【作者】 秦滢杰；

【导师】 万昌秀；

【作者基本信息】 四川大学 ， 生物医学工程， 2007， 硕士

【摘要】 理想的骨修复材料除了具有骨传导性，骨诱导性以及良好的生物相容性外，还必须具有可生物降解性。本论文作为国家自然科学基金项目“分子自组装RADA16-RGD／CPP纳米仿生基质骨修复材料的研究（50472091）”的一部分，欲研究新型骨修复材料掺锶聚磷酸钙的降解性能。近年来，聚磷酸钙（CPP）作为一种无机聚合物，具有可控降解速率、生物相容性好和足够的机械强度，越来越受到人们的重视。同时，锶作为治疗骨质疏松症的药物也逐渐受到广泛的关注，适量的锶能促进成骨细胞的增殖，抑制骨吸收，从而加速骨修复，并能一定程度提高骨骼的强度和韧性。本文从掺锶聚磷酸钙（SCPP）的制备工艺条件入手，在溶液介导的降解层面探讨保温时间即聚合度和煅烧温度对SCPP降解性能的影响，确定最优工艺条件。同时从巨噬细胞和破骨细胞两方面深入探讨了细胞介导的降解，最后探索了动物体内降解试验，初步确定SCPP具有良好的生物降解性能。本论文制备了不同工艺条件的SCPP材料。利用IR探讨了SCPP的化学结构，利用液相³¹PNMR对不同保温时间的SCPP的聚合度进行表征，结果显示SCPP的聚合度随保温时间的延长而增大。通过在不同温度下煅烧，制备了7种工艺条件的SCPP，XRD显示均为β-SCPP，SEM结果显示材料形貌随工艺条件不同而变化，700℃下煅烧的SCPP表面致密，晶界不明显，而800℃和900℃下煅烧的SCPP表面有微孔，晶界清晰。本论文在溶液介导的降解层面探讨了保温时间即聚合度和煅烧温度对SCPP降解性能的影响。研究结果表明：SCPP降解速率并不随聚合度的增大而减小，相同煅烧温度下，保温时间为5h的SCPP（聚合度为13）降解速率最快，其次是保温时间为3h的SCPP（聚合度为9），降解最慢的是保温时间为7h的SCPP（聚合度为19）；在SCPP的熔融温度范围内，相同保温时间下，SCPP的降解速率随煅烧温度的升高而增大，7种工艺条件SCPP的降解速率的顺序为7H900C＞5H800C＞5H700C＞3H800C＞3H700C＞7H800C＞7H700C；SEM显示降解速率最快的7H900C，5H800C和5H700C表面被Ca-P沉积物覆盖：综合考虑煅烧工艺及降解速率，选取5H800C的SCPP为最优工艺条件。本论文在细胞介导的降解层面研究了巨噬细胞对SCPP降解性能的影响，并探讨了Sr对巨噬细胞功能的影响。通过对材料失重，培养液中离子浓度的检测表明细胞组SCPP的降解速率略大于CPP，与单纯SCPP组相比巨噬细胞能明显促进SCPP的降解速率；ICP结果显示：6w内Sr的释放累计量为10μg，即1×10^-4mmol，远小于人体内Sr的含量3.5mmol，属于正常的生理范围。MTT和扫描电镜等结果进一步表明Sr的存在不会影响巨噬细胞的活性及形貌。本论文在细胞介导的降解层面初步研究了破骨细胞对SCPP降解性能的影响，并探讨了Sr对破骨细胞的影响。通过TRAP染色，骨陷窝检测，细胞形貌观察鉴定所培养细胞为破骨细胞，成功建立了破骨细胞的培养方法。SCPP组吸收陷窝数量，细胞数量明显少于CPP组，表明SCPP组破骨细胞介导的降解速率小于CPP组；扫描电镜显示CPP组细胞数量多于SCPP组表明Sr抑制破骨细胞的增殖。本论文初步进行了1％SCPP，CPP在犬股骨和兔胫骨中的体内降解试验。通过对Masson染色图片的图像分析表明：犬股骨内钻孔植入时SCPP的降解速率小于CPP；兔体内整段缺损植入时SCPP的降解速率大于CPP，HA。本论文首次在体外模拟体内生物学环境，从溶液介导的降解和细胞介导的降解两个层面研究了新型骨修复材料掺锶聚磷酸钙（SCPP）的降解性能，并进行了体内降解实验的探索。体外溶液介导的降解实验表明工艺条件不同，SCPP降解速率不同；体外细胞介导的降解实验表明：巨噬细胞和破骨细胞促进SCPP的降解，但由于Sr抑制破骨细胞增殖，细胞介导的SCPP降解过程中巨噬细胞发挥主要作用。动物体内降解实验表明：不同动物，不同部位，由于微环境中细胞种类不同，降解结果将完全不同。在犬体内是钻孔植入使得降解过程中主要依靠破骨细胞，导致SCPP的降解速率小于CPP；而在兔体内是整段植入使得体液，巨噬细胞降解过程中作用增大，导致SCPP降解速率大于CPP。体内外结果综合考虑，SCPP的降解方式主要有体液介导，巨噬细胞和破骨细胞介导三种，当体液，巨噬细胞对降解贡献大时SCPP的降解速率大于CPP，反之SCPP的降解速率小于CPP。总的来说SCPP显示出良好的生物降解性能。本论文对SCPP降解性能的研究提供了重要的数据，同时也为SCPP的临床应用提供了可行性依据。更多还原

