Targeting Tendon Calcification

Tendon calcification is a common clinical disease, often complicated after tendon injury or surgery, or as a special manifestation of tendinopathy. Tendon calcification often occurs in the rotator cuff, achilles tendon and flexor tendon. As one histological feature of tendinopathy, tendon calcification is commonly found in severe tendinopathy in the rotator cuff. There is no epidemiological data of tendon calcification in China. In European populations, the epidemiological of tendon calcification in the rotator cuff is 2.7-22%, which occurs most often in women aged 35-50, and 10% of cases occur in bilateral tendon. Tendon calcification after Achilles tendinopathy occurs commonly in young athletes, or older, sedentary or obese patients. The incidence of tendon calcification secondary to tendinopathy is not clear, the literature reports that the rate was 5%. As one of manifestation of acquired heterotopic ossification, the similarities and differences of pathological features between tendon calcification and other forms of acquired heterotopic ossification is unclear. In addition, clinical retrospective study suggests that tendon calcification may cause pain, tendon weakness, local edema and varying degrees of activity limitation. However, there is no direct evidence indicate that tendon calcification cause any clinical symptoms. Pathogenesis of tendon calcification is unclear, at present several hypotheses about pathogenesis exist, and several factors are involved in the process of tendon calcification. Some scholars believe that after tendon injury, local stem cells are involved in the process of tendon repair and calcification; and after the injury, the local stem cells should conduct tenogenic differentiation to participate in the repair process, however local microenvironment of tendon changed due to injury, resulting in local stem cells into cartilage/bone differentiation. However, which population of stem cells involved in the calcification remains unclear. After calcification, the only treatment is surgery if conservative treatment failed. So comparing with treatment after tendon calcification formed, prevention of tendon calcification is more important. However, there is no drug for tendon calcification prevention in clinical treatment. Part one Development and validation of animal model of tendon calcificationObjective:To evaluate the Achilles tendon calcification model induced by Achilles tenotomy is stable and reliable, and provide the basic information for later study using this model.Methods:C57 mice were anesthetized under sterile conditions. Then 1 cm longitudinal incision in the skin of right heel was made to expose muscle membrane and Achilles tendon. Achilles tendon and plantar tendon were transected in the middle of the tendon. Mice were sacrificed and followed by μCT scanning to detecte calcification volume at 10 weeks postoperatively. After paraffin sections were made HE and Alcian blue staining were stained to investigate histological changes.Results:A large number of inflammatory cell infiltration, minority tendon cells, less extracellular matrix were found in the middle of the Achilles tendon 1 week post operation.4 weeks after surgery, inflammatory cells almost disappeared, replaced by a large number of tendon cells, extracellular matrix obviously increased comparing with 1 week, but the structure of cells and neo-tendon was disorder.6 weeks after surgery, no significant inflammatory cells were found, extracellular matrix continued to increase, but the structure of tendon cells and neo-tendon remains disordered.10 weeks after surgery, tendon structure was more completely, extracellular matrix was significantly increased, the structure of cells and neo-tendon tendon was more in order.1 week after surgery, a small amount of Alcian blue staining in the tendon insertion end was found. Positive Alcian blue staining and the structure of cartilage-like cells can be seen clearly at 6 weeks postoperatively.10 weeks after surgery, ossified tissue can be found in the insertion end of tendon. After 10 weeks Achilles tendon calcification were found mainly in the tendon insertion end by μCT scanning. There was no significant difference among the four groups after quantitative analysis of calcification volum.Summary:The histological examination confirmed that the Achilles tendon calcification model induced by tenotomy is a typical insertional tendon calcification, and the pathological process is endochondral