Study And Applicance Of Repair Periodontium With PRP/nHA/Co Compound Material

Periodontitis is one of the most common diseases in oral medicine, and the repair of periodontal defect is targeted to periodontal membrane, cementum and alveolar ridge. Tissue engineering, which three main elements are seeding cells, degradable vector material and regulating factors, provides new method of treatment for periodontal defect regeneration.Generally, the tissue engineering of periodontics also includes the three main methods of Growth factor-Vector, Seeding cell-Vector and Seeding cell-growth factor- vector. There are many clinical problems to be solved for the later two kinds (the origin of seeding cells and its immunology etc.). So the key research field is to accelerate the periodontal regeneration with appropriate vector containing foreign growth factors.The ideal vector materials should have the following characters: (1) good tissue compactibility; (2) bio-degradebiligy and the control for their degradation;(3)good bone conductibility and bone inductivity; (4) without antigen; (5) The surface should be in favor of maintaining the cell morphology and mould, and help the cell in adherence and proliferation; (6) the vector should have three-dimensional, multi-hole structure, and the size of holes should be in favor of the transportation of nutrients and metabolic products; (7) the vector should has its intensity; (8) the vector should take growth factors and other active materials; (9) the vector should be easy to be sterilized.PRP(Platelet Rich Plasma) is that of platelet concentrates,have a large number of platelets and rich growth factors.Transforming growth factor-β(TGF-β) and Platelet-derived growth factor (PDGF) are mainly in the plateletαParticles,when PRP mixing of CaCl2 and zymoplasm,the growth factors will release and promote bone tissue regeneration and repair.Hydroxyapatite(HA)is the main abio-element in marrow and teeth of animals. The collagen fibers is regularly arranged around hydroxyapatite, and the alveolar ridge be up to 97% of hydroxyapatite. HA ceramics has very good bio-compatibility and bone conductibitlity, be widely used in bone regeneration. The conductibitlity of HA can provide good environment for cell growth, while its multi-hole structure can be benefit for cell growth, tissue regeneration and vascularization, which is fit for the require of bone tissue engineering. Nano-HA ceramics is similar with natural bone, can improve the mechanical character of bone implants and be used as the vector of PRP.Collagen is a kind of fiber protein. CollagenⅠhas antitension and can be found in all parts of the body. Collagen can not be only formed the framework of extra-cellular matrix, but can interact with cells to influence the morphology, proliferation and differentiation of cells. Collagen can be used as the effective vector of PRP, and its degradability makes it an important material in tissue engineering.Objectives of this research: To composite PRP, nHA and collagen together, and evaluate the bio-characteristics of this compound material. In this way, we want to use the advantage of every one to make it a new substitute of bone regeneration and vector material.Methods:Synthesize of nHA: The nHA was synthesized with precipitation. The Ca(OH)2 and H3PO4 was mixed together in the ratio of Ca/P as 1.67, and the H3PO4 solution was put into Ca(OH)2 glycol solution under mixed. After 2h's mixed, they were filtrated, drying and then calcined for 2h under 800℃respectively.Extraction of CollagenⅠ: Adult cattle Achilles's tendon was first frozened, and then cut into 0.1mm slice. It was put into 0.05 moL? L-1 acetic acid(pH3.2), and digested with pepsin(100mg?g-1), and then filtrated and centrifuged. The upper layer was salt outed with NaCl, and centrifuged after the separation of white deposition substance. The deposited collagen was dissolved in acetic acid, and dialysed with 0.01 moL? L-1 pH7.4 PBS for 96h, then dialysed with distilled water until there was no Na+,Cl- in the solution. The dialysed collagen was dehydrated with PEG and concentrated as liquid collagen, which was then coupleted with glutaraldehyde.Preparation of nHA/Co Membrane: 0.5g nHA powder was surged with ultrasonic for 10min, and dissolved in 75ml,0.1mol? L-1 HCl. Then, 225ml collagen type I was added into the former solution and whisked and diluted with distilled water to 800ml, then 0.025mol? L-1 NaOH (PH=12.5)was put into it until the pH was about 6.2, and then remained at 7.2 for 10min. The upper solution was wiped off by eccentricity, and the sample was frozen dried for all kinds of test. Preparation of PRP:Whole blood was drawn from dog , after two times of centrifugation ( first time 1500r/min,10min;second time 3000r/min,8min),then red blood cells and platelet poor plasma was separated with PRP. platelet count was carried out.Preparation of nHA/Co/ PRP Membrane : Mix PRP and( 10%CaCl2,zymoplasm with 6∶1), then add to the nHA/Co compound membrane。Animal experiment:4 adult male dogs were selected in this experiment. A"U"like bone defect about 5mm was prepared on the buccal side of molar area until 5mm under the enamel-cemtum junction. The exposed periodontal membrane and cementum was scraped and planned. The PRP/nHA/Co membrane was put on right side and the left side was empty as control side. Two animals were sacrificed 4, 8 weeks after the surgery respectively. Sixty-four row CT ray and Three-Dimensional Reconstruction observation was taken showed the new bone formation, and routing HE staining,BMP-2,bFGF Immunohistochemistry and TRAP methods was made to observed under microscope.Results:Electron Microscope result : (1)The size of hole of the synthesized nHA resembling with that of natural bone; (2) The surface hole and size of compound material was symmetrical, and nHA granule was equally distributed to the matrix of collagen and combined with collagen finely, with the mean size of granule 60nm; (3) The chemical composition of compound material was similar with that of natural bone, mainly by collagen and carbonic nHA.PRP count result: PRP was prepared successfully and the platelet count was 5.14 times of that of the whole blood and the concentration. Naked eye result:The animals grow healthy, with the gingival healing well and PD<1.2mm.CT and Three-Dimensional Reconstruction observation:new alveolar bone formation and the ridge height growed to normal. While , the ridge height was not growed in the control group.Tissue observation:Control Group: HE staining found a lot of lymphocyte cells and neutrophile cells could be seen around the defect area,no cartilage tissue formation, the junctional epithelium grow。Immunohistochemistry staining found BMP-2,bFGF is negative.TRAP staining found the positive cells were more than experimental group.Experimental Group: HE staining found a lot of cartilage-like matrix,cartilage around compound membrane. Then the new bone and connective tissue were generated and no junctional epithelium grow。Immunohistochemistry staining found BMP-2,bFGF is positive in the new bone and connective tissue and osteoblast,bone cells around them。TRAP staining found the positive cells were less than control group。The upper experiments showed that:1,nHA/Co has good bio-compatibility, without any cell toxicity and can be used as the bone substitute material.2,PRP activated by the cow zymoplasm in the early release of a large number of growth factors , and then the platelet will continue to release.3,PRP/nHA/Co could be completely degraded 6-8 weeks after implantation and accelerate the periodontal regeneration.Conclusions: PRP/nHA/Co compound membrane can be used as a new bio-active material and vector material as bone substitution in tissue engineering.