Constructing a biofunctionalized 3D-printed gelatin/sodium alginate/chitosan tri-polymer complex scaffold with improvised biological andmechanical properties for bone-tissue engineering

Sodium alginate(SA)/chitosan(CH)polyelectrolyte scaffold is a suitable substrate for tissue-engineering application.The present study deals with further improvement in the tensile strength and biological properties of this type of scaffold to make it a potential template for bone-tissue regeneration.We experimented with adding 0%–15%(volume fraction)gelatin(GE),a protein-based biopolymer known to promote cell adhesion,proliferation,and differentiation.The resulting tri-polymer complex was used as bioink to fabricate SA/CH/GEmatrices by three-dimensional(3D)printing.Morphological studies using scanning electron microscopy revealed the microfibrous porous architecture of all the structures,which had a pore size range of 383–419μm.X-ray diffraction and Fourier-transform infrared spectroscopy analyses revealed the amorphous nature of the scaffold and the strong electrostatic interactions among the functional groups of the polymers,thereby forming polyelectrolyte complexes which were found to improve mechanical properties and structural stability.The scaffolds exhibited a desirable degradation rate,controlled swelling,and hydrophilic characteristics which are favorable for bone-tissue engineering.The tensile strength improved from(386±15)to(693±15)kPa due to the increased stiffness of SA/CH scaffolds upon addition of gelatin.The enhanced protein adsorption and in vitro bioactivity(forming an apatite layer)confirmed the ability of the SA/CH/GE scaffold to offer higher cellular adhesion and a bone-like environment to cells during the process of tissue regeneration.In vitro biological evaluation including the MTT assay,confocal microscopy analysis,and alizarin red S assay showed a significant increase in cell attachment,cell viability,and cell proliferation,which further improved biomineralization over the scaffold surface.In addition,SA/CH containing 15%gelatin designated as SA/CH/GE15 showed superior performance to the other fabricated 3D structures,demonstrating its potential for use in bone-tissue engineering.

3D Nanocomposite Hydrogel Scaffolds Fabricated by Rapid Prototyping for Bone Tissue Engineering

Colloidal gels made of oppositely charged nanoparticles are a novel class of hydrogels and can exhibit pseudoplastic behavior which will enable them to mold easily into specific shapes.These moldable gels can be used as building blocks to self-assemble into integral scaffolds from bottom to up through electrostatic forces.However,they are too weak to maintain scaffold morphology just depending on interparticle interactions such as Van der Waals attraction and electrostatic forces especially for bone tissue engineering.In this study,oppositely charged gelatin nanoparticles were firstly prepared by two-step desolvation method,followed by the mixture with water to form colloid gels.To solve the problem of weak mechanical performance of colloid gels, gelatin macromolecules were introduced into the prepared gels to form blend gels.The blend gels can be easily processed into three-dimensional( 3D) porous scaffolds via motor assisted microsyringe( MAM)system,a nozzle-based rapid prototyping technology,under mild conditions.After fabrication the scaffolds were crosslinked by glutaraldehyde( GA,25% solution in water by weight),then the crosslinked gelatin macromolecules network could form to improve the mechanical properties of colloid gels.The average particle size and zeta potential of gelatin nanoparticles were measured by NanoZS instrument.The morphology and microstructures of scaffolds were characterized by macroscopic images.The mechanical properties of the scaffolds were studied by a universal material testing machine.

3D Bioplotting of Gelatin/Alginate Scaffolds for Tissue Engineering:Influence of Crosslinking Degree and Pore Architecture on Physicochemical Properties

Gelatin/Alginate hydrogels were engineered for bioplotting in tissue engineering. One major drawback of hydrogel scaffolds is the lack of adequate mechanical properties. In this study, using a bioplotter, we constructed the scaffolds with different pore architectures by deposition of gelatin/alginate hydrogels layerby-layer. The scaffolds with different crosslinking degree were obtained by post-crosslinking methods. Their physicochemical properties, as well as cell viability, were assessed. Different crosslinking methods had little influence on scaffold architecture, porosity, pore size and distribution. By contrast, the water absorption ability, degradation rate and mechanical properties of the scaffolds were dramatically affected by treatment with various concentrations of crosslinking agent （glutaraldehyde）. The crosslinking process using glutaraldehyde markedly improved the stability and mechanical strength of the hydrogel scaf- folds. Besides the post-processing methods, the pore architecture can also evidently affect the mechanical properties of the scaffolds. The crosslinked gelatin/alginate scaffolds showed a good potential to encap-sulate cells or drugs.

