Three-dimensional bio-printing

Three-dimensional(3D) printing technology has been widely used in various manufacturing operations including automotive, defence and space industries. 3D printing has the advantages of personalization, flexibility and high resolution, and is therefore becoming increasingly visible in the high-tech fields. Three-dimensional bio-printing technology also holds promise for future use in medical applications. At present 3D bio-printing is mainly used for simulating and reconstructing some hard tissues or for preparing drug-delivery systems in the medical area. The fabrication of 3D structures with living cells and bioactive moieties spatially distributed throughout will be realisable. Fabrication of complex tissues and organs is still at the exploratory stage. This review summarize the development of 3D bio-printing and its potential in medical applications, as well as discussing the current challenges faced by 3D bio-printing.     

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.     

Bio-microarray fabrication techniques--a review

Microarrays with biomolecules (e.g., DNA and proteins), cells, and tissues immobilized on solid substrates are important tools for biological research, including genomics, proteomics, and cell analysis. In this paper, the current state of microarray fabrication is reviewed. According to spot formation techniques, methods are categorized as "contact printing" and "non-contact printing." Contact printing is a widely used technology, comprising methods such as contact pin printing and microstamping. These methods have many advantages, including reproducibility of printed spots and facile maintenance, as well as drawbacks, including low-throughput fabrication of arrays. Non-contact printing techniques are newer and more varied, comprising photochemistry-based methods, laser writing, electrospray deposition, and inkjet technologies. These technologies emerged from other applications and have the potential to increase microarray fabrication throughput; however, there are several challenges in applying them to microarray fabrication, including interference from satellite drops and biomolecule denaturization.     

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.     

Synchrotron radiation X-ray fluorescence microscopy reveals a spatial association of copper on elastic laminae in rat aortic media

Copper, an essential trace metal in humans, plays an important role in elastic formation. However, little is known about the spatial association between copper, elastin, and elastin producing cells. The aorta is the largest artery; the aortic media is primarily composed of the elastic lamellae and vascular smooth muscle cells, which makes it a good model to address this issue. Synchrotron radiation X-ray fluorescence microscopy (SRXRF) is a new generation technique to investigate the spatial topography of trace metals in biological samples. Recently, we utilized this technique to determine the topography of copper as well as other trace elements in aortic media of Sprague Dawley rats. A standard rat diet was used to feed Sprague Dawley rats, which contains the normal dietary requirements of copper and zinc. Paraffin embedded segments (4 μm of thickness) of thoracic aorta were analyzed using a 10 keV incident monochromatic X-ray beam focusing on a spot size of 0.3 μm × 0.2 μm (horizontal × vertical). The X-ray spectrum was measured using an energy-dispersive silicon drift detector for elemental topography. Our results showed that phosphorus, sulfur, and zinc are predominately distributed in the vascular smooth muscle cells, whereas copper is dramatically accumulated in elastic laminae, indicating a preferential spatial association of copper on elastic laminae in aortic media. This finding sheds new light on the role of copper in elastic formation. Our studies also demonstrate that SRXRF allows for the visualization of trace elements in tissues and cells of rodent aorta with high spatial resolution and provides an opportunity to study the role of trace elements in vasculature.     

Multizone paper platform for 3D cell cultures

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules, and to other environmental factors (temperature, mechanical stress, etc). The majority of high-throughput assays based on 3D cultures, however, can only detect the average behavior of cells in the whole 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning and micromanipulation. Based on a procedure reported previously ("cells-in-gels-in-paper" or CiGiP), this paper describes a simple method for culture of arrays of thin planar sections of tissues, either alone or stacked to create more complex 3D tissue structures. This procedure starts with sheets of paper patterned with hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells suspended in extracellular matrix (ECM) gel onto the patterned paper creates an array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose fibers) containing cells. Stacking the sheets with zones aligned on top of one another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D culture, by peeling apart the sheets of paper, "sections" all 96 cultures at once. It is, thus, simple to isolate 200-micron-thick cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D cultures are assembled from multiple layers, the number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. This capability made it possible to compare the growth of 3D tumor models of different spatial composition, and to examine the migration of cells in these structures.     

