【作者】 张雯；

【导师】 王学川；

【作者基本信息】 陕西科技大学 ， 应用化学， 2019， 博士

【摘要】 胶原蛋白（Collagen,COL）为动物组织成分,具有良好的可降解性、生物相容性以及低抗原活性。细菌纤维素（Bacterial cellulose,BC）具有的高结晶度、三维网状结构、良好的生物相容性和可降解性使其在生物医药领域受到了普遍关注。本论文基于组织工程技术在医药领域的应用前景及多孔微球作为组织工程支架所具有的优势,以COL和BC为基体构建了具有多级结构及成分的COL/BC功能性多孔微球,对该微球药物吸附及释放行为进行了研究,并对其在骨组织工程中的应用进行了初探。主要研究内容和结果如下:（1）采用酸膨胀-胃蛋白酶水解法,从猪皮中提取I型COL,同时采用SDS-PAGE、CP/MSA ¹³C NMR、FT-IR、XRD、TGA及SEM等现代分析技术对提取产物进行性能检测及表征。最优提取工艺为:异丙醇作为脱脂剂、w_皮块:V_脱脂剂=1:10、胃蛋白酶用量5.0 wt%、胃蛋白酶水解时间60 h、盐析NaCl浓度1.1 mol/L,此条件下COL提取率为86.63%。所提取的猪皮COL具有一定纤维结构,含有COL特征官能团,由2条α₁链和1条α₂链组成[（α₁）₂（α₂）₁],符合I型COL典型肽链结构特征且具有完整的三股螺旋结构。（2）从红茶菌中分离了一株BC产量为3.56 g/L的菌株G.xylinus ZHCJ618（GenBank No.MG451840）,由菌落及细胞形态、生理生化特性及16S rRNA序列分析结果鉴定其为木葡糖醋酸杆菌（Gluconacetobacter xylinus）。CP/MSA ¹³C NMR、FT-IR、XRD、TGA及SEM等分析结果表明所发酵BC具有典型三维网状层状结构,表现出较好的耐热性以及较高的结晶度。利用响应面实验优化得出BC最优发酵培养基配方:蔗糖39.0 g/L、蛋白胨20.0 g/L、硫酸镁1.0 g/L、柠檬酸钠3.5 g/L、黄嘌呤1.0 g/L、乙醇1.0%（v/v）mL、pH 6.0。在此条件下,BC产量为6.15 g/L,较优化前提高了72.8%。（3）利用Malaprade反应,以NaIO₄为氧化剂,对BC进行氧化,制备了2,3-二醛BC（2,3-dialdehyde bacterial cellulose,DABC）。DABC最优制备工艺为:反应温度40℃、反应时间14 h、w_NaIO4:w_BC=5:10、pH 6.0、BC质量浓度0.4 wt%,在此工艺条件下所制备DABC醛基含量为2.10 mmol/100g,DABC回收率为75.2%。CP/MSA ¹³C NMR、FT-IR、XRD、TGA及SEM等分析结果表明,BC分子中的-OH能够被氧化为-CHO,氧化度随着氧化时间的延长逐渐增加,氧化过程不改变BC的Ⅰ型纤维素结晶特征,但氧化后纤维发生卷曲,纤维素分子从高度有序排列变得无序化,引起DABC结晶度降低,降低程度与氧化程度相关。同时其热稳定性有较小幅度降低。氧化后的DABC仍具有三维立体网状结构。（4）利用Schiff-base反应使DABC与COL复合制备COL/DABC复合物（CDABC）。得出CDABC最优制备工艺为:反应时间5 h、反应温度30℃、w_DABC:w_COL=1:1,此工艺条件下,DABC醛基消耗率为86.9%。CP/MSA ¹³C NMR、FT-IR、XRD、TGA及SEM等分析结果表明,DABC的-CHO与COL的-NH₂成功进行了Schiff-base反应,实现了BC与COL的化学复合。CDABC仍然保持DABC的结晶特征,但结晶度随着COL的结合有所降低。所生成CDABC孔隙减少,仍呈现立体网状结构,热稳定性未显著降低。（5）以CDABC为原料,采用模板法联合反相悬浮再生法制备了COL/BC多孔微球,利用SEM、粒径分析及N₂吸附-脱附等现代分析技术对产物进行了检测表征。得出COL/BC多孔微球最优制备工艺为:CDABC质量分数4.0wt%、聚苯乙烯模板加量2.5 wt%、w_吐温-80:w_司盘-80=1:3、表面活性剂加量为正十六烷（n-hexadecane,HD）相的100 wt%、w_IL:w_HD=1:11、乳化时间2 h,此工艺条件下所制备COL/BC多孔微球表面粗糙,布满孔洞,微球粒径8¹2μm,比表面积S_BETET 123.4 m²/g,孔容V_Poreore 0.59 cm³/g,平均孔径D_BJHJH 198.5 nm。微球对N₂分子的吸附等温线为典型的Ⅳ型,符合介孔材料吸附特征。BSA分子通过载药过程进入COL/BC多孔微球并附着于其内部孔道内壁,载药率为309.8 mg/g。（6）以牛血清白蛋白（BSA）为模型药物,研究了COL/BC多孔微球药物吸附及释放行为。结果表明,在303.15³13.15 K温度范围内,单分子层物理吸附模型Langmuir方程能较好的描述COL/BC多孔微球对BSA分子的吸附平衡关系。准二级吸附模型能够较好的解释及预测COL/BC多孔微球吸附BSA的膜扩散动力学过程。Kannan-Sundaram粒内扩散模型拟合结果表明,COL/BC多孔微球吸附BSA的动力学过程同时受到膜扩散和粒内扩散的共同影响。吸附热力学行为结果表明,微球吸附BSA分子过程中,ΔH⁰=0.10 J/mol>0,为吸热反应;283.15³13.15 K条件下吸附时,ΔG⁰<0,且随着温度的升高,|ΔG⁰|呈增大趋势,表明整个过程是自发进行的,且随着温度的升高自发趋势越大;ΔH⁰=0.10 J/mol<84 KJ/mol,ΔG⁰介于-18.47^-20.43 KJ/mol之间,表明吸附过程以物理吸附为主。微球释药行为研究结果表明,COL/BC多孔微球负载BSA以孔道内壁吸附为主,同时具有骨架型载药系统特征。组织病损引起组织液离子强度增大不利于微球中BSA的释放,温度及pH的改变对微球释药行为影响不大,其释药动力学过程均能采用一级释放模型进行较好的描述。（7）以小鼠MC3T3-E1细胞为研究对象,从细胞及分子水平对COL/BC多孔微球在骨组织工程中的应用进行了初探。结果表明COL/BC多孔微球具有良好的生物相容性。细胞在COL/BC多孔微球及COL/BC/BMP-2多孔微球上生长时,能够产生更多的钙结节,免疫印迹实验（Western blot,WB）检测结果表明Runx2,ALP,OPN,OCN及ⅠCOL等活性因子均有较高表达水平,表明所构建具有多级结构和多级成分的COL/BC/BMP-2多孔微球能够有效促进小鼠MC3T3-E1细胞粘附、增殖及成骨分化,多孔微球结构设计达到了预期效果。通过本论文的研究,制备了具有多级结构及成分且稳定性好的COL/BC多孔微球;阐明了COL/BC多孔微球药物吸附及释放机理,建立了其药物吸附/释放平衡关系;对COL/BC多孔微球在骨组织工程中的应用进行了初探。论文的完成为多孔微球类高分子材料在药物缓控释及化工吸附领域的应用提供了理论依据,为组织工程、药物缓释及化工吸附等领域提供了一种新型多孔微球材料,为COL及BC的高值转化提供了理论及技术依据。更多还原