bone formation. In this study, there were no significant difference about calcification volume among 4 groups 10 weeks postoperatively, which demonstrate that the achilles tendon calcification model induced by tenotomy is stable and reproducible, and can be used in the future studies.Part two Tendon calcification is progressive and detrimental to the tendon biomechanical propertiesObjective:To characterize tendon calcification and investigate whether it is a progressive disease, and whether it is detrimental to the tendon biomechanical properties.Methods:C57 mice were anesthetized under sterile conditions. Then 1 cm longitudinal incision in the skin of right heel was made to expose muscle membrane and Achilles tendon. Achilles tendon and plantar tendon were transected in the middle of the tendon. Mice were sacrificed and followed by μCT scanning to detecte calcification volume at 10 weeks and 6 months postoperatively. After paraffin sections were made HE and Alcian blue staining were stained to investigate histological changes.Results:After 10 weeks Achilles tendon calcification were found mainly in the tendon insertion end by μCT scanning in both 10 weeks and 6 months group. There was no significant difference among the four groups after quantitative analysis of calcification volum. Quantitative analysis for the calcification volume showed that the volume of tendon calcification after 6 months is significantly more than that of 10 weeks. The results showed that there is no significant difference between 10 weeks and 6 months group about cross-section area. Fiber alignment of 10 weeks is more disorder than that of 6months. Meanwhile, we compared the biomechanical properities of 10 weeks and 6 months group, including Grip-to-Grip modulus, Mid-substance modulus, maximum load and maximum stress. The results showed that there is no statistical difference between 10 weeks and 6 months group about Grip-to-Grip modulus. Mid-substance modulus, maximum load and maximum stress in 6 months group are significantly lower than that of 10 weeks group. We also evaluated the histology for 10 weeks and 6 months samples. Ossified tissue were found in the tendon-bone junction with a lot of cartilage-like cells surrounding and can be positive to alcian blue staining. We also found similar phenomenon in muscle-tendon junction.Summary:By comparing the calcification volume and biomechanical changes for 10 weeks and 6 months group, we found that tendon calcification is progressive and detrimental to the tendon biomechanical properties. Future exploration for a new drug to prevent tendon calcification is particularly necessary.Part three Isolation and identification of injuried tendon-derived progenitor cellsObjective:To identify stem/progenitor cells involved in tendon calcification after tendon injury and to develop new cellular therapeutic target for prevention of tendon calcification.Methods:The mice were euthanized and achilles tendon was harvested. After mincing tendon was digested by tendon digestive solution then incubated at 37℃ in water bath for 1 hour. The isolated cells were platted in 100mm dish.Results:10 days after the primary culture, we count the number of colony formation. The results showed that the cells extracted from normal tendon and injuried tendon can form colonies. However the number of colony formation from injuried tendon is more than that of normal tendon. The cells extracted from normal tendon and injuried tendon express stem cell marker Scal, CD29, CD44 and CD49e. And hematopoietic markers CD45, CD11b and Grl are negative. Thus it excludes the possibility that the cells are from hematopoietic and epithelial source. The cells isolated from injuried tendon can differentiate to chondrogenic, osteogenic and adipogenic lineage. In chondrogenic culture system, we found that the chondrogenic differentiation ability is stronger than the tendon stem/progenitor cells and bone marrow stromal cells.Summary:In this study, we isolated the cells in the neo-tendon 1 week after tendon injury. Our results show that after tendon injury, the neo-tendon contains a unique population of cells,and these cells exhibit colony formation, self-renew and multipotency. And these cells also express stem cells marker. We named this unique population of cells as injuried tendon-derived progenitor cells.Part four To explore new drug for tendon calcification prevention base on injuried tendon-derived progenitor cellsObjective:To explore the potential drug which can inhibit tendon