A Study On The Mechanism Of Thammatic Brain Injury Allecting The Speed Of Bone Fracture Healing

Background:It has been observed clinically that an overgrowth of callus and even ectopic ossification in the muscle may occur in a patient with bone fracture combined with cerebral injury, and the speed of bone healing is accelerated, yet the mechanism is not clear. It is generally believed that the process of healing after bone fracture is a recapitulation of normal development of embryo bone, including a series of changes in the bone cells and BCM (extracellular matrix), from the beginning of injury of the cells, blood vessels and bone matrix to the complete reconstruction of the bone. It is a complex process influenced in many ways and multiple aspects by the gross and local environment of the organism and involving in participation of many growth factors, forming the base of bone healing. Whatever the ways of promoting the repair of bone injury are, they are based on the promotion of the changes in the growth factors. Therefore it is worthy to make a further study for the mechanism influencing the changes in growth factor level expression, which effecting the bone healing in patient with bone fracture combined with traumatic brain injury.After bone fracture, the synthesis and secretion of vascular endothelial growth factor (VEGF) are in synchronization with the osteogenesis. VEGF could promote the multiplication of endothelial cells, forming capillaries to contribute to the bone healing. The synthesis of bFGF is occurred mainly at the early stage of osteogenesis, and it is regarded it could stimulate the bone healing through the stimulation of the multiplication of undifferentiated mecenchymall stem cells, which may have relationwith the differentiation of the mesenchymall cell to form osteoblast. The immediate early genes (IEGs) c-fos, c-jun are widely distributed in eucaryote, expressing FOS and JUN nucleoprotein under the induction of the second messenger. FOS and JUN protein could form allodiploid AP-1 through leucine zipper, as a transcription factor, the allodiploid AP-1 combine with the AP-1 combing locus of many genes or TPA (Phorbol) reaction series effecting the expression of target genes in the late stage. The cortical impact injury could change the expression of IEGs. but the definite mechanism is unclear.VEGF and bFGF has dosage dependence, and has synergism in the process of wound healing. There is a close correlation between bFGF and cancer gene c-fos and c-jun;they complement each other and act together, regulating the growth and differentiation of cells under precise control. The influence of brain injury on the bone reconstruction is very complex. As conjectured from the feature of IEGs expression in the early stage of TBI, it is a subject worthy to study whether the IEGs expressed FOS, JUN protein after TBI could effect the VEGF and bFGF expression around the traumatic lesion in the brain, and subsequently effecting the VEGF and bFGF expression at the fracture site and in turn leading to the change in the speed of bone healing.Methods:One hundred and eighteen ICR male mice bought from Zhejiang provincial Academy Medical Sciences, with body weight about 20 g, clean grade, breeding in a quiet and non- irritative environment under room temperature for more than 24h. The animals were divided randomly into 2 groups: Group A, (TBI+F) Traumatic brain injury combined with unilateral tibial fracture;Group B (F): Simple unilateral tibial fracture. Prospectively using kirschner wire for intra medullary immobilization of the tibial fracture to rule out the influence of mechanical factor. Six animals from each group were killed by rapid decapitation respectively at lh, 3h, 6h, 12h, 24h, 48h, 72h, 96h later after modeling. The brain tissue were taken through craniotomy, the cerebral hemisphere of the injured side was treated with 4% paraformaldehyde phosphate for fixation, and cut about 1 cm periosteum and muscle around the fracturesite at the same time, using 4% paraformaldehyde phosphate for fixation overnight. The section preparation was made through routine dehydration, paraffin imbedding. The main procedure of immunohisto chemistry coloration (SP method) includes deparaffin, hydration, histo antigen EDTA heat repair, endogenous peroxidase blocking agent and normal immune animal serum, CFOS, C-JUN, VEGF, bFGF mono-ante, di-ante, streptomycin affinity agent-biotin-peroxidase compound;DAB coloration;and haematoxylin recolouration. Observation for the results: The coloration positive signal appeared with brown yellow granules. The specimen were observed with blind method under light microscope, counting positive cells / 100 cells in 10 optic fields (X 400 fold), and taking the mean value. Leaving 6 animals for normal control, the other animals (Group A, n=7, group-B, n=9) were subjected to X-ray examination for the evaluation of bone healing at 1 w, 2w, 3w, 4w respectively, exposure requirement: 40Kv, 5mAs, observing the healing state of the bone fracture. The experimental data were presented by mean value ± standard deviation or the percentages of the raw data, using spsslO.O statistic sofhvare for statistical treatment. The mean value deviation was treated with pairwise comparison q-test among multiple mean values. The correlation between the FOS, JUN protein and bFGF, VEGF expression in brain and the fracture site was treated using spearman rank correlation analysis, P<0.05 was defined as having statistical significance.Results:1. In Group A (TBI+F), the ratio of bone healing at lw, 2w, 3w and 4w were 0/7, 2/7, 6/7 and 7/7 respectively, and that in Group B (F) were 0/9, 0/8, 2/7 and 7/7 respectively. The iconography showed that in Group A nearly all of the animals had bone healing at the 3rd week after the operation, with larger amount of callus, while in Group B, the bone healing resemble that of Group-A until the 41 week.2. The normal mice had a low expression of FOS and JUN protein in the cerebral cortex, the FOS, JUN protein positive expression was presented in the nucleus of nerve cells, with brown yellow color, the cytoplasm was not colored, the nucleuswas round or oval in shape, part of the nucleoli showed a deep brown color. In Group A, an increase in the FOS, JUN protein expression around the contusion lesion of the brain was noted at 1 h after TBI, which reached the high peak at 3h, began falling at 6h and reduced to the control level after 12h;there