3D打印生物墨水在组织修复与再生医学中的应用

背景：通过选用合适的生物墨水，3D打印技术可用以制造人体组织和器官的代替物，并在人体内发挥作用。近些年来3D打印技术发展迅速，在再生医学中有着巨大的应用潜力。目的：介绍3D打印用生物墨水的类型，并综述生物墨水的分类、应用、优缺点及未来愿景。方法：以“3D printing,Biological ink,Tissue engineering,hydrogel,Synthetic material,Cytoactive factor,3D打印、生物墨水、组织工程”为检索词，运用计算机检索2000-2022年以来发表在PubMed、CNKI数据库中的相关文献，最终纳入83篇进行综述。结果与结论：在过去的几十年里，生物3D打印技术发展迅速，在组织工程和生物医学等各个领域都受到了极大的关注。相对于传统生物支架制造方法在功能性及结构方面受到的限制，3D打印可以更好地模拟生物组织复杂的结构，并且具有合适的力学、流变学和生物学特性。生物墨水是3D打印中必不可少的一部分，通过生物材料制备的生物墨水，经打印后产生的生物支架在组织修复和再生医学等方面有着巨大的科研潜力及临床意义，其材料的研究本身也越来越受到...     

3D打印技术在整形外科的研究及应用进展

背景:3D生物打印利用组织工程及干细胞研究的成果,以活细胞及其他细胞活性成分作为打印原料,最终实现生物活性组织的打印和制作。目的:对3D打印技术目前在整形外科的应用及研究进展作一综述。方法:由第一作者检索从2007至2016年PubM ed、Ovid、中国知网等数据库收录的关于3D打印技术在整形外科应用及研究进展的文献。英文检索词为"three-dimensional printing,3D printing,plastic and reconstructive surgery,orthopaedic,organ printing",中文检索词为"3D打印,整形外科,修复重建,器官打印",按纳入、排除标准筛选最终纳入44篇文献进行分析、总结。结果与结论:3D打印目前在颅颌面骨重建、耳鼻再造、皮肤打印及乳房重建等方面的应用较为广泛,特别在制作骨替代品及个性化假肢、假体方面技术已相当成熟。3D打印技术经历了从假体制作到生物活性打印的发展过程,最终有望进入器官打印阶段,彻底解决自体或同种异体移植所存在的局限。     

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     

组织工程中3D生物打印技术的应用

背景：近年来,研究者将最先应用于工程领域的3D打印技术嫁接到组织工程学中,希冀利用3D生物打印技术进行体外组织、器官复制过程,并取得了一些令人惊喜的成果。 目的：从3D打印技术的原理、打印操作步骤、与组织工程学的关系、优势和难题、临床应用等方面对其目前的发展趋势做一概述。 方法：第一作者应用计算机检索2000年1月至2013年10月PubMed数据库、中国期刊全文数据库、维普中文期刊网有关3D生物打印技术在组织工程中应用的文章,英文检索词“three-dimensional bioprinting, tissue engineering, rapid prototyping technology, scaffold materials, selective laser sintering, fused deposition modeling, stereolithography ”,中文检索词“3D生物打印,组织工程学,快速成型技术,支架材料,选择性激光烧结,熔融沉积成型,立体光刻技术”,排除重复性研究。共检索到79篇相关文献,其中52篇文献符合纳入标准。 结果与结论：3D生物打印就是借助影像技术(CT、MRI)资料的辅助,应用计算机辅助设计技术虚拟出待构建体的三维结构,然后利用相应的材料,逐层创建出实体的一种组织工程学技术。其具有高精度、构建速度快,可实现按需制造等优势,但也面对力学、生物学等方面的难题,临床应用前景广阔。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

