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本帖首发于茶馆，大胆在此一稿多投 7 n; G9 n: O+ @# F( H9 Z1 e) w4 |
, W5 O4 E7 A9 L3 c- e背景：葡萄在3D打印机基础的帖子里贴了关于细胞打印的新闻，在查找原始来源时，发现发表该研究的杂志同时发表了4篇生物领域3D打印的文章，大大拓展了我对这个领域的认知。不敢专享，特翻译了一下摘要，与大家共享。+ {( s' ?) ~* |5 O4 F+ k8 ?8 N
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声明：本人单纯医学背景，无理工和生技背景，所以以下的翻译和解读不见得正确，如有谬误，欢迎指出，也欢迎讨论。
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! B X+ m' O; k2 b' I来源：Biofabrication Volume 5 Number 1, March 2013（未来尚未到来，网络出版倒是赶在时间前面，原因嘛，大概是因为这是季刊，所以具体写该季度的几月份都无所谓了...）) t+ N& m; s1 Y) @- K7 |# C
http://iopscience.iop.org/1758-5090/5/18 w0 Y/ i' V1 S: [- r
4 O1 B j' w7 L/ ^! ]- z% ?7 j& }9 J第一篇：
: ?- G( |+ u' ^( [Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications (有全文)
' ^9 w0 I) I# O2 s用混合打印法制造机械性能和生物学性能改进、可用于软骨组织工程应用的组织结构) P) t" q7 J) m' r& l! ?0 \
, i( J6 o7 t% ]) a发表单位：Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences
$ J; j3 A0 N8 y* H S8 J" `（网上查到的背景信息：维克森林大学 Wake Forest University一直是再生医学研究的全球领军人物，拥有美国联邦政府和国家科学委员会的巨额研究资金。维克森林大学再生医学研究院的人体组织工程学家安东尼·艾塔拉给成千上万需要膀胱移植的病人带来了福音，他在实验室造出了一个真正的人类膀胱。16年来，艾塔拉一直致力于人造器官的研究，不过直到今年4月份他才一鸣惊人，他宣布人造膀胱移植成功，这是世界上首例人造器官的成功移植。接受移植手术的7名患者来自美国波士顿儿童医院，年龄在4岁到19岁之间。http://usa.edutime.net/Universit ... UniversityInfo.aspx）
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$ G, ^( a5 j, ?4 XBioprinting is an emerging technique used to fabricate viable, 3D tissue constructs through the precise deposition of cells and hydrogels in a layer-by-layer fashion. Despite the ability to mimic the native properties of tissue, printed 3D constructs that are composed of naturally-derived biomaterials still lack structural integrity and adequate mechanical properties for use in vivo, thus limiting their development for use in load-bearing tissue engineering applications, such as cartilage.
5 Q% G, }1 }3 Z生物打印是一项新出现的技术，它通过将细胞和水凝胶一层隔一层精准沉积的方式来制造有用的3D组织结构。由天然来源的生物材料经3D打印制造的结构虽能模仿组织的自然属性，但仍缺乏结构上的完整性和活体使用所需的机械强度，因此限制了其在承重组织（如软骨）中的生物工程应用。
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Fabrication of viable constructs using a novel multi-head deposition system provides the ability to combine synthetic polymers, which have higher mechanical strength than natural materials, with the favorable environment for cell growth provided by traditional naturally-derived hydrogels. However, the complexity and high cost associated with constructing the required robotic system hamper the widespread application of this approach. Moreover, the scaffolds fabricated by these robotic systems often lack flexibility, which further restrict their applications. To address these limitations, advanced fabrication techniques are necessary to generate complex constructs with controlled architectures and adequate mechanical properties.
