【作者】 张华；

【导师】 任鹏刚；

【作者基本信息】 西安理工大学 ， 材料加工工程， 2021， 博士

【摘要】 高分子水凝胶是富含水的聚合物交联材料,类似细胞外基质结构,在组织修复与再生、组织工程、3D生物打印等领域具有突出的应用优势。然而,水凝胶材料往往存在凝胶化与自愈合慢,刚度、强度、韧性不够,易蠕变等问题,难以满足细胞生长、3D打印以及组织修复的力学性能与粘弹性需求。研究发现,向水凝胶网络中建立“非共价作用”是提高凝胶化速率、增强力学性能和改善粘弹性响应的有效策略。但如何在水凝胶网络中合理利用非共价作用来满足生物医用的性能需求,是发展生物活性水凝胶新材料的重要挑战。本论文选择生物活性多糖水凝胶为研究基体,通过向网络中引入含疏水缔合、氢键等非共价作用组分,发展了力学性能增强、粘弹性能可控的生物相容水凝胶材料,并尝试用于3D生物打印、软骨和皮肤修复等生物医学领域。核心研究内容与结论如下:（1）采用光引发聚合制备了疏水缔合丙烯酰化F127（F127DA）胶束交联的甲基丙烯酰化透明质酸（Me HA）水凝胶。F127DA胶束的可逆形变为网络抵抗应力破坏提供了能量耗散。当F127DA胶束与Me HA的含量分别为15 wt.%和1.5 wt.%时,水凝胶的压缩强度、模量和韧性分别达到3.44 MPa,312 k Pa和～407.5 k J·m^-3,接近于部分软骨组织的力学性能。胶束与Me HA分子间的共价交联赋予网络良好的耐溶胀性。水凝胶在磷酸盐缓冲液中体积轻度溶胀1.3倍,削弱了网络非共价作用,导致其压缩强度、模量和韧性降低至0.59MPa,55 k Pa和81.8 k J·m^-3,但溶胀前后均表现出良好的抗疲劳和网络自恢复能力。针对溶胀后力学性能下降的问题,进一步向水凝胶中引入二甲基亚砜/水（DMSO/H₂O）混合溶剂,增强了Me HA分子内与分子间的氢键作用,为凝胶网络提供了更多可逆交联点。随着DMSO在双溶剂中所占含量的增加,凝胶从平整孔结构逐渐转变为具有微相分离结构,其压缩力学性能相应增强。当DMSO体积分数为0.6时,凝胶的压缩强度,模量和韧性分别达到10.12 MPa,106.8 k Pa和742.1 k J·m^-3。并且,疏水缔合作用和氢键作用赋予凝胶网络优异的抗疲劳和自恢复性能。胶束交联透明质酸水凝胶可降解并具有良好的生物相容性,植入体内喉软骨缺损表现出促进软骨修复的潜能。（2）胶束共价交联透明质酸水凝胶网络一旦破坏,不具备自愈合性能,难以满足3D打印要求。因此,进一步将F127DA胶束引入到动态酰腙键交联的透明质酸水凝胶中,发现胶束作为抗形变单元不仅提高了凝胶的力学刚度,而且胶束与透明质酸分子通过亲疏水作用形成复合体,缩短了水凝胶的凝胶化时间。当F127DA含量从0%增大到7%（wt./v）时,动态凝胶的储能模量从60 Pa增大到180 Pa,凝胶化时间从60～300 s缩短到15 s以内。光引发胶束网络共价交联可进一步将凝胶模量提升至1 k Pa。动态酰腙键和亲疏水作用同时赋予水凝胶快速可逆的凝胶-溶胶转变与自愈合性能,可作为直写式3D打印“墨水”构建立体三维结构。该水凝胶生物相容,植入关节软骨缺损处能显著提升新生软骨生成,促进胞外基质氨基聚糖和II型胶原蛋白表达,展现出促进关节软骨损伤愈合的潜能。（3）透明质酸/胶束宏观水凝胶在3D打印过程中线条结构扭曲,导致精度和分辨率低。此外,研究证实多细胞聚集体对组织修复具有促进作用,但以上水凝胶不具备调控细胞聚集的生物功能。为此,设计了化学交联的甲基丙烯酰化壳聚糖（CHMA）/聚乙烯醇（PVA）水凝胶,并通过剪切作用将其转化为微凝胶。微凝胶间通过CHMA和PVA分子间的可逆氢键作用二次组装为“墨水”,既具有一定的屈服强度,又可在剪切作用下呈现塞流流动,当应力撤去后立即自愈合,并保持良好的抗蠕变性能。应用CHMA/PVA微凝胶“墨水”实现了类血管、人耳、股骨等大长径比仿生结构的高保真构建。并且,该水凝胶的化学微环境可调节干细胞聚集成高活性生长的细胞球,这对组织修复具有重要意义。（4）为了进一步探明水凝胶刚度对细胞球粘附骨架和组织修复的影响,采用光聚合和定向梯度冷冻法制备了组分恒定、化学环境稳定的CHMA/PVA水凝胶,实现了水凝胶模量从14 k Pa至61 k Pa的连续分布。成纤维细胞在CHMA/PVA水凝胶表面形成细胞聚集体,并且在高刚度（43-61 k Pa）水凝胶表面的粘附骨架比在低刚度（14-35 k Pa）水凝胶表面的大。CHMA/PVA水凝胶具有良好的体内生物相容性,14 k Pa和61 k Pa水凝胶作为敷料均显著促进皮肤表皮与真皮的生长,降低疤痕形成,缩短皮肤愈合时间近4天,但61 k Pa水凝胶较14 k Pa水凝胶更有利于胶原蛋白成熟。该生物相容水凝胶在细胞行为调控与皮肤修复方面具有潜在的应用价值。更多还原

【Abstract】 Hydrogels,a family of synthetic materials with crosslinked hydrophilic networks and high content of water,are similar to extracellular matrix and therefore are suitable for a wide range of biomedical applications,including tissue repair and regeneration,tissue engineering and 3D-bioprinting.However,the conventional hydrogels are faced with slow gelation dynamics and self-healing,low stiffness,strength and toughness,and fast creep property.Moreover,they are generally weak and brittle.These issues make hydrogels difficultly meet the mechanical and viscoelastic requirements for cell growth,3D printing and tissue repair.It has demonstrated that introducing non-covalent interactions into hydrogel network is an effective strategy for improving gelation dynamics,mechanical properties and viscoelasticity.However,it remains challenges for the development of bioactive hydrogel materials with reasonably non-covalent interactions in the hydrogel network to adapt the biomedical performance requirements.In this dissertation,we have introduced the non-covalent