Experimental Study Of Effects Of Insulin-Like Growth Factor-1(IGF-I) And Basic Fibro-Blast Growth Factor(BFGF) On Injured Tendon Healing And Establishment Of The Animal Model

Objective: (l)To establish the rat model in vitro of injured patellar tendon healing. (2)To investigate the effects of IGF-1 on early stages of tendon healing in rats.(3)To observe whether there is synergetic effect on early stages of tendon healing in rats between IGF-1 and bFGF.(4)To observe the effects of IGF-1 on later stages of tendon healing in rats.(5) To observe whether there is synergetic effect on later stages of tendon healing in rats between IGF-1 and bFGF.Methods:(1)We used Male Wistar rats ,aged 8 weeks ,and weighting 250-300g.They were anesthetized with an abdominal injection of 2.5% (w/v) pentobarbital solution in a dose of 4.5mg/kg body weight.The skin and the patellar tendon sheath were opened with a surgical blade to expose the tendon.On the experimental side,a window defect of 2 × 4~6mm was created in the mid-part of the rat patellar tendon.(2)The first part:Eighty male Wistar rats were randomly divided into 4 groups at 20 rats per group. IGF-1 dissolved in 100μl sterile distilled water were injected into the window defects at a dose of 0,10,50,500ng at 3 days postinjury.The rats were killed 7days after operation.The second part:80 Wistar rats were randomly divided into four groups at 20 rats per group.3days after operation,The rats in the first group were injected with 100ul sterile distilled water only.Those in the second group were injected with 50ng IGF- I dissolved in 100 ul sterile distilled water.Those in the third group were injected with 50ng bFGF dissolved in 100 u 1 sterile distilled water and those in the fourth group were done with 50ng bFGF+50ng IGF-1 dissolved in lOOu 1 sterile distilled water.The rats were killed 7 days after operation.The third part: 80 Wistar rats were randomly divided into four groups at 20 rats per group. IGF- I were injected into the window defects for 3 times at a dose of 0, 10, 50, 500ng at 3,10,17 days after operation separately. The rats were killed 4 weeks after operation.The fourth part: Eighty male Wistar rats were randomly divided into 4 groups at 20 rats per group. 3,10, 17 days after operation separately, The rats in the first group were injected with lOOul sterile distilled water. Those in the second group were injected with 50ng IGF-1 dissolved in 100 u 1 sterile distilled water.Those in the third group were injected with 50ng bFGF dissolved in 100u 1 sterile distilled water and those in the fourth group were done with 50ng bFGF+50ng IGF-1 dissolved in 100 u 1 sterile distilled water.The rats were killed 4 weeks after operation.(3)After the rats were killed,The injured patellar tendon were observed dirrectly through a microscope;Then,patellar tendons dissected from ten animals in each dosage group were fixed and paraffinized for irnmunohistochemical analysis of proliferating cell nuclear antigen(PCNA) and type III collagen.The other ten patellar tendons in rats were prepared for mechanical testing for ultimate stress and biochemical analysis of the pyridinoline crosslink.Results:The first part:There was a difference in the number of proliferating cells among the various treatment groups (P <0.01) .A correlation analysis showed that there was a positive associatio between the number of proliferating cells and the dosage of IGF- I (P <0.05 ). There was a difference in the level of type III collagen expressionamong the various treatment groups (P <0.01) .A correlation analysis showed that there was a positive association between the level of type III collagen expression and the dosage of IGF-1 (P <0.01) .There was not a difference in mean ultimate stress associated with dosage among the various treatment groups (P>0.05) ; There was not a difference in mean pyridinoline contents among the various treatment groups(P>0.05) .The second part: Seven days after operation,the proliferating cells in each group increased in a different degree,but the proliferating cells in that group injected with IGF-1 and bFGF was the most prominent,and there was a significant difference compared with other groups (P <0.05)o There was not significant difference in proliferating between the group injected with IGF-1 only and that with bFGF only(P>0.05)o the level of type III collagen expression in each group increased in a different degree,but the level of type III collagen expression in that group injected with IGF- I and bFGF was the most prominent,and there was a significant difference compared with other groups (P <0.05) ;There was not significant difference in the level of type III collagen expression between the group injected with IGF-1 only and that with bFGF only (P>0.05)o There was not significant difference in the mean ultimate stress of the patellar tendon among the various treatmeng groups (P>0.05 ) ; There was not a difference in mean pyridinoline contents among the various treatment groups (P>0.05) .The third part:4 weeks after operation,the number of the proliferating cells in each group was not prominent,and there was not significant difference among the various groups (P>0.05)o The level of type III collagen expression in each group was not high,and there was not significant difference among the various groups (P> 0.05 )o There was a difference in mean ultimate stress of the patellar tendons among each group (P<0.01) ,and correlation analysis showed a positive association between the mean ultimate stress of the patellar tendons and the dosage of IGF-1 (P O.01) . There was a difference in mean pyridinoline content of the patellar tendons among each group (P<0.01) , and correlation analysis showed a positive association betweenthe mean pyridinoline content of the patellar tendons and the dosage of IGF- I (P <0.01) .The fourth part: 4 weeks after operation, the number of the proliferating cells in each group was not prominent,and there was not significant difference among the various groups (P>0.05) .The level of type III collagen expression in each group was not high,and there was not significant difference among the various groups (P> 0.05) .But the mean ultimate stress and the mean pyridinoling content of the patellar tendons in each group all increased in a different degree,and the mean ultimate stress in the group injected with IGF- I and bFGF increased most obviously,and there was a significant difference compared with the other groups (P <0.05) . There was not significant difference between the group injected with IGF-1 only and that with bFGF only (P>0.05) .Conclusions:(1) The rat model of patellar tendon injuries could be a experimental one which was used to study the effect of various growth factors on the healing of tendon injuries.(2) The first part: Supplementation with IGF-1 in vivo affected the cell proliferation and the expression of type III collagen in early stages of rat patellar tendon healing,but no effects on the ultimate stress.The second part:There was synergistic effects between IGF-1 and bFGF on cell proliferations and type III collagen expression in early stages of the rat tendon injuries,but no effects on the ultimate stress.The third part:There was not obvious effects of IGF-1 on cell proliferations and type III collagen expression in later stages of the rat tendon injuries,but IGF- I could affect the mean ultimate stress .The fourth part:There was synergistic effects between IGF-1 and bFGF on the ultimate stress in later stages of the rat tendon injuries,but no effects on cell proliferations and type III collagen expression.(3) This study showed that IGF-1 and bFGF could faciliate the healing of the...