利用同轴3D打印技术构建促内皮细胞生长类血管组织工程支架

体外构建血管网络对组织工程领域厚组织与器官再生至关重要。利用同轴3D打印技术,以海藻酸钠/丝素蛋白为生物墨水,可快速制备含人脐静脉内皮细胞(HUVECs)的类血管组织工程支架。首先通过材料压缩模量和可打印性测试,优化适用于同轴系统的材料浓度;然后通过光学相干层析成像技术,研究打印参数对中空纤维丝形状的影响,优化同轴打印参数;结合模拟灌流实验,对支架内部类血管结构进行表征;最后通过细胞活、死染色和Alamar Blue法,检测支架中HUVECs生长情况。结果表明,经优化的生物墨水及打印参数能顺利制备具有内部联通性完整的类血管组织工程支架;HUVECs在体外培养时存在团聚生长现象,类血管通道的存在有利于维持组织整体活性,一周存活率在97%以上,且相比对照组能够维持较高的增殖速率。研究证明,利用同轴3D打印技术能成功构建促内皮细胞生长的类血管组织工程支架,可为厚组织及器官再生提供新的可能。     

基于同轴细胞打印双网络生物墨水优化及类血管支架的打印

文题释义：双网络生物墨水：生物墨水是指可以用于生物3D打印机的材料,具有类似细胞外基质的理化性质,可用于制造与人体器官相似的组织。双网络生物墨水内部具有两种交联网络,能使体外构建的组织具有良好的机械性能,适用于不同的应用场景。 同轴细胞打印：生物3D打印也叫细胞打印,是指操控细胞生物墨水体外构建活性组织的过程。同轴细胞打印是生物3D打印的延伸和发展,通过结合多层同轴针头可以直接快速制备含有内部连通网络的组织工程支架。 背景：细胞体外培养情况下无法在远离营养物质200 μm以上的区域存活,血管网络构建对组织工程领域厚组织和器官再生至关重要,同轴细胞打印为体外构建类血管通道提供了一种新的方式。 目的：优化生物墨水的同轴细胞打印性能,制备具有类血管结构的组织工程支架。 方法：通过间歇式巴氏灭菌制备无菌海藻酸钠溶液,冷冻保存;以脱胶蚕丝为原料制备无菌丝素蛋白冻干粉,密封保存;将丝素蛋白冻干粉加入解冻的海藻酸钠溶液中,再加入人脐静脉内皮细胞,作为生物墨水;将生物3D打印机的外轴连接生物墨水,内轴连接交联剂,同轴打印类血管支架材料,进行光学相干层析成像扫描、扫描电镜观察;拉伸测试海藻酸钠与丝素蛋白/海藻酸钠同轴打印环形试件(不含细胞)的弹性模量。采用冷冻保存7 d的海藻酸钠溶液与人脐静脉内皮细胞制作同轴打印支架,冷冻保存7 d的海藻酸钠溶液、人脐静脉内皮细胞与密封保存6个月的丝素蛋白冻干粉制作同轴打印支架,培养24 h后死活染色观察细胞存活率。设计打印串联与并联结构的类血管支架,培养1,3,7,10,14 d后检测细胞增殖情况。 结果与结论：①光学相干层析成像扫描显示,该混合生物墨水最高打印高度为9层,整体厚度约为4.4 mm;扫描电镜显示,类血管支架的中空纤维丝外壁呈无规则条状卷曲,存在微米级内部连通孔隙结构,中空纤维丝内壁具有更致密的孔隙结构;②丝素蛋白/海藻酸钠同轴打印环形试件的弹性模量大于单纯海藻酸钠同轴打印环形试件(P < 0.05);③采用保存7 d海藻酸钠溶液制作的支架细胞存活率为(86.7±3.4)%,加入丝素蛋白冻干粉支架的细胞存活率为(98.1±1.2)%,说明冷冻保存7 d的海藻酸钠溶液未染菌,丝素蛋白的保质期可达6个月;④并联结构类血管支架培养7,10,14 d的细胞增殖活性高于串联结构的类血管支架(P < 0.05);⑤结果表明,实验制备的类血管支架材料具有良好的生物相容性与机械性能。 ORCID: 0000-0002-5556-6672(张一帆) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

