| Part one:Effects of epidermal growth factor receptor signaling inhibitor gefitinib on fracture healing in ratsObjective:Establishing a rat fracture model of fumer,to observe the effect of gefitinib,an inhibitor of epidermal growth factor receptor(EGFR)signaling pathway,on the healing of femoral fractures in rats.Methods:50 Spragu-Dawley(SD)rats of 4 weeks old were selected to establish the femoral fracture models and were randomly divided into two group by random number table:the experimental group was given gefitinib(100mg·Kg-1·d-1)dissolved in 0.5%methyl cellulose by intragastric administration,and the control group was given an equal dose of 0.5%methyl cellulose.X-ray examming after 7,14,21,28 and 42 days of fracture and general observation of femoral fracture healing after femoral specimens were collected.Evaluation of fracture healing by using Lane and Sandhu radiographic scoring and the Image J software was used to evaluate callus diameter.Then the differences of bone healing between the two groups were compared by microCT analyses and biomechanical testing.The bone volume(BV),bone volume fracture(BV/TV),trabecular thickness(Tb.Th),trabecular separation(Tb.Sp),trabecular number(Tb.N),bone surface(BS),BS/BV and bonemineral density(BMD)were measured by microCT.Biomechanical test failure load,stiffness and work to failure of femur specimens.Results:The X-ray score of the experimental group were higher and the maximum diameter of callus were lower than those in the control group(P<0.05)on the 7,14 and 21 day after the fracture but not on 28 and 42 days.The results of microCT showed that in the experimental group,BV was significantly higher in the 14th day(59.30 ± 6.54mm3)and 21st day(43.08 ± 2.33mm3)than in the 14th day(42.39 ± 7.82mm3)and 21st day(33.43 ± 5.94mm3),BV/TV in the 14th,21st,28th day(0.61 ± 0.06%,0.55 ± 0.05%,0.53 ± 0.04%)was higher than those in the control group(0.48 ± 0.07%,0.44 ± 0.07%,0.43 ± 0.03%,Tb.N in the 14th,21st,28th day(2.05 ± 0.11 1/mm,1.86 ± 0.18 1/mm and 2.034 ± 0.26 1/mm)were higher than those in control group(1.63 ± 0.21 1/mm,1.32 ± 0.21 1/mm and 1.65 ± 0.081/mm),Tb.Th in the 14th,21st,28th day(0.33 ± 0.02mm,0.33 ± 0.03mm,0.27 ±0.02mm)were higher than those in the control group(0.27 ± 0.03mm,0.28 ± 0.02mm,0.23 ±0.01mm),TB.Sp in the 3rd week(0.39 ± 0.07mm)was higher than those in the control group(0.30±0.03mm),BS/BV in the 14th,21st,28th day(9.3±1.66mm-1,10.15±1.14mm-1,7.99±0.81mm-1)was higher than those in the control group(6.51±1.42mm-1,7.33±1.31mm-1.6.24±0.79mm-1)and BMD in the experimental group in the 42nd days(723±7.4mgHA/ccm)higher than that of the control group(686±21.6 mgHA/ccm)(P<0.05).Biomechanical testing showed that the stiffness of experimental group(9898 ± 1733N/mm)was higher than the control group(5492 ±940N/mm)at 21 days,the failure load of experimental group(38.65 ± 1.07N,63.63 ±7.74N)were significantly larger than that of the control group(29.47±1.00N,45.42±3.26N)(P<0.05)at 21and 28 days after fracture and the work to failure of experimental group(60.20±4.34Nmm)were significantly larger than that of the control group(37.35±12.81Nmm)at 21and 28 days after fracture(P<0.05).Conclusion:Gefitinib,an inhibitor of epidermal growth factor receptor(EGFR)signaling pathway,has an effect on fracture healing and could promote fracture healing.Part two:A preliminary study on the effect of the inhibitor of epidermal growth factor receptor signaling pathway-gefitinib on the mechanism of fracture healingObjective:On the basis of the first part of the study,to explore the primary mechanism of gefitinib that the inhibitor of epidermal growth factor receptor signaling pathway promotes fracture healing.Methods:62 Spragu-Dawley(SD)rats of 4 weeks old were selected to establish the femoral fracture models and were randomly divided into two group by random number table:the experimental group was given gefitinib(100mg·Kg-1·d-1)dissolved in 0.5%methyl cellulose by intragastric administration,and the control group was given an equal dose of 0.5%methyl cellulose.Serum,callus and femur specimens were collected at 7,14,21,28 and 42 days after operation.The HE and safranin fast green staining were used to observe the histomorphology and cartilage formation of callus.Ki-67+ cells were detected by immunohistochemistry in order to observe the cell proliferation in callus.Dynamic observation of the new osteogenesis of callus by hard tissue section.The levels of serum osteogenic markers(BALP and PINP)and osteoclast markers(TRACP-5b and CTX)were measured by Elisa.qRT-PCR was used to detect the mRNA expression of related bone turnover markers(BALP,COLlal,COL 10,COL2a1,Osteocalcin and TRAP).Moreover,collagen staining in callus tissue was observed by Masson staining,and the difference of fracture healing between the two groups was compared.Results:The HE and safranin fast green staining showed that in the experimental group the callus tissue especially cartilage callus were significantly bigger than the control group at 7,14,21 day,and the fracture healing process of the experimental group was faster than that of the control group.The number of Ki67 + proliferating cells in the experimental group increased in the 7th,14th and 21st days.The dynamic observation of hard tissue section showed that the new bone tissue in bony callus of the experimental group was more than the control group.The level of serum BALP in experimental group was higher than that of control group(2.07±0.19ng/mL,2.77±0.29ng/mL)in the 14th and 21st day(2.57±0.14ng/mL,3.47 ± 0.26ng/mL)(P<0.05).The PINP in the experimental group was higher than that of the control group(2216.50 ± 96.68pg/mL,2692.33 ± 136.76pg/mL,3196.75±221.90pg/mL)in the 7th,14th and 21st day(1969.50±89.34pg/mL,2241.33±107.86 pg/mL,2603.25 ± 361.60 pg/mL).And the levels of TRACP5b(11.58±0.47ng/mL)and CTX(8.02±0.40ng/mL)in the 7th day were higher than that of the control group(10.33±0.53ng/mL,7.11±0.36ng/mL)(P<0.05).RT-PCR showed that the expression of BALP mRNA was higher than the control group in the 14th day,and the expression of COL1a1 mRNA was higher than the control group in the 7th and 14th day.The expression of COL2a1 mRNA increased than that of the control group in the 14th and 21st day.The expression of COL10 mRNA was higher than that of the control group in the 7th,14th and 21st day.The expression of TRAP mRNA was higher than that in the control group in the 7th day,while was lower than that in the control group in the 21st day(P<0.05).And the Masson staining showed that the amount of collagen fibers in the matrix was higher in the 7th,14th and 21st day of the experimental group.Conclusion:Gefitinib,an inhibitor of the epidermal growth factor receptor(EGFR)signaling pathway,promoted the differentiation and maturation of chondrocytes and osteoblasts in callus tissue,promoted the formation of callus,and significantly affected the bone formation in fracture healing.Gefitinib promoted the early accumulation of osteoclasts at the fracture end of fracture,and promoted bone resorption and absorption of necrotic substances at the fracture end. |
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