| Background:The great development of dental surgery and implantology in recent years has been possible due to the increasing research on surgical techniques and biomaterials. Nevertheless, lack of enough bone tissue is a major problem that implantologists still have to face. Most of the patients have to accept the bone incremental surgery during the treatments, but the osteogenic performance of the bone substitutes will affect the surgical results. Through continuous clinical treatment, people can fully understand the value of guided bone regeneration and bone substituting, so they try to improve the osteoinductive and osteoconductive efficacy in the scientific research.Bone morphogenetic protein2 is the most potential grow factor which has osteoinduction nowadays.Due to it spreads rapid in body fluid and decompose by prolease easily, it is hard to maintain its therapeutic concentrations. It could not continued to stimulate target cells to accelerate bone regeneration. In order to improve osteoinduction,researcher attempted to increase rhBMP-2, but lead to a series of complications.such as causing tissue swelling, increasing inflammation, promoting ectopic bone formation which is not expected, inducing cyst-like lesions in callus.More over, rhBMP-2 is expensive and has short half-life. Therefore, to develop a stable and sustained releasing system of BMP-2 is the focus, not only for treatment, but also for reducing the complication.Jianwu Dai has develop a collagen-based targeting bone repair systema. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. They showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. Collagen-binding assay showed that more rhBMP2-h than rhBMP-2 was retained on collagen after washing.In the clinical practice, four walls bone defect which like deficiency of alveolar bone width and height is the most difficult defect for doctors to implant, clinician always practice GBR to improve the width and height of alveolar bone. The collagen membrane used in GBR has the role of isolation and mechanical barrier,but not has osteoinduction.Newly formed bone has grown only one direction from the alveolar and it is slow.Objective:Prepare collagen membrane with osteoinduction by loading collagen-targeting recombinant human bone morphogenetic protein-2 and set up the rabbit GBR model. Evaluate the osteoplastic effect of BMP2-h/collagen membrane on rabitt GBR model.Methods:1.Machined the titanium cylinder using theΦ9mm medical titanium rod. After the polishing,they would be cleaned by ultrasonography and chemical reagents., packaged. After the high temperature and high pressure disinfection, they would be dried for use.2.45 New Zealand white rabbits were 5-6 months old and 2.7-3.2kg weight. Every rabitt was implanted 4 titanium cylinder. Four Treatment group in this experiment distributed on one rabitt:A:rhBMP2-h/collagen membrane, B:free rhBMP-2/collagen membrane,C:collagen membrane,D:control group45 New Zealand white rabbits participate this surgery,and 5 rabitts have loose titanium cylinders.40 rabbits include statiscal analysis.40 New Zealand white rabbits were randomly divided into 1,2,3,4 four groups.Every groups has 10 rabitt.The experimental time in group 1 was 2 weeks,in group 2 was 4 weeks, in group 3 was 6 weeks and in group 4 was 12 weeks.Every group in every time has 10 sample.3.To set up the GBR model.Every rabbits were prepared 4 annular craniofacial bone lacuna,implant the titanium cylinder, place bone dust, given rhBMP2-h/collagen membrane, free rhBMP-2/collagen membrane, collagen membrane and none membrane. Bullosa the skin, and sutured wound closely. In three days after the surgery,40 million units of penicillin was injected intramuscularly to prevent infection. All rabbits were held in separate cages. At the 13th,14th day before the executing, the rabbits would have subcutaneous injection of tetracycline. They also had subcutaneous injection of calcein at the 3 th,4th day before the executing. After 2,4,6 and 12 weeks,the animals were sacrificed. The soft tissues and the experimental parietal bones comprising the 4 titanium cylinderswere were taken en bloc and fixated by immersion in 4% buffered formaldehyde.8 samples in every group were given plastic embedded and 2 were given paraffin embedded. Leica SP1600 saw microtome was used to obtain 150-200 u m thick sections from routine plastic embedded bone tissue with titanium cylinder.. Each section was milled to 30μm thick. The millings were stained and verified with fluorescence staining and methylene blue-fuchsin acid.Sections was observed through histology,histomorphometry and fluorescence observation.Paraffin sections were HE trained and histology histomorphometry observation.Results:1.The animals recovered well after the surgery, and the wound also grew well. Every rabbit has 4 titanium cylinder,45 rabbits have 180 titanium cylinder. The loose rate was 6.7%,2.From observation of the specimens, soft