Series Research For The Pathogenesis Of Calcific Achilles Tendinopathy

1. BackgroundThe gastrocnemius muscle merges with the soleus to form the Achilles tendon 15 cm proximal to their calcaneal insertion, which is the most massive tendon in human-being. The Achilles tendon has a round upper part and is relatively flat in its distal part. The fibres of the Achilles tendon are spiral 90°. This arrangement increases the tendon elongation and helps the release of the energy stored within the tendon during locomotion. Normal Achilles tendon has a glistening white appearance and toughness property. There are only two-layer membrane envelopes instead of a synovial sheath. The intra-tendon blood flow is considerably low especially in the calcaneal insertion. The main function of Achilles tendon is to transmit the strength of the triceps surae to maintain the standing and walking balance of the lower limbs. With the improvement of Chinese people living standard and the popularity of national fitness consensus, Achilles tendon injury and Achilles tendinopathy has emerged as the common diseases. Tendinopathy is clinically characterised by activity-related pain, focal tendon tenderness and intratendinous imaging changes. It represents a common and significant problem in the elite athletes population, however, Achilles tendinopathy is not always associated with excessive physical activity, the inactive individuals can also experience tendinopathy. Once it occurs, it may require a long recovery time with conservative management to restore the normal function of the ankle. What’s even more serious is the detrimental effect on the biomechanical properties which may lead to lower adaption to high mechanical loading and aggravate the clinical conditions of Achilles tendinopathy. Achilles tendon rupture may subsequently be happened following Achilles tendinopathy, which is considered as the death warrant for the elite athletes career. The pathophysiological mechanism of Achilles tendinopathy and its potential prevention are the forward issues in sport medicine, but unfortunately its potential mechanism has not been fully clarified, resulting in the obstacle of Achilles tendinopathy treatment development.The underlying pathology was historically believed to be one of inflammation, and thus the condition was termed ’tendinitis’. While the initiating stimulus may begin with inflammation, numerous studies have shown the established pathology to consist of tendon degeneration with a complete absence of inflammatory cells. Consequently, the term ’tendinosis’ has been recommended to describe the typical histopathological appearance of the underlying pathology. Tendinosis is defined by Jozsa and Kannus as intratendinous degeneration-that is, hypoxic, mucoid or myxoid, hyaline,fatty, fibrinoid, calcific, or some combination of these-from a variety of causes (ageing, microtrauma, vascular compromise, etc). Histologically, there is non-inflammatory intratendinous collagen degeneration with fibre disorientation and thinning, hypercellularity, scattered vascular ingrowth, and increased interfibrillar glycosaminoglycans. Macroscopically, the affected portions of the tendon lose their normal glistening white appearance and become grey and amorphous.What makes "tendinosis" differs from "tendinitis" is that tendinopathy could not be simply considered as PGE2 dependent inflammatory, It has been proposed that tendinosis is a failure of cell matrix adaptation to trauma due to an imbalance between matrix degeneration and synthesis. However, the degenerative process is active instead of inactive, with changes in cell activity and phenotype with an increased turnover of matrix and a different expression of genes compared with normal tendons. The repetitive overload mechanical stress may let the degenerative Achilles tendon go into a decompensated vicious circle and finally generate Achilles tendon rupture. It is worthy noting that some individuals were asymptomatic until Achilles tendon rupture occured. The concealed onset may let people ignore its potential perniciousness, therefore to be aware the natural history and the specific pathophysilogical of Achilles tendinopathy is necessary for avoiding the risky factors of Achilles tendinopathy and developing mechanism-based therapy strategy for its prevention and treatment.During the past decades, several studies have suggested that Achilles tendon degeneration (tendinosis) typically appears histopathologically in the pathogenesis of tendinopathy, but its pathophysiology is not fully understood. Because of the potential demand to further identify efficient medical intervention, validated animal models with Achilles tendinopathy should be developed to represent the consistent conditions and known risk factors in humans. Thus, several animal models of Achilles tendinopathy have been established in previous studies. Although these existing studies shed light on novel established models of Achilles tendinopathy, they have distinct drawbacks,such as complex inducible conditions and special treadmill instrument requirement, confounding variables interference, which may compromise its reproducible characterization and restrict its applications. Therefore, an established animal model