Application of inkjet printing to tissue engineering

Recent advances in organ printing technology for applications relating to medical interventions and organ replacement are described. Organ printing refers to the placement of various cell types into a soft scaffold fabricated according to a computer-aided design template using a single device. Computer aided scaffold topology design has recently gained attention as a viable option to achieve function and mass transport requirements within tissue engineering scaffolds. An exciting advance pioneered in our laboratory is that of simultaneous printing of cells and biomaterials, which allows precise placement of cells and proteins within 3-D hydrogel structures. This advance raises the possibility of spatially controlling not only the scaffold structure, but also the type of tissue that can be grown within the scaffold and the thickness of the tissue as capillaries and vessels could be constructed within the scaffolds. Here we summarize recent advances in printing cells and materials using the same device.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

Three-dimensional tissue constructs built by bioprinting

Bioprinting is an evolving tissue engineering technology. It utilizes computer controlled three-dimensional printers for rapid and high-precision construction of three-dimensional biological structures. We employed discrete and continuous bioprinting to build three-dimensional tissue constructs. In the former case bioink particles - spherical cell aggregates composed of many thousands of cells - are delivered one by one into biocompatible scaffolds, the biopaper. Structure formation takes place by the subsequent fusion of the bioink particles due to their liquid-like and self-assembly properties. In the latter case a mixture of cells and scaffold material is extruded from the biocartridge akin to toothpaste to arrive at the desired construct. Specifically, we built rectangular tissue blocks of several hundred microns in thickness as well as tubular structures of several millimeters in height. The physical basis of structure formation was studied by computer simulations.     

The bioink: A comprehensive review on bioprintable materials

This paper discusses “bioink”, bioprintable materials used in three dimensional (3D) bioprinting processes, where cells and other biologics are deposited in a spatially controlled pattern to fabricate living tissues and organs. It presents the first comprehensive review of existing bioink types including hydrogels, cell aggregates, microcarriers and decellularized matrix components used in extrusion-, droplet- and laser-based bioprinting processes. A detailed comparison of these bioink materials is conducted in terms of supporting bioprinting modalities and bioprintability, cell viability and proliferation, biomimicry, resolution, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and post-bioprinting maturation times, tissue fusion and formation post-implantation, degradation characteristics, commercial availability, immune-compatibility, and application areas. The paper then discusses current limitations of bioink materials and presents the future prospects to the reader.     

Actin exposure upon tissue injury is a targetable wound site-specific protein marker

BackgroundIdentification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive.MethodsIn this study, we have developed an immunohistological approach for identification of protein epitopes specifically exposed in wounded tissue sites.ResultsUsing ex-vivo tissue samples in combination with fluorescently-labeled antibodies we show that actin, an intracellular cytoskeletal protein, is specifically exposed upon injury. The targetability of actin in injured sites has been demonstrated in vivo through the specific delivery of anti-actin conjugated particles to the wounded tissue in a lethal rat model of grade IV liver injury.ConclusionsThese results illustrate that identification of injury-specific protein markers and their targetability for specific delivery is feasible.General significanceIdentification of wound-specific targets has important medical applications as it could enable specific delivery of various products, such as expression vectors, therapeutic drugs, hemostatic materials, tissue healing, or scar prevention agents, to internal sites of penetrating or surgical wounds regardless of origin, geometry or location.     

