg). After the rats were fully unconscious, the hair on the skin of both legs was shaved. After the posterior limbs were prepped with iodine, a sharp longitudinal incision was made to expose the Achilles tendon from its origin at the gastrocnemius muscle to the insertion of the calcaneus. Achilles tendons were transected at 5 mm above its insertion into the calcaneus. The tendon was repaired with 5.0 absorbable suture (Maxon, Coividien Inc., USA) by modified Kessler technique; the skin was sutured with 3.0 polypropylene (Propilen, Dogsan Inc., Turkey). As reported, the high reactivity of D-ribose has led to its widespread use as a glycating reagent for research in AGEs-related diseases [30,31,32]. This study referred to the study of Ozawa et al. [30] to determining the dosing strategy for D-ribose (mmol/L). After the operation, animals were allowed to move freely. PBS solution, 350 mmol/L d-ribose (low dose group) and 1000 mmol/L d-ribose (high dose group) were administered to the Achilles tendon by injection twice a week, and each injection is 0.2 ml.

Macroscopic evaluation and peritendinous adhesion scoring

After six weeks of continuous injections followed by a one-week rest period, the surgical site of the rats was reopened under anesthesia. The macroscopical scoring system designed by Stoll et al. [33] was used to assess the tendon repair. The degree of peritendinous adhesion grade was evaluated by the adhesion scoring system based on the surgical findings described by Ishiyama et al. [34]. The specific scoring methods were as follows: grade 1, no adhesion; grade 2, adhesion area can be separated by blunt dissection alone; grade 3, adhesion area ≤ 50% which requires sharp dissection for separation; grade 4, adhesion area of 51–97.5% which requires sharp dissection for separation; and grade 5, adhesion area of > 97.5% requiring sharp dissection for separation.

Biochemical biomarkers measurement

After harvesting the Achilles tendon specimens, the rats were sacrificed by cervical dislocation. The tendons were flash-frozen in liquid nitrogen and stored at − 80 °C. The tissue homogenate was prepared and centrifuged (r/min) at 3000 r/min for 5 min, and the supernatants were stored at − 80 °C. Total protein was determined using a BCA protein assay kit (Sigma-Aldrich, MO, USA). The levels of samples’ AGEs (Sigma-Aldrich, MO, USA), TNF-α (Sigma-Aldrich, MO, USA), IL-6 (Sigma-Aldrich, MO, USA) and collagen-I (Sigma-Aldrich, MO, USA) were quantified using the enzyme linked immunosorbent assay (ELISA) detection kit according to the manufacturer’s instructions. Each sample was run in triplicate and data were normalized to total protein.

Histological examination

The histological specimens of Achilles tendon tissue were taken from the surgical site. The 4% buffered formalin (pH 7.4) was used to fix the Achilles tendon tissues for 72 h. The specimens were embedded in paraffin. Serial sections of 5 μm were mounted onto glass slides for histopathological analysis. The histopathological sections were stained using hematoxylin and eosin (HE). CaseViewer software (version 2.4, HUNGARY) was employed to assist in the analysis of histopathological sections. Movin semi-quantitative grading scale, which considers fiber structure, fiber arrangement, rounding of the nuclei, regional variations in cellularity, increased vascularity, decreased collagen stainability and hyalinization, was used to evaluate the Achilles tendon repair [35]. Each variable is scored from 0 to 3 (0 normal, 1 slightly abnormal, 2 abnormal and 3 markedly abnormal), and a pathological score was calculated from the sum of these scores [36].

