| BackgroundTendon is a dense connective tissue connecting the bone and muscle, which has the function of kinetic energy transfer between muscle and bone, because the tendon cells surrounded by a collagen fiber bundle longitudinal arrangement, therefore tendon has stronger tensile strength.The tendon tissue is the organizational structure which has high mechanical strength and less proportion of blood vessels, so the tendon injury is not easily repaired and regeneration. The clinical treatment of acute rupture of tendon or injury often use surgical operation.With the continuous improvement of suture method, and the rapid development of suture material, now the fracture rate decreased significantly after the tendon suture. But for fixing method after the repair of tendon injury, previous clinical experience is to use non functional position for fixing, namely relaxation position. This method taking into account the main suture after early stress will make the suture tendon re rupture. But with the development of science and technology, the function of fixing is also a challenge to the traditional method. A function position fixed makes constant gentle stretch stimulation after tendon repair. Previously experimental study showed that the collagen content of Achilles, patellar tendon are increased after applied for stretch stimulation in vitro.The function positioning fixing can produce continuous tension stress and can benefits the repair of tendon, what mechanism is tacking effect in this precess? To elucidate the potential mechanism is conducive to a more extensive application of function position fixed in repair of tendon injury, it also provide a new method and means for clinical.ObjectiveAnalyze acute injury of rabbit Achilles tendon repairing postoperative functional position fixation promoting tendon repair in acute injury of rabbit Achilles and its mechanism.MethodsThis study is divided into two parts. The first part is to investigate postoperative functional position fixation in acute injury of rabbit Achilles tendon injury, detecting its biomechanics and related growth factor expression. The second part is to explore the continuous tension stress stimulation on tendon stem cells to differentiation of tendon cell.The first part, healthy adult male New Zealand rabbits were selected as the objects, New Zealand rabbits were randomly divided into 4 groups,42 rats in each group, i.e. A, B, C and D group. The A group received the broken Achilles tendon suture and fixing in the function position. B group was given the relaxation position fixation. C group wasn’t given to the fixed, free activities. D group deemed as normal control group,7d,14d,28d after operation were used to investigate the mechanical properties of Achilles tendon repair tissue, HE, picric Sirius red staining observation:repair of tendon tissue morphology and collagen distribution. Ultrasound was used to observe related indexes of the Achilles tendon tissue of each group at postoperative 7d,14d and 28d. Ultrastructure of postoperative Achilles tendon tissue of each group at 28d were detected by Transmission electron microscope. Tendon related gene and protein were detected by real-time quantitative PCR and ELISA method.The second part, isolation and culture of rabbit Achilles tendon stem cells, osteogenic, chondrogenic, adipogenic solution were used to induce TSCs, and its multilineage differentiation potential were tested by staining. Flow cytometry was used to indentify stem cell specific protein level. Applying the continuous tension stress stimulation to cultured tendon stem cells, the parameter were 2% strain,4% strain and 6% strain. After applying 24h and 48h, cell differentiation related gene and protein expression level were measured.ResultsIn the first part, the A group showed a large amount of collagen fibers formed in injury site in 28d after operation, the fibers were arranged in order, inflammatory cells were less likely to see. Rabbit Achilles tendon repair tissue of B group also showed a large amount of collagen fiber formation, fiber structure arranged, but the degree were inferior than the A group. Postoperative 7d,14,28d, four groups of rabbit Achilles tendon tensile maximum load, stiffness, maximum fracture stress differences were statistically different (P< 0.05), A group is better than B and C group (P< 0.05). Maximum load, tensile stiffness, maximum fracture stress values of A group at the 28d were 269.32+28.43 (N),25.63+1.43 and 9.78+2.53 (N/mm) (Mpa). In the 28d, rabbit Achilles tendon collagen type Ⅰ and type Ⅲ collagen ratio of group A are very close to the normal rabbit Achilles tendon. However, type I collagen ratio of B group is slightly lower than that of group A, group C was the lowest, only 86.32%.7d after operation, COL Ⅰ, COL Ⅲ, TGF-beta 1, bFGF, PDGF and expression of IGF-1 gene of group A rabbit Achilles tendon tissues were significantly elevated. Higher than that of B group, C group and D group, and VEGF expression of A group is lower than B and C group (P< 0.05). After the 28d, COL Ⅰ, COL Ⅲ, TGF-beta 1, bFGF, PDGF, VEGF and IGF-1 gene expression levels were decreased to some extent in each group, but still higher than the level of D group (P<0.05). Results of ELISA detection showed 7d after operation, COL I, TGF-beta 1, bFGF, PDGF protein levels of group A were significantly higher than those in B group and C group (P< 0.05). But VEGF and IGF-1 protein levels have no statistically significant difference between the four groups in (P>0.05). 14d after operation, COL I, bFGF and PDGF protein levels group A increased, and significantly higher than those in B group and C group (P< 0.05).In the second part, tendon stem cells showed cobblestone like tile on the bottom of the culture bottle, and a clone like growth. Safranin O, alizarin red and oil red O staining showed that tendon stem cells have to chondrogenic, osteogenic and adipogenic differentiation potential. Under 2%tensile stress, type Ⅰ collagen and type Ⅲ collagen of tendon stem cells significantly increased at the 24h, but in 48h, there was no change in the expression of these two genes (P> 0.05), while TNC and SCX two tendon cell marker gene and protein expression decreased at the 48h. In 4% continuous tension stress stimulation, COL Ⅰ, COL Ⅲ, TNC and SCX were significantly increased, and expression at 48h is higher than 24h (P< 0.05). While 6% of the continuous tension stress stimulation failed to make the tendon stem cells of COL Ⅰ, COL Ⅲ, TNC and SCX gene and protein expression significantly increased, which were significantly lower than the 2% and 4% stretch stress group (P< 0.05).Conclusions1, Rabbit acute Achilles tendon fracture and repair using functional position fixing is better than non functional fixation to promote collagen content of tendon tissue increase and fiber structure recovery.2, Rabbit acute Achilles tendon repair after functional position fixation can improve biomechanical properties of Achilles tendon repair tissue.3, Rabbit acute Achilles tendon repair after functional position fixation can promote tendon related growth factor gene and protein level in the repair of Achilles tendon tissue.4、Isolation and culture of tendon stem cells in vitro, which can be a clone like growth, have the potential of multi-directional differentiation.5,4% continuous tension stress stimulation can effective induce tendon to tendon stem cells differentiation. |
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