Three-dimensional bioprinting human cardiac tissue chips of using a painting needle method

Three-dimensional (3D) printers are attracting attention as a method for arranging and building cells in three dimensions. Bioprinting technology has potential in tissue engineering for the fabrication of scaffolds, cells, and tissues. However, these various printing technologies have limitations with respect to print resolution and due to the characteristics of bioink such as viscosity. We report a method for constructing of 3D tissues with a “microscopic painting device using a painting needle method” that, when used with the layer-by-layer (LbL) cell coating technique, replaces conventional methods. This method is a technique of attaching the high viscosity bioink to the painting needle tip and arranging it on a substrate, and can construct 3D tissues without damage to cells. Cell viability is the same before and after painting. We used this biofabrication device to construct 3D cardiac tissue (LbL-3D Heart) using human-induced pluripotent stem cell–derived cardiomyocytes. The constructed LbL-3D Heart chips had multiple layers with a thickness of 60 µm, a diameter of 1.1 mm, and showed synchronous beating (50–60 beats per min). The aforementioned device and method of 3D tissue construction can be applied to various kinds of tissue models and would be a useful tool for pharmaceutical applications.     

Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer

Background

The primary objective of Tissue engineering is a regeneration or replacement of tissues or organs damaged by disease, injury, or congenital anomalies. At present, Tissue engineering repairs damaged tissues and organs with artificial supporting structures called scaffolds. These are used for attachment and subsequent growth of appropriate cells. During the cell growth gradual biodegradation of the scaffold occurs and the final product is a new tissue with the desired shape and properties.In recent years, research workplaces are focused on developing scaffold by bio-fabrication techniques to achieve fast, precise and cheap automatic manufacturing of these structures. Most promising techniques seem to be Rapid prototyping due to its high level of precision and controlling. However, this technique is still to solve various issues before it is easily used for scaffold fabrication.In this article we tested printing of clinically applicable scaffolds with use of commercially available devices and materials. Research presented in this article is in general focused on “scaffolding” on a field of bone tissue replacement.

Results

Commercially available 3D printer and Polylactic acid were used to create originally designed and possibly suitable scaffold structures for bone tissue engineering. We tested printing of scaffolds with different geometrical structures. Based on the osteosarcoma cells proliferation experiment and mechanical testing of designed scaffold samples, it will be stated that it is likely not necessary to keep the recommended porosity of the scaffold for bone tissue replacement at about 90%, and it will also be clarified why this fact eliminates mechanical properties issue. Moreover, it is demonstrated that the size of an individual pore could be double the size of the recommended range between 0.2–0.35 mm without affecting the cell proliferation.

Conclusion

Rapid prototyping technique based on Fused deposition modelling was used for the fabrication of designed scaffold structures. All the experiments were performed in order to show how to possibly solve certain limitations and issues that are currently reported by research workplaces on the field of scaffold bio-fabrication. These results should provide new valuable knowledge for further research.

     

生物4D打印技术在心血管组织工程中的研究及应用进展

3D生物打印技术是应用包含生物材料与活细胞在内的生物墨水来构造生物医学产品的技术，近年来得到快速发展。3D打印的组织是静态的，而人体的组织则处于实时动态之中，并且随时能够发生形态及性能的变化，要提高体外环境与体内真实环境的吻合度，就需要一种能够模拟这种动态过程的体外组织构建技术。4D打印概念的提出，给实现这种复杂技术提供了一条新的思路。4D打印可理解为“3D打印+时间”，在3D打印基础上，4D打印应用一种或多种对刺激具有响应的智能材料，这种材料可以在相应的刺激下改变它们的形态、性能及功能，以满足多种需求。本文重点关注4D打印技术在心血管系统中的最新研究进展及其潜在应用领域，为该项技术的发展提供一些理论及应用参考价值。     

The “Janus face” of the thrombin binding aptamer: Investigating the anticoagulant and antiproliferative properties through straightforward chemical modifications