Biomechanical testing

The tendons used in the biomechanical experiments were not fixed with paraformaldehyde. Before testing, the tendons were thawed in saline at 4 ℃ for 24 h. The cross-sectional area (mm2) of the repaired place of the Achilles tendon was measured with a precision caliper (DL91150, Deli group, China). During the tensile test, both ends of the tendon were fixed on the material testing system (AG–X 10 kN, Shimadzu, Japan), respectively. Both ends of the Achilles tendon were wrapped in gauze to prevent slippage. Before the testing, each tendon was subjected to a pre-tension of 10 N at 1 Hz for 10 min to eliminate tendon viscoelasticity. Uniaxial tensile test with a tensile speed of 5 mm/s was used in present study. The experiment was terminated when the Achilles tendon was completely broken at the repaired site. The outcomes were recorded using Trapezium X software (Shimadzu, Japan), including the ultimate load (N), the yield load (N) and tensile stiffness (N/mm).

Statistical analysis

In this study, SPSS statistical software (version 26.0, Inc., Chicago, IL, USA) was used to analyze the experimental data, and the results were expressed as mean ± standard deviation. Shapiro-Wilk test is used to measure whether the continuous variable data conforms to the positive distribution. One-way analysis of variance was used to compare the high dose group, low dose group and PBS group, P < 0.05 was considered statistically significant. The Kruskal-Wallis test was used to compare the histological scores, adhesion scores, and macroscopic scores among the three groups. The F test function of the G*Power software (version 3.1.9, Heinrich Heine University, Düsseldorf, Germany) was used to calculate the minimum sample size (ANOVA: fixed effects, omnibus, one-way) (1-β err prob = 0.8; α = 0.05) for biomechanical experiment. Based on the pre-experimental data of the ultimate load in the high dose group, low dose group and PBS group, the calculated minimum sample size needed for the biomechanical experiment was nine. In each group, ten Achilles tendons were used in biomechanical experiments in this study.

Results

The conditions of animals

A total of 36 SD rats were included in this experiment, and there was no death after the operation. No clinical symptoms of drug toxicity were found in all the experimental rats. During the study period, there was no statistical difference in weight among the three groups.

AGEs levels in Achilles Tendon increased by injecting D-Ribose

The level of AGEs in the Achilles tendon increased with the increase of D-ribose concentration (p < 0.001). Compared with the PBS group, the content of AGEs in the high dose group and the low dose group was significantly higher (p < 0.001, p = 0.001). The content of AGEs in the high dose group was significantly higher than in the low dose group (p < 0.001) (Fig. 1).

Fig. 1
figure 1

AGEs expression during the Achilles tendon repair in different groups. ***p<0.001, ****p < 0.0001

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Macroscopic findings

The surface of the Achilles tendon in the PBS group was smooth, and the color showed pearl white, the texture of the Achilles tendon was soft and elastic. Less tissue adhesion could be observed in the operation site, and it was easy to be separated. With regard to the low dose group, the color of the Achilles tendon had yellowed notably. In the high dose group, the color of the Achilles tendon was grayish yellow or gray. The texture of the tendon was hard and tough, the boundary between the tendon and surrounding tissue was unclear, and the adhesion was difficult to separate (Fig. 2). The macroscopic score of Achilles tendon repair was significantly reduced with the AGEs accumulated (p < 0.001), and the adhesion score was increased with the AGEs accumulated (p < 0.001) (Fig. 3).

Fig. 2
figure 2

Macroscopic observation in different groups

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Fig. 3
figure 3

Macroscopic score and specific adhesion index in high dose group, low dose group and PBS group. *p < 0.05, **p < 0.01, ****p < 0.0001

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Histological examination

The pathological section is shown in Fig. 4. In the PBS group, a large amount of collagen fiber filling could be seen under light microscopy, the fibers were arranged closely and linearly, a few inflammatory cells infiltrated and capillaries were rare.

Fig. 4
figure 4

Pathological sections of the Achilles tendon in high dose group, low dose group and PBS group (HE staining, 200x). Clusters of capillaries are indicated by a black arrow. The blue box highlights the severely disrupted arrangement of collagen fibers, with most tenocytes displaying oval nuclei. In the green box, collagen fibers are neatly organized, with numerous tenocytes exhibiting long, spindle-shaped nuclei arranged in linear fashion. The red box shows an improvement in the arrangement of collagen fibers compared to the high dose group

Full size image

For the low dose group, a large number of collagen fibers were filled, the arrangement of collagen fibers was relatively loose and disordered compared with the PBS group. Most of the tenocytes in the stroma were fusiform, and the distribution regularity of tenocytes was relatively worse compared with the PBS group.