生物三维打印细胞负载水凝胶类组织的精准优化

生物三维打印技术与水凝胶的完美结合,可为制造复杂结构功能的组织器官提供一种极具吸引力的解决方案。定制打印细胞负载水凝胶类组织的内部结构,可以更好地仿生真实组织器官的三维微环境,对打印后细胞生长、组织形成和功能再生至关重要。但水凝胶理化特性多变,精准打印与设计结构匹配的多孔结构仍然极具挑战。提出基于光学相干层析成像技术(OCT)的生物三维打印细胞负载水凝胶类组织的精准优化方法,通过自制的三维扫频OCT系统无损在线成像打印组织块和定量评价结构参数,迭代降低设计与打印间的结构差异,提高细胞负载水凝胶打印的精准性和稳定性。实验结果表明,基于OCT无损定量表征结果反馈优化打印参数设置,指导打印过程,使得细胞负载水凝胶类组织的结构形态参数与设计值的偏差从40%左右控制到7%以内,包括内部孔隙尺寸、支撑尺寸、孔隙率、表面积、体积五项关键参数;细胞培养两周后的存活率从80%左右显著提高到90%以上。研究表明OCT技术为批量定制细胞负载水凝胶类组织、生物三维打印组织和器官等提供了具有潜力的精准化工具。     

三维生物打印技术在泌尿系组织工程中的研究进展

针对泌尿系疾病导致的组织器官损伤和缺失,目前临床上的治疗方法存在局限性。组织工程通过对细胞、生物支架和生物相关分子的研究,提供了一种可替代或再生受损组织器官的治疗手段。三维（3D）生物打印技术作为新兴制造技术,能对载有细胞的生物材料精确控制,进一步推动着组织工程领域的发展。本文综述了3D生物打印技术在肾脏、输尿管、膀胱、尿道组织工程中的研究进展和应用,并讨论了目前面临的主要挑战和未来展望。     

3D打印与组织工程心肌、心脏瓣膜、大血管及血管网的构建

背景：由于3D打印技术具有高度的仿生性和复制精微复杂结构的优势,被广泛应用于骨科、整形美容、医学修复与心血管疾病的诊疗中。 目的：综述3D打印技术在构建心脏瓣膜、再生血管、工程心肌组织和心血管疾病模型等方面的研究进展、优势及存在的问题,并展望其临床运用前景。 方法：由第一作者检索PubMed数据库、CNKI数据库2013年4月至2015年4月的相关文献,检索关键词为“3D printing,cardiovascular system,rapid prototyping;3D打印,心血管,快速成型技术”。 结果与结论：目前,3D打印技术涉及了心血管研究和应用的各个方面,在组织工程心肌、组织工程心脏瓣膜、组织工程大血管及血管网的构建上已有突破性进展,3D打印方案逐渐完善,其应用已从实验室研究走向临床应用。但在更广泛的运用前还有很多亟待攻克的难题,最为突出的问题之一是如何保证打印器官或组织的血供问题,尽管目前3D打印的管状结构已基本保证了组织器官的血供需求,但毛细血管的超微结构难以通过图像重建模仿,在构建局部微循环方面还有待进一步突破。 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     

种子细胞及支架材料在组织工程角膜构建中的应用

背景：在眼科角膜移植在组织工程中有很好的应用前景,但理想的支架材料一直制约组织工程角膜发展。 目的：分析各种子细胞、支架材料的应用,总结近年来组织工程角膜构建所取得的进展。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2000/2010-10)和PubMed数据库(2000/2010-10),检索词分别为“组织工程,角膜移植”和“tissue engineering, corneal transplantation”。经计算机初检得到223篇文献,纳入与组织工程角膜研究进展、应用和体外构建的相关实验研究和综述,排除重复研究类文献,对33篇汇总分析。从种子细胞、支架材料、器官构建、临床应用等方面进行总结,对组织工程角膜的研究进展和未来发展进行探讨。 结果与结论：结果表明,种子细胞和支架材料仍为组织工程研究的重点,由于眼免疫赦免现象和角膜的无血管特性,组织工程角膜移植将优于其他器官和组织的移植,成为能大量构建且易于移植的组织工程器官。目前,组织工程角膜构建已获得初步成功,但各类支架材料均存在一定缺点,寻找理想的支架材料是下一步的研究方向。     

