【作者】 王力；

【导师】 张强；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2022， 博士

【摘要】 近十几年来,为了应对能源危机和环境污染等问题,人们加大了对可再生、可降解的生物质资源的开发力度,例如纤维素、淀粉、海藻酸和甲壳素等。纳米纤维素是一种由纤维素分子精密组装形成的新型纳米材料,可以从自然界中广泛获取。因具有高横纵比、高比表面积、高亲水性和高生物相容性等特点,纳米纤维素常被用于构建生物医学材料,包括薄膜、水凝胶和气凝胶等。这些基于纳米纤维素的复合材料在药物递送、组织工程、医疗植入、伤口愈合、伤口止血以及生物传感等生物医学领域中都有着巨大的应用潜力。本论文旨在利用纳米纤维素及其优秀特性构建出同时具备超高强度、刚度、韧性的水凝胶材料以及更安全、更方便、更高效的自膨胀型止血材料,在解决医疗难题的同时,进一步挖掘纳米纤维素在生物医学领域的应用潜力和价值。研究成果总结如下:(1)纳米纤维素广泛存在于植物细胞壁以及细菌代谢产物中,是一种绿色环保的可再生资源。我们首先采用木葡糖酸醋杆菌参与的静态发酵方法合成了细菌纤维素。所得到的细菌纤维素纳米纤维呈礼带状(宽为30-100 nm,长度大于10μm),不仅具有95.17%的超高结晶度和-10～-26 m V的高表面电势,还具有超高的热稳定性以及优秀的流变性质。随后,我们通过控制羧甲基化改性水平并结合机械处理从漂白木浆粉中提取了不同取代程度的羧甲基化纤维素纳米纤维。不同取代度的羧甲基化纤维素纳米纤维具有不同的物理化学特征,随着取代度的提高,纳米纤维的尺寸、结晶度水平和热稳定性逐渐降低,而表面电势逐渐上升。此外,取代度为0.20的羧甲基化纤维素纳米纤维具有最高的粘度,并且具有稳定的凝胶性能和优秀的触变性。(2)水凝胶因其高含水量、三维网络结构和高生物相容性等特点,成为了生物医学领域最有价值的材料之一。但相较于皮肤、软骨等天然水凝胶,传统人工合成水凝胶具有较差的力学性能,这极大限制了其生物医学应用。因此,提高合成水凝胶的力学性能对拓宽水凝胶材料在生物医学领域的应用具有重要意义和价值。我们通过碱性磷酸酶催化的生物矿化方法在细菌纤维素和海藻酸-钙构成的有机双网络中诱导沉积磷酸钙颗粒,最终得到了一种具有超高强度、刚度和韧性的有机-无机复合水凝胶。在水凝胶中,刚性的细菌纤维素网络不仅作为主要的基质成分,还作为磷酸钙沉积的模板,使形成高强度的细菌纤维素/磷酸钙复合结构。柔性的海藻酸-钙离子网络可以通过离子键的断裂有效耗散水凝胶变形过程中所吸收的能量。此外,细菌纤维素/磷酸钙复合结构以曲折的路径断裂并触发更多的离子键断裂,这进一步增强了水凝胶的能量耗散能力。在以上机制的协同下,该水凝胶实现了超强的力学性能。最优组水凝胶具有48 MPa的超高断裂应力、1329 MPa的超高杨氏模量和3013 J/m~2的超高断裂能。最后,我们成功将该水凝胶用于离体软骨缺损的修复,表明该水凝胶在骨组织修复领域中有着巨大的应用前景。(3)贯穿型创伤因具有较深且不规则的创口,往往会造成难以控制的大出血,而常规的止血材料难以有效接触和压迫血管的破损位点,导致止血过程极其困难。现已报道的自膨胀型止血材料因其吸血能力有限、膨胀倍率低下、成分和制备过程复杂以及安全性不明等问题,难以用于临床研究。因此,开发更安全、更方便和更高效的贯穿伤用自膨胀型止血材料迫在眉睫。我们在湿热条件下使用柠檬酸交联羧甲基化纤维素纳米纤维制备了一种具有自膨胀性能和海绵状特性的复合气凝胶。在气凝胶中,柠檬酸不仅充当化学交联剂提高羧甲基化纤维素纳米纤维网络的强度,还提供酸性环境使羧甲基化纤维素纳米纤维表面的羧基质子化。质子化羧基之间通过形成“双氢键缔合”结构极大稳定了材料内部的微孔,使气凝胶可以通过毛细作用快速吸收水分并膨胀自身体积。稳定的微孔还允许水分在气凝胶内部自由流通,从而赋予了气凝胶可反复吸水和脱水的海绵状特性。我们在体外证明了该气凝胶具有极高的生物安全性、抑菌性能、吸血膨胀能力和凝血能力,并且表现出了极低的溶血率。因此,我们尝试将该气凝胶用于贯穿伤止血,并在兔肝穿孔模型中取得了极佳的效果,其止血时间仅为5.83 min,失血量低至0.38 g。该气凝胶因其易制备性、高生物相容性和优秀的止血效果,有望成为一种理想的贯穿伤止血产品。总而言之,我们首先采用静态发酵方法和化学-物理方法分别制备了细菌纤维素和羧甲基化纤维素纳米纤维两种纳米纤维素材料,并对它们的物理化学性质进行了详细的表征和分析。随后,我们基于细菌纤维素制备了一种超高力学性能的复合水凝胶并成功将其用于离体软骨缺损的修复。而且,我们还基于羧甲基化纤维素纳米纤维制备了一种可快速自膨胀的复合气凝胶并成功将其用于贯穿伤的止血。这些研究将为纳米纤维素复合材料在生物医药领域的应用提供一定的参考。更多还原

【Abstract】 In the past decade,in order to cope with the energy crisis and environmental pollution, people have increased the development of renewable and degradable biomass resources,such as cellulose,starch,alginic acid and chitin.Nanocellulose is a novel nanomaterial formed by the precise assembly of cellulose molecules,which can be widely obtained from nature.Due to their high aspect ratio,high specific surface area,high hydrophilicity,and high biocompatibility,nanocellulose is often used to construct biomedical materials,including films,hydrogels,and aerogels.These nanocellulose-based composites have great potential in biomedical fields such as drug delivery,tissue engineering,medical implantation,wound healing,wound hemostasis,and biosensing.This paper aims to use nanocellulose and its excellent properties to construct a hydrogel material with ultra-high strength,stiffness and toughness simultaneously,as well as a safer,more convenient and more efficient self-expanding hemostatic