For the high dose group, most of the collagen was crimped seriously, the shape was irregular, and the arrangement was disordered. Most of the tenocytes in the stroma were quasi-round or spindle-shaped, and the order of tenocytes was messy.

The comparison of Movin semi-quantitative score among different groups is shown in Fig. 5. The high dose group has the highest score compared with the low dose group and the PBS group (p < 0.001), and the low dose group has a higher score than the PBS group (p < 0.05).

Fig. 5
figure 5

Improved Movin semi-quantitative score to evaluate the tendon repair in high dose group, low dose group and PBS group. *p < 0.05, ****p < 0.0001

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Elisa test

The content of collagen-I, TNF-α, IL-6 of the repaired Achilles tendon were quantified by Elisa detection, and the results were shown in Fig. 6. The content of collagen-I decreased with the AGEs increased in the Achilles tendon (p < 0.001). With the enhancement of AGEs level, the expression of TNF-α and IL-6 in the repaired Achilles tendon increased dose-dependently (for TNF-α: p < 0.001, for IL-6: p < 0.001). The maximum effects were induced by high-dosage (1000 mmol/l d-ribose) group.

Fig. 6
figure 6

Elisa experiment results in high dose group, low dose group and PBS group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Biomechanical experiment

With the enhancement of the AGEs level, the cross-sectional area at the repair site of the Achilles tendon was reduced significantly (p < 0.001). The repair site of the Achilles tendon in the high dose group showed the smallest cross-sectional area. The cross-sectional area in the low dose group was smaller than in the PBS group (p = 0.046). The ultimate load and yield load of the repaired Achilles tendon decreased obviously with the enhancement of AGEs level (p = 0.001). The stiffness of the repaired Achilles tendon in the high dose group was lower than that in the PBS group (p = 0.04). There was no difference in the stiffness of the repaired Achilles tendon between low dose group and PBS group (Table 1).

Table 1 Biomechanical parameters of different groups
Full size table

Discussion

Previous studies have shown that D-ribose is the most active reducing sugar in protein glycosylation and could rapidly produce AGEs in vitro and in vivo [37, 38]. Yu et al. [31] reviewed the biological functions, metabolism, and cytotoxicity of D-ribose, reporting that its cytotoxic effects on cells are primarily associated with AGEs. Therefore, D-ribose is widely used as a glycating reagent for studies in AGEs-related diseases [39]. In present study, the repair model of the ruptured Achilles tendon in rats is successfully established in non-diabetic conditions. The AGEs content in the Achilles tendon is significantly increased by injecting D-ribose around the Achilles tendon in rats. The results show that when the AGEs in the Achilles tendon increase during the repair, the quality of Achilles tendon repair is compromised, the biomechanical properties are insufficient, and the adhesion around the tendon is increased. These conclusions have provided guiding directions for the treatment and future studies of injured Achilles tendons in patients with diabetes.

The proliferation of tendon fibroblasts is an essential part of tendon development, healing and adaptation [40]. The slow or unable proliferation of fibroblasts is an important reason for the development of tendon disease and the delay of tendon growth and repair [41]. Several studies have reported that the accumulation of AGEs could significantly impair the proliferation of fibroblasts [26, 28, 29]. Patel et al. [26] have found that when fibroblasts were treated with AGEs, the proliferation gene markers Mybl2 and Pcna decreased significantly [29]. Based on these genetic and cellular studies, we could speculate that AGEs may be a potential reason for the compromised tendon repair. However, to date, there is no direct pathological and mechanical evidence to prove it. Besides, it is also not known whether the effect of AGEs on Achilles tendon repair has reached the point where clinical intervention and prevention are needed.

