Injuries to joints or surrounding tissues and insufficient movement caused by inflammation or therapeutic immobilization may lead to stress shielding of the tendons, resulting in structural and biomechanical changes.1,2 Whenever feasible, tendon repair should be done as early as possible, although immobilization may still be required. Therefore, it is vitally important to investigate an effective clinical intervention to prevent or alleviate the contracture without changing the state of immobilization.
The molecular mechanisms of tendon contracture after stress shielding are unclear. Previous studies reported by Uchida et al3 and Wang et al4 showed that interleukin-1 (IL-1) secreted by tendon cells played an important role by modulating the release of other cytokines. Interleukin-1 was significantly increased in stress-shielded tendons. Thampatty et al5 and Yang et al6 reported that IL-1β strongly induced the mRNA expression levels of metalloproteinases (MMP)-1 and -3 that degraded collagen and extracellular matrix, suggesting that IL-1 aggravated the tendon degeneration by modulating MMPs. Therefore, antagonizing the effects of IL-1 may be a possible solution to inhibit tendon degrading.
Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring competitive inhibitor of IL-1–induced proinflammatory activity. The IL-1Ra gene is polymorphic, resulting in quantitative differences in IL-1Ra and IL-1β production. With similar affinity for the type I receptors of IL-1α and IL-1β, it is unable to activate the receptors and downstream kinases; hence, it acts as an antagonist against IL-1. Usually, the dynamic equilibrium between IL-1 and IL-1Ra sustains the normal physiology.7 Any abnormal increase of IL-1 or deficiency of endogenous IL-1Ra may result in harmful effects. Previous studies reported that exogenous IL-1Ra was used to treat diseases related to an abnormal increase of IL-1, such as rheumatoid arthritis,8–12 osteoarthritis,13 and intervertebral disk degeneration.14 Interleukin-1 receptor antagonist also alleviates the inflammation in Achilles tendons resulting from chronic injuries.15
However, few studies report the beneficial effects of IL-1Ra on tendon degradation. Miyatake et al16 observed the effects of IL-1Ra injected between the patellar tendon and the infrapatellar fat pad on the mechanical properties of the stress-shielded patellar tendon. They reported that IL-1Ra improved the tangent modulus and the tensile strength of the stress-shielded patellar tendons.16 This suggests that IL-1Ra likely interfered with the occurrence and development of tendon degradation, warranting further investigation. Therefore, the current study investigated the physiological mechanism of action of IL-1Ra by determining the morphological changes of the Achilles tendons and metabolic turnover of collagens and MMPs in stress-shielded tendon.
Materials and Methods
Experimental Animals and Grouping
This study was approved by the Institutional Review Board of the Affiliated Sixth People’s Hospital of Shanghai Jiaotong University, Shanghai, China.
Forty male Sprague-Dawley rats aged 8 to 10 weeks and weighing 210±10 g were provided by the animal laboratory of Shanghai Sixth People’s Hospital. Rats were divided randomly into the 2- and 4-week phosphate-buffered saline (PBS) groups (2wPBS, 4wPBS) and the 2- and 4-week IL-1Ra groups (2wIL-1Ra, 4wIL-1Ra). The Achilles tendon of each rat’s left hind limb was used as the model, with 5 of the contralateral hind limbs randomly selected from the 2wPBS group and 5 from the 2wIL-1Ra group serving as the normal controls.
A 2-0 tendon suture (Pudong Jinhuan Medical Products Co, Ltd, Shanghai, China) was inserted through the tibiofibular fork of the left hind limb and placed between the calcaneus and the plantar aponeurosis. The suture was tightened to the maximum to fix the talocrural joint in the equinus position. The sciatic nerve was exposed and transected proximal to its bifurcation into the common peroneal and tibial nerves. Penicillin was given within 3 days postoperatively to prevent infection (400,000 IU/day, intraperitoneal injection). The rats were fed for 3 days in a box and then transferred into cages, where they moved about freely.4,17
Five μg of human recombinant IL-1Ra combined with 0.1 mL of PBS18 or 0.1 mL of PBS alone were injected around the Achilles tendons in the IL-1Ra and PBS groups. The rats in the 4-week groups were administered 4 doses on day 1 after modeling and then 1, 2, and 3 weeks later, whereas the rats in 2-week groups were given 2 doses on day 1 after modeling and then 1 week later.
