高活性多孔磷钙基载药复合骨支架的制备及性能研究
发布时间：2019-06-27 19:58
【摘要】：每年有大量病人因骨产品的缺口而忍受着骨相关疾病困扰。自然骨有自我愈合的能力,但是骨缺损过大时不能自我愈合就需要进行手术移植,现在临床治疗手段包括主要自体骨和异体骨移植,但自体骨会造成新的创伤及感染,而异体骨存在着免疫排斥及疾病感染的风险,因此人工骨支架得到了科研者的关注。理想的人工骨支架需要一定的孔隙率、力学性能和细胞附着性能,并且可以提供力学性能稳定性确保骨移植后在缺损部位完成骨修复过程。本课题选择磷钙基材料制备骨支架,因为自然骨中的无机成分主要为类骨磷酸钙,此外磷酸钙具有良好的成骨作用。首先对多相磷酸钙合成和相形成条件进行了系统研究,选择合适制备方法制备出具有良好力学性能和生物学性能的多孔磷钙基支架,并对支架的物化性能和生物学性能进行了系统表征,最后对支架的成骨性能进行了评估,其主要研究内容和结果如下： 1.通过湿化学方法合成的羟基磷灰石(HA)主要为针状,而合成的磷酸三钙(TCP)为无规则颗粒。常温下主要为无定形态,在800℃温度以上煅烧后变为p-TCP。湿化学方法可以制备多相磷酸钙,可通过pH值、钙磷比和煅烧温度控制多相磷酸钙的相组成。煅烧温度在600-800℃主要为缺钙性磷灰石(CDA)相,在1200℃为多相。多相磷酸钙在pH为6.5和10.5时,随钙磷比的增加结晶度呈增加趋势,主要为HA和p-TCP两相。pH在7.5-9.5之间,钙磷比在1.55时,多相磷酸钙除了HA和β-TCP相的存在外有CDA相存在；钙磷比在1.60和1.75时,多相磷酸钙具有较好的结晶度,存在三种以上的相成分；钙磷比在1.65和1.70时,多相磷酸钙主要有HA和CDA相。多相磷酸钙在未煅烧条件下主要针状形貌,随煅烧温度增加,颗粒形貌发生变化,在600℃为棒状,在800℃下为类椭圆状,在120℃下变为无规则相貌。 2.双相磷酸钙中HA/(HA+β-TCP)比值主要受反应条件中pH值和钙磷比影响。随着pH值的增加,双相磷酸钙中的HA含量增加,反之,β-TCP含量降低。在相同pH值的条件下,钙磷比为1.65时,双相磷酸钙中的HA含量最高；钙磷比接近1.65时,双相磷酸钙中的HA含量越高；偏离1.65时,双相磷酸钙中的HA含量越低。 3.采用乳化发泡法结合循环冷冻解冻方法制备BCP/PVA支架,可以通过PVA含量调节控制支架的孔径尺寸(50-700μm)、孔隙率(73-87%)、力学性能(0.19-0.26MPa)、降解速率以及生物学性能。随着PVA含量增加,BCP/PVA支架的孔隙率、力学性能、降解速率及细胞相容性呈现降低趋势。当PVA含量为30%,BCP/PVA支架的孔径尺寸在300-500μm之间,且压缩强度最大。此外,BCP/PVA支架在模拟体液中pH变化幅度较小,保持在7.18-7.36之间。BCP/PVA支架对成骨细胞的繁殖没有抑制作用,随着PVA含量的降低,成骨细胞在支架表面的繁殖呈增加趋势,且间充质干细胞可以在PVA含量为30%的BCP/PVA支架上附着和繁殖。综上所述,制备出的BCP/PVA支架可以满足松质骨组织工程的要求。 4.采用PU泡沫复制法可以制备孔径尺寸在300-700μm的孔连通结构BCP支架,可通过选择不同ppi的PU泡沫控制BCP支架的孔径尺寸。采用HA/PLLA纳米复合物对多孔BCP支架进行涂层,未改变支架的孔径尺寸和孔连通结构,可以填补BCP支架孔壁的断裂裂纹并在孔壁上形成高分子基纤维,以提高支架的力学性能。对BCP支架进行涂层后,孔隙率有所降低,仍保持在93%以上,但压缩强度从0.31MPa提高到3.35-3.95MPa之间,可以满足松质骨压缩强度(0.02-4MPa)的要求。此外,对BCP支架涂层后,降低了支架在模拟体液中的降解速率,模拟体液的pH也没有较大幅度变化,保持在6.8-7.4之间,在30天后保持在7.4左右。采用HA/PLLA纳米复合物对多孔BCP支架进行多次涂层后减低了支架的孔隙率,但压缩强度进一步得到了提高,随涂层次数的增加,孔隙率呈降低趋势,压缩强度呈增加趋势。通过MTT实验表明,随着涂层次数的增加,支架的细胞相容性呈增加后降低趋势,其中HA/PLLA纳米复合物三次涂层的BCP多孔支架细胞相容性最好,对此组的支架进行肌肉植入实验,通过HE染色分析,发现在前期出现炎症细胞,随着时间的增加,炎症细胞消失,并出现了类骨磷灰石层和纤维组织层,表明支架具有良好的生物相容性。 5.在单倍模拟体液环境下,随着仿生沉积时间增加,HA/PLLA纳米复合物涂层BCP支架的失重比呈降低后又增加的趋势,前期以支架降解为主,后期以磷酸钙矿化物沉积为主。支架孔壁上首先有氯化钠晶体出现,后氯化钠在SBF溶液中溶解,支架孔壁表面形成纳米级磷酸钙矿化物颗粒,并伴随有针状矿化物的出现；在沉积后期,在支架表面形成均匀的沉积物涂层。在过饱和模拟体液环境下,支架的仿生沉积速度得到了提高,磷酸钙矿化物很快沉积在支架孔壁表面,随着沉积时间的增加,在支架孔壁上的沉积物形貌变得均匀,和单倍SBF环境下较为相似。对仿生沉积改性后的支架经过低温冷冻处理,在-20℃下,支架在孔壁上形成了尺寸为3-5μm大小的微孔,磷酸钙中的钙离子被镁和钾离子置换,形成生物活性类骨磷灰石。 6.通过双乳液法制备出的线性PLGA-mPEG共聚物载药微球尺寸在5-10μm,尺寸大小分布比较均匀。通过紫外分光光度计检测分析,可以确定BSA和万古霉素的标准曲线,对于BSA含量低于200mg/L时在221.6nm的拟合度较好,含量高于100mg/L时在278nm的拟合度较好；对于万古霉素,在280nm吸光度有较好的拟合度。可以通过控制共聚物的LA/GA摩尔比控制共聚物降解速率,并进一步控制共聚物载药微球的释放速率,并通过不同LA/GA摩尔比的共聚物载两种药物实现两种药物在不同阶段的控制释放。此外,制备出的载药微球可以良好地附着在HA/PLLA纳米复合物涂层BCP多孔支架上。 7.对制备出的HA/PLLA纳米复合物涂层BCP支架进行毒理性试验分析,此类支架在毒性测试中未表现出明显毒性性质,在急性毒性分级标准中为无毒性,符合国家医疗器械生物学评价标准。通过肌肉植入实验,实验动物在术后未出现活动异常,且支架在动物体内随着时间增加会形成类骨磷灰石层和纤维组织层,而对周围的肌肉组织表现出生物惰性,可知支架具有良好的生物相容性。通过骨缺损修复实验,支架植入后,在缺损部位新骨逐级形成,支架被吸收降解,在密质骨的中心区域,成熟的骨组织附近有大量的骨单位形成,新骨和周围骨组织在后期紧密连接在一块难以区分,说明制备的支架具有良好的成骨性能和骨修复效果。
[Abstract]:A large number of patients suffer from bone-related disease each year due to the gap in the bone product. The natural bone has the ability of self-healing, but when the bone defect is too large, it is necessary to carry out the operation grafting. Now the clinical treatment means include the main autograft and the allogenic bone graft, but the self-healing can cause a new wound and infection, Allogeneic bone is the risk of immune rejection and disease infection, so the artificial bone scaffold is of great concern to the researchers. The ideal artificial bone scaffold requires certain porosity, mechanical property and cell adhesion property, and can provide mechanical property stability to ensure that the bone repair process is completed at the defect site after the bone graft. In this paper, a bone scaffold is prepared by selecting a phosphate-based material, since the inorganic