【Abstract】 As a component of animal tissue,Collagen（COL）demonstrates many excellent features,such as good degradability,biocompatibility and low activity of antigen.Bacterial cellulose（BC）,with its high crystalinity,three-dimensional mesh structure,good biocompatibility and degradability,has attracted extensive attention in the fields of biological medicine.Based on the good application prospect of tissue engineering technology in the field of medicine and the advantages of porous microspheres as tissue engineering scaffolds,the COL/BC functional porous microspheres with multi-structure and multi-component were prepared using COL and BC as raw material.At the same time,the adsorption/release behaviors of the porous microspheres were studied to elucidate their mechanisms.The primary application of the porous microspheres in bone tissue engineering was also discussed.The main contents include:（1）The typeⅠCOL was extracted from pig skin using acid expansion-pepsin hydrolysis method and analyzed by modern analytical testing technology such as SDS-PAGE,FT-IR,XRD,TGA,SEM and CP/MSA ¹³C NMR.The extraction process was optimized as follows:the degreasing agent was isopropanol,w_{leather piece}:V_degreasant=1:10,w_pepsin=5.0 wt%,the hydrolysis time was 60h,and the concentration of NaCl was 1.1 mol/L.Under this condition,the extraction rate of COL was 86.63%.The extracted COL had a certain fiber structure.The chemical groups were confirmed to the characteristic functional groups of COL,which was composed of 2α1 chains and 1α₂ chain as[（α₁）₂（α₂）₁],and was consistent with the typical peptide chains structure characteristics of type I COL.（2）A strain G.xylinus ZHCJ618（GenBank no.MG451840）with the BC yield of 3.56 g/L was isolated from kombucha,and it was identified as Gluconacetobacter xylinus by morphology of colony and cell,physiological and biochemical properties and 16S rRNA sequence comparative analysis.The analysis results of CP/MSA ¹³C NMR,FT-IR,XRD,TGA and SEM showed that,the product BC had a typical three-dimensional multi-layered three-dimensional network structure,and showed good heat resistance and high crystallinity.The BC fermentation medium was optimized by the response surface experiments as:sucrose 39.0 g/L,peptone 20.0 g/L,magnesium sulfate 1.0 g/L,sodium citrate3.5 g/L,xanthine 1.0 g/L,ethanol 1.0%（v/v）mL,pH 6.0.Under this conditions,the BC yield reached 6.15 g/L,which was 72.8%higher than that before optimization.