with a simpler and more highly reproducible design could gain popularity consistent with increasing clinical interest in Achilles tendinopathy.Calcific Achilles tendinopathy is a special subtype of Achilles tendinopathy, which is characterised as Achilles tendon tendinosis with intratendon calcification or ossification. The intratendon calcification or ossification may raise the risk of rupture and require even longer recovery time. The calcification or ossification in the tendon body may increase the stiffness of Achilles tendon to compensate the decreased mechanical properties. So standing on the macro viewpoint, calcification or ossification could be considered as a pathological healing process. Even though the pathogenesis of intra-tendon ossification remains unclear, the "endochondral ossification theory" is generally accepted by most researchers. Based on this theory, the chondral metaplasia may play an important role during its pathogenesis. Any varience which influences chondral metaplasia may modulate the pathogenesis of calcific Achilles tendinopathy. On the other hand, the intra-tendon calcification or ossification could be recognized as "heterotopic ossification" in the Achilles tendon, which is defined as the formation of mature trabecular bone in sites where it is not normally presented. Any therapy which is effective for heterotopic ossification may benefit for the prevention of calcific Achilles tendinopathy.Leptin is a 16 kDa non-glycosylated hormone encoded by the gene ob, the murine homologue of the human gene LEP. It is mostly produced by adipocytes. Circulating leptin, an important endocrine factor, which is binded with the leptin receptor coded by the gene db, plays a critical role in energy and cell metabolism by binding with leptin receptor in the cell membrane. Since it has been reported in 1994, aroused interests sooner introduced it in many research fields. It has been reported that leptin could prompt the proliferation and hypertrophy of chondrocytes, modulating the endochondral ossification. However, whether it could regulate the pathogenesis of calcific Achilles tendinopathy remains unclear, calling for the extensive research.Cyclooxygenase-2(COX-2) is the key speed-limited enzyme during PGE2 synthesis, which has been considered as an essential factor during inflammation and cell metabolism. Recent studies revealed COX-2 may exert indispensible effect on heterotopic ossification development. Targeting for COX-2 to inhibit endochondral ossification may be an effective therapy for the ectopic ossification prevention. Provided calcific Achilles tendinopathy is regarded as a special type of heterotopic ossification in the Achilles tendon, that whether COX-2 plays an important role during its development, and whether selective inhibition of COX-2 will exert preventative effect on Achilles tendinopathy, is further worthy of being investigated.Recently, it has been revealed that Leptin could up-regulate the activity of COX-2 to mediate the pathogenisis of inflammation induced diseases. which aroused our interests on the crosstalk between leptin and COX-2 activity during the pathogenesis of Achilles tendinopathy. Either Leptin’s effect is COX-2 dependent, or COX-2 activity could regulate the balance between leptin and leptin receptor, should be further elucidated.2. ObjectiveOn account of the current situation of Achilles tendinopathy research, we aimed to investigate the potential pathogenesis of calcific Achilles tendinopathy and subsequently provide new evidence for its therapy development. Our study has been conducted in 4 parts as follow:1. to establish and characterize a new overuse-induced calcific Achilles tendinopathy mice model; 2. to evaluate the effect of leptin on Achilles tendinopathy in leptin receptor mutation db/db mice by employing the new mice model;3.to clarify the effect of selective COX-2 inhibitor celecoxib on Achilles tendinopathy and whether treatment dose and duration could modify its effect;4. To elucidate the crosstalk between leptin and COX-2 activity. Whether the effect of leptin is COX-2 pathway dependent, or COX-2 activity could regulate the balance between leptin and leptin receptor.3.Materials and Methods3.1.The establishment and characterization of a novel calcific Achilles tendinopathy model in mice.A total of 80 C57/B6 mice (6 wk old) were obtained from the Experimental Animal Research Center of Southern Medical University. The animals were randomly divided into two groups (n=40). All mice were anesthetized and then the experimental group underwent midpoint Achilles tenotomy on the left hind limbs through a posterior approach under aseptic conditions. The incision was routinely closed with an interrupted 4-0 silk suture whereas the control group underwent only a skin incision. The mice were kept under standard conditions. At 12 wk postoperation, all animals were sacrificed by administration of a fatally high dose of anesthetics. The right Achilles tendon tissues from the soleus muscle attachment to the calcaneus insertional end point were harvested. H&E staining, Toluiblue staining, immunohischemical staining(IHC) and immunofluosecence staining(IHF)have been conducted for the histological analysis. X-ray analysis and Micro-CT