【作者】 张莉；

【导师】 周延民； 高文信；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2010， 博士

【摘要】 目的：将富含血小板血浆(PRP)、纳米羟基磷灰石(nHA)和胶原(Co)复合,探讨复合此种材料的有效方法,并通过动物实验评价复合材料的生物学性能。方法：采用化学沉淀法制备nHA,采用乙酸溶解和蛋白酶降解消化法提取Ⅰ型Co,并把nHA和Co溶液按比例混合冻干制成nHA/Co复合材料,采用二次离心法提取PRP,将PRP与nHA/Co混合制备成PRP/nHA/Co复合材料。将nHA/Co复合材料与骨髓基质细胞复合培养和植入兔下颌骨缺损处,将PRP/nHA/Co复合材料植入兔下颌骨牵张模型中,分别观察材料性能、细胞生长情况和成骨效果。结果：nHA/Co复合材料呈三维网孔状结构,骨髓基质细胞生长良好,具有良好的生物相容性；nHA/Co复合材料能够较好地修复下颌骨缺损；PRP/nHA/Co复合材料能够促进下颌骨牵张成骨。结论：PRP/nHA/Co复合材料能够促进骨组织再生,在牵张成骨过程中可以作为一种促进骨组织形成的新型生物活性材料和支架材料。更多还原

【Abstract】 Experimental Studies of PRP/nHA/Co Compound Material on Accelerating Distraction OsteogenesisDistraction Osteogenesis is a kind of bone lengthening of the emerging technologies in recent years, in the craniomaxillofacial surgery has been widely used to obtain the conventional treatment is difficult to obtain results, when compared with other surgery has incomparable advantages, such as no bone grafting, soft tissue surrounding bone can be expanded over the same period, surgery is relatively simple, less traumatic and so on, thus there is a wider range of clinical indications. At present, the process of distraction osteogenesis, tissue engineering of bone formation and maturity is the research focus.In the bone tissue engineering, the use of suitable carrier material carrying the exogenous growth factors to promote bone tissue regeneration is the hot spot. Platelet-rich plasma (PRP, Platelet-Rich Plasma) is concentrate of self-body’s autologous platelet, rich in growth factors, transforming growth factor-β(TGF-β) and platelet-derived growth factor-(PDGF) play the role major, those factors will release that promote bone tissue regeneration and repair. Hydroxyapatite(HA)is the main abio-element in nature bone, the alveolar ridge be up to 90% of hydroxyapatite. HA ceramics has very good biocompatibility and bone conductibility, be widely used in bone regeneration. Nano-HA ceramics is similar with natural bone, can improve the mechanical character of bone implants. Collagen is a kind of fiber protein. Collagen (Co) is a major component of bone tissue can be divided intoⅠ,Ⅱ,Ⅲ,collagen I which the most extensive, the main function is as an organization supports, gives organizations tension, promote cell proliferation and differentiation. Degradation of collagen can make to slow the release of growth factors has antitension and can be found in all parts of the body, Collagen degradability makes it an important material in tissue engineering.The purpose of this study:The platelet-rich plasma (PRP) and nano-hydroxyap-atite (nHA)/collagen (Co) composite, composite of such materials to explore effective ways to make the composite material not only has good biocompatibility and has a strong promote regeneration of tissue defects of the mandible, making it becomes new bioactive materials and scaffold material in the way of promoting the formation of new bone tissue in tissue engineering, lay the foundation for its clinical application.Experiment I Preparation of Compound Bio-MembraneExtraction of Collagen I:Adult cattle Achilles’tendon was first frozen, and then cut into 100μm slice.0.05 moL/L acetic acid and bovine tendon sheet integration, making the collagen-the final concentration of acetic acid and 25g/L, and then digested with pepsin, two high-speed centrifugation, dialysis and dehydrated extract purified, refined liquid collagen.Synthesize of nHA:the Ca(OH)2 and H3PO4 dissolved in anhydrous ethylene glycol mixed solution prepared, at room temperature, stirring, after 4h, filtered and dry aging are the dried gel, and finally at 300℃, 600℃and 800℃will muffle furnace under the conditions of dry gel calcined 2h standby.Preparation of nHA/Co materials:0.5g hydroxyapatite powder dissolved in 75mL 0.1moL/L HCl, respectively, and 300mL I get 150,225 to join the collagen solution and add distilled water diluted to 800mL, room temperature Drop one under 0.025moL/L NaOH (pH12.5), when the pH value rose to 7.2 centrifugal removal of the supernatant obtained nHA/Co composite materials composite materials.Results:(1) the synthesis of HA after 300℃,600℃and 800℃calcined, the results show that the temperature of 800℃when the HA particle size and most similar to natural bone.(2) Molecular weight collagen extracted by the detection of that collagenⅠ(3) nHA/Co materials simulate natural bone chemical composition and structural characteristics, the ultra structural observations indicate that HA particles evenly distributed in the collagen matrix, the average size of particles 60nm bonding well with the collagen.ExperimentⅡPreparation and Detection Platelet-Rich Plasma(1)Preparation of PRP:Extract 5ml blood from the central artery of rabbit ear by 10ml syringe, into centrifuge tubes, centrifuged twice. The first centrifugation speed 1500r/min for 10 minutes. Abandoned to the lower part of the red blood cells in the centrifuge tube, to collect all the plasma to another centrifuge tube, the second centrifugation, the speed 3000r/min for 8 minutes, after the second centrifugation, the plasma is divided into the upper platelet plasma (platelet poor plasma, PPP) and lower the PRP, abandoned on the top of the PPP (about 70% of the total volume of plasma), the remaining liquid is about 1.5ml, shaking, it is just PRP.