Transmission Electron Microscopy
All rats were sacrificed by an overdose of ketamine. Passive movement was performed to determine whether the left ankle was fixed in the equinus position. Rats were excluded if the left ankle moved passively. No fixation failure was encountered. Two samples of rat tissue from each group were randomly chosen for transmission electron microscopy examination. Each sample was cut to a size of 1×1×2 mm and fixed in 2% glutaraldehyde. Following fixation, Achilles tendon tissues underwent dehydration, replacement, soaking, embedding, sectioning by LKB-V ultramicrotome (Pharmacia LKB Biotechnology AB, Uppsala, Sweden), and staining with lead citrate. A CM120 transmission electron microscope (Royal Philips Electronics, Amsterdam, The Netherlands) provided by Shanghai Jiaotong University School of Medicine was used for observation.
Enzyme-linked Immunosorbent Assay
Collagens I and III and tissue inhibitors of metalloproteinase (TIMP)-1 levels were measured by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). The MMP-1 and -3 levels were measured with avidin-biotin complex ELISA. The supernatant was obtained after Achilles tendon tissues underwent grinding, lysis, and centrifugation. One hundred μl of the supernatant was added to the antibody-coated ELISA plate. After complete reaction, the first antibody, the enzyme-linked second antibody, substrate, and stop buffer were added and washed sequentially and the plates were read at 492 nm. The concentration was determined using the standard curve.
Enzyme-linked immunosorbent assay kits were provided by R&D Systems, Inc. (Minneapolis, Minnesota). Phenylmethylsulfonyl fluoride, protease inhibitor, and radioimmunoprecipitation assay lysis buffer were provided by Beyotime (Shanghai, China).
The data were analyzed using SPSS version 13.0 software (SPSS Inc, Chicago, Illinois). Results were reported as mean±SD. The t test was used to assess the differences between groups. Significance was set at P=.05.
Gross Morphology of the Achilles Tendons
The Achilles tendons in the control group had smooth, gleaming surfaces without adherence to other tissues, and those in the PBS groups were thicker and bleaker with extensive adherence to surrounding tissues. The Achilles tendons in the IL-1Ra groups were thicker and bleaker than the controls, but thinner and smoother than those in the PBS groups, with partial adherence to surrounding tissues (Figure 1).
Figure 1: Gross morphology of the Achilles tendons. Sample A, 4-week phosphate-buffered saline (PBS) group; sample B, 4-week interleukin-1 receptor antagonist (IL-1 Ra) group; sample E, control group (A). Sample C, 2-week PBS group; sample D, 2-week IL-1Ra group; sample E, control group (B).
Transmission Electron Microscopy Observation
Comparison of PBS Groups and Control Group. Collagen fibrils in the normal control group were arranged regularly, densely, and similarly, with cross-sectional images showing normal collagen fibrils with different diameters (Figures 2A, B). In comparison, collagen fibrils in the 2wPBS group were irregularly arranged, with a curved and loose structure (Figures 2C, D). Those in the 4wPBS group were completely disordered and had a staggered, distorted broken structure with an apparent increase in the number of small-diameter fibrils (Figures 2G, H).
Figure 2: Lead citrate electronic staining (×17,500). Longitudinal (A) and transverse (B) transmission electron microscopy (TEM) (scale, 2000 nm) imaging of the control group. Longitudinal (C) and transverse (D) TEM imaging of the 2-week phosphate-buffered saline (PBS) group. Longitudinal (E) and transverse (F) TEM imaging of the 2-week interleukin-1 receptor antagonist (IL-1 Ra) group. Longitudinal (G) and transverse (H) TEM imaging of the 4-week PBS group. Longitudinal (I) and transverse (J) TEM imaging of the 4-week IL-1Ra group.
Comparison of IL-1Ra Groups and Control Group. Compared with the normal collagen fibrils (Figures 2A, B), those in the IL-1Ra groups were arranged loosely and similarly, and the fibrils with different diameters were well distributed (Figures 2E, F, I, J).
