| Objective:To study the expression of VEGF in serum during tibial process in New Zealand rabbits with different rates of bone lengthening,and to investigate the relationship between transport rates and callus formation in combination with histology and imaging,so as to determine whether VEGF has the potential to regulate transport rates.Methods: The animal model of tibial bone lengthening was established by using Rauch F tibial bone lengthening methods.The experiment consists of two parts: in the first part,12 rabbits were randomly selected by number table method from 48 New Zealand white rabbits and divided into four groups: A,B,C and D,Group A was the control group(n=3):After 5mm osteotomy of the middle tibia fixation by external fixator;In group B rabbits(n=3)were subjected to tibia lengthening by the Ilizarov technique at a rate of 0.5 mm/day in 1 step after 5days’ latency period;In group C rabbits(n=3),lengthening in 2 steps at a rate of 1.0 mm/day after 5 days latency period;In group D rabbits(n=3),lengthening in 3 steps at a rate of 1.5 mm/day after 5 days latency period;The final degree of shin bone lengthening in groups B,C and D was 30 mm.The blood was taken before the surgery,5 days after the surgery,shin bone lengthening 6mm,12 mm,18mm,24 mm,30mm,10 days after end of distraction period,20 day after end of distraction period and 30 day after end of distraction period.Serum concentrations of VEGF was measured by ELISA.Radiology and histology were used to assess callus formation.The blood drawing time,radiological and histological detection time of group A were consistent with that of group C.In the second part,the remaining 36 New Zealand white rabbits were randomly divided into 4 groups with 9 rabbits in each group.Group A was the control group(n=9):After 5mm osteotomy of the middle tibia fixation by external fixation bracket;B,C and D are experimental groups with different distraction rates at 0.5 mm/day,1 mm/day,1.5 mm/day after 5 days latency period;One rabbit was killed randomly and tibia was taken for HE staining when tibia lengthening 6mm,12 mm,18mm,24 mm,30mm,10 days after end of distraction period,20 days after end of distraction period and 30 day after end of distraction period.The data was input by Excel and analyzed by SPSS 25.0,Significance of between-group differences at different stages of observation were evaluated using repeated measures ANOVA.Results: the results of ELISA showed that the concentration of serum VEGF in the control group A increased gradually after operation,reached the highest level(138.73% before operation)17 days after operation,then decreased gradually,and decreased to a slightly higher level after the end of the distraction period,the average value was 552.42 ± 8.64 ng / L;In group B(0.5mm/d),the concentration of serum VEGF increased gradually and increased significantly after distraction,and reached the highest level(182.28% before moving)11 days after operation,and then the concentration decreased gradually,but remained at a higher level until the end of moving,the average value was 638.11 ± 10.67ng/l,85.69(95% CI: 2.71-168.66)ng / L higher than that in group A(P < 0.05);The concentration of serum VEGF concentration in group C increased gradually after operation,the peak value(171.74% before operation)appeared on the 17 day(12mm)after operation,and remained at a high level until the end of distraction period,the average value was 655.32 ±11.76ng/l,which was 102.91(95% CI:57.80-148.00)ng /L higher than that in group A(P < 0.05);There was significant difference in serum VEGF between group B,C,D and group A(P < 0.05),while there was no significant difference in serum VEGF between group B,C and D(P >0.05).Bone mineral density(BMD)measurement showed that BMD of group B,C and D began to increase after distraction to 6mm,In group B,the BMD increased the fastest,decreased slightly at the later stage of distraction period,and increased again at the later stage of consolidation period,with a mean value of 0.159 ±0.007g/cm~3;The BMD of group D increased slowly,which was significantly lower than group B and group C during the distraction period,and increased significantly in the later stage of consolidation period,with a mean value of 0.093± 0.002g/cm~3.The difference of BMD between group B,C and group D was statistically significant(P < 0.05).The correlation analysis of serum VEGF concentration with bone mineral density and bone mineral content in group B,C and D Results show that There was no significant correlation between serum VEGF concentration and bone mineral density at each test point(P>0.05);There was no significant correlation between serum VEGF and bone mineral content(P > 0.05).The results of X-ray showed that the primary callus was seen on the 11 th day after operation in the group A,and then the amount of callus increased gradually.There was callus connection at the osteotomy end on the 29 day after operation,and the callus filled the gap of bone defect on the 35 day after operation.In group B,the primary callus began to appear at 6 mm(17 days postoperatively),and increased gradually at 12 mm(29 days postoperatively)to 30 mm(65 days postoperatively).The callus grew from the two ends of the bone to the interval.At 75 days postoperatively,the callus filled the interval.In group C,primary callus was formed and increased gradually when it distraction to 12mm(17 days postoperatively).Callus also grew from both ends of osteotomy to the distraction space.45 days postoperatively,callus filled the interval,the content of callus in each test point was less than that in group B.In group D,a small amount of callus could be seen at 18 mm(17 days after operation),Callus formation is slow,and the formation speed of callus was significantly less than that of group B and group C with the same detection point.HE staining showed that in group A chondrocytes began to appear 11 days after operation,and a large number of disorganized woven bone formation was observed.Part of the new bone trabecula were formed under the periosteum 17 days after operation,osteoblasts and osteocytes were transformed,29 days after operation,the bone trabeculae were connected,a small number of chondrocytes were found in the defect space and cartilage callus was replaced by new bone trabeculae.In group B,sparse woven bone and collagen fibers were found at 6mm(17 days postoperatively),part of the trabecular connections were seen at 12mm(29 days postoperatively),and a large number of chondrocytes were still found in the space between the two ends.When moved to 18 mm(41 days after operation),the bone trabeculae were connected,and a small amount of new blood vessels were found.When group D distraction to 12 mm,a large number of mesenchymal cells and fibrous tissue could be seen in the osteotomy segment.To24 mm,a large number of collagen fibers could be seen in the woven bone.Conclusion:1.The experiment found that the most suitable tibial lengthening speed was 1mm/d.2.It was found in the experiment that the tibial bone lengthening can stimulate the body to produce VEGF,and the expression of VEGF is continuously high in the process of tibial lengthening.3.The experiment found that there was no significant difference in the average serum VEGF concentration after tibia in rabbits with different tibial lengthening speeds,so it was impossible to regulate the bone lengthening speed according to the serum VEGF concentration.4.The experiment found that there was no significant correlation between VEGF expression and callus formation in tibial serum of rabbit limbs with different rates of bone lengthening. |
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