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。     

生物打印在肌肉骨骼界面重建中的应用

文题释义：生物打印：是一种能够在数字三维模型辅助下,根据增材制造原理定位装配生物材料或细胞单元,从而制备组织工程支架和组织器官等制品的一种新兴技术。 肌肉骨骼界面：是指肌肉骨骼系统中存在的一系列结构、功能和工程相似的部位,其通过肌肉和骨骼附着实现平滑连接,通常这些界面主要包括骨-肌腱、骨-韧带和骨-软骨等。其工作原理和潜在机制使它们成为组织的独特分支,其在细胞组分上显著不同,但在结构和功能上是一致的。 背景：肌肉骨骼损伤和退行性疾病的手术治疗常涉及肌肉骨骼界面的重建,而实现肌肉骨骼界面与周围宿主组织的生物整合的关键是制造具有精确结构和不同材料的替代物。生物打印技术获得的人工组织可与天然肌肉骨骼界面组织具有相似的物理结构和生物活性。 目的：介绍肌肉骨骼界面组织的结构和生物功能特性,以及生物打印技术在肌肉骨骼界面重建中的应用。 方法：由第一作者以“bioprinting, musculoskeletal interface,生物打印,肌肉骨骼界面”为关键词,检索2005至2019年期间PubMed、Web of Science、Springerlink、Medline、万方、CNKI数据库中的相关文献。初检文章201篇,筛选后对60篇文章进行分析。 结果与结论：理想的生物打印肌肉骨骼界面移植物必须结构上与原界面组织相对应,以维持体内多变的生物力学环境;其次,植入之后必须保持这些植入物的生物活性,以开始修复和替换缺陷区域的功能。生物打印技术的发展为解决肌肉骨骼界面的重建带来了希望,但其仍然存在许多挑战：仿生功能性界面结构机械性能的提高、多个仿生结构的整合、生物打印结构的血管化,以及对力学刺激在界面组织发育和再生中的作用缺乏深入的研究。对于未来界面组织工程的研究方向,可以预料的是将种子细胞、细胞因子和基因治疗,以及生物反应器纳入界面组织工程支架中的一大热点,为解决界面组织整合这一难题提供创新性的解决方案。 ORCID: 0000-0002-6668-5036(张君伟) 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     

Inkjet printing for high-throughput cell patterning

The adaptation of inkjet printing technology to the complex fields of tissue engineering and biomaterial development presents the potential to increase progress in these emerging technologies through the implementation of this high-throughput capability via automated processes to enable precise control and repeatability. In this paper, a method of applying high-throughput inkjet printing to control cellular attachment and proliferation by precise, automated deposition of collagen is presented. The results indicate that commercial inkjet printing technology can be used to create viable cellular patterns with a resolution of 350 microm through the deposition of biologically active proteins. This method demonstrates a combination of off-the-shelf inkjet printing and biomaterials and has potential to be adapted to tissue engineering and colony patterning applications. Adapting this method into the three-dimensional construction of cellular structures for eventual high-throughput tissue engineering using a bottom-up approach is possible.     

Biocompatible inkjet printing technique for designed seeding of individual living cells

Inkjet printers are capable of printing at high resolution by ejecting extremely small ink drops. Established printing technology will be able to seed living cells, at micrometer resolution, in arrangements similar to biological tissues. We describe the use of a biocompatible inkjet head and our investigation of the feasibility of microseeding with living cells. Living cells are easily damaged by heat; therefore, we used an electrostatically driven inkjet system that was able to eject ink without generating significant heat. Bovine vascular endothelial cells were prepared and suspended in culture medium, and the cell suspension was used as "ink" and ejected onto culture disks. Microscopic observation showed that the endothelial cells were situated in the ejected dots in the medium, and that the number of cells in each dot was dependent on the concentration of the cell suspension and ejection frequency chosen. After the ejected cells were incubated for a few hours, they adhered to the culture disks. Using our non-heat-generating, electrostatically driven inkjet system, living cells were safely ejected onto culture disks. This microseeding technique with living cells has the potential to advance the field of tissue engineering.     