【Abstract】 The ideal bone repair materials must have osteoconduction, osteoinduction and good biocompatibility, besides biodegradability. As a part of National Science Foundation of China ’The research of molecular self-assemble nano RADA16-RGD/CPP compounds for bone repair （50472091）’, the aim of this study is to study the biodegradation of strontium-doped calcium polyphosphate for bone repair materials.Recently, calcium polyphosphate （CPP）, as a kind of inorganic polymers, has drawn attention due to its controllable degradability, outstanding biocompatibility and enough strength. Meanwhile, strontium has been gradually recognized during the research of treatment for osteoporosis. It enhances the replication of pre-osteoblastic cells and decreases bone resorption. In this study, started with the research of Strontium-doped calcium polyphosphate （SCPP）, it is included the parts as follows: the preparation condition; the effect of holding temperature time namely polymerization degree and sintering temperature on degradation of SCPP from solution-driven level; obtain the optimal preparation condition. Secondly, cell-driven degradation of SCPP including macrophage cell-driven and osteoclast cell-driven were studied. Finally, implantation experiment into animal was carried to explore the biodegradability of SCPP.In this study, SCPP with different preparation condition were prepared. Chemistry structure of SCPP was studied by IR, and the polymerization degree of SCPP with different holding temperature time was studied by ³¹pNMR. The result of ³¹PNMR showed the polymerization degree of SCPP didn’t increase with prolonging holding temperature time. 7 kind of SCPP were obtained by controlling different sintering temperature, XRD showed all these SCPP wereβ-SCPP. SEM showed morphologies changed with sintering temperature, SCPP stintered at 700℃showed compact surface and unclear crystal boundary, while SCPP stintered at 800℃and 900℃showed lacunaris surface and clear crystal boundary.In this study, the effect of holding temperature time namely polymerization degree and sintering temperature on degradation of SCPP from solution-driven level was studied. Results showed the degradation velocity of SCPP didn’t decrease with increasing of its polymerization degree. At the same sintering temperature, SCPP, holding temperature time is 5h, degraded the fastest in all. The degradation velocity of SCPP whose holding temperature time is 3h was middle, while SCPP whose holding temperature time is 7h was the last. In the range of SCPP’s melt temperature, at the same holding temperature time, the degradation velocity of SCPP increased with increasing of sintering temperature. The degradation velocity sequence of SCPP as follows: 7H900C＞5H800C＞5H700C＞3H800C＞3H700C＞7H800C＞7H700C. Some precipitate formed on the surface of 7H900C, 5H800C and 5H700C and IR showed that the precipitate was a new apatite contended carbonate group. Considered sinter technics and degradation velocity, 5H800C was selected as the optimal preparation condition.In this study, macrophage cell-driven degradation of SCPP and the effect of Sr on the function of macrophage were studied. Results of weight change and ion consistence change showed SCPP degraded faster than CPP in cell group and macrophage accelerated degradation of SCPP compared with simplex SCPP group. ICP showed the quantity of Sr was 10μg in 6w, namely 1×10^-4mmol, which far less than the quantity in body （3.5 mmol）, so the quantity of Sr was in the range of normal level. MTT and SEM showed existing of Sr didn’t effect the activity and morphologies of macrophage.In this study, osteoclast cell-driven degradation of SCPP and the effect of Sr on the osteoclast were studied, osteoclast cell was identified by TRAP stain, bone lacuna and cell morphology and the cultured method of osteoclast was established. The resorption lacunas quantity and cell quantity of SCPP was less than CPP, which showed degradation velocity of SCPP by osteoclast was slower than CPP. SEM showed cell quantity in CPP group was more than in SCPP group, revealed the existence of Sr decreased multiplication of osteoclast.In this study, implantation experiment into rabbit’s cannon bom and dog’s thighbone were carried to explore the biodegradability of SCPP in vivo. Masson picture analysis showed in dog group CPP degraded faster than SCPP, while in rabbit group degradation velocity of SCPP in vivo was faster than CPP and far faster than HA, revealed the degradability of SCPP was better than CPP and HA.In this study, biological experiment was simulated in vitro. Degradation of SCPP was studied from solution-driven degradation and cell-driven degradation, moreover degradation of SCPP in vivo was studied. The result of solution-driven degradation showed degradation velocity of SCPP was different at different preparation condition. Cell-driven degradation showed macrophage and osteoclast cell could accelerate degradation of SCPP, however macrophage cell played primary role because Sr restrained multiplication of osteoclast cell. Degradation of SCPP in vivo showed dagradation results were different as different animal, different part and different kinds of cells in biological experiment. In dog group, osteoclast cell played primary role in degradation process so CPP degraded faster than SCPP; while in rabbit group body fluid and macrophage cell played primary role leading SCPP degraded faster than CPP. Considering in vitro and in vivo condition, the dagradation method of SCPP including solution-driven degradation, macrophage cell-driven and osteoclast cell-driven degradation. When body fluid and macrophage cell contributed more to degradation, SCPP degraded faster than CPP, contrarily SCPP degraded slower than CPP.In conclusion, SCPP displayed good biodegradability in this study. This study offered important data to degradation study of SCPP and offered feasibility basis to clinical application of SCPP at the same time.更多还原