中空纤维生物反应器的构建及其体外灌流实验

设计原代培养的乳猪肝细胞构建生物反应器,并观察肝细胞生长特点,进行体外灌流实验。采用改良的原位胶原酶灌流法分离乳猪肝细胞及间质细胞,在中空纤维舱的纤维外间隙中共培养,并间断旋转抑制细胞贴壁。用该生物反应器建立人工肝系统,对肝硬化病人的腹水进行体外灌流实验。结果表明,乳猪原代肝细胞平均产量为(6.29±0.37)×108细胞,肝细胞的活性为84%;在纤维外间隙内间断转动培养3 h后肝细胞聚合成球。电镜可见,多个肝细胞聚合成团生长,培养的肝细胞有功能性结合,并向组织型转化。测定中空纤维舱内连续48 h培养液尿素浓度,证实肝细胞聚合体有很好的尿素合成功能。体外灌流后,生物反应器组腹水总胆红素下降,A ST升高,与对照组相比有显著性差异。生物反应器组葡萄糖浓度下降,对照组无显著变化,两组间有显著性差异。通过本组实验,证实了我们设计的生物反应器所构成的生物型人工肝(BAL)是有效的。     

生物细胞三维打印技术与材料研究进展

生物细胞三维(3D)打印是一种新型的制造技术,通过该技术可以将细胞以及细胞支撑材料打印成复杂的具有3D结构与功能的组织。与其他3D打印技术相比,生物细胞3D打印需要对打印过程以及打印材料的生物环境进行研究。针对生物细胞3D打印的特点,本文主要讨论了生物细胞3D打印技术的发展现状,重点从打印技术与打印材料两个方面展开介绍。其中,针对现有打印技术,本文重点介绍了喷墨法、挤出沉积法、光固化成型法以及激光辅助法的精度、制备过程、材料要求以及对细胞状态影响,并在此基础上比较了各自的优势和局限性;针对常用的打印材料,本文重点介绍并对比了其交联方式、生物相容性以及应用场合等。生物细胞3D打印技术目前仍主要在实验室发展阶段,对现阶段生物3D打印技术原理与发展进行回顾总结不仅有助于思考如何将这一技术尽快投入实用,也有助于更好地规划生物3D打印的未来发展方向。     

3D生物打印的肝结构体植入兔纤维化肝的研究

通过将三维(3D)生物打印的类肝水凝胶结构体,经肝表面贴覆法植入纤维化肝兔模型,探讨改善肝纤维化以及形成类肝组织结构的可行性。采用兔离体肝脏胶原酶消化法提取兔原代肝细胞,并采用四氯化碳(CC1₄)皮下注射诱导兔肝纤维化模型。选取30只肝纤维化兔,随机分为实验组、对照组、假手术组,每组10只。3D生物打印兔肝细胞-海藻酸钠-明胶3D水凝胶网格状结构体片段,经肝表面贴覆法植入兔纤维化肝,为实验组。植入16 d后,检测兔肝功能生化指标及肝脏组织病理学变化,观察肝纤维化的发展程度,以及类肝组织结构形成情况。按照设计的参数,打印出含有兔原代肝细胞的网格状水凝胶结构体。通过LIVE/DEAD双荧光染色观察,打印后肝结构体的细胞存活率为82%±3%。兔肝细胞水凝胶支架体内植入16天后,可见每个植入物的大部分支架材料尚未降解,未降解的植入物与肝脏贴合紧密,融合生长。通过检测兔肝功能生化指标及植入物之下的肝脏组织病理学观察,实验组兔肝功能生化指标分别为:ALT(90.26±13.05)U/L,AST(75.37±13.45)U/L,γGT(16.62±6.72)U/L,ALB(32.48±4.43)g/L,与对照组、假手术组之间比较均P>0.05。实验组、对照组、假手术组的肝纤维化程度病理学检查评分结果分别为2.95±0.50、3.11±0.58、3.02±0.62,各组之间比较均P>0.05。兔肝细胞-3D水凝胶支架的植入,使肝功能生化指标及兔肝脏纤维化程度稍有改善,但各组之间比较均无统计学意义。组织学观察结果表明,实验组植入物中肝细胞均匀分布,未见细胞变性及死亡,可形成类肝组织结构。3D生物打印的肝结构体具有再造肝样组织片段和实现部分肝功能的潜力,将在基础医学和临床之间搭建桥梁,为肝脏再生打下基础。     