9 H( V; l3 x( J* M6 L' R. V( g采用新型多喷头沉积系统来制作组织结构，在使用传统天然来源的水凝胶以提供合适细胞生长的环境的同时，可以合并使用合成聚合物，后者比天然成分具有更好的机械强度。然而，这种制造方式需要的机器人系统的复杂性和高成本阻碍了其的广泛应用。而且，这种机器人系统制造的骨架常缺乏弹性，这进一步限制了它的应用。考虑到这些限制，需要开发更高级的制造技术来生产同时满足可控性架构和合适机械强度的复杂组织结构。
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In this study, we describe the construction of a hybrid inkjet printing/electrospinning system that can be used to fabricate viable tissues for cartilage tissue engineering applications. Electrospinning of polycaprolactone fibers was alternated with inkjet printing of rabbit elastic chondrocytes suspended in a fibrin–collagen hydrogel in order to fabricate a five-layer tissue construct of 1 mm thickness. The chondrocytes survived within the printed hybrid construct with more than 80% viability one week after printing. In addition, the cells proliferated and maintained their basic biological properties within the printed layered constructs. Furthermore, the fabricated constructs formed cartilage-like tissues both in vitro and in vivo as evidenced by the deposition of type II collagen and glycosaminoglycans. Moreover, the printed hybrid scaffolds demonstrated enhanced mechanical properties compared to printed alginate or fibrin–collagen gels alone. This study demonstrates the feasibility of constructing a hybrid inkjet printing system using off-the-shelf components to produce cartilage constructs with improved biological and mechanical properties.
3 A% B6 P$ N, k5 @5 u6 r) D* u5 D在本研究中，我们描述了一种混合喷墨打印/静电纺丝系统，该系统可用于制造软骨组织工程所需的组织。该系统分别以聚己内酯纤维进行静电纺丝，和以采用混悬在纤维蛋白-胶原蛋白凝胶中的兔弹性软骨细胞进行喷墨打印，两者交替，最终制作出1mm厚、5层的组织结构。在混合打印的组织里，1周后仍有80%的软骨细胞存活，细胞可在这种层状打印组织中继续增殖并保存基本的生物学特性。而且，这种组织在体内、体外均可形成软骨样组织，II型胶原和氨基葡聚糖的沉积证明了这一点。此外，这种打印制作的混合骨架相对于单纯海藻酸钠或纤维蛋白-胶原蛋白凝胶打印的组织，具有更好的机械强度。本研究证明了构建! j. }% r* Y6 ]: R4 ]
一个混合喷墨打印系统、采用现成材料制作具有更好生物学特性和机械性能的软骨组织的可行性。: ^. ~( E1 b- k4 [9 {) ~2 W- M' O
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% Q6 Y! a* T# K" e5 u说明及个人理解：: P' C/ y* A6 n2 E3 u% T
这篇文章网站有全文可下，上图显示了这种打印软骨组织的基本机理：铺一层聚己内酯纤维（就像无纺布一样？），再铺一层细胞，这样铺成五夹板，既有一定的强度，又能给软骨细胞提供生存的空间，让其可以增殖和分泌细胞外基质。软骨组织需要有足够的强度（抗冲击）、柔韧度（抗牵拉）、光滑度和润滑性（方便关节运动），还要具有一定的耐磨损和自我修复能力。虽然现在的3D打印技术制作出来的组织还非常初级，离实际应用估计还有很远的路程要走，但是我们在这里看到的是，不断的努力加上不同领域各项技术的结合，在推动着这项技术向实用阶段不断前行。. s, a- _. n: [
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第二篇
) d: ^7 i$ F- }7 V* A: OLaser-assisted printing of alginate long tubes and annular constructs5 b! u/ T3 n D0 R- K( |
用海藻酸钠激光支持打印制作长管和环状结构2 s% l t0 r; }$ ~9 p7 V
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发表单位：Department of Mechanical Engineering, Clemson University, Clemson, SC 29634, USA0 v' I- C1 W# [
Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA& n! f1 ^$ I8 M3 c
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Laser-assisted printing such as laser-induced forward transfer has been well studied to pattern or fabricate two-dimensional constructs. In particular, laser printing has found increasing biomedical applications as an orifice-free cell and organ printing approach, especially for highly viscous biomaterials and biological materials. Unfortunately, there have been very few studies on the efficacy of three-dimensional printing performance of laser printing. This study has investigated the feasibility of laser tube printing and the effects of sodium alginate concentration and operating conditions such as the