Preparation and Endothelialization of Multi-level Vessel-like Network in Enzymated Gelatin Scaffolds

Loss of function of large tissues is an urgent clinical problem. Although the artificial microfluidic network fabricated in large tis- sue-engineered constructs has great promise, it is still difficult to develop an efficient vessel-like design to meet the requirements of the biomimetic vascular network for tissue engineering applications. In this study, we used a facile approach to fabricate a branched and multi-level vessel-like network in a large muscle scaffolds by combining stereolithography （SL） technology and enzymatic crosslinking mechanism. The morphology of microchannel cross-sections was characterized using micro-computed tomography. The square cross-sections were gradually changed to a seamless circular microfluidic network, which is similar to the natural blood vessel. In the different micro-channels, the velocity greatly affected the attachment and spread of Human Umbilical Vein Endothelial Cell （HUVEC）-Green Fluorescent Protein （GFP）. Our study demonstrated that the branched and multi-level microchannel network simulates biomimetic microenvironments to promote endothelialization. The gelatin scaffolds in the circular vessel-like networks will likely support myoblast and surrounding tissue for clinical use.

3D PLLA/Nano-hydroxyapatite Scaffolds with Hierarchical Porous Structure Fabricated by Low-temperature Deposition Manufacturing

A new Precision Extrusion nozzle based ball screw transmission was developed. 3D hierarchical porous PLLA/nano-Hydroxyapatite（PLLA/nHA） scaffolds were fabricated by low-temperature deposition manufacturing. Scaffolds with macropores of 200-500 rtm and micropores about 10 pm were fabricated through a thorough study and control of the processing parameters, in which the processing path and speed of material extrusion determine the macropores and there is a suitable temperature zone for fabricating qualified macropores. Micropore morphology can be controlled by adjusting supercooling of solvent crystallization or adding water into the solvent system. The compressive modulus of the scaffolds in air and phosphate buffer solution was measured, which increased with HA addition. In-vitro cell culture results showed a ~ood biocomoatibilitv of PLLA/HA scaffolds with the ore-osteoblastic MC3T3-E1 cells.

Human dental pulp stem cell is a promising autologous seed cell for bone tissue engineering

Background The seed cell is a core problem in bone tissue engineering research. Recent research indicates that human dental pulp stem cells （hDPSCs） can differentiate into osteoblasts in vitro, which suggests that they may become a new kind of seed cells for bone tissue engineering. The aim of this study was to evaluate the osteogenic differentiation of hDPSCs in vitro and bone-like tissue formation when transplanted with three-dimensional gelatin scaffolds in vivo, and hDPSCs may become appropriate seed cells for bone tissue engineering. Methods We have utilized enzymatic digestion to obtain hDPSCs from dental pulp tissue extracted during orthodontic treatment. After culturing and expansion to three passages, the cells were seeded in 6-well plates or on three-dimensional gelatin scaffolds and cultured in osteogenic medium. After 14 days in culture, the three-dimensional gelatin scaffolds were implanted subcutaneously in nude mice for 4 weeks. In 6-well plate culture, osteogenesis was assessed by alkaline phosphatase staining, Von Kossa staining, and reverse transcription-polymerase chain reaction （RT-PCR） analysis of the osteogenesis-specific genes type I collagen （COL I）, bone sialoprotein （BSP）, osteocalcin （OCN）, RUNX2, and osterix （OSX）. In three-dimensional gelatin scaffold culture, X-rays, hematoxylin/eosin staining, and immunohistochemical staining were used to examine bone formation. Results In vitro studies revealed that hDPSCs do possess osteogenic differentiation potential. In vivo studies revealed that hDPSCs seeded on gelatin scaffolds can form bone structures in heterotopic sites of nude mice. Conclusions These findings suggested that hDPSCs may be valuable as seed cells for bone tissue engineering. As a special stem cell source, hDPSCs may blaze a new path for bone tissue engineering.

