Few studies emphasize the collagen metabolism-related cytokines and ultrastructure of the completely stress-shielded Achilles tendon. In this study, we used a rat model with complete stress shielding of the Achilles tendon to observe the changes in the ultrastructure of the Achilles tendon and concentration of IL-1 and TGF-β 3 weeks after stress shielding.
The model group comprised 12 male Sprague-Dawley rats. The stress of the Achilles tendon of the left hind limb was shielded through tendon cerclage combined with sciatic nerve transection, and the right served as a normal control. Three weeks later, the ultrastructure of the Achilles tendon was observed under electron microscopy and IL-1 and TGF-β levels were determined by enzyme-linked immunosorbent assay. Compared with the control side, collagen fibrils of the shielded Achilles tendons were irregularly arranged and loose. The number of small-diameter collagen fibrils increased significantly with the decrease of the average diameter of collagen fibrils. At the same time, IL-1 concentrations increased significantly in the model group as compared to that in the control group, but no significant difference was found in TGF-β levels.
These results suggest that IL-1 may play an important role in the change of ultrastructure after stress shielding.
Contracture of periarticular tendon tissues is an important cause of stiffness after joint immobilization and is unfavorable for the recovery of joint function. Less immobilization and early joint activity are generally thought to be effective ways for preventing contracture.1 However, immobilization for 2 to 4 weeks is sometimes necessary for treatment and tissue healing. Although sometimes no external fixation restricts joint activities, joint stiffness can occur owing to the lack of full range of joint motion caused by pain, swelling, or psychological reasons. Therefore, there is a need to explore additional methods for preventing contracture.
Researchers began to study the effects of immobilization on joints and tendons in the 1960s,2 mostly using animal models of simple joint immobilization.3-8 Since the isometric contraction of periarticular muscles produces stress on the tendons, simple joint immobilization cannot completely shield tendons from stress. Hayashi9 reported that stress deprivation of 70% induced minimal and insignificant decrease in the structural strength (maximum load) of the patellar tendon, and 100% stress shielding decreased the strength of the tendon markedly. Majima et al10 found that the mechanical properties of rabbit patellar tendon after completely stress shielding decreased more significantly than after partially stress shielding. Thus, we speculate that marked changes of the ultrastructure and collagen metabolism-related cytokines would be detected if we use a complete stress deprivation animal model.
Rabbit and rat models with completely stress-shielded patellar tendons have been designed by Yamamoto et al11 and Uchida et al12 according to the anatomical features of the patellapatellar tendontibial complex. In these models, the patellar tendon was released from tension completely by stretching a flexible wire installed between the patella and the tibial tubercle. A review of the literature showed that studies of stress shielding have focused on rabbit or rat patellar tendons, with no research on complete stress shielding of the Achilles tendon. Palmes et al13 developed a new mouse model with the Achilles tendon immobilized through tendon cerclage combined with sciatic nerve transection for the study of tendon healing. In theory, stress shielding with this method may be more complete because the impact of the nerve on tendon stress is eliminated after the sciatic nerve was transected.
Although the biomechanical properties of the stress-shielded tendon changed significantly,10,11,14-16 few studies have focused on the underlying mechanism or influencing factors. Changes in mechanical properties may be attributed to the changes in the ultrastructure,17 which may be related to collagen metabolism. Studies have shown that interleukin-1 (IL-1) and transforming growth factor-β (TGF-β) play an important role in collagen decomposition and synthesis, respectively.18-22 Uchida et al12 found increased IL-1, TGF-β, and tumor necrosis factor expression in stress-shielded rat patellar tendons whose mechanical properties deteriorated.
In the present study, we used a rat model with complete stress shielding of the Achilles tendon to observe the changes in the ultrastructure of the Achilles tendon and concentration of IL-1 and TGF-β 3 weeks after stress shielding.
Materials and Methods
Twelve male Sprague-Dawley rats weighing 180±10 g (aged 6-8 weeks) were provided by the animal laboratory of Shanghai Sixth Peoples Hospital. The Achilles tendon of each rats left hind limb was used as the model, and the contralateral served as the internal control.