（3）BC was oxidized to prepare 2,3-dialdehyde bacterial cellulose（DABC）by NaIO₄ using malaprade reaction.The preparation process of DABC was optimized by orthogonal experiment as:reaction temperature 40℃,reaction time 14 h,w_NaIO4:w_BC=5:10,w_BC=0.4 wt%,pH 6.0,under this conditions,the molar concentration of the aldehyde group was 2.10 mmol/100 g,the recovery rate of DABC was 75.2%.Analyzing results of CP/MSA ¹³C NMR,FT-IR,XRD,TGA and SEM showed that-OH in BC molecules was successfully oxidized into-CHO,and the degree of oxidation of BC increased with the extension of oxidation time.The crystal characteristics of BC was not influenced by oxidation process,however partial cellulose chain of BC was fractured,the fibers began to curl,and molecules of cellulose became disordered,which caused crystallinity decreased,and the reduction degree of crystallinity was associated with the degree of oxidation.Also the thermal stability of BC was reduced after oxidation,but not by much.And it still had a three-dimensional network structure.（4）COL/DABC composite material（CDABC）was prepared by schiff-base reaction.The preparation process was optimized by orthogonal experiment as:reaction time 5 h,reaction temperature 30℃,w_DABC:w_COL=1:1,under this conditions,the consumption rate of aldehyde group was 86.9%.Analyzing results of CP/MSA ¹³C NMR,FT-IR,XRD,TGA and SEM showed that schiff-base reaction was conducted successfully between DABC and COL.The crystallization characteristics of CDABC remained same as DABC,but the crystallinity was reduced.The number of holes in CDABC was reduced,however it still presented a three-dimensional network structure.And the thermal stability of CDABC was was not significantly reduced.（5）COL/BC porous microspheres were prepared using CDABC as raw material by template method combined with reverse phase suspension regeneration method and analyzed by SEM,particle size analysis and N₂adsorption–desorption experiments.The preparation process was optimized as:w_CDABC=4.0 wt%,w_{polystyrene template}=2.5 wt%,w _tween-80:w_span-80=1:3,w_surfactant=100wt%,w_IL:w_HD=1:11,emulsifying time 2 h,the surface of COL/BC porous microspheres prepared under this condition was rough and full of pores,and there were a large number of pore structures in the interior,the particle sizes of COL/BC porous microspheres were ranging from 8 to 12 m,S_BET=123.4 m²/g,V_Pore=0.59 cm³/g,D_BJH=198.5 nm.The adsorption isotherm to N₂ was belonged to typeⅣ,which conformed to the adsorption characteristics of mesoporous materials.And BSA molecules could be spread into the internal and attach to the inner wall of their pores through the process of drug loading with a drug-loading rate of 309.8 mg/g.（6）The porous microspheres were loaded with BSA as a model drug.The adsorption/release behavior of COL/BC microspheres was studied with drug-BSA as a model protein.The fitting results showed that,within the temperature range of 303.15³13.15 K,the linear Langmuir equation could better describe the adsorption equilibrium relationship between COL/BC porous microspheres and BSA molecules.The pseudo-second model can explain and predict the membrane diffusion kinetic process of microsphere adsorption on BSA better.Fitting curve of