calcification base on injuried tendon-derived progenitor cells.Methods:The mice were euthanized and achilles tendon was harvested. After mincing tendon was digested by tendon digestive solution then incubated at 37℃ in water bath for 1 hour. The isolated cells were platted in 100mm dish and P3 cells were used for following experiments. Evaluate how LDN-193189, RAR-y agonist, GANT 58 and celecoxib affect the chondrogenic and osteogenic ability of injuried tendon-derived progenitor cells.Results:In chondrogenic culture system, LDN-193189, RAR-γ agonist, GANT 58 and celecoxib significantly reduced the concentration of Alcian blue positive staining. LDN-193189, RAR-γ agonist, GANT 58 and celecoxib also inhibited the mRNA expression of Agg, Col2 and Sox9. In osteogenic culture system, LDN-193189, RAR-γ agonist, GANT 58 and celecoxib significantly reduced the concentration of Alizarin red positive staining. LDN-193189, RAR-γ agonist, GANT 58 and celecoxib also inhibited the mRNA expression of Runx2 and Osx.Summary:LDN-193189, RAR-y agonist, GANT 58 and celecoxib significantly inhibited chondrogenic and osteogenic ability of injuried tendon-derived progenitor cells, which indicated that these four drugs potentially inhibit tendon calcification.Part five Evaluation the effectiveness of drugs for inhibition of tendon calcification in vivoObjective:Using tenotomy induced tendon calcification model to evaluate the effectiveness of drug for prevention tendon calcification.Methods:C57 mice were anesthetized under sterile conditions. Then 1 cm longitudinal incision in the skin of right heel was made to expose muscle membrane and Achilles tendon. Achilles tendon and plantar tendon were transected in the middle of the tendon. After surgery, LDN-193189、NRX 204647、GANT 58 and celecoxib were administered. Mice were sacrificed and followed by μCT scanning to detecte calcification volume at 10 weeks postoperatively. mRNA was extracted from injuried tendon after 3 weeks to evaluate the safety of the drugs.Results:The results showed that LDN-193189, GANT 58 and Celecoxib significantly reduce the volume of tendon calcification in 10 weeks after surgery. However, there is no significant difference between control and RAR-γ agonist treated group about tendon calcification volume. The redness and swelling was found in the skin of heel after two weeks RAR-y agonist treatment, which indicated that RAR-y agonist induces inflammation. The results showed that LDN-193189, RAR-y agonist and GANT 58 significantly decrease the expression of Coll, Col3, Sex and Tnmd after 3 weeks. However, there is no significant difference between control and Celecoxib treated group for the expression of Coll, Col3, Scx and Tnmd.Summary:In this part, we evaluated the effectiveness of LDN-193189, NRX 204647, GANT 58 and celecoxib in the prevention of tendon calcification. Our results indicated that LDN-193189, GANT 58 and celecoxib can effectively suppress the occurrence of tendon calcification. We further compare the tendon-related gene expression in each group, the results suggest GANT 58 and LDN-193189 may affect tendon repair. Therefore Celecoxib is the mostly safe and effective drug for inhibition of tendon calcification.Part six The optimal treatment duration of Celecoxib for inhibition of tendon calcificationObjective:In this study, we will systematic evaluate the optimal treatment duration of Celecoxib for inhibition of tendon calcification.Methods:C57 mice were anesthetized under sterile conditions. Then 1 cm longitudinal incision in the skin of right heel was made to expose muscle membrane and Achilles tendon. Achilles tendon and plantar tendon were transected in the middle of the tendon. After surgery the mice were treated with celecoxib for 0-2 weeks,0-6 and 0-10 weeks. Mice were sacrificed and followed by μCT scanning to detecte calcification volume at 10 weeks postoperatively.Results:The results showed that the volume of tendon calcification in Celecoxib 0-6 weeks treated group and Celecoxib 0-10 weeks treated group were significantly lower than control group. However there is no significant difference between Celecoxib 0-2 weeks treated and control group about the volume of tendon calcification.Summary:In this study, we used tenotomy induced tendon calcification model to evaluate the optimal treatment duration of Celecoxib for inhibition of tendon calcification. The results show that middle and long-term administration of celecoxib can inhibit tendon calcification but not short term.