【作者】 满毅；

【导师】 宫苹；

【作者基本信息】 四川大学 ， 口腔临床医学， 2007， 博士

【摘要】 在骨缺损修复材料的应用上，已从惰性材料发展到生物活性材料；在操作技术上，则从创伤性手术向微创技术发展。然而现在口腔临床上使用的骨修复材料多为颗粒型，虽然具有一定的疗效，但其可塑性、机械性能、操作性能差，并且由于材料缺乏骨诱导活性常常需要配合生物膜使用，治疗周期长。为了减轻患者痛苦、缩短治疗周期、降低医疗资源消耗以及减少术后并发症。因此在骨缺损修复治疗领域，寻求可在原位固化，具有良好操作性能的磷酸钙骨水泥材料成为当前的研究热点。目前已有磷酸钙骨水泥产品开始应用于临床，但是现有的材料抗弯强度低，脆性大，在生理环境中的疲劳与破坏强度不高，尤其是在湿环境下断裂韧性很低，属于一种典型的脆性材料，因此，在没有增韧的条件下，它只能用于不承受负荷或仅承受纯压力负荷的情况。然而在口腔中，颌骨和牙体、种植体周围的牙槽骨由于咀嚼功能等原因处于周期性的拉力和压力环境，而且磷酸钙骨水泥的生成产物主要为羟基磷灰石，其通过骨传导的方式促进骨的形成，骨的生成速度慢，使磷酸钙骨水泥的应用受到很大的限制。因此有必要从磷酸钙骨水泥的生物力学性能和骨诱导性两个方面进行改性和不断的完善。本研究首先通过湿法反应制备α-TCP磷酸钙骨水泥，在磷酸钙骨水泥中添加天然材料蚕丝丝素增强磷酸钙材料的机械性能，弥补磷酸钙材料脆性大、抗弯强度低的缺点；然后在磷酸钙骨水泥调和液中添加地塞米松、β-甘油磷酸钠、L-抗坏血酸做为骨诱导剂增强材料的骨诱导活性，获得改良的磷酸钙材料，并进行了细胞、分子生物学检测，为动物试验、临床应用打下基础。本研究结果及提示：1．通过湿法反应法，以碳酸钙和磷酸为原料，可以获得高纯、微细的α—TCP粉末。以磷酸氢钙、碳酸钙为辅料的α—TCP体系骨水泥，采用0.25MNaH₂PO₄／Na₂HPO₄为骨水泥调和液，其初凝时间在7min左右，终凝时间在17min左右，pH值接近中性，抗压强度为7.4628MPa，抗弯强度为5.1629MPa，基本满足临床要求。骨水泥粉体颗粒水化后，在SBF中转化为细小的磷灰石晶体。2．在胰酶浓度为0.25％，温度37℃，脱胶时间12h情况下，可以良好的除去蚕丝丝胶，并且保持蚕丝优良的机械性能。对蚕丝丝素纤维进行编织，以3mm的长度加入磷酸钙骨水泥，不影响磷酸钙骨水泥的凝固性能，抗压强度增加到12.541MPa，抗弯强度增加到8.8822MPa。3．在骨水泥调和液中加入骨诱导配方不影响骨水泥的性能，不同复合方式的骨水泥（骨水泥组；骨水泥复合丝素组；骨水泥复合骨诱导配方组；骨水泥复合丝素、骨诱导配方组）与骨髓基质干细胞共培养，在细胞增殖活性和空白对照组没有统计学差异，细胞粘附形态上与对照组相似。4．不同复合方式的骨水泥与细胞共培养后，RT—PCR检测均表达成骨相关基因ALP、COL—Ⅰ和OCN，空白对照组表达ALP、COL—Ⅰ，不表达OCN；ELISA检测添加了骨诱导配方的骨水泥在细胞与材料共培养早期表达成骨特异性蛋白OCN，未添加组在2周后表达OCN蛋白，空白组不表达OCN蛋白，添加骨诱导配方的磷酸钙骨水泥与未添加骨诱导配方的磷酸钙骨水泥在骨特异性蛋白的表达上有显著性差异。综上所述，本研究通过湿法反应法制备了高纯，微细的α-TCP粉末，以磷酸氢钙、碳酸钙为辅料获得了基本满足临床要求的磷酸钙骨水泥材料；在磷酸钙骨水泥中添加天然材料蚕丝丝素增强磷酸钙材料的抗弯、抗压性能；在磷酸钙骨水泥调和液中添加地塞米松、β-甘油磷酸钠、L-抗坏血酸做为骨诱导剂增强材料的骨诱导性能，最终获得具有骨诱导性和一定韧性的磷酸钙骨水泥材料。更多还原

【Abstract】 Nowadays, with the development of biomaterial science, the bone defects repair materials developed from the traditional mechanical fixation to biomaterial implantation and the technique of surgery developed from traumatic to minimal invasive technology. However, most of the biomaterials used in oral clinical is particle and not good in shaping, biomechanics, handling and lack of osteoinductivity which always have to be used with biomembrane. In order to relieve the pain of patients and reduce the syndromes, seeking for a more ideal material such as calcium phosphate cement which could owns the properties of self-setting in situ, easy handling and bone inductivity became the most attractive point.Some of the calcium phosphate cement （CPC） has been used in clinic; however, the relatively low strength and susceptibility to brittle catastrophic fracture of the cement have severely limited its use to only non load bearingm applications. CPC sets to form hydroxyapatite which lack of osteoinductive ability, So we have to improve the the biomechanic and osteoinductive ability of the CPC.The aim of the study is to synthesize the CPC with improved mechanics and osteoinductivity. First theα-TCP particle and the CPC based onα-TCP was synthesized. Then the degummed silk is used to improve the strength and fracture resistance of materials. Ascorbic acid,β-glycerophosphate and dexamethasone are added in CPC liquid to improve the osteoinductivity of the materials. The results of the study show that:1.α-TCP was synthesized by the reaction between phosphoric acid and CaCO₃ slurry. The assist agents of CPC are CaCO₃ and DCPD. The miture powders were mixed with 0.25M 0.25MNaH₂PO₄/Na₂HPO₄. The initial setting time is about 7 min, the final setting time is about 17min. The compressive strength reached 7.46MPa and the bending strength reached 5.16MPa.2. Trypsin was picked out as degumming reagent for the ability of preserving the mechanic integrity of silk. The examination of degummed silk suggested that the sericin can be totally removed under our degumming protocols.The simple twisting method was adopted by us for the sake of adding the degummed silk in 3mm length. The CPC added with degummed silk can reach 12.54MPa of compressive strength and 8.882MPa of bending strength. The setting time is almost the same between the CPC group and the CPC added with degummed silk and osteoinductive agents.3. In vitro experiments were designed and carried out to evaluate the biological reaction of the CPC. The BMSCs cells were cultured on CPCsurfaces in vitro. Cell attachment, spreading, cell proliferation and differentiation were evaluated and there is no statistic difference between CPC and control group.4. The mRNA level of osteoblast-related genes ALP, Col-I and OCNwere examined using RT-PCR; The mRNA of ALP, Col-I and OCNwere expressed in all CPC groups and wasn’t expressed in control group. The protein level of OCN was examined using ELISA, there is statistic difference between CPC and CPC+osteoinductive group. With the limitation of these findings,α-TCP with the assist agents of CaCO₃ and DCPD can almost meet the clinic demands. The compressive and bending stress were improved with the adding degummed silk fiber to CPC and the osteogenic ability of the CPC were improved by adding the osteoinductive agents to CPC.更多还原