BackgroundThe thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative activities. Its chemico-physical and/or biological properties can be tuned by the site-specific replacement of selected residues.MethodsFour oligodeoxynucleotides (ODNs) based on the TBA sequence (5′-GGTTGGTGTGGTTGG-3′) and containing 2′-deoxyuridine (U) or 5-bromo-2′-deoxyuridine (B) residues at positions 4 or 13 have been investigated by NMR and CD techniques. Furthermore, their anticoagulant (PT assay) and antiproliferative properties (MTT assay) have been tested and compared with two further ODNs containing 5-hydroxymethyl-2′-deoxyuridine (H) residues in the same positions, previously investigated.ResultsThe CD and NMR data suggest that all the investigated ODNs are able to form G-quadruplexes strictly resembling that of TBA. The introduction of B residues in positions 4 or 13 increases the melting temperature of the modified aptamers by 7 °C. The replacement of thymidines with U in the same positions results in an enhanced anticoagulant activity compared to TBA, also at low ODN concentration. Although all ODNs show antiproliferative properties, only TBA derivatives containing H in the positions 4 and 13 lose the anticoagulant activity and remarkably preserve the antiproliferative one.ConclusionsAll ODNs have shown antiproliferative activities against two cancer cell lines but only those with U and B are endowed with anticoagulant activities similar or improved compared to TBA.General significance:The appropriate site-specific replacement of the residues in the TT loops of TBA with commercially available thymine analogues is a useful strategy either to improve the anticoagulant activity or to preserve the antiproliferative properties by quenching the anticoagulant ones.     

Flexible Pseudocapacitive Electrochromics via Inkjet Printing of Additive‐Free Tungsten Oxide Nanocrystal Ink

Direct inkjet printing of functional inks is an emerging and promising technique for the fabrication of electrochemical energy storage devices. Electrochromic energy devices combine electrochromic and energy storage functions, providing a rising and burgeoning technology for next‐generation intelligent power sources. However, printing such devices has, in the past, required additives or other second phase materials in order to create inks with suitable rheological properties, which can lower printed device performance. Here, tungsten oxide nanocrystal inks are formulated without any additives for the printing of high‐quality tungsten oxide thin films. This allows the assembly of novel electrochromic pseudocapacitive zinc‐ion devices, which exhibit a relatively high capacity (≈260 C g^?1 at 1 A g^?1) with good cycling stability, a high coloration efficiency, and fast switching response. These results validate the promising features of inkjet‐printed electrochromic zinc‐ion energy storage devices in a wide range of applications in flexible electronic devices, energy‐saving buildings, and intelligent systems.     

Versatile and inexpensive Hall-Effect force sensor for mechanical characterization of soft biological materials

Mismatch of hierarchical structure and mechanical properties between tissue-engineered implants and native tissue may result in signal cues that negatively impact repair and remodeling. With bottom-up tissue engineering approaches, designing tissue components with proper microscale mechanical properties is crucial to achieve necessary macroscale properties in the final implant. However, characterizing microscale mechanical properties is challenging, and current methods do not provide the versatility and sensitivity required to measure these fragile, soft biological materials. Here, we developed a novel, highly sensitive Hall-Effect based force sensor that is capable of measuring mechanical properties of biological materials over wide force ranges (μN to N), allowing its use at all steps in layer-by-layer fabrication of engineered tissues. The force sensor design can be easily customized to measure specific force ranges, while remaining easy to fabricate using inexpensive, commercial materials. Although we used the force sensor to characterize mechanics of single-layer cell sheets and silk fibers, the design can be easily adapted for different applications spanning larger force ranges (>N). This platform is thus a novel, versatile, and practical tool for mechanically characterizing biological and biomimetic materials.     

脱细胞基质生物墨水制备方法及应用进展

组织器官脱细胞后制备成的脱细胞基质 (Decellularized extracellular matrix,dECM) 含有许多蛋白质和生长因子,不仅能够为细胞提供三维支架还能够调控细胞再生,是目前最具有生物结构的生物材料。3D生物打印可以层层打印dECM和自体细胞的组合,构建载细胞组织结构。文中综述了不同来源的组织器官脱细胞基质生物墨水制备方法,包括脱细胞、交联等,以及脱细胞基质生物墨水在生物打印中的应用,并展望了其未来的应用前景。     

