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。     

3D生物打印在软骨修复中的应用*

关节软骨损伤后的自我修复是医学界一直在研究和探讨的难题。3D生物打印技术可以精准的分配载细胞生物材料,构建复杂的三维活体组织,在优化软骨缺损修复组织的内部结构、机械性能以及生物相容性上有很大优势,因此近年来成为软骨修复组织工程领域的研究热点。重点介绍了软骨生物3D生物打印的最新进展,包括软骨生物打印“墨水”材料的选择、种子细胞的来源以及3D生物打印技术的发展。此外,还阐述了3D生物打印技术在组织工程学应用上的部分局限性,并对其在软骨修复领域的发展与应用进行了预测。     

Three-dimensional bioprinting in tissue engineering and regenerative medicine

With the advances of stem cell research, development of intelligent biomaterials and three-dimensional biofabrication strategies, highly mimicked tissue or organs can be engineered. Among all the biofabrication approaches, bioprinting based on inkjet printing technology has the promises to deliver and create biomimicked tissue with high throughput, digital control, and the capacity of single cell manipulation. Therefore, this enabling technology has great potential in regenerative medicine and translational applications. The most current advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review, including vasculature, muscle, cartilage, and bone. In addition, the benign side effect of bioprinting to the printed mammalian cells can be utilized for gene or drug delivery, which can be achieved conveniently during precise cell placement for tissue construction. With layer-by-layer assembly, three-dimensional tissues with complex structures can be printed using converted medical images. Therefore, bioprinting based on thermal inkjet is so far the most optimal solution to engineer vascular system to the thick and complex tissues. Collectively, bioprinting has great potential and broad applications in tissue engineering and regenerative medicine. The future advances of bioprinting include the integration of different printing mechanisms to engineer biphasic or triphasic tissues with optimized scaffolds and further understanding of stem cell biology.     

The use of bacterial polysaccharides in bioprinting

Additive manufacturing or 3D printing has spearheaded a revolution in the biomedical sector allowing the rapid prototyping of medical devices. The recent advancements in bioprinting technology are enabling the development of potential new therapeutic options with respect to tissue engineering and regenerative medicines. Bacterial polysaccharides have been shown to be a central component of the inks used in a variety of bioprinting processes influencing their key features such as the mechanical and thermal properties, printability, biocompatibility, and biodegradability. However, the implantation of any foreign structure in the body comes with an increased risk of bacterial infection and immunogenicity. In recent years, this risk is being potentiated by the rise in nosocomial multidrug-resistant bacterial infections. Inks used in bioprinting are being augmented with antimicrobials to mitigate this risk. The applications of bacterial polysaccharide-based bioinks have the potential to act as a key battlefront in the war against antibiotic resistance. This paper reviews the range of bacterial polysaccharides used in bioprinting and discusses the potential of various bioactive polysaccharides to be integrated into these inks.     

细胞打印技术及应用

细胞打印技术是一种在体外构造具有生物活性的三维多细胞体系的先进技术。近年来,有关细胞打印技术的研究引起广泛的关注,其原因在于该领域具有明显的学科交叉与渗透融合的特点,它处于生命科学与快速成型技术、生物制造技术、生物科学和材料科学的交汇点。更加值得关注的是它为组织工程学突破二维研究的局限性,在三维尺度上精确控制与人体组织或器官相似的三维构造体方面的研究提供了一种新的思路。基于这一技术不仅在三维组织工程,还对细胞生物学、高通量药物筛选及细胞传感器等方面的前沿问题均有广阔的研究应用前景,介绍了近年来开发用于细胞打印的技术及其潜在的应用。     

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。     

Cover Picture: Biotechnology Journal 10/2014

This regular issue of BTJ includes the new section “Biotech Methods”, and features articles on nanofibers and biofilms. The cover illustrates the technique of electrospinning, which is applied for the production of artificial filaments that can be organized to mats, which has many applications such as wound coverning or for stabilization of bioprinting/3D tissue units. It is described that silica [bio‐silica] converts those mats to morphogenetically active materials. Image by Werner Mülller. See the article by Müller et al. 10.1002/biot.201400277     