Biofabrication: an overview of the approaches used for printing of living cells

The development of cell printing is vital for establishing biofabrication approaches as clinically relevant tools. Achieving this requires bio-inks which must not only be easily printable, but also allow controllable and reproducible printing of cells. This review outlines the general principles and current progress and compares the advantages and challenges for the most widely used biofabrication techniques for printing cells: extrusion, laser, microvalve, inkjet and tissue fragment printing. It is expected that significant advances in cell printing will result from synergistic combinations of these techniques and lead to optimised resolution, throughput and the overall complexity of printed constructs.     

Technical note: The use of 3D printing in dental anthropology collections

Objectives

Rapid prototyping (RP) technology is becoming more affordable, faster, and is now capable of building models with a high resolution and accuracy. Due to technological limitations, 3D printing in biological anthropology has been mostly limited to museum displays and forensic reconstructions. In this study, we compared the accuracy of different 3D printers to establish whether RP can be used effectively to reproduce anthropological dental collections, potentially replacing access to oftentimes fragile and irreplaceable original material.

Methods

We digitized specimens from the Yuendumu collection of Australian Aboriginal dental casts using a high‐resolution white‐light scanning system and reproduced them using four different 3D printing technologies: stereolithography (SLA); fused deposition modeling (FDM); binder‐jetting; and material‐jetting. We compared the deviations between the original 3D surface models with 3D print scans using color maps generated from a 3D metric deviation analysis.

Results

The 3D printed models reproduced both the detail and discrete morphology of the scanned dental casts. The results of the metric deviation analysis demonstrate that all 3D print models were accurate, with only a few small areas of high deviations. The material‐jetting and SLA printers were found to perform better than the other two printing machines.

Conclusions

The quality of current commercial 3D printers has reached a good level of accuracy and detail reproduction. However, the costs and printing times limit its application to produce large sample numbers for use in most anthropological studies. Nonetheless, RP offers a viable option to preserve numerically constraint fragile skeletal and dental material in paleoanthropological collections.

     

Endomicroscopic optical coherence tomography for cellular resolution imaging of gastrointestinal tracts

Our ability to detect neoplastic changes in gastrointestinal (GI) tracts is limited by the lack of an endomicroscopic imaging tool that provides cellular‐level structural details of GI mucosa over a large tissue area. In this article, we report a fiber‐optic‐based micro‐optical coherence tomography (μOCT) system and demonstrate its capability to acquire cellular‐level details of GI tissue through circumferential scanning. The system achieves an axial resolution of 2.48 μm in air and a transverse resolution of 4.8 μm with a depth‐of‐focus (DOF) of ~150 μm. To mitigate the issue of limited DOF, we used a rigid sheath to maintain a circular lumen and center the distal‐end optics. The sensitivity is tested to be 98.8 dB with an illumination power of 15.6 mW on the sample. With fresh swine colon tissues imaged ex vivo, detailed structures such as crypt lumens and goblet cells can be clearly resolved, demonstrating that this fiber‐optic μOCT system is capable of visualizing cellular‐level morphological features. We also demonstrate that time‐lapsed frame averaging and imaging speckle reduction are essential for clearly visualizing cellular‐level details. Further development of a clinically viable μOCT endomicroscope is likely to improve the diagnostic outcome of GI cancers.      

胚胎肝细胞用于肝组织工程的体外培养研究

目的:观察三维受控组装系统下,胚胎肝细胞在三维立体结构的体外生长状态,探讨胚胎肝细胞在肝组织工程中应用的可行性。方法:用清华大学机械工程系研制的"三维受控组装系统",将第15 d小鼠胚胎肝细胞作为肝组织工程的种子细胞,与以明胶为主的复合材料混合,构建成复杂三维立体结构,观察其体外生长发育状态。对体外培养1周及4周的三维类肝组织标本进行苏木精-伊红(HE)染色,免疫组织化学方法检测甲胎蛋白(AFP)及白蛋白(ALB)的表达,并对体外培养4周的三维类肝组织用PAS显色法检测肝糖原表达。结果:HE染色结果显示体外培养的胚胎肝细胞在三维支架材料中,可形成含有类血管和肝组织样结构;体外培养1周的类肝组织AFP表达呈阳性,体外培养4周的三维类肝组织ALB表达呈阳性,PAS显色亦呈阳性。结论:在三维受控组装系统的构建下,呈立体状生长的胚胎肝细胞,可逐渐形成肝组织样结构,并显示一定的肝脏功能。     