生物3D打印仿生支架促进肩袖损伤后的愈合

背景:大多数肩袖损伤发生于冈上肌腱,由于肌腱组织缺少血管以及肩袖复杂的解剖结构,临床治疗效果非常有限。组织工程技术和干细胞生物学的快速发展为提高肌腱修复质量带来了新的希望。目的:通过生物3D打印技术制备人脐带间充质干细胞/甲基丙烯酸酐化明胶复合支架,观察该支架修复肩袖损伤的效果。方法:(1)体外细胞实验:制备明胶微载体,将人脐带间充质干细胞接种于明胶微载体表面构建组织工程化干细胞。制备甲基丙烯酸酐化明胶水凝胶打印墨水,使用甲基丙烯酸酐化明胶水凝胶打印墨水重悬组织工程化干细胞,放入3D打印机的生物墨水容器中进行打印,蓝光照射固化5 min后即得人脐带间充质干细胞/甲基丙烯酸酐化明胶复合支架。通过死活染色、CCK-8实验检测支架内人脐带间充质干细胞的活性。(2)动物体内实验:采用随机区组设计方法将24只SD大鼠随机4组,每组6只:正常组不进行任何处理,肩袖损伤组、单纯支架组、细胞支架组建立冈上肌腱撕裂的肩袖损伤模型,单纯支架组、细胞支架组造模后分别将甲基丙烯酸酐化明胶支架、人脐带间充质干细胞/甲基丙烯酸酐化明胶复合支架植入肌腱损伤处。术后4周,分别进行大鼠行为学测试及肩袖冈上肌腱组织学病理组织形态观察。结果与结论:(1)体外细胞实验:死活染色结果显示,明胶微载体可减轻3D打印过程对人脐带间充质干细胞造成的损伤,随着培养时间的延长,支架内的人脐带间充质干细胞存活率升高;CCK-8实验结果显示,随着培养时间的延长,支架内的人脐带间充质干细胞活性无明显变化。(2)动物体内实验:行为学测试结果显示,相较于肩袖损伤组、单纯支架组,细胞支架组大鼠四肢运动功能明显改善;肩袖冈上肌腱苏木精-伊红与Masson染色结果显示,相较于肩袖损伤组、单纯支架组,细胞支架组肌纤维排列较规律,无明显的炎性细胞浸润,胶原容积百分比降低;免疫荧光染色结果显示,相较于肩袖损伤组、单纯支架组,细胞支架组肩袖冈上肌腱内白细胞介素6、肿瘤坏死因子α蛋白表达明显降低。(3)结果表明,生物3D打印的人脐带间充质干细胞/甲基丙烯酸酐化明胶复合支架可促进肩袖损伤组织修复再生。

甲基丙烯酰明胶水凝胶作为细胞三维培养支架在骨组织工程中的应用

背景:骨缺损的治疗一直是临床医生亟待解决的临床难题,用甲基丙烯酰明胶进行细胞体外3D培养,可以为大面积骨缺损的治疗提供新的方向。目的:文章综述了甲基丙烯酰明胶作为3D细胞培养支架在骨组织工程中的研究进展,以期为临床骨缺损修复提供进一步的参考。方法:应用计算机检索中国知网及PubMed数据库1986年1月至2023年8月收录的文献,中英文检索词分别为“骨缺损,骨组织工程,生物支架材料,水凝胶,光交联水凝胶,甲基丙烯酰明胶,三维培养,细胞培养”和“Bone defect,Bone tissue engineering,Biomaterial scaffold,Hydrogel,Methylacrylyl photocrosslinked hydrogel,Three-dimensional culture;Cell culture”,最终纳入68篇文献进行综述分析。结果与结论:①同二维培养相比,3D培养可以在无菌条件下,构建立体三维空间,更好地模拟体内环境,为细胞提供合适的温度、酸碱度及足够的营养,使细胞能够在体外正常生长增殖并保持其正常结构与功能,具有独特优势。②在骨组织工程生物支架材料的选择中,水凝胶因其良好的生物相容性、可降解性及具有立体三维网络结构等优点,已被广泛应用于骨再生的研究。③甲基丙烯酰明胶的物理及生物性能受到浓度、光照时间及光引发剂种类以及反应体系等因素的影响,而这些性能均能对细胞的黏附、生长及增殖,甚至细胞形态及功能产生一定的影响。④甲基丙烯酰明胶因具有良好的生物相容性、物理可调节性、可注射性及光敏性能,已被广泛应用于3D细胞封装、3D生物打印及基于数字光处理的立体光刻技术等细胞3D培养体系中。⑤应用各类复合甲基丙烯酰明胶进行细胞的3D培养,可以更好地促进血管形成及骨再生,为临床骨缺损的治疗提供更多可能。⑥目前甲基丙烯酰明胶的来源、合成的方法以及安全性尚没有健全的标准,需要进一步加强研究,对甲基丙烯酰明胶在3D细胞培养领域的应用进行更深入的完善。

