【作者】 何宇；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 1、普通型材料的制备目的制备温敏凝胶载体材料,调解其LCST使之符合体内植入载体的基本要求。方法应用传统的化学合成方法制备温敏凝胶载体材料,以引发剂和交联剂实现NIPAAm单体之间的引发、聚合和交联。通过在化学反应体系中添加NHMPA制得不同配方的温敏凝胶材料,以溶涨率试验、DSC等方法测定不同配方比例材料的LCST,探究最符合要求的材料配方。结果制得的P(NIPAAm-co-NHMPA)温敏材料可以在人体正常体温范围稳定存在,其LCST略高于人体正常体温。结论应用传统的化学合成方法可以制得符合体内植入要求的温敏凝胶载体材料。2、改良快速反应型材料的制备目的通过添加一系列的化学组分,寻找最合适的植入材料配方。方法在传统的化学合成方法制备可植入式温敏凝胶载体材料的基础上,通过在反应体系中添加致孔剂(NaHCO3)、改性剂(无机粘土),改善凝胶的物理性能,使其在获得温敏快速反应性能的同时兼有足够的机械,以扫描电镜、机械性能检测仪器等对改进后材料的性能进行评价,寻找最合适的植入材料配方。结果经过摸索,得到改进的优化材料配方,材料的温敏反应速度加快,力学性能得到强化,符合植入材料的要求。结论通过在化学合成反应体系中添加合适的化学组分,可以实现对材料的改行,使材料更符合其作为体内植入的要求。3、两种材料的缓释性能目的研究植入式温敏凝胶载体材料的缓释性能。方法测定阿霉素体位释放曲线及模拟体内释放曲线。研究载体材料对小分子物质的缓释性能,同时比较不同温度条件下材料释放速率的变化,初步考察材料的温敏性能对材料缓释性能的影响。结果植入式温敏凝胶材料对其负载药物或小分子物质的释放速率与其所处的温度环境有关,并在LCST下发生极大的变化。当环境温度低于LCST时,材料温和缓释药物,当环境温度超过LCST时,材料突然剧烈暴释。结论该温敏缓释特性为材料在临床中的应用奠定了广阔的基础。4、普通型材料的生物相容性目的对植入式温敏凝胶载体材料体内外生物相容性进行检测,初步评价其作为医学植入物的安全性。方法根据中国标准出版社第一编辑室编写的《医疗器械生物学评价标准汇编》,选择细胞毒性试验、细胞与材料的复合试验、溶血试验、热原试验、过敏试验、急性全身毒性试验、遗传毒性试验以及体内植入试验等常规生物相容性评价程序对植入式温敏凝胶载体材料体内、体外的生物相容性进行综合评价。结果材料的毒性评级为0～1级;扫描电镜下观察,细胞与材料表面紧密贴合,形态良好;溶血率为2.54%;无热原反应;过敏试验为0～1级;无急性全身毒性症状;无遗传毒性;体内植入试验显示随着时间的延长,材料周围逐渐形成纤维包裹,无特殊不良反应。结论制得的植入式温敏凝胶材料是一种生物惰性材料,符合临床应用的要求。5、普通型材料的应用初探目的观察负载阿霉素的普通温敏型水凝胶聚-(N-异丙基丙稀酰胺/N-羟甲基丙烯酰胺) P(NIPAAm-co-NHMPA)对小鼠骨肉瘤的治疗效果。方法制备水凝胶;负载阿霉素并记录载药量;小鼠荷瘤成功后分为两组,治疗组填塞载药胶,对照组填塞空胶,观察一般情况及肿瘤生长情况,绘制肿瘤生长曲线;制作冰冻切片镜下观察。结果载药凝胶填塞法对小鼠体内骨肉瘤的生长有一定抑制作用;冰冻切片镜下观察,载药胶对肿瘤组织有杀伤作用。结论温敏凝胶载药后对肿瘤组织有一定的杀伤作用,其机制与凝胶局部缓释阿霉素有关,是一种有潜力的医用植入材料。更多还原

【Abstract】 1. Construction of thermosensitive hydrogel as an embedding carrierBACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use. OBJECTIVE: To construct a thermosensitive hydrogel and regulate it LCST for proper use as an embedding carrier.DESIGN:In vitro study UNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N’-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., other reagent were analytical pure.METHODS: We used APS and TEMED as an oxidoreduction initiation system and MBAAm as cross-linking agent to implement the polymerization, some amount of NHMPA was added into reaction system to regulate LCST which was determined by shrinking tests. MAIN OUTCOME MEASURES:Correlation analysis was made between the LCST and the amount of NHMPA used to yield a linear relationship.RESULTS:Synthesized Hydrogel showed thermosensitive character as expected and the LCST was 38.5℃while some amount of NHMPA were added into the reaction system. CONCLUSION:The thermosensitive hydrogel P(NIPAAm-co-NHMPA) has a proper LCST of 38.5℃and might become a good medical implant for clinical use.2. Improving thermosensitive hydrogel by adding several chemical componentsBACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use. OBJECTIVE: To improve the thermosensitive hydrogel for faster thermo-responsibility and better mechanical function by adding several chemical components for proper use as an embedding carrier.DESIGN:In vitro studyUNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N’-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., NaHCO3 and inorganic clay were analytical pure.METHODS:We used APS and TEMED as an oxidoreduction initiation system and MBAAm as cross-linking agent to implement the polymerization, some amount of NHMPA was added into reaction system as the experiment in part I to make sure a LCST proper for implantation, then some amount of NaHCO3 and inorganic clay was added into reaction system separately to evaluate the improvement of thermo-responsibility by NaHCO3 and amendment of mechanical function by inorganic clay. Finally, NHMPA, NaHCO3 and inorganic clay were added together to evaluate the best prescription.MAIN OUTCOME MEASURES:Thermo-responsibility and mechanical function was recorded to decide the best prescription of thermosensitive hydrogel as a medical embedding carrier.RESULTS:After NaHCO3 added, the hydrogel got a better thermo-responsibility than original hydrogel but a poor mechanical function; after inorganic clay added into the reaction system, the mechanical function