| Objective: Compromised Achilles tendon healing in patients is one of the complications of diabetes.During the development of diabetes,a large number of advanced glycation end products(AGEs)accumulate in the Achilles tendon.This study aims to establish a rat Achilles tendon healing model to observe the effect of AGEs on Achilles tendon healing,to explore whether AGEs is an important reason for the compromised Achilles tendon healing in diabetic patients,and to provide a theoretical basis for the treatment of Achilles tendon rupture and related studies in diabetic patients.Methods: 1.Thirty-six male 8-week-old Specific Pathogen Free(SPF)Sprague Dawley(SD)rats were selected.The Achilles tendons were transected sharply with scalpel at the upper 5mm of the calcaneus of both hind limbs,and sutured with modified Kessler method.After operation,the rats were randomly divided into low dose AGEs group,high dose AGEs group and PBS control group.The low dose and high dose groups were injected around the Achilles tendon with 350mmol/L(low dose)and1000mmol/L(high dose)glycosylated reagent D-ribose 0.2ml respectively to increase the level of AGEs in Achilles tendon while the control group was given the same amount of PBS twice a week for 6 weeks.After a week of rest,the rats were killed and the Achilles tendon specimens were removed.2.Evaluate the general condition,general macroscopic situation and adhesion of Achilles tendon.3.The healing site of Achilles tendon was made into tissue homogenate and the contents of AGEs,type Ⅰ collagen,TNF-α and IL-6 in the healed Achilles tendon were detected by ELISA method.4.The tissue of Achilles tendon was fixed with 4% Paraformaldehyde for 48 hours,and then made the pathological sections.The morphological changes and collagen regeneration of Achilles tendon were observed by HE staining and Masson staining,and the pathological changes of Achilles tendon were evaluated by modified Movin scale.5.The cross-sectional area of Achilles tendon was measured,and then the two ends of Achilles tendon were trimmed and fixed on the universal material tensile testing machine for biomechanical testing,including maximum load,yield load and stiffness.Results: 1.General situation: there was no death in all rats before and after operation.The wound sutures gradually fell off,the skin incision healed well,and the activity of rats returned to normal 3-4 weeks after operation.2.The level of AGEs in Achilles tendon increased with the increase of D-ribose concentration(p<0.0001).Macroscopic condition and adhesion evaluation: The macroscopic score of Achilles tendon healing in high dose AGEs group was significantly lower than that in low dose AGEs group and PBS group(p<0.0001,p<0.0001).The adhesion index around Achilles tendon in high dose AGEs group was significantly higher than that in low dose AGEs group and PBS group(p=0.004,p<0.0001).Compared with PBS group,the general score of Achilles tendon healing in low dose AGEs group was lower(p=0.039),and the peri tendon adhesion index was higher(p=0.047).3.With the increase of the level of AGEs,the levels of inflammatory factors such as TNF-α and IL-6 in Achilles tendon increased,and the production of type Ⅰ collagen decreased.The difference was statistically significant(p<0.0001).4.Histopathological changes of healed Achilles tendon: In PBS group,the fibers were arranged tightly and neatly,collagen staining was dark red,and a small amount of inflammatory cell infiltration could be seen with HE staining.Under Masson staining,the blue collagen fiber bundles were flat,regular and dense,the collagen fibers were produced more,the tendon structure was intact,the direction of the tendon bundle was the same as the longitudinal axis of the Achilles tendon,the collagen content was high.In low dose AGEs group,the arrangement of collagen fibers was relatively messy.There was a gap between a small number of collagen fibers and a small amount of inflammatory cell infiltration with HE staining.There was no difference in collagen content between low dose AGEs group and PBS group with Masson staining,but collagen fiber bundles were relatively messy,crisscross and uneven arrangement in low dose AGEs group.The lesions that were not completely repaired could be seen in the high dose AGEs group under HE staining.Most of the collagen was crimped seriously,the appearance was slender,the shape was irregular,the arrangement was disordered,the color was pink,there were more gaps between collagen,and the distribution of inflammatory cells and neovascularization was more than that in the low dose AGEs group and PBS group.The content of collagen in the high dose AGEs was lower than that of the other two groups.The collagen fibers were crimped seriously,and the continuity was worse than that of the other two groups.The fibers were disorderly and uneven,and the appearance was slender and wavy.The modified Movin semi-quantitative score showed that the high dose AGEs group had the highest score compared with the low dose group and PBS group(p=0.012,p<0.0001),and the low dose group scored higher than the PBS group(p=0.024).5.Biomechanical Tests: With the increase of AGEs concentration,the cross-sectional area,maximum load,yield load and stiffness of Achilles tendon decreased(p<0.0001).Conclusions: Injection of D-ribose around rat Achilles tendon can increase the content of AGEs in Achilles tendon.The accumulation of AGEs in Achilles tendon is an important factor leading to compromised healing,weakened biomechanical properties,aggravated stiffness and increased adhesion of Achilles tendon after rupture of Achilles tendon in diabetic patients. |
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