component in the natural bone is mainly calcium-like calcium phosphate, and the calcium phosphate has good osteogenic effect. In this paper, a systematic study on the synthesis and phase formation conditions of multi-phase calcium phosphate is carried out, and a porous phosphocalcium-based scaffold with good mechanical properties and biological properties is prepared by a suitable preparation method, and the physical and chemical properties and biological properties of the scaffold are systematically characterized. Finally, the osteogenic properties of the stent were evaluated. The main contents and results were as follows: 1. The hydroxyapatite (HA) synthesized by the wet chemical method is mainly needle-like, and the synthesized tricalcium phosphate (TCP) is non-regular. And the particle is mainly in a non-fixed form at normal temperature, and then is changed into a p-TC after the temperature of 800 DEG C or more is burned. P. The wet chemical method can prepare the multi-phase calcium phosphate, and can control the phase group of the multi-phase calcium phosphate through the pH value, the calcium-phosphorus ratio and the coal-fired temperature control. The heating temperature is 600-800 DEG C and is mainly a calcium-deficient apatite (CDA) phase, and is more than 1200 DEG C When the pH is 6.5 and 10.5, the increase of the crystallinity of the calcium-phosphorus ratio increases with the increase of the calcium-phosphorus ratio, mainly for HA and p-TCP. When the pH is between 7.5 and 9.5, the calcium-phosphorus ratio is at 1.55, the polyphasic calcium phosphate has a CDA phase in addition to the presence of the HA and the HCO3-TCP phase; when the calcium-phosphorus ratio is 1.60 and 1.75, the multi-phase calcium phosphate has good crystallinity, and there are more than three phase components; and the calcium-phosphorus ratio is between 1.65 and 1.70. The multi-phase calcium phosphate is mainly HA and CDA. Phase. The main needle-like appearance of the multi-phase calcium phosphate in the unfired condition, with the increase of the sintering temperature and the change of the morphology of the particle, is in the form of a rod at the temperature of 600 DEG C, is an elliptic shape at the temperature of 800 DEG C, and becomes a non-regular phase at the temperature of 120 DEG C. Appearance.2. The ratio of HA/ (HA + HCO3-TCP) in the two-phase calcium phosphate is mainly controlled by the pH value and the calcium-phosphorus in the reaction condition. The HA content in the two-phase calcium phosphate increased with the increase of the pH value. The amount of HA in the two-phase calcium phosphate is the highest when the ratio of the calcium and phosphorus to 1.65 is 1.65, and the higher the HA content in the two-phase calcium phosphate when the ratio of the calcium to phosphorus is about 1.65; when the calcium-phosphorus ratio is 1.65, the HA in the two-phase calcium phosphate 3. The BCP/ PVA stent was prepared by using the emulsion foaming method in combination with the circulation freeze-thaw method, and the pore size (50-700. mu.m), the porosity (73-87%), the mechanical property (0.19-0.26 MPa), the degradation rate, and the mechanical properties (0.19-0.26 MPa) of the control stent can be adjusted by the PVA content. With the increase of PVA content, the porosity, mechanical properties, degradation rate and cell compatibility of BCP/ PVA stent The trend is now reduced. When the PVA content is 30%, the pore size of the BCP/ PVA stent is between 300 and 500. m u.m, and the pressure In addition, that change in pH of the BCP/ PVA stent in the simulated body fluid was small and remained at 7.18-7. .36. The BCP/ PVA stent did not inhibit the