was much better and finally proper for clinical use.CONCLUSION : The hydrogel we finally obtained has both good function of thermo-responsibility and mechanical function, and its LCST remains above normal body temperature, and that enable its further use in clinical medicine.3. Slow-release function of two thermosensitive hydrogel BACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use.OBJECTIVE: To evaluate the slow release function of the thermosensitive hydrogel so as to decide its carrier ability.DESIGN:In vitro comparison studyUNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N’-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., NaHCO3, inorganic clay , other reagents were analytical pure.METHODS:The in vitro release experiments were carried out at 37℃and 42℃respectively to investigate the effect of temperaturesensitive property of the hydrogel on adriamycin release profiles. Release experiments were conducted by immersing the swollen adriamycin loaded hydrogel in a glass tube, filled with a 25mL PBS (0.1M, pH 7.4) at 37℃(below LCST) or 42℃(above LCST) with a shaking rate of 80 rpm. At a predetermined period of in vitro release experiment, 2mL aliquots of the buffer medium was removed from the glass tube and the concentration of adriamycin in that aliquot was measured by using a UV spectrophotometer at 475 nm while 2 mL fresh buffer solution was added back to the glass tube to maintain the same total solution volume. For the comparison purpose, hydrogel without Clay and NaHCO3 was used as a control. Then we established a tissue modeling system by agrarose to describe the in vivo release curve. Adriamycin loaded hydrogel was put in center of agrarose, and the adriamycin released was measured and recorded by spectrophotometer to draw a release curve in vivo.MAIN OUTCOME MEASURES: The concentration of adriamycin in that aliquot was measured by UV spectrophotometer (475 nm) to study the slow release function in vitro of thermosensitive hydrogel under different temperature and the in vivo release function was modeled by agarose modeling system.RESULTS:The hydrogel could slow release adriamycin, and the release velocity showed temperature sensitivety. When surrounding temperature rised LCST, the release velocity of hydrogel splendid jumped to a much higher platform at the same time.CONCLUSION:P(NIPAAm-co-NHMPA) has good slow release function and its release function was temperature responsiveness, that character enabled the hydrogel to become a good medical implant for clinical use. persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., NaHCO3, inorganic clay , other reagents were analytical pure.METHODS:The in vitro release experiments were carried out at 37℃and 42℃respectively to investigate the effect of temperaturesensitive property of the hydrogel on adriamycin release profiles. Release experiments were conducted by immersing the swollen adriamycin loaded hydrogel in a glass tube, filled with a 25mL PBS (0.1M, pH 7.4) at 37℃(below LCST) or 42℃(above LCST) with a shaking rate of 80 rpm. At a predetermined period of in vitro release experiment, 2mL aliquots of the buffer medium was removed from the glass tube and the concentration of adriamycin in that aliquot was measured by using a UV spectrophotometer at 475 nm while 2 mL fresh buffer solution was added back to the glass tube to maintain the same total solution volume. For the comparison purpose, hydrogel without Clay and NaHCO3 was used as a control. Then we established a tissue modeling system by agrarose to describe the in vivo release curve. Adriamycin loaded hydrogel was put in center of agrarose, and the adriamycin released was measured and recorded by spectrophotometer to draw a release curve in vivo.MAIN OUTCOME MEASURES: The concentration of adriamycin in that aliquot was measured by UV spectrophotometer (475 nm) to study the slow release function in vitro of thermosensitive hydrogel under different temperature and the in vivo release function was modeled by agarose