Low-temperature culturing improves survival rate of tissue-engineered cardiac cell sheets

Assembling three-dimensional (3D) tissues from single cells necessitates the use of various advanced technological methods because higher-density tissues require numerous complex capillary structures to supply sufficient oxygen and nutrients. Accordingly, creating healthy culture conditions to support 3D cardiac tissues requires an appropriate balance between the supplied nutrients and cell metabolism. The objective of this study was to develop a simple and efficient method for low-temperature cultivation (< 37 °C) that decreases cell metabolism for facilitating the buildup of 3D cardiac tissues. We created 3D cardiac tissues using cell sheet technology and analyzed the viability of the cardiac cells in low-temperature environments. To determine a method that would allow thicker 3D tissues to survive, we investigated the cardiac tissue viability under low-temperature culture processes at 20–33.5 °C and compared it with the viability under the standard culture process at 37 °C. Our results indicated that the standard culture process at 37 °C was unable to support higher-density myocardial tissue; however, low-temperature culture conditions maintained dense myocardial tissue and prevascularization. To investigate the efficiency of transplantation, layered cell sheets produced by the low-temperature culture process were also transplanted under the skin of nude rats. Cardiac tissue cultured at 30 °C developed denser prevascular networks than the tissue cultured at the standard temperature. Our novel findings indicate that the low-temperature process is effective for fabricating 3D tissues from high-functioning cells such as heart cells. This method should make major contributions to future clinical applications and to the field of organ engineering.     

Three-Dimensional (3D) cell culture monitoring: Opportunities and challenges for impedance spectroscopy

Three-dimensional (3D) cell culture has developed rapidly over the past 5–10 years with the goal of better replicating human physiology and tissue complexity in the laboratory. Quantifying cellular responses is fundamental in understanding how cells and tissues respond during their growth cycle and in response to external stimuli. There is a need to develop and validate tools that can give insight into cell number, viability, and distribution in real-time, nondestructively and without the use of stains or other labelling processes. Impedance spectroscopy can address all of these challenges and is currently used both commercially and in academic laboratories to measure cellular processes in 2D cell culture systems. However, its use in 3D cultures is not straight forward due to the complexity of the electrical circuit model of 3D tissues. In addition, there are challenges in the design and integration of electrodes within 3D cell culture systems. Researchers have used a range of strategies to implement impedance spectroscopy in 3D systems. This review examines electrode design, integration, and outcomes of a range of impedance spectroscopy studies and multiparametric systems relevant to 3D cell cultures. While these systems provide whole culture data, impedance tomography approaches have shown how this technique can be used to achieve spatial resolution. This review demonstrates how impedance spectroscopy and tomography can be used to provide real-time sensing in 3D cell cultures, but challenges remain in integrating electrodes without affecting cell culture functionality. If these challenges can be addressed and more realistic electrical models for 3D tissues developed, the implementation of impedance-based systems will be able to provide real-time, quantitative tracking of 3D cell culture systems.     