laser fluence and laser spot size on the printing quality during laser-assisted printing of alginate annular constructs (short tubes) with a nominal diameter of 3 mm. It is found that highly viscous materials such as alginate can be printed into well-defined long tubes and annular constructs. The tube wall thickness and tube outer diameter decrease with the sodium alginate concentration, while they first increase, then decrease and finally increase again with the laser fluence. The sodium alginate concentration dominates if the laser fluence is low, and the laser fluence dominates if the sodium alginate concentration is low.5 B/ x# ^/ R6 [9 c# O! J E* @! I: W
; _5 {/ \2 f0 r1 l激光支持打印如激光诱导向前转移技术在仿造或制作二维结构中已被深入研究。而且激光打印技术用于无孔细胞或组织的打印还扩展了其在生物医学领域的应用，特别适用于高粘性生物材料。但是关于激光三维打印性能研究还很少。本项目研究了激光打印管状物的可行性、海藻酸钠浓度的影响以及操作条件如激光能量密度、光斑大小对打印质量的影响（用海藻酸钠激光支持打印直径3mm的环状结构/短管）。研究发现，可以使用高粘度材料如海藻酸钠打印制作明确定义的长管和环状结构。管壁的厚度与外径随海藻酸钠浓度降低而减，而其与激光能量密度的关系则是先升、后降、再升。当激光能量密度低时，海藻酸钠浓度占" ]3 l4 R8 E' v& f! F
主导地位，而海藻酸钠浓度低时，激光能量密度占主导地位。
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说明及个人理解：
% Q6 r: b4 U$ r% X! @看来强大的激光打印技术在生物3D打印领域也有一席之地，不过这是对无细胞的生物材料，而对于采用细胞做原材料的打印，似乎还是基于所谓的喷墨打印技术。
" O, t8 `9 U- i. _虽然单纯的非细胞组织在人体内非常有限，但是在人造器官、组织里，这样的组织估计还是有很大的价值，比如作为包膜、支架、管道等等。相信将来激光打印会和其他技术互相结合和促进，各展所长，造福人类。
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Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid
, v0 |, g- l! b, M# i; D) r在疏水性高密度液体内使用富含干细胞的水凝胶进行三维打印* j o% e& _. |) F1 [6 M! ~
4 g8 c. U# z) ^ M) J9 u1 COver the last decade, bioprinting technologies have begun providing important tissue engineering strategies for regenerative medicine and organ transplantation. The major drawback of past approaches has been poor or inadequate material-printing device and substrate combinations, as well as the relatively small size of the printed construct. Here, we hypothesise that cell-laden hydrogels can be printed when submerged in perfluorotributylamine (C12F27N), a hydrophobic high-density fluid, and that these cells placed within three-dimensional constructs remain viable allowing for cell proliferation and production of extracellular matrix. Human mesenchymal stem cells and MG-63 cells were encapsulated into agarose hydrogels, and subsequently printed in high aspect ratio in three dimensional structures that were supported in high density fluorocarbon. Three-dimensional structures with various shapes and sizes were manufactured and remained stable for more than six months. Live/dead and DAPI stainings showed viable cells 24 h after the printing process, as well as after 21 days in culture. Histological and immunohistochemical analyses after 14 and 21 days revealed viable cells with marked matrix production and signs of4 J5 J( B) y! i& \1 I( @ X, _
proliferation. The compressive strength values of the printed gels consequently increased during the two weeks in culture, revealing encouraging results for future applications in regenerative medicine.
0 _8 o' Z1 d! N' g' Z, b, h在过去10年中，生物打印技术开始为再生医学和器官移植提供重要的组织工程路线。既往技术的主要缺点包括缺乏合适的材料-打印设备和基材的组合，以及打印结构的尺寸偏小。在本研究中，我们假设在浸没于全氟三丁胺（C12F2N7，一种疏水性高密度液体）的情况下，可以使用富含细胞的水凝胶进行打印，而这些位于打印出的三维结构中的细胞可以保持增殖和分泌细胞外间质的能力。研究首先将人间充质干细胞和MG-63细胞用琼脂凝胶包裹，之后将其打印在具有高纵横比的、支持于上述高密度氟碳液中的三维结构上。以此法打印制作了各种形状和尺寸的三维结构，这些结构在六个月后仍能保持稳固。活/死细胞检测和DAPI染色等显示打印后24小时打印结构中有可用性细胞存活，21日后培养在培养液中的打印结构中同样显示有细胞存活。打印后14天和21天的组织学和免疫组化分析显示有可用性细胞存活并有明显的间质生成以及细胞增殖的现象。在培养液培养的2周中，这种凝胶打印产物的抗压强度持续增加，这一结果为这项技术未来在再生医学中的应用揭示了令人鼓舞的前景。: P4 k L( X& _' L" g. T( t