口腔种植的新型钛材料生物安全性研究

目的:在动物体内进行新型钛材料的生物安全性研究。方法:采用TAI纯钛板材(Ti≥99.9%,宝鸡英耐特有色金属有限公司),通过对钛材料的喷砂、酸蚀、碱热等处理后,构建微纳双级骨仿生结构。对处理材料进行扫描电镜观察后,再经过兔皮下实验、小鼠急性溶血性实验确定此种材料的体内安全性。结果:1.扫描电镜结果显示:经过喷砂酸蚀碱热处理后的实验组具有微纳复合结构,与骨组织表面形态更为接近,从仿生学出发,更利于骨组织愈合与抑制骨组织周围炎的发生。2.皮下植入实验结果:经过喷砂酸蚀碱热处理后的实验组与只经过喷砂酸蚀的对照组对生物组织无明显刺激,未见明显的周围组织炎。3.急性毒性实验:未见经过喷砂酸蚀碱热处理后的实验组与只经过喷砂酸蚀的对照组出现明显的生物学反应,未见明显体重下降。结论:新型表面处理钛材料在生物安全性的动物体外实验方面无异常,可以进行下一步实验。     

Early stiffening and softening of collagen: interplay of deformation mechanisms in biopolymer networks

Collagen networks, the main structural/mechanical elements in biological tissues, increasingly serve as biomimetic scaffolds for cell behavioral studies, assays, and tissue engineering, and yet their full spectrum of nonlinear behavior remains unclear. Here, with self-assembled type-I collagen as model, we use metrics beyond those in standard single-harmonic analysis of rheological measurements to reveal strain-softening and strain-stiffening of collagen networks both in instantaneous responses and at steady state. The results show how different deformation mechanisms, such as deformation-induced increase in the elastically active fibrils, nonlinear extension of individual fibrils, and slips in the physical cross-links in the network, can lead to the observed complex nonlinearity. We demonstrate how comprehensive rheological analyses can uncover the rich mechanical properties of biopolymer networks, including the above-mentioned softening as well as an early strain-stiffening, which are important for understanding physiological response of biological materials to mechanical loading.     

康复矫形器的设计与材料的应用现状

分析矫形器的产品设计以及材料的应用现状,探讨矫形器的研究方向。以患者的需求和矫形器目前的发展状况为依据,探讨了矫形器设计中的重点问题,并对矫形器材料的应用展开了分析和论述。矫形器的设计应该从患者需求出发,满足生理需求和心理需求,坚持设计以人为中心的指导思想,同时注意安全性、舒适性、艺术性、定制化和智能化问题;目前矫形器的材料主要是是合成高分子材料及其复合材料,它们以良好的机械性能和生物性能广泛应用于矫形器领域。     

生物组织弹性成像方法及其新技术进展

弹性是一种描述物质物理意义的重要参数,在描述物质在热力学和动力学的变化过程中有着重要的意义。在医学上,弹性的变化往往和病变联系在一起。然而,绝大多数生物组织在他们的力学特性上所表现出的复杂性并不是弹性模量一项参数就可以完全表述的,在对于他们的粘弹性表征和流变学行为的描述中,粘滞性往往和弹性一样的重要。现在被广泛用来对生物组织机械特性表征的成像技术是弹性成像,其基本原理是给组织施加一个激励,组织会产生一个响应,而该响应的分布结合技术的处理方法,可以反映出其弹性模量等力学属性的差异。本文介绍了生物组织常见的弹性成像方法:超声弹性成像,磁共振弹性成像以及光学相干弹性成像;详细阐述了新发展起来的技术-光声弹性成像和光声粘弹成像,并讨论分析其应用前景。     

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting^1-4 (extrusion, dip pen and soft lithography), contactless bioprinting^5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization⁸. It can be used for many applications such as tissue engineering^9-13, biosensor microfabrication^14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types¹⁷. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 °C and a solid above its gelation temperature ~20 °C for 24.5% w/v solutions¹⁸. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.     