Kannan-Sundaram diffusion model showed that during the adsorption process,the adsorption rate was affected by both membrane and intra-particle diffusion.The results of adsorption thermodynamics indicated that,during the adsorption process,ΔH⁰=0.10 J/mol>0,which showed that the adsorption process is endothermic reaction.Within the adsorption temperature range of 283.15³13.15 K,ΔG⁰<0,and|ΔG⁰|showed a trend of increase with the increase of temperature,showed that the process is spontaneous,and the spontaneous tendency became greater with the increase of temperature.ΔH⁰=0.10 J/mol<84 KJ/mol,ΔG⁰ was between 18.47²0.43 KJ/mol,which showed that the adsorption was mainly a physical adsorption process.The results of release behavior of COL/BC porous microspheres showed that most of BSA loading into COL/BC porous microspheres gave priority to adsorbing on the inner wall of the pores,and the other was wrapped inside the mesh of polymer material,which was similar to framework drug loading system.The release of BSA in COL/BC porous microspheres would be affected along with the increase of ion strength in the damaged tissue changes of ionic strength,while the small change of temperature and pH of damaged tissue fluid had little influence on the drug release behavior of porous microspheres,and their drug release kinetic processes could be well described with the first-order release model.（7）The primary application of the COL/BC porous microspheres in bone tissue engineering was discussed using mouse MC3T3-E1 cells.The results showed that the COL/BC porous microspheres had a good biocompatibility.When the MC3T3-E1 cells were grown on COL/BC porous microspheres and COL/BC/BMP-2 porous microspheres,more calcium nodules were generated,the protein expression level of active factors such as Runx2,ALP,OPN,OCN andⅠCOL was higher.Which showed that the COL/BC/BMP-2 porous microspheres with a multistage structure and component could effectively promote the adhesion,proliferation and bone differentiation of MC3T3-E1 cells,the structure design of the porous microspheres confirmed to desired effect well.Through this study,the COL/BC/BMP-2 porous microspheres with a multistage structure and component was designed and prepared;The mechanism of drug adsorption/release of COL/BC porous microspheres was clarified,and the equilibrium relationship between drug adsorption and release was established;The primary application of the COL/BC porous microspheres in bone tissue engineering was discussed.The study provides a theoretical basis for the application of porous microspheres in the slow/controlled drug release technology and chemical adsorption technology.A new type of porous microsphere material was provided for tissue engineering,drug sustained release,chemical adsorption and other fields,and a theoretical and technical basis were also provided for high value transformation of COL and BC.更多还原