multiplication of the osteoblast, and with the decrease of the PVA content, the propagation of the osteoblast on the surface of the scaffold was increased, and the mesenchymal stem cells could be on the BCP/ PVA stent with a PVA content of 30%. To sum up, the prepared BCP/ PVA stent can meet the cancellous bone tissue 4. The PU foam replication method can be used to prepare the BCP support with the pore size of 300-700 & mu; m, and the BCP can be controlled by selecting PU foam with different ppi. The pore size of the stent is reduced. The porous BCP stent is coated with the HA/ PLLA nano composite, the pore size and the hole-connecting structure of the stent are not changed, the fracture crack of the hole wall of the BCP bracket can be filled, and the high-molecular-based fiber is formed on the hole wall so as to improve The mechanical properties of the stent. After the BCP stent is coated, the porosity is reduced, and the porosity is still above 93%, but the compressive strength is increased from 0.31 MPa to 3.35-3.95MPa, and the compressive strength of the cancellous bone can be met (0.02-4 MPa). In addition, after the BCP stent was coated, the rate of degradation of the stent in the simulated body fluid was reduced, and the pH of the simulated body fluid was not significantly changed, maintained between 6.8 and 7.4, and maintained at 30 days The porosity of the stent was reduced after multiple coating of the porous BCP scaffold with HA/ PLLA nanocomposite, but the compressive strength was further improved, with the increase of the number of coatings, the decrease of porosity, the compression, The results of MTT showed that with the increase of the number of coatings, the cell compatibility of the stent decreased, and the cell compatibility of the BCP porous scaffold of the HA/ PLLA nanocomposite was the best. After HE staining, the inflammatory cells were found in the early stage, with the increase of time, the inflammatory cells disappeared and the bone-like apatite layer and the fibrous tissue layer appeared, indicating that the stent was good. 5. In the single simulated body fluid environment, with the increase of the biomimetic deposition time, the weight loss ratio of the HA/ PLLA nano-composite coating BCP scaffold decreased after the decrease of the biomimetic deposition time. and the surface of the support pore wall forms the nano calcium phosphate mineralizer particles and is accompanied by the appearance of the needle-like mineralizer; and in the later stage of the deposition, in the surface of the support, in the supersaturated simulated body fluid environment, the bionic deposition speed of the support is improved, the calcium phosphate mineralizer is quickly deposited on the surface of the wall of the support wall, and the sediment appearance on the wall of the support hole becomes uniform as the deposition time is increased, and the single S and the calcium ion in the calcium phosphate is replaced by the magnesium and the potassium ions, the size of the linear PLGA-mPEG copolymer drug-loaded microsphere prepared by the double-emulsion method is 5-10 & mu; m and the size and the size distribution are relatively uniform. The standard curve of BSA and vancomycin can be determined by the detection and analysis of the ultraviolet spectrophotometer, the fitting degree of the BSA and the vancomycin is good when the content of the BSA is lower than 200 mg/ L, the fitting degree of the content is higher than 100 mg/ L, the fitting degree of 278 nm is good; for vancomycin, at 280n, the invention can control the degradation rate of the copolymer by controlling the LA/ GA molar ratio of the copolymer, and further control the release