scanning have been accomplished for the radiological assessment. Scanning electron microscopy observation and transmission electron microscopy observation have been performed for the detection of intratendon collagen fiber disorganization. Moreover, biomechanical analysis was conducted to test the biomechanical properties changes during Achilles tendinopathy development. The characterization of this novel mice Achilles tendinopathy mice model has been validated from the animal level and tissue level.3.2.The effect of leptin on Achilles tendinopathy in db/db leptin insufficient mice by employing the new mice model.SPF db/db mice(n=40,6-week old) and SPF wild type mice(n=40,6-week old) have been employed as experimental group and control group respectively in the part 2 of this study. All mice were anesthetized and then underwent midpoint Achilles tenotomy on the left hind limbs through a posterior approach under aseptic conditions. The incision was routinely closed with an interrupted 4-0 silk suture. At 12 weeks postoperation, the right Achilles tendon tissues from the soleus muscle attachment to the calcaneus insertional end point were harvested. H&E staining, Safranin-O and fast green staining, immunohischemical staining have been conducted for the histological analysis. X-ray analysis and Micro-CT scanning have been accomplished for the radiological assessment. To evaluate whether the function defects of leptin receptors will contribute to the calcific Achilles tendinopathy prevention.3.3.The effect of selective COX-2 inhibitor celecoxib on Achilles tendinopathy and whether celecoxib treatment dose and duration could modify its effect.A total of 80 SD rat (6 wk old) were obtained from the Experimental Animal Research Center of Southern Medical University. All mice were anesthetized and then underwent midpoint Achilles tenotomy on the left hind limbs through a posterior approach under aseptic conditions. The incision was routinely closed with an interrupted 4-0 silk suture The animals were randomly divided into eight groups (n =10) as follow:1. Celecoxib single dose group:celecoxib saline solution oral treatment (10mg/kg/day) for 12 weeks postoperation.2. Celecoxib double dose group:celecoxib saline solution oral treatment (20mg/kg/day) for 12 weeks postoperation.3.3 weeks group:celecoxib saline solution oral treatment (10mg/kg/day) for first 3 weeks postoperation.4.6 weeks group:celecoxib saline solution oral treatment (10mg/kg/day) for first 6 weeks postoperation.5. Post 9 weeks group:celecoxib saline solution oral treatment (10mg/kg/day) from 3 weeks postoperative to 12 weeks postoperation.6. Post 6 weeks group:celecoxib saline solution oral treatment (10mg/kg/day) from 6 weeks postoperation to 12 weeks postoperation.7. Positive control group:without any treatment during the 12 weeks postoperation.8.Placebo group:the same volume saline solution oral treatment for 12 weeks postoperation. All animals were sacrificed at 12 weeks postoperation. The left Achilles tendon tissues from the soleus muscle attachment to the calcaneus insertional end point were harvested. H&E staining, immunohischemical staining have been conducted for the histological analysis. X-ray analysis and Micro-CT scanning have been accomplished for the radiological assessment. To clarify the effect of selective COX-2 inhibitor celecoxib on Achilles tendinopathy and whether different doses and durations of celecoxib treatment could modify its effect.3.4. The crosstalk between leptin signaling and COX-2 activity during the calcific Achilles tendinopathy developmentThe primary rat Achilles tenocytes have been isolated and cultured in vitro. The third passaged cells were seeded in several 24-well plates. Once confluence was obtained, the prepared reagents was added to the cell culture medium to divided into 3 groups as follow:Group A, Cox-2 selective inhibitor NS398 treatment group, the final concentrations of NS398 were 0.2uM,2uM,20uM. Group B, Recombined leptin protein treatment group, the final concentrations of recombined leptin protein were 50ng/ml,250ng/ml. Group C, NS398+recombined leptin protein treatment group, the final concentration of NS398 was 20uM, the final concentrations of recombined leptin protein were 50ng/ml,250ng/ml. The medium was changed every 3 days. After 7 days incubation, the lysed cell protein samples were harvested. The COX-2, leptin and leptin receptor expression was detected by Western blot assay. Each experiment has been repeated 3 times. To elucidate the crosstalk between leptin signaling and COX-2 activity during the calcific Achilles tendinopathy development4. Results4.1. Successful establishment of a novel calcific Achilles tendinopathy mice model.After 12 wk of unilateral Achilles tenotomy, the contralateral Achilles tendon in the experimental group presented with severe edema and grey, losing its normal glistening white gloss, there were obvious nodes detected intra-tendon. Histological analysis demonstrated that hypercellularity and neovascularization in the contralateral Achilles tendon, IHC and IHF staining indicated fibrocartilage metaplasia and endochondral ossification in contralateral Achilles tendon after 12 wk unilateral tenotomy. Scanning electron microscopy observation and