A Research On The Function And Mechanism Of Setting Broken Bone Mixture In Repairing Defect Of Homologous Spongy Bone

Objective: This dissertation is to discuss the function and mechanism of setting broken bone mixture (rhizome of drynaria 250g; Himalayan Teasel Root 250g ;pyrite 400g; ground beetle 100g; common forage 99kg). It can promote blood circulation to dispel blood stasis, in repairing the defect of homologous spongy bone, and to probe into its possible mechanism by observing BMP-2 in the repairing area of bone. Methods: 70 adult rabbits, weighed from 2.0kg to 2.5kg without limitation of sex, were used in this test. 60 of them were randomly divided into 5 groups. And then each group was divided into a test team and a controled team, and 6 rabbits in each team of this group. The other ten rabbits offer homologous spongy bone. They were killed by pouring 10ml of air into the vein of each rabbit's ear. Spongy bone of bilaterally ilium was taken to make 1x2x2mm sponge bone particle with rongeur and scissor. Rabbits in both teams were first anaesthetized intravenously with 20mg/kg of barbital sodium, and then 1/3 of their middle and lower part of radius were exposed in asepsis environment after the skin. Then a 20mm standard bone defect model with a bone grinding machine was made, and the left and right radical bone defect with above sponge bone particles was repaired. The test team was fed with setting broken bone mixture and the controled team was fed with neither common forage nor setting broken bone mixture. In order to prevent rabbits from infection, they were injected with 0.1 g/kg of cefazolin sodium into muscle for 4 days in a row. After they were killed separately in the 1st,2nd,4th,6th,8th week, their radius were x-rayed. Two rabbits were taken from each team to be the specimen for the electron microscope and other two from each team for histological observation, situ hybridization as well as electron microscope observation. Then the effect of Setting Broken Bone Mixture towards BMP-2 gene was analyzed in the process of repairing bone defect with homologous spongy bone. Results: X-Ray observation: For the test team: There was no obvious change in the first week. There was a little porosis in second week; an obvious increase of porosis appeared in the fourth week. The bone defect blurred during the sixth week. In the eighth week, there were a lot of porosis on the bone defect, and the bone defect vanished. The porosis was less in controled team synchronically. Absorption ratio of X-Ray was better in the test team than it is in the controled team synchronically. Histological observation: Part of the osteocyte necrosed in implanted bone; some nuclei of osteocyte of implanted bone concentrated, and even vanished. There was a proliferation of fibroblasts in haematoma with inflammation. During the second and fourth week, a few cartilage cells and a lot of blood vessels formed with new bone growing into implanted bone. There were also some cells concentrated and fragmented. During the fourth and eighth week, a lot of blood vessels formed around the implanted area of the bone. The network of new bone formed and turned into marrow cavity. There was no obvious change in the controled team in the first week. A few cartilage cells and blood vessels formed during second and fourth week. During sixth and eighth week, there were an increase of porosis and blood vessels, but the marrow cavity had not formed yet. Synchronically compare, the percentage of cross-sectional area of blood vessels in test team was always higher than that in controled team. The electron microscope observation: In the first week, there were destruction on part of the bone trabecula and some sphacelus with a little inflammatory cell. In the second week, there was a fusiform of mesenchymal cells with a few fibers in the test team,. And the nuclei of osteocyte was concentrated while the chromatin agglomerated aside. In the controled team, there was no obvious fusiform cell. Some collagen fibers were found around osteocyte. Nucleocytoplasmic fibers concentrated around cells. In the fourth week, there were more capillaries in the test team than there were in the controled team. There were formation of bone trabecula and bone corpuscle in the test team, too. In the controled team, there were formation of some bone matrix, but there were lesser capillaries. In the sixth week, in the test team, the bone matrix and chromocenter were obvious, and there was an increase of the endoplasmic reticulum. While in the controled team, osteocyte, bone trabecula and bone canaliculi just formed partly. In the eighth week, there were more osteoblasts and woven bone in the test team. Osteoblasts and an increase of sclerotin are lesser in the controled team, and the woven bone was not obvious, either. Situ hybridization observation : In the test team, BMP-2 was negative after one week. It was weakly positive after two weeks and was strongly positive after 4 weeks. It was weakly positive in the sixth and eighth week. However, BMP-2 was negative in the controled groups after one week and it was positive after 4 weeks. And it was weakly positive after 2,6,8 weeks. RT-PCR result: Under the condition of 94℃, 30 second, 55-65℃, 30 second, 30 Cycles 72℃, 1 min/kbp, Target gene of BMP-2 in the controled team were: 2547±2 copies in the first week, 4972±1 copies in the second week, 1230±3 copies in the fourth week, 411±2 copies in the sixth weeks, and 856±4 copies in the eighth week. Under the same condition in the test team, target gene of BMP-2 were: 5347±3 copies in the first week, 3171±2 copies in the second week, 1981±2 copies in the fourth weeks, 1978±4 copies in the sixth week and 501±2 copies in the eighth week. Take copy number of the first week as considered reference, the ratio of copy number of the controled team and the test team in the first, second, fourth, sixth and eighth week were 1.00±0.00,2.19±0.01,3.2±0.03,2.29±0.02,1.42±0.01 in the controled team and 8.83±0.02,3.44±0.03,6.50±0.01,6.00±0.02,2.50±0.03 in the test team. Result of SAS system statistics was 0.0427, P<0.05. Conclusion: Setting broken bone mixture, which promote blood circulation to dispel blood stasis, can remarkably increase the quality of repair of bone defect with homologous spongy bone. It can enhance the repair of bone tissue and has a dramatic influence on the releasing level of Bone Morphogenetic Protein 2(BMP-2), the induction of bone formation, and revascularization.