Adapting Mechanical Characterization of a Biodegradable Polymer to Physiological Approach of Anterior Cruciate Ligament Functions

Background and objectiveOver the past decades, anterior cruciate ligament injuries have become a considerable public health issue. Due to specific physiological conditions such injuries often demand replacement surgery and can take up to two years to a complete recovery. Using biomaterials able to accelerate the healing process could represent a remarkable progress in the field. The main goal of this article is, therein, to evaluate the mechanical properties of poly(ε-caprolactone) (PCL) fibers with biological properties enhanced by poly(sodium styrene sulfonate) (PNaSS) grafting when subjected to mechanical stress in different conditions.Materials and methodsPCL fibers were thermal grafted with PNaSS. The grafting density was estimated by the toluidine blue colorimetric assay (TB). The influence of the grafting on in vitro primary ACL fibroblast behavior was evaluated by cell proliferation and fluorescence microscope images. The mechanical behavior was evaluated by tensile experiments in air and water, fatigue experiments and simulated walk efforts.ResultsThe results show that poly(ε-caprolactone) bundles have their mechanical behavior changed by the different surface treatments and nature of mechanical stress. Although, compared with the values of the natural ligament, the poly(ε-caprolactone) has shown superior mechanical properties (Young's Modulus, elastic deformation and ultimate tensile stress) in all studied scenarios. In addition, the pNaSS-grafted surfaces presented a positive influence in the cell proliferation and morphology.ConclusionThe pNaSS-grafted PCL has responded mechanical and biological requests for suitable ligament prosthesis material and could be considered as a promising alternative for ACL reconstruction.     

计算机辅助设计修复先天性手部畸形的临床报告

目的:探讨计算机辅助设计修复先天性手部畸形的临床效果。方法:2015年2月到2019年4月选择在本院进行住院治疗的先天性手部畸形患者72例,根据随机数字表法分为研究组与对照组,各36例,对照组给予腹部带蒂复合组织瓣修复治疗,研究组在对照组修复的基础上给予基于3D打印技术的计算机辅助设计修复治疗,记录与报告两组预后。结果:所有患者都皮瓣修复成功,成功率为100.0%。研究组术后3个月的拇指侧偏、虎口挛缩、感染、血管危象等并发症发生率为5.6%,显著低于对照组的30.6%(P0.05)。研究组术后3个月的手部旋转功能优良率为97.2%,显著高于对照组的80.6%(P0.05)。研究组术后3个月的手部疼痛、功能活动和自我感受评分显著高于对照组(P0.05)。结论:基于3D打印技术的计算机辅助设计修复先天性手部畸形成功率高,能减少患者并发症的发生,提高患者的手部旋转功能优良率与手部综合功能。     

A computational avenue towards understanding and design of zwitterionic anti-biofouling materials

ABSTRACT

This review introduces a computational avenue towards understanding and design of zwitterionic anti-biofouling materials. Biofouling means nonspecific adsorption of proteins, cells, and bacteria on materials and devices. It can sabotage materials functions and bring detrimental effects on the biological systems. Various anti-biofouling materials have been developed and zwitterionic materials emerge as promising candidates recently. The design and understanding of zwitterionic anti-biofouling materials rely on the answers of three fundamental questions: (a) what molecular properties govern the anti-biofouling performance of materials, (b) what is the structure–property-anti-biofouling relationship for materials, and (c) how to identify anti-biofouling materials based on the revealed mechanisms? This paper discussed the efforts of answering the three questions using molecular simulations. We first discuss the simulations that revealed the importance of hydration in the anti-biofouling performance of materials and why this mechanism leads to the discovery of zwitterionic anti-biofouling materials, then the simulations that investigated structure-properties-performance relationships of zwitterionic anti-biofouling materials, and the development of computational approaches that can identify zwitterionic anti-biofouling molecules. Finally, we discuss the opportunities in understanding and design of anti-biofouling biomaterials using computer simulations.