rate of the copolymer drug-loaded microspheres, In addition, the prepared drug-loaded microspheres can be well adhered to the HA/ PLLA nano composite 7. The prepared HA/ PLLA nano-composite coating BCP stent was subjected to toxic and rational test analysis, and no obvious toxic property was shown in the toxicity test, and it was non-toxic and in accordance with the acute toxicity grading standard. The biological evaluation standard of the national medical device. The experimental animals did not show any abnormal activity after operation through the muscle implantation experiment, and the scaffold in the animal body increased with the time to form the bone-like apatite layer and the fibrous tissue layer, and the surrounding muscle tissue exhibited biological inertia, The stent has good biocompatibility. After the stent is implanted through the bone defect repair experiment, after the stent is implanted, the new bone is gradually formed at the defect site, the stent is absorbed and degraded, a large number of bone units are formed in the vicinity of the mature bone tissue in the central region of the dense bone, and the new bone and the surrounding bone tissue are The late tight connection is difficult to distinguish, and the prepared stent has the advantages of
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08
[Abstract]:A large number of patients suffer from bone-related disease each year due to the gap in the bone product. The natural bone has the ability of self-healing, but when the bone defect is too large, it is necessary to carry out the operation grafting. Now the clinical treatment means include the main autograft and the allogenic bone graft, but the self-healing can cause a new wound and infection, Allogeneic bone is the risk of immune rejection and disease infection, so the artificial bone scaffold is of great concern to the researchers. The ideal artificial bone scaffold requires certain porosity, mechanical property and cell adhesion property, and can provide mechanical property stability to ensure that the bone repair process is completed at the defect site after the bone graft. In this paper, a bone scaffold is prepared by selecting a phosphate-based material, since the inorganic component in the natural bone is mainly calcium-like calcium phosphate, and the calcium phosphate has good osteogenic effect. In this paper, a systematic study on the synthesis and phase formation conditions of multi-phase calcium phosphate is carried out, and a porous phosphocalcium-based scaffold with good mechanical properties and biological properties is prepared by a suitable preparation method, and the physical and chemical properties and biological properties of the scaffold are systematically characterized. Finally, the osteogenic properties of the stent were evaluated. The main contents and results were as follows: 1. The hydroxyapatite (HA) synthesized by the wet chemical method is mainly needle-like, and the synthesized tricalcium phosphate (TCP) is non-regular. And the particle is mainly in a non-fixed form at normal temperature, and then is changed into a p-TC after the temperature of 800 DEG C or more is burned. P. The wet chemical method can prepare the multi-phase calcium phosphate, and can control the phase group of the multi-phase calcium phosphate through the pH value, the calcium-phosphorus ratio and the coal-fired temperature control. The heating temperature is 600-800 DEG C and is mainly a calcium-deficient apatite (CDA) phase, and is more than 1200 DEG C When the pH is 6.5 and 10.5, the increase of the crystallinity of the calcium-phosphorus ratio increases with the increase of the calcium-phosphorus ratio, mainly