中国人肝细胞系1运用于生物人工肝的生物代谢功能

背景：前期研究发现,中国人肝细胞系1细胞分化程度高且生物代谢功能良好, 并且中国人肝细胞系1细胞组织学上来源于正常肝组织,较其他来源于肿瘤源性的肝细胞系更为安全。 目的：探讨中国人肝细胞系中国人肝细胞系1细胞在混合型生物人工肝中的生物代谢功能。 方法：15只食蟹猴随机分成对照组(n=5)和治疗组(n=10),均建立急性肝功能衰竭模型,治疗组接受以全接触灌流型生物反应器接种微载体微重力中国人肝细胞系1细胞建立的人源细胞混合型生物人工肝进行治疗。 结果与结论：急性肝功能衰竭食蟹猴血清谷氨酸转氨酶、总胆红素、总胆汁酸、尿素氮、肌酐、血氨均明显上升,而白蛋白、Fischer指数则显著下降;人源细胞混合型生物人工肝治疗后,急性肝功能衰竭食蟹猴血清谷氨酸转氨酶、总胆红素、总胆汁酸、尿素氮、肌酐、血氨和白蛋白均恢复。提示中国人肝细胞系1细胞在混合型生物人工肝中生物代谢功能良好,表现出良好的肝特异性生物合成及生物代谢功能。     

大孔明胶微载体的制备及肝细胞附着生长的研究

急性肝功能衰竭是一种致死率非常高 (>60 %)的疾病[1,2 ] 。人们仿照人工肾的原理 ,研制了许多化学吸附材料。然而由于肝脏高度复杂的代谢功能 ,简单应用现存的血液灌流技术在临床上效果不够理想。生物人工肝支持系统是以培养的肝细胞为主要材料 ,它不同于以往的化学吸附材料 ,具有生物合成和解毒两种功能 ,被认为是治疗肝衰的一种有效方法 ,因此成为国际上的一个研究热点[3] 。因为肝细胞是一种贴壁依赖性细胞 ,在人工肝的研究工作中提高肝细胞的培养密度是一个难题。利用微载体较大的比表面积可以提高肝细胞培养密度 ,但是已开发的微载体…     

3D打印器官芯片研究进展

器官芯片是一种将生物体活细胞植入精准设计的微流体芯片内,可特定再现生物体组织器官功能的仿生的微生理系统,在疾病模拟、毒性检测、新药研发、精准医疗等许多方面具有独特应用前景。3D生物打印技术与器官芯片技术相结合制作3D打印器官芯片,可实现芯片制造工艺的简易化和低成本化,同时满足对复杂异质三维微环境的精细需求。综述3D打印器官芯片在打印方式、打印墨水等方面的研究进展,阐述其最新生物医学应用,总结器官芯片结构和功能单元的打印方式和打印墨水,探讨基于现有打印工艺实现器官芯片一体化制造的潜在可行性,概述3D打印器官芯片技术在心、肝、肺、肾、神经、肿瘤等组织和器官结构和功能仿生方面的最新进展,最后展望3D打印器官芯片技术领域的发展趋势和有待解决的关键问题。     