Applications of stem cells and bioprinting for potential treatment of diabetes

Currently, there does not exist a strategy that can reduce diabetes and scientists are working towards a cure and innovative approaches by employing stem cellbased therapies. On the other hand, bioprinting technology is a novel therapeutic approach that aims to replace the diseased or lost β-cells, insulin-secreting cells in the pancreas, which can potentially regenerate damaged organs such as the pancreas. Stem cells have the ability to differentiate into various cell lines including insulinproducing cells. However, there are still barriers that hamper the successful differentiation of stem cells into β-cells. In this review, we focus on the potential applications of stem cell research and bioprinting that may be targeted towards replacing the β-cells in the pancreas and may offer approaches towards treatment of diabetes. This review emphasizes on the applicability of employing both stem cells and other cells in 3 D bioprinting to generate substitutes for diseased β-cells and recover lost pancreatic functions. The article then proceeds to discuss the overall research done in the field of stem cell-based bioprinting and provides future directions for improving the same for potential applications in diabetic research.     

3D‐Printed Conical Arrays of TiO2 Electrodes for Enhanced Photoelectrochemical Water Splitting

Control over the topography of semiconducting materials can lead to enhanced performances in photoelectrochemical related applications. One means of implementing this is through direct patterning of metal‐based substrates, though this is inadequately developed. Conventional techniques for patterned fabrication commonly involve technologically demanding and tedious processes. 3D printing, a form of additive fabrication, enables creation of a 3D object by deposition of successive layers of material via computer control. In this work, the feasibility of fabricating metal‐based 3D printed photoelectrodes is explored. Electrodes comprised of conical arrays are fabricated and the performance for photoelectrochemical water splitting is further enhanced by the direct growth of TiO2 nanotubes on this platform. 3D metal printing provides a flexible and versatile approach for the design and fabrication of novel electrode structures.     

Scaffold‐free inkjet printing of three‐dimensional zigzag cellular tubes

The capability to print three‐dimensional (3D) cellular tubes is not only a logical first step towards successful organ printing but also a critical indicator of the feasibility of the envisioned organ printing technology. A platform‐assisted 3D inkjet bioprinting system has been proposed to fabricate 3D complex constructs such as zigzag tubes. Fibroblast (3T3 cell)‐based tubes with an overhang structure have been successfully fabricated using the proposed bioprinting system. The post‐printing 3T3 cell viability of printed cellular tubes has been found above 82% (or 93% with the control effect considered) even after a 72‐h incubation period using the identified printing conditions for good droplet formation, indicating the promising application of the proposed bioprinting system. Particularly, it is proved that the tubular overhang structure can be scaffold‐free fabricated using inkjetting, and the maximum achievable height depends on the inclination angle of the overhang structure. As a proof‐of‐concept study, the resulting fabrication knowledge helps print tissue‐engineered blood vessels with complex geometry. Biotechnol. Bioeng. 2012; 109: 3152–3160. © 2012 Wiley Periodicals, Inc.     

Processing silk hydrogel and its applications in biomedical materials

This review mainly introduces the types of silk hydrogels, their processing methods, and applications. There are various methods for hydrogel preparation, and many new processes are being developed for various applications. Silk hydrogels can be used in cartilage tissue engineering, drug release materials, 3D scaffolds for cells, and artificial skin, among other applications because of their porous structure and high porosity and the large surface area for growth, migration, adhesion and proliferation of cells that the hydrogels provide. All of these advantages have made silk hydrogels increasingly attractive. In addition, silk hydrogels have wide prospects for application in the field of biomedical materials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:630–640, 2015     

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.     