material,while solving medical problems,further explore the application potential and value of nanocellulose in the field of biomedicine.The research results are summarized as follows:(1)Nanocellulose is widely present in plant cell walls and bacterial metabolites,and is a green and environmentally friendly renewable resource.We first synthesized bacterial cellulose by static fermentation involving Gluconacetobacter xylinum.The resulting bacterial cellulose nanofibrils were ribbon-shaped(30-100 nm wide,>10μm long),not only possessing a high crystallinity of 95.17%and a high zeta potential of-10 to-26 m V,but also ultra-high thermal stability and excellent rheological properties.Subsequently,we extracted carboxymethylated cellulose nanofibrils with different degrees of substitution from bleached wood pulp powder by controlling the level of carboxymethylation modification combined with mechanical treatment.With the increasing degree of substitution,the size,crystallinity level,and thermal stability of carboxymethylated cellulose nanofibrils gradually decreased,while the zeta potential gradually increased.In addition,carboxymethylated cellulose nanofibrils with a degree of substitution of 0.20 had the highest viscosity and exhibited stable gel properties and excellent thixotropy.(2)Hydrogels have become one of the most valuable materials in the biomedical field due to their high water content,three-dimensional network,and high biocompatibility.Compared with natural hydrogels such as skin and cartilage,traditional synthetic hydrogels have poor mechanical properties,which greatly limits their biomedical applications.Therefore,improving the mechanical properties of synthetic hydrogels is of great significance and value to broaden the application of hydrogel materials in the biomedical field.We induced the deposition of calcium phosphate particles in an organic double network composed of bacterial cellulose and alginate-calcium by an alkaline phosphatase-catalyzed biomineralization approch,and finally obtained an organic-inorganic composite hydrogel with ultra-high strength,stiffness and toughness.In the hydrogel,the rigid bacterial cellulose network not only serves as the main matrix component,but also serves as a template for calcium phosphate deposition,enabling the formation of a high-strength bacterial cellulose/calcium phosphate composite structure.The flexible alginate-calcium ion(II)network can effectively dissipate the energy absorbed during the deformation of the hydrogel through the unzipping of ionic bonds.In addition,the bacterial cellulose/calcium phosphate composite structure breaks in a tortuous path and triggers more ionic bond breaks,which further enhances the energy dissipation capability of the hydrogel.Under the synergy of the above mechanisms,the hydrogel achieves superior mechanical properties.The optimal group of hydrogels had an ultra-high fracture stress of 48 MPa,an ultra-high Young’s modulus of 1329 MPa,and an ultra-high fracture energy of 3013 J/m~2.Finally,we successfully used the hydrogel for the repair of cartilage defects ex vivo,indicating that the hydrogel has great application prospects in the field of bone tissue repair.