The Preliminary Study Of The Relationship Between Different Interface Treatment And Tendon-to-bone Healing And The Mechanism Of TNC

Introduction:Tendon-to-bone healing is a serious issue on the clinic.The problems exist in all tendon-to-bone connections of the body,in which the rotator cuffs,the Achilles tendon were particularly evident.11%-94%of rotator cuff injury reconstructed patients failure within 2 years,which observed on the imaging.These are a huge burden on society and the family.Research about enthesis repairment and reconstruction were the major needs to the country in the field of population and health.Different treatment of interfaces before the repairing affected the quality of tendon-to-bone healing.The traditional view is that the tendon-to-bone interface must be completely rejuvenated before it is repaired,and the tendon-to-bone interface freshness includes both the cortical removal and the preserved cortex.However,the relationship between different interface treatment and tendon-to-bone healing needs to be further explored,and the mechanism is not clear.The main factors affecting tendon-to-bone healing included growth factors,gene therapy,periosteal closed tendon,osteogenic induction material,cell therapy,biodegradable scaffolds and biomimetic patches et al.These methods are quite different from normal tendon-to-bone interface at both functional and biological.Previous studies have shown that tenascin-C(TNC)may be associated with tendon-to-bone healing processes and that muscle and tendon injuries are associated with a large number of TNC protein expression;TNC may be one of the key factors affecting tendon-to-bone healing.The aims of this study were to develop a rat model of acute Achilles tendon failure repaired by different interface treatments,and looking for the relationship between TNC and tendon-to-bone healing of different interface treatment.Tendon stem cells(TSCs)and bone marrow mesenchymal stem cells(BMSCs)were mixed co-cultured with different ratio in order to simulate the situation of two kinds cell met at the tendon-to-bone interface.The effects of TNC on proliferation and differentiation of TSCs,BMSCs and different proportional mixed co-culture cells were tested.Methods:Part ?:The animal model of rat Achilles tendon acute rupture repair was made,and the Achilles tendon was treated by preserving the original fibrocartilage layer,removing the original fibrocartilage layer and bone tunnel.Through the maximum failure load and stiffness,biomechanical test indexes were used to detect the quality and intensity of tendon-to-bone healing.The bone mineral density(BMD)and BV/TV were measured by microCT scanning in order to observe the changes of bone mass under the tendon-to-bone interface.HE,Masson,and Safranine O staining were used to know tendon-to-bone interface morphology.Part ?:Immunohistochemical method was used to analyze by semi-quantitative analysis of Osteocalcin,SOX-9 and TNC of the tendon-to-bone interface and Achilles tendon's muscle-tendon junction by different interface treatments.The tendon stem cells and bone marrow derived mesenchymal stem cells were Mixed and co-cultured to simulate the two kind cells meeting in the tendon-to-bone healing interface.The TNC was added to evaluate the effect of proliferation of different proportions of mixed co-cultured tendon stem cells,tendon stem cells and bone marrow derived mesenchymal Stem cells,and the effect of genetic markers expression of adipogenesis(PPAR?),osteogenesis(RUNX2),tenogenesis(SCX)and chondrogenesis(SOX-9)of tendon stem cells,bone marrow mesenchymal stem cells and 1:1 ratio co-cultured cells in fixed culture time(3d)and immobilized TNC dose(1?g/ml).Results:Part ?1.Through the rat Achilles tendon-calcaneal complex mechanics test of healing by the different interface treatments:the Maximum failure load of the rat Achilles tendon-calcaneal complex repaired by without removal the original fibrocartilage tissue was significantly lower than self-control at postoperative 4 weeks(34.42±4.65 N vs 60.78±6.63N)and 8 weeks(41.21±3.38 N vs 60.05±5.25 N).After postoperative 8 weeks,the stiffness of each group was significantly lower than self-control(G1:33.96±5.59 N vs58.59±6.54 N/mm;G2:25.96±2.80 N vs 50.20±8.29 N/mm;G3:32.73±5.20 N vs 59.12±10.17 N/mm).There was no significant difference between the groups in the operation groups and between the control groups.2.The BMD(4w:0.378±0.041 g/cm~3 vs 0.808±0.014 g/cm~3;8w:0.349±0.009g/cm~3 vs 0.795±0.030 g/cm~3)and BV/TV(4w:33.82±4.35%vs 76.24±1.77%;8w:37.33±5.80 vs 70.28±2.39%)of the healing interface of removal the