纳米复合甲基丙烯酰明胶水凝胶在不同骨缺损环境中应用的价值

背景:近年来,纳米复合甲基丙烯酰明胶水凝胶在修复骨组织损伤方面的研究备受关注。目的:综述应用于骨组织工程的纳米复合甲基丙烯酰明胶水凝胶近年来的最新进展,举例说明纳米复合甲基丙烯酰明胶水凝胶在不同骨缺损环境中的应用。方法:应用计算机检索中国知网、万方、PubMed和Web of Science数据库2016-2023年发表的相关文献,中文检索词为:“明胶,甲基丙烯,纳米,骨,骨组织工程,骨再生,成骨”,英文检索关键词为“gelatin,methacryl*,nano*,bone,bone tissue engineering,bone regeneration,osteogenesis”。结果与结论:①当前作为甲基丙烯酰明胶填料的纳米材料包括无机纳米材料、有机纳米材料以及有机-无机纳米杂化材料。②无机纳米材料可有效提高水凝胶的力学强度、触变性能,调节其降解时间,同时还可通过调节自身表面电荷、搭载药物/因子、自身降解释放金属离子等方式,实现水凝胶的抗菌、促骨形成、免疫调节、促血管生成等功能。③有机纳米材料、有机-无机纳米杂化材料复合甲基丙烯酰明胶水凝胶是两类新兴的材料,目前研究相对较少,但从已发表的研究来看,相比无机纳米材料复合甲基丙烯酰明胶水凝胶,有机纳米材料复合甲基丙烯酰明胶水凝胶的生物相容性及载药性能更佳,纳米相与有机聚合物相间的相互作用更强,纳米粒子的分散性更好。④有机-无机纳米杂化材料复合甲基丙烯酰明胶水凝胶结合了前二者的优势,且金属离子释放可控性更佳,具有巨大的研究潜力。⑤纳米材料可增强甲基丙烯酰明胶水凝胶的抗菌、免疫调控、促成骨等生物学性能,以促进感染性骨缺损、伴糖尿病骨缺损、骨肉瘤切除术后等复杂骨缺损环境下的骨再生。然而,纳米复合甲基丙烯酰明胶水凝胶用于骨缺损修复中的研究还仅限于动物实验,仍需要进行更多的安全性评价和临床试验。

壳聚糖-明胶网络/羟基磷灰石复合材料支架的研究——制备及形貌

目的 制备壳聚糖 -明胶网络 /羟基磷灰石 (CS- Gel/ HA)复合材料多孔支架 ,并考察组分和制备条件对其微观形貌的影响。方法 采用相分离法制备 CS- Gel/ HA多孔支架 ;利用扫描电镜观察微观形貌 ;液体替代法测孔隙率。结果 采用相分离法 ,通过控制组分配比和预冷冻温度可制备不同密度和孔隙率的 CS- Gel/ HA多孔支架。结论 CS- Gel/