In this study, the effects of AGEs on the macroscopic, pathological, metabolic and biomechanical evaluation of Achilles tendon healing are further discussed. A previous in vitro study immersed the rat tail tendon in AGEs and found that the tendon texture became stiff and rigid, and the color of the Achilles tendon was changed from pearl white to gray-yellow or gray-white [42]. The Achilles tendon in current study has shown the same texture and color in high dose group, which is consistent with clinical experience with tissues in diabetic patients [43]. Furthermore, severe swelling, distinctive adhesions with the paratenon, fascia and subcutaneous tissue are visible in the repaired Achilles tendons of the high dose group. These facts may inhibit their frictionless sliding in the course of motion, thus damaging their function [44]. This may be related to the fact that AGEs can age and oxygenize the collagen and modify the collagen surface, which is known to affect cell-matrix interactions in a manner leading to exacerbated inflammation and may inhibit tendon repair [45, 46].

The precise linear arrangement of collagen fibers is correlated with the integrity of the tissue, and the arrangement of collagen fibers along the direction of tension determines the mechanical function of the tendon [47]. The pathological section of HE in present study has shown that compared with the PBS group, most of the collagen fibers in high dose group are crimped seriously, the shape is irregular, and the arrangement is disordered. In general, tenocytes are embedded between extracellular matrix fiber bundles, and their uniaxial arrangement has to be re-established during the tendon repair stage [47]. The results of this study have shown that the tenocytes at the Achilles tendon repair site in the high dose group were more immature, the tenocytes were arranged more randomly and irregularly. The histological score has gone up with the increase of AGEs in the Achilles tendon, which indicates that AGEs have caused impaired tendon repair, and the mechanical function of the tendon might also be affected.

The biomechanical properties of a tendon also depend on its composition, and the most abundant component in tendon tissue is collagen fibrin [48]. Among them, collagen-I fibrin is a strong fibrin subtype, accounting for about 95% of the total collagen in the extracellular matrix of normal tendons [49]. In the stage of remodeling and maturation of Achilles tendon repair, collagen-I mainly establishes the recovery of tendon mechanical strength [50]. Patel et al. [26]. have noted that AGEs-treated fibroblasts could decrease the expression of collagen-I. Likewise, the results of this study have also proved that the levels of collagen-I in repaired Achilles tendons in the high dose group were much lower than those in the PBS group. Actually, the repair of tendon rupture is strongly affected by post-traumatic inflammation, in which TNF-α and IL-6 may play a key role [51, 52]. In addition, TNF-α and IL-6 have also been shown to down-regulate the expression of collagen-I [47]. A study by Raghav et al. [46] has shown that AGEs can significantly up-regulate the levels of TNF- α and IL-6 in the tissues of diabetic patients. In present study, the contents of TNF- α and IL-6 in the Achilles tendon treated with AGEs are significantly increased. The compromised repair of the Achilles tendon and the increase of peritendinous Adhesion caused by AGEs might be related to the oxidative stress and inflammatory response induced by the AGE-RAGE axis [45].

In order to determine whether the compromised tendon repair caused by AGEs is worthy of proceeding clinical intervention and further study in the future, additional biomechanical evaluation is still needed. In current study, AGEs have been found to reduce the cross-sectional area at the repair site of the Achilles tendon, which is probably related to the decrease of collagen production. In the process of tendon repair, the recovery of tensile strength mainly depends on collagen-I fibers [53]. In agreement with the measured data of collagen-I content, the ultimate load and yield load of the repaired Achilles tendon are decreased significantly with the AGEs increased in the tendon. Furthermore, the tendon stiffness in the high dose group was significantly lower than that in the PBS group, and tendons with reduced stiffness may result in less efficient force transfer.