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 so as 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. The sciatic nerve of the control side was merely exposed without treatment. Penicillin was given to prevent infection within 3 days postoperatively. The rats were fed for 3 days in a box and then transferred into cages for 3 weeks where they could move about freely.
All rats were sacrificed by an overdose of ketamine. Passive movement was performed to determine if the left ankle was fixed in the equinus position. The rat would be excluded if its left ankle could be moved passively. In this study, no failure to fix was found. Bilateral Achilles tendons of 12 rats were removed. Two pairs of them were randomly chosen for transmission electron microscopy examination. Other specimens were taken for IL-1 and TGF-β determination.
Following fixation, Achilles tendon tissues underwent dehydration, replacement, soaking, embedding, sectioning by LKB-V ultratome, and staining with lead citrate. A Philips CM120 TEM (Philips Healthcare, Best, The Netherlands) was used for observation.
Analysis of Collagen Fibril Diameter
Collagen fibril diameter was measured using Image-Pro Plus 6.0 software (Media Cybernetics Inc, Bethesda, Maryland). The software scale was corrected before selecting the collagen fibrils for maintaining consistency with the scale of the electron microscope, such that the diameter obtained was the actual diameter of the collagen fibrils. Four cross-section images from 2 pairs of tendons were analyzed blindly. Each image was divided into 4 areas. After randomly choosing 1 collagen fibril at the center of 1 area, 200 surrounding collagen fibrils were marked the edge of the cross-section, the diameter was calculated, and the fibrils were classified into 11 scales according to diameter. The average diameters of the collagen fibrils were from a summary of 8 areas in each group.
IL-1 and TGF-β Test
The specimens were tested for IL-1 and TGF-β by enzyme-linked immunosorbent assay after grinding, lysis, and ultracentrifugation at low temperatures. Phenylmethyl sulfonyl fluoride protease inhibitor and radio immunoprecipitation assay lysis buffer were provided by Beyotime Institute of Biotechnology (Jiangsu, China).
The levels of IL-1 and TGF-β are reported as mean±standard deviation. Paired t test was used to assess the differences between the stress-shielded and contralateral tendons. A significance level was set at P=.05.
Collagen fibrils of the shielded Achilles tendons were irregularly arranged, and the number of tendon cells increased in combination with cell shrinkage and mitochondrial swelling (Figure 1). Cross-sectional images of the normal Achilles tendon showed that collagen fibrils with different diameters were well distributed. In the shielded Achilles tendons, however, collagen fibrils with different diameters were distributed unevenly, and the number of small-diameter fibrils increased significantly (Figure 2). Longitudinal section images showed that collagen fibrils had the same arrangement and were denser in normal Achilles tendon, whereas the collagen fibrils of stress-shielded Achilles tendon were disordered and had a staggered, curled, loose structure (Figure 3).
|Figure 1: Number of cells in the Achilles tendon of each study group (×4200; scale, 5000 nm). The number of tendon cells in the model group (right) increased compared to that in the control group (left), combined with cell shrinkage and mitochondrial swelling. |
|Figure 2: Transverse plane imaging of the Achilles tendon in each study group using transmission electron microscopy (×17,500; scale, 2000 nm). In the model group (right), collagen fibrils of different diameters were distributed unevenly, and the number of small-diameter fibrils increased significantly compared to that in the control group (left). |
|Figure 3: Longitudinal plane imaging of the arrangement of collagen fibrils in each study group using transmission electron microscopy (×17,500; scale, 2000 nm). In the model group (right), collagen fibrils were disordered compared to the control group (left). |
Sixteen hundred collagen fibrils were measured in each group. Large-diameter fibrils (>100 nm) accounted for 56.5% of the normal Achilles tendon, and small-diameter fibrils (<100 nm) accounted for 43.5%. The fibrils of stress-shielded Achilles tendons were mainly small-diameter fibrils. The average diameter of collagen fibrils in the model group was significantly decreased compared to that in the control group (45.534±9.398 nm vs 101.705±24.570 nm, respectively; t=30.198; P<.01). The proportion of collagen fibril diameter in normal Achilles tendons and stress-shielded tendons is shown in Figure 4.
|Figure 4: Histogram of the proportion of collagen fibril diameter distribution. |
The results showed that 3 weeks after stress shielding the Achilles tendons, IL-1 concentrations increased significantly in the model group compared to that in the control group. In contrast, no significant difference was observed in TGF-β concentrations between the 2 groups (Table).