Synergistic action of fibroblast growth factor-2 and transforming growth factor-beta1 enhances bioprinted human neocartilage formation

Bioprinting as a promising but unexplored approach for cartilage tissue engineering has the advantages of high throughput, digital control, and highly accurate placement of cells and biomaterial scaffold to the targeted 3D locations with simultaneous polymerization. This study tested feasibility of using bioprinting for cartilage engineering and examined the influence of cell density, growth, and differentiation factors. Human articular chondrocytes were printed at various densities, stimulated transiently with growth factors and subsequently with chondrogenic factors. Samples were cultured for up to 4 weeks to evaluate cell proliferation and viability, mechanical properties, mass swelling ratio, water content, gene expression, ECM production, DNA content, and histology. Bioprinted samples treated with FGF-2/TGF-β1 had the best chondrogenic properties among all groups apparently due to synergistic stimulation of cell proliferation and chondrogenic phenotype. ECM production per chondrocyte in low cell density was much higher than that in high cell seeding density. This finding was also verified by mechanical testing and histology. In conclusion, cell seeding density that is feasible for bioprinting also appears optimal for human neocartilage formation when combined with appropriate growth and differentiation factors.     

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices^1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.Direct-write assembly^4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 μm). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (< 1 μm) can be patterned and assembled into larger arrays and multidimensional architectures.In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.     

Progress in scaffold‐free bioprinting for cardiovascular medicine

Biofabrication of tissue analogues is aspiring to become a disruptive technology capable to solve standing biomedical problems, from generation of improved tissue models for drug testing to alleviation of the shortage of organs for transplantation. Arguably, the most powerful tool of this revolution is bioprinting, understood as the assembling of cells with biomaterials in three‐dimensional structures. It is less appreciated, however, that bioprinting is not a uniform methodology, but comprises a variety of approaches. These can be broadly classified in two categories, based on the use or not of supporting biomaterials (known as “scaffolds,” usually printable hydrogels also called “bioinks”). Importantly, several limitations of scaffold‐dependent bioprinting can be avoided by the “scaffold‐free” methods. In this overview, we comparatively present these approaches and highlight the rapidly evolving scaffold‐free bioprinting, as applied to cardiovascular tissue engineering.     

Atomic Layer Deposition as a General Method Turns any 3D‐Printed Electrode into a Desired Catalyst: Case Study in Photoelectrochemisty

3D‐printing technologies have begun to revolutionize many manufacturing processes, however, there are still significant limitations that are yet to be overcome. In particular, the material from which the products are fabricated is limited by the 3D‐printing material precursor. Particularly, for photoelectrochemical (PEC) energy applications, the as‐printed electrodes can be used as is, or modified by postfabrication processes, e.g., electrochemical deposition or anodization, to create active layers on the 3D‐printed electrodes. However, the as‐printed electrodes are relatively inert for various PEC energy applications, and the aforementioned postfabrication processing techniques do not offer layer conformity or control at the Ångström/nano level. Herein, for the first time, atomic layer deposition (ALD) is utilized in conjunction with metal 3D‐printing to create active electrodes. To illustrate the proof‐of‐concept, TiO₂ is deposited by ALD onto stainless steel 3D‐printed electrodes and subsequently investigated as a photoanode for PEC water oxidation. Furthermore, by tuning the TiO₂ thickness by ALD, the activity can be optimized. By combining 3D‐printing and ALD, instead of other metal deposition techniques, i.e., sputtering, rapid prototyping of electrodes with controllable thickness of the desired material onto an as‐printed electrodes with any porosity can be achieved that can benefit a multitude of energy applications.     

Dosimetric characterization of 3D printed bolus at different infill percentage for external photon beam radiotherapy

Background and purpose3D printing is rapidly evolving and further assessment of materials and technique is required for clinical applications. We evaluated 3D printed boluses with acrylonitrile butadiene styrene (ABS) and polylactide (PLA) at different infill percentage.Material and methodsA low-cost 3D printer was used. The influence of the air inclusion within the 3D printed boluses was assessed thoroughly both with treatment planning system (TPS) and with physical measurements. For each bolus, two treatment plans were calculated with Monte Carlo algorithm, considering the computed tomography (CT) scan of the 3D printed bolus or modelling the 3D printed bolus as a virtual bolus structure with a homogeneous density. Depth dose measurements were performed with Gafchromic films.ResultsHigh infill percentage corresponds to high density and high homogeneity within bolus material. The approximation of the bolus in the TPS as a homogeneous material is satisfying for infill percentages greater than 20%. Measurements performed with PLA boluses are more comparable to the TPS calculated profiles. For boluses printed at 40% and 60% infill, the discrepancies between calculated and measured dose distribution are within 5%.Conclusions3D printing technology allows modulating the shift of the build-up region by tuning the infill percentage of the 3D printed bolus in order to improve superficial target coverage.     