crosslinking systems including hydrophobic association and hydrogen bonding into polysaccharide-based hydrogels to prepare biocompatible hydrogels with high mechanical properties and controlled viscoelasticity.The hydrogels are clinically promising for 3D printing,cartilage damage repair and skin wound healing.The main contents and conclusions are summarized as follows:（1）Novel hyaluronate hydrogels were fabricated through in-situ photo-initiated free radical copolymerization of methacrylated hyaluronate acid（Me HA）and Pluronic F127 diacrylate.The reversible hydrophobic association of F127DA micelles provides an energy dissipation mechanism to resist deformation.The optimized hydrogel with 15 wt.%of F127DA micelles and1.5 wt.%of Me HA(F₁₅H_1.5)exhibited a high compressive strength（3.44 MPa）and modulus（312k Pa）,and strong compressive toughness(407.5 k J·m^-3),which matches those of cartilage tissues.On the other hand,the covalently cross-linked F127DA and Me HA chains improved the stability of the network against swelling.The hydrogels showed a low swelling ratio（1.3 times of volume expansion）in phosphate buffered saline solution.After swelling,the compressive strength（0.59MPa）,modulus（55 k Pa）and fracture energy(81.8 k J·m^-3)of hydrogel dramatically decreased but still had excellent anti-fragility and self-recovery capability.Furthermore,the mechanical defects of FH hydrogel caused by swelling could be remedied by introducing organic solvents into the hydrogel.It was found that DMSO can induce direct strong hydrogen bonding interactions among Me HA chains replaced from the secondary structure of Me HA in water with water bridged multiple hydrogen.Therefore,the original soft hydrogel with a high swelling degree gradually became stiff with a decreased swelling degree as the DMSO contents in binary solvents increased.the gels show the maximum strength（10.12 MPa）,modulus（106.8 k Pa）and toughness(742.1 k J·m^-3)in DMSO with a volume fraction of around 0.6.Moreover,the FH gels display a rapid recoverability under cyclic loading-unloading stressing particularly in the presence of DMSO within the network due to their dual-dynamic dissipation networks including hydrogen-bonded Me HA and hydrophobically assembled F127DA micelles.The micelle-crosslinked hyaluronate hydrogel is degradable and biocompatible.In vivo studies showed that the implantation of hydrogel in thyroid cartilage defects of rabbit larynx effectively promoted the regeneration of cartilage.（2）The covalently micelle-crosslinked hyaluronate hydrogels do not self-healing after destruction,which limited them for direct 3D printing application.In this part,F127DA micelles were introduced into dynamically hydrazone-crosslinked hyaluronic acid hydrogels.It was found that micelles as a deform-resistant unit not only improved the stiffness of hydrogels,but also shortened the gelation time of hydrogel preparation due to a complex formation between hyaluronic acid and micelles through hydrophilic and hydrophobic interactions.The storage moduli of dynamic hydrogels increase from<60 Pa to 180 Pa and the