颅骨修补材料研究现状及3D打印技术应用前景

BACKGROUND: Skull repair materials cannot only restore the normal shape of the skull, but also play an important role in brain functional recovery. OBJECTIVE: To summarize the research status of polyetheretherketone (PEEK), titanium alloy and tissue engineering technique in cranioplasty and the prospect of three-dimensional (3D) printing technology. METHODS: Literatures related to skull repair materials were retrieved in databases of CNKI and PubMed published from 1995 to 2016, using the keywords of “bone regeneration material in calvarial, 3d printing bone scaffold” in Chinese and English, respectively.  RESULTS AND CONCLUSION: Although titanium and PEEK have been used in clinic, titanium holds conductivity, thermal conductivity, while PEEK that may be displaced or lost is not involved in osseointegration. Tissue engineering technology participates in the skull tissue reconstruction, achieving satisfactory repair outcomes, but the problems of scaffold selection and preparation, seed cell obtainment, and growth factor release need to be overcomed. 3D printing technology can print personalized shape, fit the defect precisely, but the raw materials should have good biocompatibility and biomechanical property. Combination of tissue engineering technology with 3D printing technology shows a broad prospect in cranioplasty.     

Laser printing of pluripotent embryonal carcinoma cells

A technique by which to print patterns and multilayers of scaffolding and living cells could be used in tissue engineering to fabricate tissue constructs with cells, materials, and chemical diversity at the micron scale. We describe here studies using a laser forward transfer technology to print single-layer patterns of pluripotent murine embryonal carcinoma cells. This report focuses on verifying cell viability and functionality as well as the ability to differentiate cells after laser transfer. We find that when cells are printed onto model tissue scaffolding such as a layer of hydrogel, greater than 95% of the cells survive the transfer process and remain viable. In addition, alkaline comet assays were performed on transferred cells, showing minimal single-strand DNA damage from potential ultraviolet-cell interaction. We also find that laser-transferred cells express microtubular associated protein 2 after retinoic acid stimulus and myosin heavy chain protein after dimethyl sulfoxide stimulus, indicating successful neural and muscular pathway differentiation. These studies provide a foundation so that laser printing may next be used to build heterogeneous multilayer cellular structures, enabling cell growth and differentiation in heterogeneous three-dimensional environments to be uniquely studied.     

Inkjet printing of macromolecules on hydrogels to steer neural stem cell differentiation

Inkjet printing allows for the rapid and inexpensive printing of cells, materials, and protein molecules. However, the combination of inkjet printing and control of neural stem cell (NSC) multipotency and differentiation has remained unexplored. We used an inkjet printer (Canon BJC-2100) to print biologically active macromolecules on poly-acrylamide-based hydrogels (HydroGel(TM)), which were subsequently seeded with primary fetal NSCs. NSCs cultured on areas printed with fibroblast growth factor-2 (FGF2) remained undifferentiated, consistent with the effects of FGF2 when administered in solution. NSCs cultured in parallel on the same hydrogels but in areas printed with ciliary neurotrophic factor (CNTF) or fetal bovine serum (FBS) displayed a rapid induction of markers for astrocytic (glial fibrillary acidic protein, GFAP) or smooth muscle (smooth muscle actin, SMA) differentiation, respectively. These results are consistent with known actions of CNTF and FBS on NSCs. Importantly, NSCs cultured on a printed gradient of increasing levels of CNTF showed a linear increase in numbers of cells expressing GFAP, demonstrating a functional gradient of CNTF. Lastly, genetically modified NSCs proved to respond properly to printed macromolecules, suggesting that inkjet printing can successfully be combined with gene delivery to achieve effective control of stem cell differentiation.     

医学3D打印技术在视网膜修复的研究进展

生物3D打印技术为视网膜受损修复这一难题的解决提供了可能。用海藻酸钠水凝胶打印一个用于细胞生长的支架,利用3D打印的超高精度将视网膜的神经节细胞和神经胶质细胞植入支架,并加入一定浓度的生长因子,诱导细胞向特性方向生长,用这种方法打印视网膜细胞将实现形态可控、强度可控、梯度释放,有望实现体外视网膜细胞的精确打印,解决临床视网膜神经缺损修复的难题。本文在对这些成果进行系统总结的基础上,简要评析了研究现状并且提出了亟待解决的问题,以期为该领域的进一步研究提供参考。     