Differentiation potential of human adipose stem cells bioprinted with hyaluronic acid/gelatin‐based bioink through microextrusion and visible light‐initiated crosslinking

Bioprinting has a great potential to fabricate three‐dimensional (3D) functional tissues and organs. In particular, the technique enables fabrication of 3D constructs containing stem cells while maintaining cell proliferation and differentiation abilities, which is believed to be promising in the fields of tissue engineering and regenerative medicine. We aimed to demonstrate the utility of the bioprinting technique to create hydrogel constructs consisting of hyaluronic acid (HA) and gelatin derivatives through irradiation by visible light to fabricate 3D constructs containing human adipose stem cells (hADSCs). The hydrogel was obtained from a solution of HA and gelatin derivatives possessing phenolic hydroxyl moieties in the presence of ruthenium(II) tris‐bipyridyl dication and sodium ammonium persulfate. hADSCs enclosed in the bioprinted hydrogel construct elongated and proliferated in the hydrogel. In addition, their differentiation potential was confirmed by examining the expression of pluripotency marker genes and cell surface marker proteins, and differentiation to adipocytes in adipogenic differentiation medium. Our results demonstrate the great potential of the bioprinting method and the resultant hADSC‐laden HA/gelatin constructs for applications in tissue engineering and regenerative medicine.     

Three-dimensional printing biotechnology for the regeneration of the tooth and tooth-supporting tissues

The tooth and its supporting tissues are organized with complex three-dimensional (3D) architecture, including the dental pulp with a blood supply and nerve tissues, complex multilayer periodontium, and highly aligned periodontal ligament (PDL). Mimicking such 3D complexity and the multicellular interactions naturally existing in dental structures represents great challenges in dental regeneration. Attempts to construct the complex system of the tooth and tooth-supporting apparatus (i.e., the PDL, alveolar bone, and cementum) have made certain progress owing to 3D printing biotechnology. Recent advances have enabled the 3D printing of biocompatible materials, seed cells, and supporting components into complex 3D functional living tissue. Furthermore, 3D bioprinting is driving major innovations in regenerative medicine, giving the field of regenerative dentistry a boost. The fabrication of scaffolds via 3D printing is already being performed extensively at the laboratory bench and in clinical trials; however, printing living cells and matrix materials together to produce tissue constructs by 3D bioprinting remains limited to the regeneration of dental pulp and the tooth germ. This review summarizes the application of scaffolds for cell seeding and biofabricated tissues via 3D printing and bioprinting, respectively, in the tooth and its supporting tissues. Additionally, the key advantages and prospects of 3D bioprinting in regenerative dentistry are highlighted, providing new ideas for dental regeneration.     

3D‐printed individual labware in biosciences by rapid prototyping: In vitro biocompatibility and applications for eukaryotic cell cultures

Three‐dimensional (3D) printing techniques are continuously evolving, thus their application fields are also growing very fast. The applications discussed here highlight the use of rapid prototyping in a dedicated biotechnology laboratory environment. The combination of improving prototypes using fused deposition modeling printers and producing useable parts with selective laser sintering printers enables a cost‐ and time‐efficient use of such techniques. Biocompatible materials for 3D printing are already available and the printed parts can directly be used in the laboratory. To demonstrate this, we tested 3D printing materials for their in vitro biocompatibility. To exemplify the versatility of the 3D printing process applied to a biotechnology laboratory, a normal well plate design was modified in silico to include different baffle geometries. This plate was subsequently 3D printed and used for cultivation. In the near future, this design and print possibility will revolutionize the industry. Advanced printers will be available for laboratories and can be used for creating individual labware or standard disposables on demand. These applications have the potential to change the way research is done and change the management of stock‐keeping, leading to more flexibility and promoting creativity of the scientists.