original fibrocartilage tissue were significantly lower than self-control group(P<0.05)at postoperative 4 weeks and 8 weeks,which showed bone mass was significantly reduced.3.There were degradation and reconstruction process of the original tough ligament structure of without removal the original fibrocartilage tissue.It performed that fibrous structure disorder,the original point of hypertrophic chondrocytes gradually reduced in the first postoperative 4 weeks then gradually increased.After repairing,the healing interface of the removal of the original fibrocartilage tissue appeared nest-like chondrocytes,Sharpey's fiber were appeared at the tendon-to-bone interface,fiber arrangement more orderly.There was more safranine O staining of chondrocytes at the tendon-to-bone interface of the removal of the original fibrocartilage tissue repair method.Part ?1.At postoperative 4 weeks and 8 weeks,the average optical density of osteocalcin in the bone under the tendon-to-bone interface of both two groups was lower than the control group(4w control vs G1 vs G2:0.0438±0.0062 vs 0.0116±0.0009 vs 0.0067±0.0008;8w:0.0837±0.0244 vs 0.0269±0.0066 vs 0.0066±0.0005).2.The average optical density of SOX-9 of cartilage-like cells at the interface of the tendon-to-bone interface of both two groups was significantly higher and reached the peak at the first week after surgery(Control vs G1 vs G2:0.039±0.004 vs 0.068±0.006 vs 0.069±0.006),and gradually declined at postoperative 2 weeks,4 weeks and 8 weeks,respectively(2w:0.057±0.007 vs 0.045±0.002 vs 0.027±0.004;4w:0.021±0.002 vs0.040±0.002 vs 0.023±0.003;8w:0.029±0.008 vs 0.024±0.006 vs 0.035±0.003).3.TNC immunohistochemical at the Achilles tendon proximal muscle-tendon junction of the two kinds of interface treatment of rat Achilles tendon point repairing showed different changes tendency at different time points:The retained the original fibrous cartilage group was at the peak level(0.119±0.001)at postoperative 4 weeks and then declined.The removal original fibrocartilage group were treated as the peak(0.082±0.003)at 2 weeks after operation,and then declined.Its peak level was the lowest among the three groups.The control group showed that the peak(0.159±0.006)at 2 weeks after the operation,and then declined.There was a significant difference between the control group and the operation group at postoperative 8 weeks(Control vs G1vs G2:0.013±0.001vs 0.038±0.005 vs 0.040±0.006).4.The proliferation rate of the BMSCs was lower than the TSCs.The proliferation rate of mixed co-cultured cells was as fast as the TSCs after 3 days cultured.At 2 days and3 days,TNC significantly affected 1:1 mixture Co-cultured cells'proliferation with different doses(1?g/ml?10?g/ml and 100?g/ml).5.The TNC could promote expression of RUNX2 and SOX-9 genes in direct 1:1mixed co-culture model,and TNC has the effect of inhibiting SCX expression in TSCs.The genetic markers expression had no significant changes in indirect co-culture model.6.Y-27632,PQ 401 and PD 98059 could affect the effects of TNC that promoted the RUNX2 and SOX-9 expression in direct mixed co-culture model and inhibited the SCX expression.Conclusions:1.The healing qualities of animal models by repairing after removed fibrocartilages and the bone tunnel are better than repairing without removing original fibrocartilages.Repairing after removed fibrocartilages will cause osteopenia after the surgery for a while,but the change was not the determinate factor to affect the healing quality of tendon-to-bone.The use of removal of the original fibrocartilage repair would lead to the reconstruction of the enthesis's bone continued,significantly reduced.The treatment of removal of the oringinal fibrocartilage layer had better interface morphology during the healing process.2.The healing modes of different interfaces after the acute ruptures of rat's Achilles tendon caused expression differences expression of osteocalcin and SOX-9 in the tendon-to-bone interface,TNC in the muscle-tendon junction of Achilles tendon,the differences may be relevant to the formation of fibrocartilage in the tendon-to-bone interface.3.1:1 direct mixed co-culture can change the response of BMSCs and TSCs to TNC stimulation.Under the condition of 3 days,the dose of TNC was 1?g/ml,TNC could infunce the proliferation of 1:1 direct mixed co-culture cells,and promote RUNX2 and SOX-9 gene expression of the 1:1 direct mixed co-culture cells,as well as inhibite the SCX gene expression of TSCs.The effects of TNC,which influnced the gene expression,have a relationship with signaling pathways involved in ROCK,IGF-1R and ERK.