Comparison of IL-1Ra Groups and PBS Groups. The collagen fibrils in the 2wIL-1Ra group were arranged more regularly in the longitudinal section than those in the 2wPBS group (Figures 2C, E), and those in the 4wIL-1Ra group were arranged more regularly in both longitudinal and transverse sections compared with the 4wPBS group (Figures 2G–J), which indicated that IL-1Ra interfered with the deterioration of collagen fibrils after stress shielding.
Levels of Collagen I and III
The collagen I level in the 2wPBS and the 2wIL-1Ra groups increased significantly compared with those in the control group (2wPBS, 633.12±64.78 vs control, 402.99±45.45, P<.001; 2wIL-1Ra, 672.27±66.79 vs control, 402.99±45.45, P<.001). The collagen I level in the 4wPBS group was significantly less than that in the control group (327.18±69.18 vs 402.99±45.45, respectively; P=.023), whereas that in the 4wIL-1Ra group was close to that in the control group (423.30±75.56 vs 402.99±45.45, respectively; P=.506), with significant differences between the 4wPBS group and the 4wIL-1Ra group (327.18±69.18 vs 423.30±75.56, respectively, P=.031) (Figure 3A).
Figure 3: Enzyme-linked immunosorbent assay test of collagen I (A), collagen III (B), metalloproteinase (MMP)-1 (C), MMP-3 (D), and tissue inhibitors of metalloproteinase (TIMP)-1 (E) (n=8). Abbreviations: IL-1ra, interleukin-1 receptor antagonist; PBS, phosphate-buffered saline. *P<.05 (treated groups vs normal control group); ΔP<.05 (IL-1Ra groups vs PBS groups); #P<.05 (4-week groups vs 2-week groups).
The collagen III level in the PBS groups and the IL-1Ra groups increased significantly compared with that in the normal group (P<.001), and those in the 4-week groups was less than those in the 2-week groups (P<.001). No significant difference in collagen III content existed between the PBS and IL-1Ra groups (Figure 3B).
Levels of MMP-1 and -3 and TIMP-1. After 2 weeks of stress shielding, MMP-1 levels in the PBS and the IL-1Ra groups increased dramatically, with a statistically significant difference (352.37±29.17 vs 283.01±11.33, respectively; P<.001). At 4 weeks, MMP-1 decreased but was still higher compared with the control group, with no significant difference (213.90±38.94 vs 233.93±20.99, respectively; P=.228) (Figure 3C).
The MMP-3 level in the PBS groups was significantly less compared with that in the control group (P<.001) and continued to decrease with time (2wPBS, 39.81±2.10 vs 4wPBS, 36.40±2.49, P=.023). The MMP-3 level in the 2wIL-1Ra group decreased less compared with those in the 2wPBS group (44.71±2.01 vs 39.81±2.10, respectively; P=.001). However, the MMP-3 level increased in the 4wIL-1Ra group (Figure 3D).
The TIMP-1 level continued to increase in both the PBS and the IL-1Ra groups, and no significant difference existed (Figure 3E).
Previous studies indicated a relationship between tendon degradation and the degree of stress shielding.19 The current stress-shielding model eliminates the stress on the Achilles tendon caused by the activities of the gastrocnemius and anterior tibial muscle. At the same time, the cerclage of the ankle limits passive activity, such that the Achilles tendon is in an approximately 100% nonstress environment, which may not be commonly noticed in clinical practice but allows easy detection of a significant change of degradation, eliminating the relative errors caused by different shielding levels.
In the current study, IL-1Ra prevented the morphological deterioration of Achilles tendons after stress shielding and intervened in the metabolic turnover of the collagen I and MMP-1 and -3 enzymes.
Previous studies showed that collagen fibrils were arranged irregularly and small-diameter fibrils significantly increased 3 weeks after stress shielding.4 In the current study, collagen fibrils 2 weeks after stress shielding were arranged irregularly and appeared broken, with significant deterioration in 4 weeks after stress shielding. The IL-1Ra treatment restored the fibrils to normal morphology as seen in the images in longitudinal and transverse planes. Therefore, IL-1Ra was able to successfully prevent the morphological deterioration of Achilles tendons after stress shielding.