三维打印技术在骨组织工程领域的应用研究进展

综述了三维(3D)打印技术的出现、分类与优势等.介绍了该技术在骨组织工程领域的应用,包括光固化立体印刷、熔融沉积成型、选择性激光烧结和3D喷印的工作原理、存在的优缺点以及国内外学者在该领域的研究进展.目前骨组织工程支架的制备大多应用了3D打印技术,以生物可降解的活性材料为原料制备而成.在我国该领域虽然发展迅速,利用3D打印技术进行人工骨合成、骨科术前模拟等已经越来越普遍,亦取得了令人满意的效果,但要研发出合适的生物材料以及设备精度的改进仍是亟待解决的问题.目前,仿生器官的功能化已成为3D打印技术领域的一大困难,其中多细胞共培养、血管化及支架的制备是实现功能化必须克服的问题,相信通过努力,该项技术将会为器官的再生与修复带来更多令人瞩目的成果.     

Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing

Organ printing, a novel approach in tissue engineering, applies layered computer-driven deposition of cells and gels to create complex 3-dimensional cell-laden structures. It shows great promise in regenerative medicine, because it may help to solve the problem of limited donor grafts for tissue and organ repair. The technique enables anatomical cell arrangement using incorporation of cells and growth factors at predefined locations in the printed hydrogel scaffolds. This way, 3-dimensional biological structures, such as blood vessels, are already constructed. Organ printing is developing fast, and there are exciting new possibilities in this area. Hydrogels are highly hydrated polymer networks used as scaffolding materials in organ printing. These hydrogel matrices are natural or synthetic polymers that provide a supportive environment for cells to attach to and proliferate and differentiate in. Successful cell embedding requires hydrogels that are complemented with biomimetic and extracellular matrix components, to provide biological cues to elicit specific cellular responses and direct new tissue formation. This review surveys the use of hydrogels in organ printing and provides an evaluation of the recent advances in the development of hydrogels that are promising for use in skeletal regenerative medicine. Special emphasis is put on survival, proliferation and differentiation of skeletal connective tissue cells inside various hydrogel matrices.     

The integration of 3-D cell printing and mesoscopic fluorescence molecular tomography of vascular constructs within thick hydrogel scaffolds

Developing methods that provide adequate vascular perfusion is an important step toward engineering large functional tissues. Meanwhile, an imaging modality to assess the three-dimensional (3-D) structures and functions of the vascular channels is lacking for thick matrices (>2 ≈ 3 mm). Herein, we report on an original approach to construct and image 3-D dynamically perfused vascular structures in thick hydrogel scaffolds. In this work, we integrated a robotic 3-D cell printing technology with a mesoscopic fluorescence molecular tomography imaging system, and demonstrated the capability of the platform to construct perfused collagen scaffolds with endothelial lining and to image both the fluid flow and fluorescent-labeled living endothelial cells at high-frame rates, with high sensitivity and accuracy. These results establish the potential of integrating both 3-D cell printing and fluorescence mesoscopic imaging for functional and molecular studies in complex tissue-engineered tissues.     

Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing

Organ or tissue printing, a novel approach in tissue engineering, creates layered, cell-laden hydrogel scaffolds with a defined three-dimensional (3D) structure and organized cell placement. In applying the concept of tissue printing for the development of vascularized bone grafts, the primary focus lies on combining endothelial progenitors and bone marrow stromal cells (BMSCs). Here we characterize the applicability of 3D fiber deposition with a plotting device, Bioplotter, for the fabrication of spatially organized, cell-laden hydrogel constructs. The viability of printed BMSCs was studied in time, in several hydrogels, and extruded from different needle diameters. Our findings indicate that cells survive the extrusion and that their subsequent viability was not different from that of unprinted cells. The applied extrusion conditions did not affect cell survival, and BMSCs could subsequently differentiate along the osteoblast lineage. Furthermore, we were able to combine two distinct cell populations within a single scaffold by exchanging the printing syringe during deposition, indicating that this 3D fiber deposition system is suited for the development of bone grafts containing multiple cell types.