modeling system.RESULTS:The hydrogel could slow release adriamycin, and the release velocity showed temperature sensitivety. When surrounding temperature rised LCST, the release velocity of hydrogel splendid jumped to a much higher platform at the same time.CONCLUSION:P(NIPAAm-co-NHMPA) has good slow release function and its release function was temperature responsiveness, that character enabled the hydrogel to become a good medical implant for clinical use. MAIN OUTCOME MEASURES:L929 cells were maintained and the cytotoxicity test was carried out by normal MTT method; the cells/scaffolds interaction test was detected by microscopic; the hemolysis test was meatured by hemolysis rate; pyrogen test was meatured by bacterial endotoxin detection. The erythema and edema of bilateral spine of guinea pigs were recorded in sensitization test; the general state of each mouse in acute systemic toxicity test were recorded 4, 24, 48 and 72h after injection; all mice in genetic toxicity test were executed six hours after injection and marrow polychromatic erythrocyte(PCE)micronucleus was counted under microscope; 1, 4 and 8 weeks after implantation, two rabbits were executed respectively and the muscle about 0.5cm around the material was cut, made into sections and observed under light microscope.RESULTS:In cytotoxicity test, the toxicity rate was 0-1. Through the scanning electron microscopy, cells and scaffolds were tightly joined, producing a perfect interface, and the shape of cells was all right. Hemolysis rate was 2.54%. No pyrogen reaction. In sensitization test, there was no erythema and edema occuranced after leaching liquor and saline injected while positive result was seen after formaldehyde injected; in acute systemic toxicity test, the symptom of toxicity was not found in neither experimental group nor control group but formaldehyde injection group showed positive result; the genetic toxicity test suggested no difference between experimental group and negative control group while control group with cyclophosphamide injection showed positive result; the implantation test showed that the inflammation around the material was mild.CONCLUSION:P(NIPAAm-co-NHMPA) has good biocompatibility in vitro/vivo and might become a good medical implant for clinical use.5. Therapeutical effect of thermosensitive adriamycin slow-release hydrogel against mouse osteosarcoma in vivo BACKGROUND: Thermosensitive hydrogel Poly-(N-isopropylacrylamide) (P(NIPAAm)) and its ramifications are widely used in drug delivery, cell culture and tissue engineering, but have not been evaluated as medical implant for clinical use.OBJECTIVE: To study the therapeutical effect of a novel thermosensitive adriamycin slow-release hydrogel P(NIPAAm-co-NHMPA) against mouse osteosarcoma in vivo.DESIGN:Random, no-blind, group control, animal experimental study UNIT:Orthopaedics department of Union Hospital, Tongji Medical College, Huazhong University of Science & TechnologyMATERIALS:NIPAAm monomer and NHMPA monomer from Aldrich Co., crosslinking agent N, N’-methylene bisacrylamide (MBAAm) from Fluka Co., initiator ammonium persulfate (APS) and accelerating agent tetramethyl ethylene diamine (TEMED) from Sigma Co., other reagent were analytical pure.METHODS:The tumor bearing mice were divided into two groups: drug containting hydrogel was embedded into experimental group and empty hydrogel was embedded into control group.MAIN OUTCOME MEASURES:General state of animal health was observed and tumor growth state was recorded to draw a tumor growth curve. At the end of observation, frozen sections were made for microscope observation. RESULTS:Imbedding drug containing hydrogel could inhibit the growth of mouse osteosarcoma in vivo; at the contact surface between drug containing hydrogel and tumor tissue, cell necrosis was found under microscope.CONCLUSION:P(NIPAAm-co-NHMPA) could slow release adriamycin and inhibit the growth of mouse tumor in vivo, that might become a good medical implant for clinical use.更多还原