Using radioactive labeling studies, Amiel et al20 measured the collagen mass of rabbit’s medial collateral ligament after immobilization and observed the effects of stress shielding on the metabolic turnover of collagen. Nine weeks after immobilization, the total mass of collagen showed no significant change, although the newly synthesized collagen increased by nearly 13% whereas the original collagen decreased by approximately 14%. After 12 weeks, new collagen increased by 1.2%, original collagen decreased significantly to 27.8% and the total collagen mass was also reduced, suggesting that the synthetic ability of collagen decreased and its degrading ability increased with prolonged immobilization.20
Type I collagen is the main component of the tendons. Collagen I and III levels increased at the early stage of stress shielding and decreased over time. At 2 weeks, collagen I increased in the PBS and the IL-1Ra groups. Collagen I decreased much more slowly in the 4wIL-1Ra group than in the 4wPBS group, indicating the important role of IL-1 in the downregulation of collagen I at a later stage instead of the upregulation of collagen I at an early stage, suggesting the ability of IL-1Ra to prevent the downregulation of collagen I. No differences in collagen III levels existed between the PBS and the IL-1Ra groups, indicating that IL-1 and IL-1Ra did not affect collagen III metabolism, consistent with another study.5
Metalloproteinase-1 was reported to cleave collagen I.21,22 Due to the lack of homology of MMP-1 between rodents and humans,23 MMP-1 levels are not measured directly by quantitative polymerase chain reaction. Instead, MMP-13, which has a similar immunohistochemistry and function as MMP-1, was tested. In the denervation-induced stress deprivation model of the patellar tendon in mice, Mori et al24 found that MMP-13 expression dramatically increased up to day 14, followed by a decrease. Similarly, differences in MMP-1 levels between the IL-1Ra and the PBS groups appeared at 2 weeks instead of 4 weeks, indicating that the effects of IL-1 and IL-1Ra on MMP-1 occur during the early stage of stress shielding.
The Mori et al24 study did not report any change in MMP-2 expression and concluded that the modulation of MMP-13 expression was specific. In contrast, the MMP-3 levels continued to significantly decrease after stress shielding. Metalloproteinase-3 catalyzes several substrates, including proteoglycan, laminin, and fibronectin.25 The decline of MMP-3 expression was also found in the broken or degraded Achilles tendon,26–29 although its effects on the matrix metabolism are unknown. A previous study4 showed that IL-1 increased after stress shielding. The current study showed that the decrease of MMP-3 in the stress-deprived tendon was antagonized by IL-1Ra. Therefore, IL-1 inhibited MMP-3 expression, which appears to contradict the results of Thampatty et al.5 The contradiction may be attributed to in vitro experimental conditions. In the study by Thampatty et al,5 the metrocytes were arising from normal Achilles tendons, and the experiment was in vitro.
Tissue inhibitors of metalloproteinase exist in relative equilibrium.30 In the current study, MMP-1 and TIMP-1 increased after stress shielding but asynchronously, with the MMP-1 levels higher at 2 weeks than at 4 weeks, whereas the TIMP-1 levels changed in the opposite way. Interleukin-1β was reportedly unable to enhance TIMP expression,31–33 and in the current study, the level of TIMP-1 was not altered by IL-1Ra. Therefore, the increase of TIMP was not related to IL-1.
Although many studies showed that the diameter of stress-shielded fibrils was reduced, the mechanism is still unclear.4,24,34 Mori et al24 reported that the reduction of the collagen fibril diameter was attributed to the transient increase of MMP-13 expression induced by osteopontin that partially altered collagen assembly. Also, osteopontin was considered to enhance the release of IL-1.35,36 It is reasonable to speculate that IL-1 is required for the reduction of the collagen fibril diameter and IL-1Ra prevented the reduction by inhibiting IL-1β–stimulated effects and regulating MMP levels.
In the current study, the effect of denervation of Achilles tendons on collagen metabolism was unclear. However, it was reported that denervation does not change the ratio of collagen I and III mRNA expression in the extracellular matrix of muscle.37
In the current study, IL-1Ra treatment prevented the morphological deterioration and collagen metabolism of the denervated Achilles tendons after stress shielding, probably by inhibiting the decline of MMP-3 and increasing MMP-1 levels at an early stage, which indicates the important role of IL-1 in degradation of the stress-shielded tendon. This study highlights a potential therapeutic application of IL-1Ra.
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