Experimental Study On The Expression Of VEGF In Fracture Healing After Brain Injury

Objectives: To explore the mechanism of vascular endothelial growth factor(VEGF) enhanced osteogenesis on SD rats fracture healing after brain injury by investigate the distinct expression of VEGF in fracture only and in fracture with brain injury.Theory basis was afforded for the treatment of refractoriness fracture for clinical selectivity use of gene therapy.Method: 66 SD rats were randomly divided into 3 groups: normal control(n=6), fracture group (F group,n=30),fracture with brain injury group(TBI+F group,n=30)Brain injury model of rats were striked by using of modified Marmarou installation in order to make diffuse midrange brain injured model.The methods of fracture model of rats: Knee joint of left lower extremity was depilated and sterilized and opened skin in length wise to reveal and decoherence ligament of knee joint. Condyles of tibia was perforated and inserted into about 25 millimeter asepsis Kirschner wire along cavum pulpi direction. Decoherencd passivitily muscle and anadesma in above 1/3 of shin bine and fracted transversally shin bone and closed cut one by one. When SD rats were numbered and weighed, they were fixed on the specially made operation table after effective anesthesia by intraperitoneal injection in 10% chloral hydrate (3ml/kg). Operation process was all carried out in condition of strict asepsis and then put them into cages to freely run and take food and water.6 rats in each of F group and TBI+F group were put to death in the third day, first week, second week, twenty-forth day, sixth week after the operation. The parts of fracture were examined with X-rays to observe the reparative process and effect. Osteotylus samples were obtained about one centimeter away from broken ends of fractured bone, then got a half of the total preserve in liquid nitrogen after removaled quickly soft tissue and cleaned by cold normal saline for RT-PCR detection and another half for observation of light microscope immunohistochemistry staining.Result:1 X-rays1.1 F group:In the third day, first week and second week there are no callus existing and fracture lines are clear. In the twenty-forth day there are some callus existing and fracture lines are clear. In the sixth week there are fracture lines but no clear and a great quantity callus surrounding the fracture parts.1.2 TBI+F group:In the third day and first week there are no callus existing and fracture lines are clear. In the second week there are some callus existing and fracture lines are clear. In the twenty-forth day there are fracture lines but no clear and more callus surrounding the fracture parts. In the sixth week the fracture lines disappears and there are a great quantity callus surrounding the fracture parts that partly thickening and bone density is higher.2 RT-PCR2.1 F group: In the third day there were no espression of VEGF. In the first week there were some expression of VEGF and after this increases gradually. In the twenty-forth day expression of VEGF achieves peak. In the sixth week there were no espression of VEGF.2.2 TBI+F group: In the third day there were some expression of VEGF and after this increases gradually. In the second week expression of VEGF achieves peak and keeps on to the twenty-forth day. In the sixth week there were no espression of VEGF.3 Light microscope observation3.1 F group: In the third day, some granulation tissue were investigated.In the first week granulation tissue grew into the fracture parts and cartilage cells were formed. In the second week neogenesis bone trabecula were formed. After the twenty-forth day bone trabecula starts to link each other and sclerotomal cells convert to bone cells. In the sixth week bone trabecula gradually start to moulding and converts to lamellar bone.3.2 TBI+F group: In the third day, granulation tissue were investigated in the fracture parts. In the first week granulation tissue grows into the fracture parts and cartilage cell and individually newly formed bone trabecula are formed. In the second week newly formed bone trabecula are formed. In the twenty-forth day bone trabecula stsrt to link and moulding each other. In the sixth week bone trabecula start to convert to lamellar bone .4 Immunohistochemistry staining4.1 F group: In the third day, some VEGF positive cells were investigated in granulation tissue occasionally. In the first week, VEGF positive expression were investigated in cartilage matrix, bone cell, chondroblast, vascular endothelial cell and fibroblast in granulation tissue. In the second week , VEGF positive expression were investigated in osteoblast, cartilage cells,and fibroblast .After the twenty-forth day , VEGF positive expression were investigated in osteoblast, cartilage cells,and vascular endothelial cell. In the sixth week, VEGF positive expression could only investigated in some cartilage cells.4.2 TBI+F group: In the third day, VEGF positive cells were investigated in granulation tissue . In the first week, VEGF positive expression were investigated in cartilage matrix, bone cell, chondroblast, vascular endothelial cell and fibroblast in granulation tissue. In the second week, VEGF powerful positive expression were investigated in osteoblast, chondroblast, cartilage cells,and fibroblast .After the twenty-forth day , VEGF positive expression were investigated in osteoblast, cartilage cells,and vascular endothelial cell. In the sixth week, VEGF positive expression could only investigated in some cartilage cells.5 Immunohistochemistry staining image analysis:The expression of VEGF of F group were started to appear in the first week and keep on to the peak about in the third week. The expression of VEGF of TBI+F group were started to appear in the third day and reachs the peak and keep on to about the third week. In the third and 7th day, the expression of VEGF in TBI+F group was obviously higher than in F group(P﹤0.01) and has statistical significance. In the second week expression of VEGF in F group was higher than in TBI+F group(P﹤0.01) and has statistical significance. In the 24th day, the expression of VEGF in F and TBI+F group has not obvious difference(P﹥0.05) and not statistically significant. And to the the sixth week both of groups have not expressed.Conclusion:1 Multiple factor have participated and coordinated the union of fracture and VEGF may be produce a marked effect as a important factor in fracture healing.2 Expression crest-time of VEGF is about in the third week in fracture healing but is two to three week in fracture healing combined with traumatic brain injury which persistence time is longer and ahead of schedule.3 More quick healing and more callus of rats fracture after central nerve injury shows that central nerve injury has promoted union of fracture.That may be relevant to longer persistence time and ahead of schedule in expression crest-time of VEGF.4 It offers original pathway and theory basis to treat refractoriness fracture for clinical use safely and efficiently of VEGF.