A stress-shielding rat Achilles tendon 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 the Achilles tendon is in an approximately 100% non-stress environment. No loosening of the fixation was noted over the 3 weeks of the study, and all the left ankles remained fixed in the equinus position.
Some studies have found that the cross-sectional area of the tendon increases after stress shielding.10,12,13,23 In our study, 3 weeks after stress shielding, the arrangement of collagen fibrils was observed to be significantly disordered and loosely structured under transmission electron microscopy. These findings may partly explain the thickness of the tendon bundle after stress shielding.
By analyzing the collagen diameter, we found that the average diameter of the Achilles tendon collagen fibrils became smaller, and small-diameter fibrils were significantly increased 3 weeks after stress shielding, a finding similar to that reported in Majima et als study17 on the effects of stress shielding on collagen fibril diameter in rabbit patellar tendon. In their study, the average fibril diameter showed significant changes 6 weeks after stress shielding. In our model, however, significant changes were observed 3 weeks after stress shielding.
The increase in small-diameter collagen fibrils may be related to collagen metabolic turnover during stress shielding. Some studies have shown that small-diameter collagen fibrils composed of type III collagen fibrils may be newly synthesized.21 Other studies have reported that an increase in relevant collagenase leads to enhanced catabolism and thus to a smaller diameter of collagen fibrils.17,24 In the present study, collagen fibrils under transmission electron microscopy did not appear aligned, but staged, curled, and segmentalized.
Interleukin-1, a cytokine with various biological activities, is secreted by tendon cells. It is thought to induce catabolic effects in tendon matrix by downregulating type-1 collagen gene expression and upregulating matrix metalloproteinase gene expression.25 Transforming growth factor-β is the main cytokine that can stimulate type I and type III collagen gene transcription, as well as the synthesis of other protein components in the extracellular matrix.26-28 In the present study, we found that IL-1 concentrations increased significantly 3 weeks after stress shielding, a finding consistent with the study of Uchida et al.12 However, no significant changes in TGF-β were observed in our study. Therefore, we speculated that the increase in small fibrils 3 weeks after stress shielding was related primarily to increased IL-1 concentrations and its enhanced function.
Using radioactive labeling studies, Amiel et al29 measured the collagen mass of rabbit 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 a marked increase (approximately 13%) was found in the newly synthesized collagen, along with a decrease (approximately 14%) in the original collagen. After 12 weeks, new collagen increased by only 1.2%, original collagen decreased significantly to 27.8%, and the total collagen mass also reduced, suggesting that the synthetic ability of collagen decreased and its degrading ability increased with prolonged immobilization time. Uchida et al12 compared the increase in IL-1 and TGF-β concentrations in the patellar tendon 2 and 6 weeks after stress shielding and found that at 2 weeks, IL-1 increased significantly compared to TGF-β, and at 6 weeks, TGF-β increased significantly compared to IL-1. These results suggest that the decomposition and synthesis of collagen change after immobilization, which may be related to the degree of stress shielding, stress loss time, and other unknown factors.
A limitation of the present study was the lack of dynamic observation of tendons after immobilization. No significant change in TGF-β was found 3 weeks after stress shielding; however, it is unclear whether the TGF-β concentrations increased significantly during early immobilization (within 3 weeks). In addition, as the sciatic nerves were disconnected in the model group, Achilles tendons were in a denervated state, and local inflammatory or healing response may occur in the surgical site. Although little research was conducted on the effects of neuronal factors on tendon contracture, accumulating evidence has demonstrated that the neuronal system plays an active role in tendon repairing30 through the local expression of neuropeptides31,32 such as substance P, calcitonin gene-related peptide, and galanin. Further investigation is needed to find whether the lack of nerve would interfere with the process of tendon contracture.