(2) Counting of whole blood and PRP platelet:extract PRP and whole blood each 20ul,adding 0.38ml platelet diluents, collecting gently shake up after the lessons of platelet suspension 15μl was injected into blood count room, put it aside for 15 minutes, then put it in the high-powered light microscope for the platelet counting(3) PDGF concentration determination:ELISA method used in PRP and whole blood concentrations of PDGF was determined separately.Results:(1)whole blood and PRP platelet concentrations were 398.4G/L and 1974.9G/L, PRP platelet concentration is 4.96 times as it in whole blood,in line with the requirements of PRP.(2)whole blood and PRP in the PDGF concentrations were 35.86 ng /ml and 143.4 ng/ml, PRP platelet concentration is 4.02 times as it in whole blood, results show that PRP does contain beneficial growth factor for bone Formation.ExperimentⅢBiocompatibility of Compound Membrane(1)Extraction and culture of BMSCs:Experiments used New Zealand white rabbits as the target collection of bone marrow stoma cells. Sumianxin anesthesia injected animals, routine preoperative skin preparation, sterile laboratory in the inner thigh in animals with bilateral femur bone marrow aspiration, and immediately turn down to prevent blood coagulation in vitro, the collected bone marrow stoma cells into the contained 15% fetal calf serum DMEM complete medium, wind and percussion with a suction pipette several times, the cells suspended in culture medium. Hatched box in the CO2 temperature for 5 days, half of the cell medium was changed, the initial medium change after 3 days medium was changed again the whole time. Thereafter, once every 3 days medium was changed to 15 days, adherent bone marrow stoma cells covered the bottom of the flask. When 90% of the bottom flask wall was covered with BMSCs, will remove the culture medium, slow flushing bone marrow stoma cells, the use of EDTA digestion with trypsin the cells were collected and observed under inverted microscope cell growth, the wall of wind and percussion BMSCs transferred down to the tube, discard the supernatant after centrifugation, the bottom wall of the collected cells were cultured.(2) Co-culture of Osteoblast and nHA/Co:Before culture nHA/Co composite film with Co60 irradiation sterilization, the bone marrow stoma cells combined with the composite film culture, cell attachment on the material, remove the culture membrane rinsing with PBS, conventional HE staining, optical microscopy, histological observation, and using scanning electron microscopy observation of the sample were extracted and organizational structure in order to detect bone marrow stoma cells were cultured with the process of composite materials have changed, and the proliferation of changes, which evaluate the biocompatibility of composite material.Results:(1) The cells showed positive staining of Von Kossa after 21 days of induction, proving that it had the ability of osteogenic.(2) The co-culture:Light microscope showed that cells adhered on the surface and margin of membrane and linked to form a slice; SEM showed that many BMSCs adhered on the margin of membrane and there was some matrices in some area. So the membrane had good biocompatibility and no toxic to the cells.Experiment IV Experimental Repair of Mandible Defect with Compound MembraneMethods:20 healthy adult New Zealand rabbits were prepared rectangle bone defect of about 2.0cm×1.0cm×0.5cm at middle part of bottom edge of both mandibles. The right side was the experimental one, whose defect was covered with PRP/nHA/Co compound membrane; while the left group was sutured without any membrane. The animals were sacrifi-ced 4w,8w after surgery respectively, the both mandibles were taken soft X-ray; and routine HE staining for light microscope examination. Results: Naked eyes observed healthy rabbits with the incision healing well.Soft X ray showed: Control Group:there was still the nick 8 weeks after the surgery. PRP/nHA/Co:Bone-like density in the bone defect area 4 weeks. Bone density increased and the defect area healed completely 8 weeks.Tissue Observation:Control Group:Lots of fiber tissue and some new trabicular bone and osteoblast after 4 weeks some layer bone increased after 8 weeks.nHA/Co Group:There was a still lot of membrane in the defect area and the new trabicular bone linked to form reticular structure 4 weeks. The membrane had degraded completely