静电纺PLA/丝素明胶管状支架的结构与性能

为研制组织工程小口径血管,以良好生物相容性和生物可降解性的聚乳酸(PLA)、丝素蛋白、明胶为原料,通过静电纺丝法,以高速旋转的滚轴为收集装置,构建了外层为PLA纤维和内层为丝素明胶纤维的PLA/丝素明胶复合管状支架(直径为4.5 mm)。采用扫描电镜观察该管状支架的形貌结构;测定其孔隙率及生物力学性能,并在该支架上进行人脐静脉内皮细胞(HUVECs)培养实验。结果表明:PLA/丝素明胶复合管状支架具有较高的断裂强度和较好的柔软性,爆破强度远高于人体的正常血压;支架具有多孔结构,SEM照片显示HUVECs在支架上分化、增殖、生长状态良好。

磷酸钙骨水泥/明胶复合组织工程支架材料的制备及其结构与性能

磷酸钙骨水泥组织工程支架材料具有良好的生物相容性和骨传导性,是一种良好的骨组织工程支架材料,但是这种材料存在力学性能差的缺点,限制了它的应用。本文采用生物相容性良好的可降解明胶材料与磷酸钙骨水泥支架进行复合,制备出的明胶/磷酸钙骨水泥复合支架材料,其压缩强度可达3．7MPa,比复合前磷酸钙支架材料的强度提高了37倍,而且材料具有良好的柔韧性,适合用作为非承重部位骨组织缺损修复用组织工程支架材料。

壳聚糖-明胶-磷酸三钙多孔复合体作为骨组织工程支架的实验研究

目的探索壳聚糖-明胶-磷酸三钙(CS-Gel/TCP)多孔复合体作为骨组织工程支架材料的可行性。方法采用二次冻干技术制备CS-Gel/TCP复合体,为三维立体孔径200～400μm的海绵状支架材料。将兔骨髓基质细胞(MSC)进行体外培养、扩增,并植入该支架,检测支架的细胞相容性。皮下植入观察支架材料的体内降解及组织相容性。结果MSC植入支架后,其噻唑蓝(MTT)活性与碱性磷酸酶(ALP)活性与对照组比较差异无统计学意义。在植入早期可观察到一过性免疫排斥反应,支架在体内12周基本降解吸收。

KGM/明胶/nano HAP椎间盘纤维环组织工程支架的制备与研究

椎间盘退变性疾病已经成为严重影响人们工作和生活的疾病,生物组织工程技术的出现为椎间盘疾病的治疗提供了新的思路和方法。本研究以魔芋葡甘聚糖(Konjac Glucomannan,KGM)、明胶和纳米羟基磷灰石(nano HAP)为原料,分别采用湿法纺丝法和卷膜法构建了椎间盘纤维环组织工程支架。采用扫描电子显微镜(SEM)、X射线衍射仪(XRD)和傅里叶变换红外光谱仪(FT-IR)对支架的成分、结构和形貌进行了分析,并测试了纤维环支架的抗压强度、吸水率、孔隙率、体外降解性及细胞毒性。实验结果表明,得到的椎间盘纤维环组织工程支架具有各向异性的结构特点,与天然椎间盘纤维环结构极为类似,且为多孔结构。支架中加入nano HAP可以提高其强度;湿法纺丝法比卷膜法制备的纤维环支架强度高;戊二醛交联比氨水交联的纤维环支架强度高,但降解速率快;此外,采用戊二醛作交联剂可对nano HAP进行更好的包裹,从而更好地提高支架的强度。支架的吸水率均在700%以上,孔隙率为66%~75%。该研究为研发新型椎间盘纤维环组织工程支架材料提供了一种思路以及一定的实验和理论依据。

应用人恒牙牙髓干细胞与明胶支架构建组织工程骨的研究

目的构建由人恒牙牙髓干细胞和三维明胶支架组成的组织工程骨,并验证其成骨效果。方法从正畸治疗减数拔除的恒前磨牙牙髓组织中应用酶消化法获得牙髓细胞,单抗Stro-1标记、免疫磁珠阳性分选系统分选获得牙髓干细胞。第3代细胞被分别接种到6孔板或三维明胶支架上进行成骨向诱导培养,诱导14天后支架组移植入裸鼠背部皮下。应用ALP染色、Von Kossa染色验证牙髓干细胞的体外成骨能力;应用X线、HE染色和免疫组化验证组织工程骨的体内成骨效果。结果牙髓干细胞体外成骨向诱导后7、14天ALP染色呈阳性;14、21天Von Kossa染色形成矿化结节。在体内组织工程骨成骨明显,X线显示实验侧支架内有团块状高密度阻射影;HE染色显示大量的骨组织形成;免疫组化显示骨结构区域骨桥蛋白、骨涎蛋白和骨钙素阳性表达。结论人恒牙牙髓干细胞和明胶支架构建形成了组织工程骨。