(3)Penetrating wounds often cause uncontrollable massive bleeding due to deep and irregular wounds.However,conventional hemostatic materials are difficult to effectively contact and compress the damaged site of the blood vessel,resulting in extremely difficult hemostasis.The self-expanding hemostatic materials that have been reported are difficult to be used in clinical research due to their limited blood-sucking capacity,low expansion rate,complex composition and preparation process,and unclear safety.Therefore,the development of safer,more convenient and more efficient self-expanding hemostatic materials for penetrating wounds is imminent.We fabricated a composite aerogel with self-expanding properties and sponge-like properties using citric acid to crosslink carboxymethylated cellulose nanofibrils under moist heat conditions.In the aerogel,Citric acid not only acts as a chemical crosslinker to enhance the strength of the carboxymethylated cellulose nanofibrils network,but also provides an acidic environment to protonate the carboxyl groups on the surface of carboxymethylated cellulose nanofibrils.The protonated carboxyl groups greatly stabilize the micropores inside the material through the formation of a“double hydrogen bond association”structure,so that the aerogel can quickly absorb water and expand its volume through capillary action.The stable micropores allow the free flow of water inside the aerogel,giving the aerogel its sponge-like properties that can be repeatedly rehydrated and dehydrated.We had demonstrated in vitro that the aerogel possessed extremely high biosafety,antibacterial properties,blood-sucking and swelling ability,and coagulation ability,and exhibited a very low hemolysis rate.Therefore,we tried to use the aerogel for penetrating wound hemostasis,and achieved excellent results in a rabbit liver perforation model,with a hemostasis time of only 5.83min and a blood loss as low as 0.38 g.The aerogel is expected to be an ideal hemostasis product for penetrating wounds due to its easy preparation,high biocompatibility and excellent hemostatic effect.All in all,we first prepared two nanocellulose materials,bacterial cellulose and carboxymethylated cellulose nanofibrils,by static fermentation method and chemical-physical method,respectively,and characterized and analyzed their physicochemical properties in detail.Subsequently,we prepared a composite hydrogel with ultra-high mechanical properties based on bacterial cellulose and successfully used it for the repair of cartilage defects ex vivo.Furthermore,we also prepared a fast self-expanding composite aerogel based on carboxymethylated cellulose nanofibrils and successfully used it for hemostasis of penetrating wounds.These studies will provide some reference for the application of nanocellulose composites in the field of biomedicine.更多还原