培养于TECA型生物人工肝的猪肝细胞生物转化功能的测定

了解培养于TECA型生物人工肝(BALSS)中猪肝细胞活性和生物转化功能的变化.将新鲜分离的特种猪肝细胞培养于TECA-BALSS的生物反应器中,通过BALSS中空纤维管膜与含盐酸利多卡因的RPMI-1640培养液进行6h交叉灌流.对照组BALSS内无猪肝细胞.采用胎盘蓝染色法测定肝细胞活率.用高压液相法测定利多卡因浓度.结果显示:猪肝细胞在TECA-BALSS中培养6h时,其细胞活率达89.7%;随着交叉灌流时间的增加,其体液环路中的盐酸利多卡因浓度逐步下降,6h时的转化率达80%,显著高于无肝细胞对照组的转化率2.5%(P＜0.01).这表明采用TECA-BALSS可大量的培养猪肝细胞,不仅肝细胞能维持较高的存活率,并能发挥肝细胞的生物转化功能.     

生物人工肝的支架材料研究进展

用于构建生物人工肝的关键材料包括生物材料及支架材料，前者主要指肝细胞，肝非实质细胞等细胞材料，后者主要指用于构建生物反应系统的膜或其它支架材料，此类材料的性能直接关系到肝细胞的生长及代谢功能，与人工肝的支持效果密切相关，本文将近年研究较多的生物人工肝支架材料作一综述。     

Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery

This study offers a novel 3D bioprinting method based on hollow calcium alginate filaments by using a coaxial nozzle, in which high strength cell-laden hydrogel 3D structures with built-in microchannels can be fabricated by controlling the crosslinking time to realize fusion of adjacent hollow filaments. A 3D bioprinting system with a Z-shape platform was used to realize layer-by-layer fabrication of cell-laden hydrogel structures. Curving, straight, stretched or fractured filaments can be formed by changes to the filament extrusion speed or the platform movement speed. To print a 3D structure, we first adjusted the concentration and flow rate of the sodium alginate and calcium chloride solution in the crosslinking process to get partially crosslinked filaments. Next, a motorized XY stages with the coaxial nozzle attached was used to control adjacent hollow filament deposition in the precise location for fusion. Then the Z stage attached with a Z-shape platform moved down sequentially to print layers of structure. And the printing process always kept the top two layers fusing and the below layers solidifying. Finally, the Z stage moved down to keep the printed structure immersed in the CaCl₂ solution for complete crosslinking. The mechanical properties of the resulting fused structures were investigated. High-strength structures can be formed using higher concentrations of sodium alginate solution with smaller distance between adjacent hollow filaments. In addition, cell viability of this method was investigated, and the findings show that the viability of L929 mouse fibroblasts in the hollow constructs was higher than that in alginate structures without built-in microchannels. Compared with other bioprinting methods, this study is an important technique to allow easy fabrication of lager-scale organs with built-in microchannels.     

3D bioprinting in orthopedics translational research

Abstract

The repair of critical-size bone defect remains a challenge for orthopedic surgeons. With the advent of an aging society and their accompanying chronic diseases, it is becoming more difficult to treat bone defects, especially large segmental bone defects that are caused by trauma, tumors, infections, and congenital malformations. New materials and technologies need to be developed to address these conditions. 3D bioprinting is a novel technology that bridges the biomaterial and living cells and is an important method in tissue engineering projects. 3D bioprinting has the advantages of replacing or repairing damaged tissue and organs. The progress in material science and 3D printing devices make 3D bioprinting a technology which can be used to create various scaffolds with a large range of advanced material and cell types. However, in regard to the widespread use of bioprinting, biosafety, immunogenicity and rising costs are rising to be concerned. This article reviews the developments and applications of 3D bioprinting and highlights newly applied techniques and materials and the recent achievements in the orthopedic field. This paper also briefly reviews the difference between the methods of 3D bioprinting. The challenges are also elaborated with the aim to research materials, manufacture scaffolds, promote vascularization and maintain cell viability.     