for HA and p-TCP. When the pH is between 7.5 and 9.5, the calcium-phosphorus ratio is at 1.55, the polyphasic calcium phosphate has a CDA phase in addition to the presence of the HA and the HCO3-TCP phase; when the calcium-phosphorus ratio is 1.60 and 1.75, the multi-phase calcium phosphate has good crystallinity, and there are more than three phase components; and the calcium-phosphorus ratio is between 1.65 and 1.70. The multi-phase calcium phosphate is mainly HA and CDA. Phase. The main needle-like appearance of the multi-phase calcium phosphate in the unfired condition, with the increase of the sintering temperature and the change of the morphology of the particle, is in the form of a rod at the temperature of 600 DEG C, is an elliptic shape at the temperature of 800 DEG C, and becomes a non-regular phase at the temperature of 120 DEG C. Appearance.2. The ratio of HA/ (HA + HCO3-TCP) in the two-phase calcium phosphate is mainly controlled by the pH value and the calcium-phosphorus in the reaction condition. The HA content in the two-phase calcium phosphate increased with the increase of the pH value. The amount of HA in the two-phase calcium phosphate is the highest when the ratio of the calcium and phosphorus to 1.65 is 1.65, and the higher the HA content in the two-phase calcium phosphate when the ratio of the calcium to phosphorus is about 1.65; when the calcium-phosphorus ratio is 1.65, the HA in the two-phase calcium phosphate 3. The BCP/ PVA stent was prepared by using the emulsion foaming method in combination with the circulation freeze-thaw method, and the pore size (50-700. mu.m), the porosity (73-87%), the mechanical property (0.19-0.26 MPa), the degradation rate, and the mechanical properties (0.19-0.26 MPa) of the control stent can be adjusted by the PVA content. With the increase of PVA content, the porosity, mechanical properties, degradation rate and cell compatibility of BCP/ PVA stent The trend is now reduced. When the PVA content is 30%, the pore size of the BCP/ PVA stent is between 300 and 500. m u.m, and the pressure In addition, that change in pH of the BCP/ PVA stent in the simulated body fluid was small and remained at 7.18-7. .36. The BCP/ PVA stent did not inhibit the multiplication of the osteoblast, and with the decrease of the PVA content, the propagation of the osteoblast on the surface of the scaffold was increased, and the mesenchymal stem cells could be on the BCP/ PVA stent with a PVA content of 30%. To sum up, the prepared BCP/ PVA stent can meet the cancellous bone tissue 4. The PU foam replication method can be used to prepare the BCP support with the pore size of 300-700 & mu; m, and the BCP can be controlled by selecting PU foam with different ppi. The pore size of the stent is reduced. The porous BCP stent is coated with the HA/ PLLA nano composite, the pore size and the hole-connecting structure of the stent are not changed, the fracture crack of the hole wall of the BCP bracket can be filled, and the high-molecular-based fiber is formed on the hole wall so as to improve The mechanical properties of the stent. After the BCP stent is coated, the porosity is reduced, and the porosity is still above 93%, but the compressive strength is increased from 0.31 MPa to 3.35-3.95MPa, and the compressive strength of the cancellous bone can be met (0.02-4 MPa). In addition, after the BCP stent was coated, the rate of degradation of the stent in the simulated body fluid was reduced, and the pH of the simulated body fluid was not significantly changed, maintained between 6.8 and 7.4, and maintained at 30 days The porosity of the stent was reduced after multiple coating of the porous BCP scaffold