transmission electron microscopy observation showed that fiber disarrangement and collagen fusion in the contralateral Achilles tendon. Radiological analysis suggested mineralization in the contralateral Achilles tendon and calcaneus deformity. Biomechanical analysis confirmed poor biomechanical properties in contralateral Achilles tendon after 12 wk unilateral Achilles tenotomy.4.2. The function defects of leptin receptors inhibit the development of calcific Achilles tendinopathy.Radiological analysis indicated no mineralization detected in the bilateral Achilles tendon of db/db mice after 12 wk unilateral tenotomy. Histological assessment demonstrated fiber disorganization and hypercelluarity in the contrallateral Achilles tendon after 12 wk unilateral Achilles tenotomy in db/db mice. Mild blood vessel ingrowth could also be indentified in the contralateral Achille tendon of db/db mice. There were large numbers of adipocytes infiltration in the paratendon tissue. IHC staining showed that COX-2 expression is relatively low in the experimental group compared to control group.4.3. COX-2 activity is essential for the calcific Achilles tendinopathy pathogenesis.Oral celecoxib treatment for 12 weeks postoperation could significantly reduce the incidence of heterotopic ossification and the volume of the ectopic ossification tissue after Achilles tenotomy in rat. There was no difference beween single dose group and double dose group. Other treatment options in part 3 of this study could not significantly reduce the incidence of heterotopic ossification after Achilles tenotomy in rat compared to the positive control group and placebo group. There was no difference beween 3 weeks group and positive control group on the volume of the ectopic ossification tissue intra Achilles tendon. Less volume of ectopic ossification tissue intra Achilles tendon has been detected in the 6 weeks group compared to 3 weeks group, of which is more than post 6 weeks group. Less volume of ectopic ossification tissue intra Achilles tendon has also been detected in the post 9 weeks group compared to 6 weeks group, of which is less than post 6 weeks group. Histological analysis showed that there was no mature woven bone with bone marrow cavity in the individuals of celecoxib treatment group without intratendon ossification, the tendon fiber disorganization, hypercelluarity and neovascularization with chondrocyte-like cells and regenerated cartilage have been detected in these 2 groups. In the other 6 groups, tendon fiber disorganization, large amount of hypertrophic chondrocytes infiltration, endochondral ossification has been indentified from the H&E staining analysis. Less hypertrophic chondroncytes were detected in the 6 weeks group compared to the 3 weeks group, meanwhile less hypertrophic chondroncytes were observed in the post 9 weeks group than post 6 weeks. There is no difference between post 9 weeks group and 6 weeks group. IHC staining showed leptin was widely expressed in the Achilles tendon with tenotomy.4.4. Leptin facilitates calcific Achilles tendinopathy development via COX-2 dependent pathwayThe specific inhibition of COX-2 activity by NS398 in rat Achilles tenocytes could consequently result in the leptin expression reduction in vitro in a dose dependent manner, as well as the leptin receptor expression. On the other hand, recombined leptin protein could induce the COX-2 expression of rat Achilles tenocytes in a dose dependent manner, as well as the as the expression of the leptin receptors.20uM NS398 treatment could reverse the COX-2 expression induced by recombined leptin protein, as well as the increased expression of leptin and leptin receptors of the tenocytes.5. Conclusions5.1. In this study, we introduce a novel, simple, but reproducible spontaneous contralateral calcific Achilles tendinopathy mice model induced by unilateral Achilles tenotomy, which represents overuse conditions during tendinopathy development in humans. It should be a useful tool to further study the underlying pathogenesis of calcific Achilles tendinopathy.5.2. Leptin receptor mutation may compromise calific Achilles tendinopathy development in db/db mice. Leptin plays an essential role in the endochrondral ossification.5.3. COX-2 selective inhibitor celecoxib could prevent heterotopic ossification in the rat model with Achilles tenotomy. The potential inhibition effect may probably result from the inhibition of tendon stem cell erroneous differentiation and chondrocytes hypertrophy. Therapies targeting COX-2 activity could contribute to the treatment of calcific Achilles tendinopathy.5.4 Recombined leptin could induce the COX-2 activity to provoke the degradation of extracelluar tendon matrix and intratendon chondral metaplasia. The increased leptin receptor expression by leptin treatment may further augment the leptin intracellular signalings. COX-2 activity blocking could inhibit the anabolism effect of leptin on tenocytes and impair the leptin intracellular signaling by decreasing the leptin receptors expression. The leptin-leptin receptors signaling is correlated with COX-2 activity during the pathogenesis of calcific Achilles tendinopathy. Leptin promote the calcific Achilles tendinopathy via a COX-2 dependent manner.