tissue fibers can be seen to be closely wrapped around the titanium cylinders without dead space. Titanium cylinders were so solid that they provided an ideal space for the new bone growth.3.The new bone were attached by fluorescent dye, so that they could issue a green and yellow fluorescence. The fluorescent height and area of each groups were clearly contrast, and the result also had good comparability. More new bone volume in rhBMP2-h/collagen membrane group at 6 and 12 weeks than the other three group4.By the methylene blue-fuchsin acid train, we could clearly see that the news bone had grown into the titanium cylinder. Mature bone was dyed garnet, osteoid and fibrous tissue was stained purplish blue.2 weeks later, there were less new bone and no trabecular bone was found near the top of the titanium cylinder.4 weeks later, there were more trabecular bone formation, Early trabecular bone was found near the top of the titanium cylinder in rhBMP2-h/collagen membrane group but not free rhBMP-2/collagen membrane group,collagen membrane group and none membrane group.6 weeks later, the osteoid had been fully occupied all the space in titanium cylinder. There was a great quantity of mature bone near the top of the titanium cylinder in rhBMP2-h/collagen membrane group but not found in other groups.12 weeks later, the mature bone reach the half of the titanium cylinder and.the trabecular bone was thick. rhBMP2-h/collagen membrane group has much more bone than the other group although a few bone near the top of the titanium cylinder was absorbed.5.HE train:2 weeks later, a lot of artery and fibroblast grew into the titanium cylinder,specially in rhBMP2-h/collagen membrane group. Collagen membrane incomplete degradation.4 weeks later, most of the membranes were degradated.Near the top of the titanium cylinder,there were many osteoid and artery in rhBMP2-h/ collagen membrane group but fibrous tissue in others group.6 weeks later, intensive trabecular bone arrange near the top of the titanium cylinder, this phenomenon was not found in other group.12 weeks later, many mature trabecular gather at the top of the titanium cylinder in rhBMP2-h/collagen membrane group and fibrous tissue gathe in other group.6.Statistical analysis.Factorial classification:the results were statistically significant between time group and between treatment group (F=2.008, P=0.045); time and treatment have effects of interaction.2 weeks later,average area fraction of newly formed bone (2.45±1.88)% rhBMP2-h/collagen membrane group, (2.18±1.81)%free rhBMP-2/collagen membrane,(1.62±1.01)%collagen membrane group,(1.79±2.04)%control group, the results were no statistically significant (F=0.372, P=0.774).4 weeks later, average area fraction of newly formed bone(7.53±2.80)%rhBMP2-h/ collagen membrane group,(6.54±2.28)%free rhBMP-2/collagen membrane,(5.97±2.93)% collagen membrane group,(4.74±1.51)% control group, the results were no statistically significant (F=1.810,P=0.168).6 weeks later, average area fraction of newly formed bone(12.29±2.42). %rhBMP2-h/collagen membrane group,(8.40±3.62)%free rhBMP-2/collagen membrane,(7.03±1.17)% collagen membrane,(6.27±1.89)% none membrane group (F=9.619, P=0.000). Average area fraction of newly formed bone:rhBMP2-h/ collagen membrane group>free rhBMP-2/collagen membrane group≈collagen membrane group≈no membrane.12 weeks later, average area fraction of newly formed bone (16.50±4.75)% rhBMP2-h/collagen membrane group, (11.34±4.72)%free rhBMP-2/collagen membrane,(10.01±2.18)%collagen membrane, (9.12±4.06)% no membrane, the results were statistically significant (F=5.279, P=0.005). Average area fraction of newly formed bone:rhBMP2-h/collagen membrane group> free rhBMP-2/collagen membrane group≈collagen membrane group≈no membrane.Conclusion: 1.The rabbit GBR model provide a stable space to explore the osteoplastic effect of bone substitutes. Author improve the GBR model with the collagen membrane placing at the top of titanium cylinders,which was more resemble to clinical practice. This animal model is more simple, and the trauma is smaller and it can make the experimental result more reliable,objective and comparable.2.Collagen membrane obtain osteoinduction by loading collagen-targeting recombinant human bone morphogenetic protein-2. It has not only conductive effect but also effect of osteoinduction,can prevent the fibrous tissue growing into titanium cylinder,maintain the growth gap, and promote newly bone to regenerate.Collagen membrane loading with collagen-targeting rhBMP-2 sustained release rhBMP-2.6 weeks later, many mature trabecular gather at the top of the titanium cylinder, and the measurement results of trabecular area show rhBMP2-h/collagen membrane group> free rhBMP-2/collagen membrane group≈collagen membrane group≈control.3.RhBMP2-h/collagen membrane have stronger osteoconduction than free rhBMP-2/collagen membrane.
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