was an agreement in time-process between the FOS and JUN expression. Statistical analysis showed in Group A, the comparison between Group A and the control group at lh, 3h and 6h had significant difference (/><0.05), the pairwise comparison between these 3 time point had significant differences, p<0.05. In Group B, the FOS, JUN protein expression was slightly stronger in comparison with the control group, but had no statistical significance (P>0.05).3. The bFGF expression of the cerebral cortex in normal mice showed a slight positive coloration, appeared mainly in the cytoplasm with brown yellow granules. There was a change in the strength of bFGF reaction around the injured brain lesion at 1 h after TBI in Group A;the strength increased obviously at 3h, became stronger continually, reached the highest point at 12h and then began falling, after that reduced to control level at 72h. Statistical analysis revealed the comparison at lh, 3h, 6h, 12h, 24h, 48h between Group A and control group had significant difference (P<0.05), and the pairwise comparison between these 6 time-point had significance (P<0.05). In Group B, the bFGF of the cerebral cortex showed a low level expression and had no significant difference in comparison with the control group (P>0.05).4. The VEGF protein expression of cerebral cortex in normal mice was negative. The positive coloration expression was mainly in cytoplasm with brown yellow granules. In Group A, there was significant difference of VEGF expression around the contusion lesion of the brain after TBI. It began increasing at 6h after injury, reached high peak at 24h and then began falling, hen reduced control level at 72h. Statistical analysis showed in Group A, the comparison at 6h, 12h, 24h, 48h had significant difference in comparison with the control group (/><0.05), and the pairwise comparison between these 4 time-point had significant difference (P<0.05). In group B, there was a low level expression of VEGF inthe cerebral cortex, and had no significant difference in comparison with the control group (P>0.05).5. The normal mice had a low expression of FOS and JUN protein in the periosteum and muscle. In Group A and Group B, an increase in the FOSn JUN protein expression around the fracture site was noted at 3h after injury, which reached the high peak at 6h, began falling at 6h and reduced to the control level after 24h;the comparison between Group A, Group B and the control group at 3h, 6h and 12h had significant difference (/><0.05), the pairwise comparison between these 3 time point had significant differences, p<0.05.6. The bFGF expression in the periosteum and muscle in normal mice was slightly positive. The bFGF expression around the fracture site after TBI in Group A: it began at l-3h after the injury, and became stronger at 6h, and reached high peak at 12h and still maintained at high level at 72-96h. In Group B, the bFGF expression around the fracture site increased at 6-12h after the injury, reached ■ high peak at 24h and maintained a high level at 72-96h. The statistical analysis showed that the comparison at 3h, 6h, 12h, 24h, 72h, 96h in Group A and 6h, 12h, 24h, 48h, 72h, 96h in Group B had significant difference as compared with the control group (P<0.05);and the pairwise comparison between 3h, 6h, 12h and 48h time-point had significant difference (/><0.05);and the pairwise comparison ' between the other time-point had no significance (P>0.05).7. In normal mice, the VEGF protein expression of periosteum and muscle was negative. In Group A, the VEGF expression around the fracture site after TBI increased at 6h, reached peak value at 24h. In Group B, the VEGF expression increased at 12-24h, reached high peak at 48h. Both groups had VEGF protein expression maintained at high level for more than 96h. Statistical analysis revealed that in comparison with control group, the Group A at 6h, 12h, 24h, 48h, 72h, 96h and Group B at 12h, 24h, 48h, 72h, 96h had significant difference (P< 0.05), and the pairwise comparison between 6h, 12h, 24h time-point had significant difference (P<0.05), and the pairwise comparison between the other time-point had no significance (P>0.05).8. Spearman rank analysis was carried out to study the correlation between FOS,JUN protein and bFGF,VEGF expression in both groups, which showed a positive correlation between the FOS and bFGF,bFGF and VEGF expression around the injured brain lesion in Group A with correlation coefficient for 0.896,0.832, respectively, p<0.05, around the fracture site with correlation coefficient for 0.938,0.945, respectively, p<0.05;In Group B, the FOS and bFGF,bFGF and VEGF expression around the fracture site had positive correlation with correlation coefficient for 0.833,0.914 respectively, P<0.05. In Group A, the FOS,JUN expression of the brain and that of expression at the fracture site had a positive correlation with correlation coefficient for 0.913 > 0.907 respectively, P<0.05. In Group B, the expression correlation of FOS,JUN protein between and fracture site had no statistical significance.Conclusions:1) The experimental animal model (TBI + Long bone fracture) of closed unilateral tibial fracture combined with traumatic brain injury on fixed head of free-falling body impact model is an ideal experimental animal model for the evaluation of the effect of brain injury on the bone healing.2) In TBI, the high expression of FOS, JUN protein around the injured lesion cause ah evident increase in bFGF expression in the contused brain tissue, effecting the increase in VEGF expression level and subsequently playing a synergism role in the repair of tissue.3) The high expression of FOSJUN around the contused brain after TBI could directly and/or indirectly promote the expression of FOSJUN at the fracture site, thus enhancing the osteogenic action of VEGF and bFGF and accelerating the healing of fracture.4) After brain injury, the earlier appearance and increase of the peak value of VEGF, bFGF expression at the fracture site are helpful for the promotion of multiple cells chemotaxis, multiplication and differentiation, thus accelerating the osteogenesis.5) The high expression of VEGF -^ bFGF around the contused brain after TBI could accelerating the healing of fracture through peripheral blood.6) The early optimal immobilization of bone fracture after TBI is reasonable based on the mechanism underlying the acceleration of osteogenesis, and is helpful for the promotion of bone healing.