The Experimental Study Of Nano-hydroxyapatite DCSI On Rats’ Bone Defect

Objective:Observing the nano-hydroxyapatite/deproteinised calf blood serum injection (NHA/DCSI) in treatment of the bone defect and comparing it with the control group, we make an elementary discussion about the repairing effects of different doses of DCSI on bone defect, and whether the amount of new vessels and the formation of new bones are related to local DCSI doses. It provides the experimental basis and theoretical foundations for further research and clinical application of the bone tissue engineering materials, and provides a new way for the treatment of bone defects.Method:Mix respectively the nano-hydroxyapatite powder with the10mg and20mg DCSI and prepare the mixture into the compound artificial bones, and make use of the random numerical table method to feed respectively the Wistar rats in3rat cages,15ones per cage, and the cages are labeled with1-3, in which No.1is as the blank control group and No.2and No.3are as the experimental groups. Implant the nano-hydroxyapatite materials only into the mice of No.1cage, implant NHA/10mgDCSI into the mice of No.3cage, and implant NHA/20mgDCSI into the mice of No.3cage. In the4th,8th and12th week after operation, we select randomly5rats to put them to death, intercept the bone ends of the femoral bone defect to remove the soft tissue of muscle and tendon, retain of poroma as the labaratory specimens, and all the specimens should be preserved for use in the liquid nitrogen immediately after marking the time and names. Select each group of animal specimens, stain with hematoxylin eosin, and observe the tissue repair of the bone defect and its differences under the microscope. Take advantage of the immunohistochemistry vascular staining to examine the number of vascular in the tissue, and to judge the bone-formation activity and strength of this tissue. The positive staining granules in sight which appear yellow or brown put color on vascular endothelial cell plasma, selecting randomly2of100-times fields to count for each slice and averaging to observe it the number of vascular is different among the groups. Adopt RT-PCR technology to detect the vascular endothelial growth factor (VEGF) gene expression of the bone defect area in each group. The experimental data obtained is measurement data, expressing with mean±standard deviation (x±S). The three samples are compared by one-way analysis of variance, and the two samples are compared by independent SNK-q test, if P<0.05, it means that there is a significant difference.Result:Bone densitometry:In the4th,8th and12th week after operation, use the dual energy X-ray absorptiometry to measure each group’s bone density. In the4th week after operation, the variance analysis F=5.46, in the12th week after operation, F=41.74, which shows that the time is longer, the difference is more obvious. SNK-q test shows: in the8th and12th week after operation, compare P<0.05in each group, which has statistical differences.Histological examination:By Lane-Sandhu histological score, with the time goes on, the score value of the experimental group and the control group is higher, while for each time point, the score of the control group is the lowest, in the4th week after operation, the variance analysis F=10.706, in the12th week after operation, F=13.000, which shows that the time is longer, the difference is more obvious. SNK-q test shows: in the4th and8th week after operation, compare P<0.05between the control group and the experimental group, and in the12th week after operation, compare P<0.05in each group.Immune vessel count:The compound artificial bone materials have angiogenesis in Week4, which is significantly higher than the control group. With the extension of the time, the new vessels increase, and in all experimental animals, the number of vascular of the compound20mg artificial bone material animal is the most. In the4th week after operation, the variance analysis F=8.486, in the8th week after operation, F=30.636, and in the12th week after operation, F=34.667. SNK-q test shows:in the4th week after operation, compare P<0.05between the control group and the experimental group, and in the8th and12th week after operation, compare P<0.05in each group.Vascular endothelial growth factor gene expression:The expression of vascular endothelial growth factor gene is not obvious in Week4, obvious in Week8, and to the peak in Week12. In the4th week after operation, the variance analysis F=4.192, in the8th week after operation, F=36.400, and in the12th week after operation, F=30.682. SNK-q test shows:in the4th week after operation, compare P<0.05between the control group and the10mg experimental group, which has statistical difference between the20mg compound group and the other groups, and in the8th and12th week after operation, compare P<0.05in each group.Conclusion: NHA/DCSI artificial bone is better than the simple application of NHA to repair the bone defect, which is favorable for the formation of the early vascularization and new bones in bone defect areas. With the increase of the compound dose, the measured value of the bone density, the formation amount of the new bones, the amount of the neovascularization, and the gene expression of the vascular endothelial growth factor increase correspondingly. Because of the massive ingrowth of the blood vessel, the artificial bones are vascularized, which provides the required mesenchymal cells and osteogenic cells for the bone repairing so as to promote the formation of the new bone tissue. This study only focuses on two doses of experiment, and is just the results of animal experiments. Although the experimental results show that as composite dose increases, the number of the angiogenesis and the amount of new bones formation increase, doses cannot be increased without limits, because too large dose may result in the formation of local hemangioma, systemic toxic reaction and other complications. However, the safe and effective dose of the local drug and the suitable dose for human body still need further research.