Our study found that small-diameter collagen fibrils increased significantly 3 weeks after complete stress shielding. In addition, IL-1 concentrations increased significantly, while TGF-β concentrations did not change significantly. These preliminary findings suggested that the increase in small-diameter collagen fibrils after stress shielding may be closely related to the increase in IL-1 concentrations. Further study is needed to determine the effects of anti-IL-1 treatment, which may provide a new way to prevent the degrading caused by stress deprivation.
- Braddom RL. Physical Medicine and Rehabilitation. 3rd ed. Philadelphia, PA: WB Saunders; 2006.
- Evans EB, Eggers GWN, Butler JK, Blumel J. Experimental immobilization and remobilization of rat knee joints. J Bone Joint Surg Am. 1960; 42(5):737-758.
- Amiel D, Woo SL, Harwood FL, Akeson WH. The effect of immobilization on collagen turnover in connective tissue: a biochemical-biomechanical correlation. Acta Orthop Scand. 1982; 53(3):325-332.
- Laros GS, Tipton CM, Cooper RR. Influence of physical activity on ligament insertions in the knees of dogs. J Bone Joint Surg Am. 1971; 53(2):275-286.
- Ma YH, Cao CF, Tang XY, Zhou JJ, Ding ZX, Wang W. Changes in morphology and mechanical characteristics of contractural Achilles tendon in rabbits. Chinese Journal of Rehabilitation. 2009; (24):7-9.
- Noyes FR. Functional properties of knee ligaments and alterations induced by immobilization: a correlative biomechanical and histological study in primates. Clin Orthop Relat Res. 1977; (123):210-242.
- Tipton CM, James SL, Mergner W, Tcheng TK. Influence of exercise on strength of medial collateral knee ligaments of dogs. Am J Physiol. 1970; 218(3):894-902.
- Woo SL, Gomez MA, Sites TJ, Newton PO, Orlando CA, Akeson WH. The biomechanical and morphological changes in the medial collateral ligament of the rabbit after immobilization and remobilization. J Bone Joint Surg Am. 1987; 69(8):1200-1211.
- Hayashi K. Biomechanical studies of the remodeling of knee joint tendons and ligaments. J Biomech. 1996; 29(6):707-716.
- Majima T, Yasuda K, Fujii T, Yamamoto N, Hayashi K, Kaneda K. Biomechanical effects of stress shielding of the rabbit patellar tendon depend on the degree of stress reduction. J Orthop Res. 1996; 14(3):377-383.
- Yamamoto N, Ohno K, Hayashi K, Kuriyama H, Yasuda K, Kaneda K. Effects of stress shielding on the mechanical properties of rabbit patellar tendon. J Biomech Eng. 1993; 115(1):23-28.
- Uchida H, Tohyama H, Nagashima K, et al. Stress deprivation simultaneously induces over-expression of interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta in fibroblasts and mechanical deterioration of the tissue in the patellar tendon. J Biomech. 2005; 38(4):791-798.
- Palmes D, Spiegel HU, Schneider TO, et al. Achilles tendon healing: long-term biomechanical effects of postoperative mobilization and immobilization in a new mouse model. J Orthop Res. 2002; 20(5):939-946.
- Miyatake S, Tohyama H, Kondo E, Katsura T, Onodera S, Yasuda K. Local administration of interleukin-1 receptor antagonist inhibits deterioration of mechanical properties of the stress-shielded patellar tendon [published online ahead of print December 11, 2007]. J Biomech. 2008; 41(4):884-889.
- Yamamoto E, Hayashi K, Yamamoto N. Mechanical properties of collagen fascicles from stress-shielded patellar tendons in the rabbit. Clin Biomech (Bristol, Avon). 1999; 14(6):418-425.
- Yamamoto E, Tokura S, Yamamoto N, Hayashi K. Mechanical properties of collagen fascicles from in situ frozen and stress-shielded rabbit patellar tendons. Clin Biomech (Bristol, Avon). 2000; 15(4):284-291.
- Majima T, Yasuda K, Tsuchida T, et al. Stress shielding of patellar tendon: effect on small-diameter collagen fibrils in a rabbit model. J Orthop Sci. 2003; 8(6):836-841.
- Cutroneo KR. How is Type I procollagen synthesis regulated at the gene level during tissue fibrosis. J Cell Biochem. 2003; 90(1):1-5.