and the defect area was completely substituted with new bone and the healing is finished 8 weeks.Experiment V PRP/nHA/Co composite materials on Accelerating Distraction OsteogenesisMethods:36 healthy adult big ears rabbits of New Zealand, bilateral mandibular cortical bone cut depth of less than lmm, implanting PRP/ nHA/Co composite membrane material in the corresponding position, nHA/Co group implanted nHA/Co composite membrane, the control group does not place any material, fixed distraction device, layered suture. After 4weeks,8weeks, animals were killed to take bilateral mandibular, Line X ray photography; to do routine HE stained sections, BMP-2 and bFGF immunohistochemical staining observed under light microscope.Results:The eye observation:the survival situation and condition of experimental animals is well, distracter solid and the incision healed well.Soft X-ray observation:control group after fixed stretched8weeks, the place stretched apart is still showing a flaky low-density shadow.nHA/Co Group fixed stretched after 8weeks; the stretched gap was significantly narrowed. The density is higher then before.PRP/nHA/Co group fixed stretched for 4 weeks, the distance was short between the both sides of the bone stump, a part show a higher density areas. Fixed stretched for 8 weeks, the density in the areas is similar to the normal bone around, the continuity of the lower edge of mandibular recovery well.Density Analysis:nHA/Co group values in bone mineral density higher than the control group with a fixed period, there are significant differences;PRP/nHA/Co group values in bone mineral density higher than the control group with a fixed period, there are significant differences.HE histological observation:At the 4 weeks, a chain opened district is filled with granulation tissue. at the 8 weeks, trabecular bone formation, but the disarray. nHA/Co group at the 4 weeks, some regions have formed osteoid, bone cells have been buried in them. At the 8 weeks, the chain opened district trabecular bone thickening and thicker, narrowing the distance between the two stump. PRP/nHA/Co group at the 4 weeks, the chain opened district can be seen a lot of trabecular bone area. The distraction group at 8 weeks, new bone tissue make up the most part of the chain opened district. The original woven bone was changing into the lamellar bone.Immunohistochemical observation:BMP-2:control group 4weeks, hyperplasia active mesenchymal cells and fibroblasts in the place stretched apart could be seen strong positive staining.8 weeks, fibrous connective tissue weakened into a weak positive staining. nHA/Co group at thee 4 weeks, osteoblasts at the edge of new woven bone could be seen positive staining (figure) in the place stretched apart.8 weeks, fibrous connective tissue weakened into a weak positive staining. PRP/nHA/Co group at 4 weeks, the fibrous tissue around the newborn trabecular bone showed positive staining in the place stretched apart.8 weeks, the new bone matrix and osteoblasts around can be seen the weak positive staining.bFGF:control group 4 weeks, bFGF in hyperplasia active mesenchymal cells in the place stretched apart could be seen positive staining.8 weeks, fibrous connective tissue shows weak positive staining. nHA/Co group 4 weeks, positive staining in the place stretched apart was mainly located in osteoblasts at the edge of new bone.8 weeks, is mainly change is the positive staining in the mesenchymal cells and fibroblast cells weaken. PRP/nHA/Co group:4 weeks, osteoblasts at the edge of new woven bone could be seen positive staining in the place stretched apart.8 weeks, it shows weak positive staining in the not fully calcified bone.Conclusion::1. The compound HA granule was similar with that of natural bone, and fit the require of nano-size for HA in natural bone; the extracted collagen is highly purified with low antigen city; The HA could combine with collagen to form tightness bonding.2. nHA/Co has good biocompatibility, without any cell toxicity and can be used as the bone substitute material.3. Second centrifugation used in extraction of PRP is simply and effective to operate, and the content of PRP platelets extracted by the method is high.4. PRP in the early release of a large number of growth factors, and then the platelet will continue to release.5. PRP/nHA/Co composite materials implanted in mandible distraction model can contribute to distraction osteogenesis, improving the quality of newly formed bone.6. Detected BMP-2,bFGF by immunohistochemistry, confirmed the PRP/nHA/Co composite materials can contribute to distraction osteogenesis.PRP/nHA/Co composite materials can promote bone regeneration, it can be used as New bio-active materials and scaffold material in the Promotion of the formation of new bone tissue in the process of distraction osteogenesis.更多还原