Prospect and retrospect of 3D bio-printing

3D bioprinting has become a popular medical technique in recent years. The most compelling rationale for the development of 3D bioprinting is the paucity of biological structures required for the rehabilitation of missing organs and tissues. They're useful in a multitude of domains, including disease modelling, regenerative medicine, tissue engineering, drug discovery with testing, personalised medicine, organ development, toxicity studies, and implants. Bioprinting requires a range of bioprinting technologies and bioinks to finish their procedure, that Inkjet-based bioprinting, extrusion-based bioprinting, laser-assisted bioprinting, stereolithography-based bioprinting, and in situ bioprinting are some of the technologies listed here. Bioink is a 3D printing material that is used to construct engineered artificial living tissue. It can be constructed solely for cells, but it usually includes a carrier substance that envelops the cells, then there's Agarose-based bioinks, alginate-based bioinks, collagen-based bioinks, and hyaluronic acid-based bioinks, to name a few. Here we presented about the different bioprinting methods with the use of bioinks in it and then Prospected over various applications in different fields.     

3D生物打印技术及其在组织工程中的应用

3D生物打印是在3D打印(three-dimensional printing,3DP)和组织工程等多学科融合的基础上发展出来的一种新兴应用技术,该技术能够根据数字化的模型设计将生物材料和/或细胞打印成三维生物功能体,被广泛应用于血管、骨骼等组织的再生与重建领域.     

Tissue vascularization through 3D printing: Will technology bring us flow?

Background: Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi‐scale vasculature seen in in vivo systems remains elusive. Results: 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. Conclusions: As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases. Developmental Dynamics 244:629–640, 2015. © 2015 Wiley Periodicals, Inc.     

3D打印技术在骨科的应用研究

3D打印呈现井喷式的发展,该技术在骨科的应用是一个热门的研究方向。本文阐述了3D打印技术在骨科方面的应用近况,主要对该技术在术前规划、术中导航、临床教学、医患沟通、康复支具、骨科内植物、生物打印等多个方面的应用进行综述,归纳了3D打印技术运用于骨科的优势,总结了目前存在的一些技术难点,并对3D打印技术在大块骨缺损的修复、假肢等方面的应用进行了展望。     

3D bioprinting of neural stem cell-laden thermoresponsive biodegradable polyurethane hydrogel and potential in central nervous system repair

The 3D bioprinting technology serves as a powerful tool for building tissue in the field of tissue engineering. Traditional 3D printing methods involve the use of heat, toxic organic solvents, or toxic photoinitiators for fabrication of synthetic scaffolds. In this study, two thermoresponsive water-based biodegradable polyurethane dispersions (PU1 and PU2) were synthesized which may form gel near 37 °C without any crosslinker. The stiffness of the hydrogel could be easily fine-tuned by the solid content of the dispersion. Neural stem cells (NSCs) were embedded into the polyurethane dispersions before gelation. The dispersions containing NSCs were subsequently printed and maintained at 37 °C. The NSCs in 25–30% PU2 hydrogels (∼680–2400 Pa) had excellent proliferation and differentiation but not in 25–30% PU1 hydrogels. Moreover, NSC-laden 25–30% PU2 hydrogels injected into the zebrafish embryo neural injury model could rescue the function of impaired nervous system. However, NSC-laden 25–30% PU1 hydrogels only showed a minor repair effect in the zebrafish model. In addition, the function of adult zebrafish with traumatic brain injury was rescued after implantation of the 3D-printed NSC-laden 25% PU2 constructs. Therefore, the newly developed 3D bioprinting technique involving NSCs embedded in the thermoresponsive biodegradable polyurethane ink offers new possibilities for future applications of 3D bioprinting in neural tissue engineering.