Overall, AGEs could be a crucial factor to explain the higher disability rate and re-rupture rate of injured Achilles tendons in patients with diabetes. This indicates the necessity for targeted interventions to mitigate the effects of AGEs on tendon healing in diabetic individuals with Achilles tendon injuries. More importantly, diabetes is not the singular reason for the accumulation of AGEs. Skovgaard et al. [54] have found that an AGEs-rich diet is an important source of AGEs accumulation in tendon tissue, especially in the Achilles tendon. Besides, related studies have also suggested that AGEs accumulate in tendons with aging [55, 56]. Therefore, clinicians and researchers should pay enough attention to the effect of AGEs on Achilles tendon repair. To date, using AGE-RAGE axis blockers and avoiding AGEs-rich diets may be potential ways to avoid AGEs-related complications. Besides, prior study has established that the activation of PPAR-γ can reduce the production of inflammatory factors provoked by AGEs across diverse cell categories [57]. Furthermore, it possesses the ability to reduce the levels of RAGE expression and hinder its subsequent signal transmission, thus mitigating oxidative stress and inflammation within tissues [58]. Xu et al. [59] demonstrated that pioglitazone enhanced cellular autophagy and mitigated the apoptotic effects induced by AGEs in tendon-derived stem cells. Future studies could integrate the findings of this study with those of previous studies to further investigate strategies for mitigating the effects of AGEs on Achilles tendon healing.

There are some limitations in this study. (1) This study was carried out based on the model of acute rupture of Achilles tendon in rats, while the rupture of Achilles tendon in diabetic patients may result from a long-term injury process. (2) In this study, the ankle joint was not fixed after the operation, but clinically, the ankle joint fixation after Achilles tendon repair was routine. (3) Incorporating gait analysis can further evaluate the mobility and pain of rat joints, providing more references for clinical reference [60]. This study did not perform functional tests such as Achilles functional index by walking track analysis. However, the significant adhesion of the Achilles tendon in the high dose group indicates a potential impact of AGEs on rat mobility [44]. (4) Utilizing D-ribose to induce AGEs may not completely simulate the impact of diabetes on tissues. This may limit the interpretation and clinical application of the results.

Conclusions

In current study, the compromised tendon repair model induced by AGEs was successfully established in rat. This study found that AGEs can impair the repair of the Achilles tendon.

Data availability

The dataset supporting the conclusions of this article is available at our institution contacting the corresponding author.

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Acknowledgements

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Funding

This study was supported by the Key Clinical Support Specialty of Hongkou District Health Commission of Shanghai—Geriatric Medicine (HKLCFC202412), Shanghai Hongkou District Health Committee of Medical Scientific Research Subject Major Projects (Hongwei-2301-03) and Hospital Foundation of Shanghai Fourth People’s Hospital Affiliated to Tongji University (sykyqd06401).

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Authors and Affiliations

  1. Department of Geriatrics, Shanghai Fourth People’s Hospital Affiliated to Tongji University, No.1279 Sanmen Road, Shanghai, 200434, China

    Juan Yang & Ling Yang

  2. The Second Clinical Medical College, Lanzhou University, No. 82 Cuiyingmen, Chengguan District, Lanzhou City, 730030, Gansu Province, China

    Jirui He

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Contributions

J.Y. Conceptualization, Methodology, Formal analysis, Writing-original draft. J.H. Conceptualization, Methodology, Formal analysis, Visualization. L.Y. Conceptualization, Methodology, Visualization, Resources, Supervision. All authors read and approved the final manuscript.

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Correspondence to Jirui He or Ling Yang.

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All methods were carried out in accordance with relevant guidelines and regulations. This study is reported in accordance with the ARRIVE guidelines. The animal study protocol was approved by the Institutional Review Board of Lanzhou university second hospital (D2022-118, 14 February 2022).

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Yang, J., He, J. & Yang, L. Advanced glycation end products impair the repair of injured tendon: a study in rats. BMC Musculoskelet Disord 25, 700 (2024). https://doi.org/10.1186/s12891-024-07760-z

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Keywords

BMC Musculoskeletal Disorders

ISSN: 1471-2474

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