with HA/ PLLA nanocomposite, but the compressive strength was further improved, with the increase of the number of coatings, the decrease of porosity, the compression, The results of MTT showed that with the increase of the number of coatings, the cell compatibility of the stent decreased, and the cell compatibility of the BCP porous scaffold of the HA/ PLLA nanocomposite was the best. After HE staining, the inflammatory cells were found in the early stage, with the increase of time, the inflammatory cells disappeared and the bone-like apatite layer and the fibrous tissue layer appeared, indicating that the stent was good. 5. In the single simulated body fluid environment, with the increase of the biomimetic deposition time, the weight loss ratio of the HA/ PLLA nano-composite coating BCP scaffold decreased after the decrease of the biomimetic deposition time. and the surface of the support pore wall forms the nano calcium phosphate mineralizer particles and is accompanied by the appearance of the needle-like mineralizer; and in the later stage of the deposition, in the surface of the support, in the supersaturated simulated body fluid environment, the bionic deposition speed of the support is improved, the calcium phosphate mineralizer is quickly deposited on the surface of the wall of the support wall, and the sediment appearance on the wall of the support hole becomes uniform as the deposition time is increased, and the single S and the calcium ion in the calcium phosphate is replaced by the magnesium and the potassium ions, the size of the linear PLGA-mPEG copolymer drug-loaded microsphere prepared by the double-emulsion method is 5-10 & mu; m and the size and the size distribution are relatively uniform. The standard curve of BSA and vancomycin can be determined by the detection and analysis of the ultraviolet spectrophotometer, the fitting degree of the BSA and the vancomycin is good when the content of the BSA is lower than 200 mg/ L, the fitting degree of the content is higher than 100 mg/ L, the fitting degree of 278 nm is good; for vancomycin, at 280n, the invention can control the degradation rate of the copolymer by controlling the LA/ GA molar ratio of the copolymer, and further control the release rate of the copolymer drug-loaded microspheres, In addition, the prepared drug-loaded microspheres can be well adhered to the HA/ PLLA nano composite 7. The prepared HA/ PLLA nano-composite coating BCP stent was subjected to toxic and rational test analysis, and no obvious toxic property was shown in the toxicity test, and it was non-toxic and in accordance with the acute toxicity grading standard. The biological evaluation standard of the national medical device. The experimental animals did not show any abnormal activity after operation through the muscle implantation experiment, and the scaffold in the animal body increased with the time to form the bone-like apatite layer and the fibrous tissue layer, and the surrounding muscle tissue exhibited biological inertia, The stent has good biocompatibility. After the stent is implanted through the bone defect repair experiment, after the stent is implanted, the new bone is gradually formed at the defect site, the stent is absorbed and degraded, a large number of bone units are formed in the vicinity of the mature bone tissue in the central region of the dense bone, and the new bone and the surrounding bone tissue are The late tight connection is difficult to distinguish, and the prepared stent has the advantages of
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08
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6 王sセ，
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