gelation time decreases from 60～300 s to less than 15 s as the content of F127Da increases from 0 to 7%（wt./v）.Moreover,the modulus of the hydrogel can be further increased to 1 k Pa by micelle crosslinking initiated by UV-light.The dynamic hydrazones and hydrophobic associations in the network endowed the hydrogels with reversible gel-sol transitions and rapid self-healing,allowing for direct extrusion-based 3D printing.Moreover,the dynamical hydrogels are biocompatible.They can significantly promote the cartilage regeneration and enhance glycosaminoglycan and collagen-II matrix deposition in vivo,implying that such biocompatible hydrogel has promising applications in cartilage repair and regeneration.（3）The 3D printed fibers of macroscopic hyaluronic acid/micelle hydrogels were distorted,resulting in low precision and resolution.On the other hand,it has been demonstrated that the cell spheroids are better for tissue repair than dissociative cells.However,the above hydrogels cannot regulate cell aggregation.Therefore,novel covalently crosslinked chitosan methacrylate（CHMA）and polyvinyl alcohol（PVA）hybrid hydrogels were developed.This bulk hydrogel was controllably fractured into microparticles.The particles are able to associate into hydrogels through extensive hydrogen bonding between CHMA and PVA chains.Such particulate hydrogels showed a self-supportive yield strength and experienced a plug flow when injected through a syringe and subsequently self-healed into gels as shear forces are removed.Moreover,these gels behaved as a viscoelastic solid with outstanding creep resistance.Those characteristics enabled the particulate hydrogels with excellent printability.Diverse biomimetic constructs with very high aspect ratio including blood vessel,human ear,and rat thigh-bone were directly printed by using the microparticle hydrogels.The CHMA/PVA scaffolds supported the growt.h of bone marrow-derived mesenchymal stem cells and formation of cell spheroids,which are most important for tissue repair and regeneration.（4）In order to further investigate the influence of stiffness on the cytoskeleton of cell spheres and tissue repair,Novel hybrid hydrogels consisting of CHMA and PVA were prepared by two-step crosslinking through photo-polymerization and directional freezing-thawing process.The modulus of CHMA/PVA network can be modulated from 14 k Pa to 61 k Pa.Such CHMA/PVA hydrogels can induce the formation of fibroblast microaggregates in vitro.Importantly,the cytoskeleton adhered on the hydrogels with high stiffness（43-61 k Pa）surface was larger than that adhered on the hydrogel with low stiffness（14-35 k Pa）.CHMA/PVA hydrogels are biocompatible in vivo.Both hydrogels with low and high stiffness（14 k Pa and 61 k Pa）as wound dressing could significantly accelerated the re-epithelialization and reduce scar formation but more collagen matured in 61 k Pa hydrogel group.The hydrogel group exhibited 4 d faster wound closure than the control group.We envision that such biocompatible CHMA/PVA hydrogel can be used for regulating cell behaviors and enhancing wound healing.更多还原