一种新型肝组织工程支架的制备及生物相容性评价

目的:构建一种新型肝组织工程支架,并对其生物相容性进行评价,探讨其作为肝组织工程支架的可行性.方法:选用壳聚糖及明胶材料,通过微制造技术制备一种新型壳聚糖/明胶肝组织工程支架,并与大鼠原代肝细胞共培养,采用HE染色、扫描电镜、MTT法及肝细胞功能学指标等多种检测方法,对支架的细胞毒性及生物相容性进行评价.结果:成功制备了一种新型壳聚糖/明胶复合肝组织工程支架,其孔隙率高、表面积大,有利于肝细胞的黏附与生长,种植在新型壳聚糖/明胶肝组织工程支架上的肝细胞呈现良好的生长增殖趋势,保持良好的生理功能,新制备的支架呈缓慢匀速降解,保持良好的空间结构.结论:制备的壳聚糖/明胶肝组织工程支架具有良好的生物相容性及低细胞毒性,满足肝组织工程的要求.

电纺丝复合纤维的制备及其影响因素探讨

合适的支架是进行组织工程研究必需的条件之一．作者运用实验室自行研制的可控电纺丝系统，对具有良好生物相容性的明胶和生物可降解并具有良好成纤性能的聚己内酯（PCL）的复合电纺丝纤维的制备及影响因素进行了详细的探讨．实验结果表明：明胶在与PCL混合的情况下成纤能力得到较大提高，并分别制得了直径介于250~660nm、均匀无珠状物的多孔纤维膜．由于明胶具有良好的生物和组织相容性，PCL和明胶的混合不仅明显提高了二者的成纤能力，改变了各自都不容易被电纺的缺陷，更重要的是这二者的复合纤维兼具天然高分子和合成聚合物的优点，将为进一步对食道和血管组织工程的研究提供合适的支架样品和技术服务，对组织工程领域的研究起到促进作用．

明胶基生物材料在组织工程中的应用

本文主要对明胶及其复合材料在组织工程领域的研究进展作了评述,表明明胶基生物材料作为种子细胞和信号分子的载体具有广阔的应用前景。

热致相分离法制备明胶多孔支架及其性能研究

本文对热致相分离法制备明胶多孔支架及其性能进行了系统的研究。采用冷冻致孔真空干燥法制备了明胶基组织工程多孔支架,并对影响其结构与性能的因素(如明胶溶液浓度、交联剂用量、体系pH值等)进行了考察。实验结果表明:真空冷冻干燥法制备的明胶基多孔组织工程支架都具有三维孔洞结构;所制备的支架平均孔径可达100μm。

羟基磷灰石/羧甲基壳聚糖/明胶复合支架的制备及表征

采用原位共沉淀结合冷冻干燥工艺制备羟基磷灰石/羧甲基壳聚糖-明胶复合支架材料,然后利用X射线衍射仪(XRD)、傅立叶红外光谱仪(FTIR)检测其组成,扫描电子显微镜(SEM)观察微观结构,模拟体液浸泡法测试其降解性能,直接接触法观察其细胞毒性。结果表明:羟基磷灰石通过化学键与羧甲基壳聚糖和明胶结合;支架为相互连通的多孔结构,孔径尺寸介于100μm到300μm之间;纳米羟基磷灰石呈针状,均匀分布在支架中。与纯羟基磷灰石相比,复合支架降解性更好,更利于细胞黏附和增殖。实验结果同时表明支架材料的微观结构、降解性和细胞毒性可以通过改变明胶的含量进行调节,以满足不同修复部位的需求,是一种理想的骨组织工程用支架材料。