- Sporn MB, Roberts AB, Wakefield LM, Assoian RK. Transforming growth factor-beta: biological function and chemical structure. Science. 1986; 233(4763):532-534.
- Tohyama H, Yasuda K, Onodera S. Effects of cytokines on the mRNA of interstitial collagenase and collagens in extrinsic fibroblasts infiltrating into the necrotized patellar tendon. Transactions of the 48th Annual Meeting of Orthopaedic Research Society. 2002; (48):432.
- Tsuzaki M, Guyton G, Garrett W, et al. IL-1 beta induces COX2, MMP-1, -3 and -13, ADAMTS-4, IL-1 beta and IL-6 in human tendon cells. J Orthop Res. 2003; 21(2):256-264.
- Verrecchia F, Mauviel A. TGF-beta and TNF-alpha: antagonistic cytokines controlling type I collagen gene expression. Cell Signal. 2004; 16(8):873-880.
- Fujie H, Yamamoto N, Murakami T, Hayashi K. Effects of growth on the response of the rabbit patellar tendon to stress shielding: a biomechanical study. Clin Biomech (Bristol, Avon). 2000; 15(5):370-378.
- Thornton GM, Shao X, Chung M, et al. Changes in mechanical loading lead to tendon-specific alterations in MMP and TIMP expression: influence of stress deprivation and intermittent cyclic hydrostatic compression on rat supraspinatus and Achilles tendons [published online ahead of print September 18, 2008]. Br J Sports Med. 2010; 44(10):689-703.
- Thampatty BP, Li H, Im HJ, Wang JH. EP4 receptor regulates collagen type-I, MMP-1, and MMP-3 gene expression in human tendon fibroblasts in response to IL-1 beta treatment [published online ahead of print September 15, 2006]. Gene. 2007; 386(1-2):154-161.
- Brenmoehl J, Miller SN, Hofmann C, et al. Transforming growth factor-beta 1 induces intestinal myofibroblast differentiation and modulates their migration. World J Gastroenterol. 2009; 15(12):1431-1442.
- Katsura T, Tohyama H, Kondo E, Kitamura N, Yasuda K. Effects of administration of transforming growth factor (TGF)-beta1 and anti-TGF-beta1 antibody on the mechanical properties of the stress-shielded patellar tendon [published online ahead of print October 7, 2005]. J Biomech. 2006; 39(14):2566-2572.
- Phillips AO, Topley N, Steadman R, Morrisey K, Williams JD. Induction of TGF-beta 1 synthesis in D-glucose primed human proximal tubular cells by IL-1 beta and TNF alpha. Kidney Int. 1996; 50(5):1546-1554.
- Amiel D, Akeson WH, Harwood FL, Frank CB. Stress deprivation effect on metabolic turnover of the medial collateral ligament collagen. A comparison between nine- and 12-week immobilization. Clin Orthop Relat Res. 1983; (172):265-270.
- Ackermann PW, Salo PT, Hart DA. Neuronal pathways in tendon healing. Front Biosci. 2009; (14):5165-5187.
- Ackermann PW, Li J, Lundeberg T, Kreicbergs A. Neuronal plasticity in relation to nociception and healing of rat Achilles tendon. J Orthop Res. 2003; 21(3):432-441.
- Ackermann PW, Ahmed M, Kreicbergs A. Early nerve regeneration after Achilles tendon rupturea prerequisite for healing? A study in the rat. J Orthop Res. 2002; 20(4):849-856.
Messrs Wang, Tang, Zhang, Yan, and Ma are from the Department of Rehabilitation Medicine, the Affiliated Sixth Peoples Hospital of Shanghai Jiaotong University, Shanghai, China.
Messrs Wang, Tang, Zhang, Yan, and Ma have no relevant financial relationships to disclose.
The authors thank Dr Zanxian Ding, head of the Department of Animal Laboratory of the Affiliated Sixth Peoples Hospital of Shanghai Jiaotong University, for his valuable suggestions and technical assistance.
Correspondence should be addressed to: Yanhong Ma, MS, Department of Rehabilitation Medicine, the Affiliated Sixth Peoples Hospital of Shanghai Jiaotong University, Shanghai 200233, China (email@example.com).