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说明及个人理解：
$ G5 \( M9 c$ \+ `9 F3 Q$ }Viable的一个翻译是“能自行生产发育的”，在这里暂时翻译成“可用性”，意思是有用处的，即可以执行分泌间质的功能、可以增殖的功能能正常的细胞。5 l' g6 M0 s' h
作为一个外行，个人猜测这个研究的意义在于，将打印组织的支架和打印过程都放在一个高密度液体中，增加了对支架和打印后组织的支撑作用，使得打印更大尺寸的组织成为可能。3 i+ y" ]2 s% C$ ?' B
对我这样看热闹的人来说，一个深刻的、再次加深的感觉是：在3D打印技术的发展过程中，多领域技术互相支撑和交融，使得我们在估测其发展前景时，不能仅仅考虑打印技术的本身，而要认识到，在它的发展过程中将汇聚无数尖端技术，这将产生巨大的合力，我们完全有可能看到令人目眩的飞跃。' g4 @2 F5 V7 G" O
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第四篇：
, U+ H$ @! u- @Development of a valve-based cell printer for the formation of human embryonic stem cell spheroid aggregates （有全文）
1 p3 g( ?$ x; V7 k/ ^开发一项基于阀门技术的细胞打印技术用于制作人胚胎干细胞的球形体聚集, I% I5 o) U0 n: E# U+ E4 v3 t. q
% U0 n7 |# r. y }% tIn recent years, the use of a simple inkjet technology for cell printing has triggered tremendous interest and established the field of biofabrication. A key challenge has been the development of printing processes which are both controllable and less harmful, in order to preserve cell and tissue viability and functions. Here, we report on the development of a valve-based cell printer that has been validated to print highly viable cells in programmable patterns from two different bio-inks with independent control of the volume of each droplet (with a lower limit of 2 nL or fewer than five cells per droplet). Human ESCs were used to make spheroids by overprinting two opposing gradients of bio-ink; one of hESCs in medium and the other of medium alone. The resulting array of uniform sized droplets with a gradient of cell concentrations was inverted to allow cells to aggregate and form spheroids via gravity. The resulting aggregates have controllable and repeatable sizes, and consequently they can be made to order for specific applications. Spheroids with between 5 and 140 dissociated cells resulted in spheroids of 0.25–0.6 mm diameter. This work demonstrates that the valve-based printing process is gentle enough to maintain stem cell viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency. This study includes the first analysis of the response of human embryonic stem cells to the printing process using this valve-based printing setup.0 X/ r6 @$ Y4 g+ e- e2 ?1 I) T
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近年来，采用简单的喷墨打印技术进行细胞打印引发了研究者浓厚的兴趣，开创了生物制造这个新领域。该技术的一个主要挑战是需要开发出既可控又损伤小的打印工艺，以保存细胞和组织的活性和功能。在本研究中，我们报告了一种基于阀门技术、在可编程模式下打印高可用性细胞的细胞打印机，它可以使用2种不同的生物墨水、独立控制每滴墨水的量（每滴可小于2nL或少于5个细胞）。人胚胎干细胞的球形体打印是通过套印两种浓度梯度相反的生物墨水：一种是溶于介质的人胚胎干细胞，另一种是单纯的介质。这样造成的同样大小、细胞浓度梯度相反的墨滴，使得细胞可以聚集，并通过重力形成球形体。这一打印程序导致的细胞聚集体其尺寸可控，且可复制，可定制用于特定用途。这种由5到140个分散细胞组成的球形体直径在0.25-0.6mm。这项工作表明，基于阀门技术的细胞打印具有保持干细胞活性的轻柔度、适合制作统一标准球形体的精确度，而且打印后细胞仍保持其多能性。本研究还首次分析了人胚胎干细胞经该基于阀门技术的打印设备打印后的反应。2 H' B2 O3 [6 `) y0 z# G
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说明及个人理解：' h+ w! ^6 b0 |: |, k% _
这篇就是葡萄提到的那个研究的原文摘要和相关图表，本文有网上全文，感兴趣者可以去网站上看。
& e2 U: Y$ \/ n) q: {其实我不明白为什么国外新闻网站选择这篇研究进行报道，虽然看不太懂，但个人感觉这个技术还非常初级：作者是通过重力的作用，使细胞团可以聚集成球体形状，这是一种“排列”细胞使其构成特定形状的一种尝试。但是这样的细胞团并没有形成一个固定的结构，所以我不能理解它究竟有多大的价值。
4 F5 D& d. C# x+ |/ g- @: n+ m但是呢，我们这次又看到3D打印技术和其他技术的交叉和结合，所以，还是非常有意义滴：） |
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发表于 2013-2-12 23:08:22
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