High-resolution computed tomography of single breast cancer microcalcifications in vivo

Microcalcification is a hallmark of breast cancer and a key diagnostic feature for mammography. We recently described the first robust animal model of breast cancer microcalcification. In this study, we hypothesized that high-resolution computed tomography (CT) could potentially detect the genesis of a single microcalcification in vivo and quantify its growth over time. Using a commercial CT scanner, we systematically optimized acquisition and reconstruction parameters. Two ray-tracing image reconstruction algorithms were tested: a voxel-driven "fast" cone beam algorithm (FCBA) and a detector-driven "exact" cone beam algorithm (ECBA). By optimizing acquisition and reconstruction parameters, we were able to achieve a resolution of 104 μm full width at half-maximum (FWHM). At an optimal detector sampling frequency, the ECBA provided a 28 μm (21%) FWHM improvement in resolution over the FCBA. In vitro, we were able to image a single 300 μm × 100 μm hydroxyapatite crystal. In a syngeneic rat model of breast cancer, we were able to detect the genesis of a single microcalcification in vivo and follow its growth longitudinally over weeks. Taken together, this study provides an in vivo "gold standard" for the development of calcification-specific contrast agents and a model system for studying the mechanism of breast cancer microcalcification.     

Printing multistrain bacterial patterns with a piezoelectric inkjet printer

Many studies involving interacting microorganisms would benefit from simple devices able to deposit cells in precisely defined patterns. We describe an inexpensive bacterial piezoelectric inkjet printer (adapted from the design of the POSaM oligonucleotide microarrayer) that can be used to "print out" different strains of bacteria or chemicals in small droplets onto a flat surface at high resolution. The capabilities of this device are demonstrated by printing ordered arrays comprising two bacterial strains labeled with different fluorescent proteins. We also characterized several properties of this piezoelectric printer, such as the droplet volume (of the order of tens of pl), the distribution of number of cells in each droplet, and the dependence of droplet volume on printing frequency. We established the limits of the printing resolution, and determined that the printed viability of Escherichia coli exceeded 98.5%.     

以胶原膜为支架的心肌细胞体外三维培养

将从新生乳鼠心室肌组织获取的心肌细胞接种于鼠尾胶原膜三维支架和组织培养板,以细胞形态、细胞搏动、葡萄糖比消耗率(q_glu)、乳酸比产率(q_lac)、乳酸转化率(Y_lac/glu)、肌酸激酶及乳酸脱氢酶的活力为观察指标,比较心肌细胞在鼠尾胶原膜中三维(3D)培养和组织培养板中二维(2D)培养的差异。培养于鼠尾胶原膜的乳鼠心肌细胞在第5天形成闰盘连接,形成面积约为80mm³、肉眼可见自律性同步收缩的心肌细胞3D培养物。3D培养体系中乳鼠心肌细胞的q_glu、q_lac和Y_lac/glu的均值分别为7.37 μmol/10.6cells/d、2.92 μmol/106cells/d和0.38 μmol/μmol;2D培养体系中乳鼠心肌细胞的q_glu、q_lac和Y_lac/glu的均值分别为7.59 μmol/10.6cells/d、3.83 μmol/10.6cells/d和 0.51 μmol/μmol。两种培养体系中乳鼠心肌细胞的肌酸激酶及乳酸脱氢酶的活力无明显差别。实验结果表明：培养于鼠尾胶原膜的心肌细胞保持正常心肌细胞的代谢活力和收缩功能。     

Enabling personalized implant and controllable biosystem development through 3D printing

The impact of additive manufacturing in our lives has been increasing constantly. One of the frontiers in this change is the medical devices. 3D printing technologies not only enable the personalization of implantable devices with respect to patient-specific anatomy, pathology and biomechanical properties but they also provide new opportunities in related areas such as surgical education, minimally invasive diagnosis, medical research and disease models. In this review, we cover the recent clinical applications of 3D printing with a particular focus on implantable devices. The current technical bottlenecks in 3D printing in view of the needs in clinical applications are explained and recent advances to overcome these challenges are presented. 3D printing with cells (bioprinting); an exciting subfield of 3D printing, is covered in the context of tissue engineering and regenerative medicine and current developments in bioinks are discussed. Also emerging applications of bioprinting beyond health, such as biorobotics and soft robotics, are introduced. As the technical challenges related to printing rate, precision and cost are steadily being solved, it can be envisioned that 3D printers will become common on-site instruments in medical practice with the possibility of custom-made, on-demand implants and, eventually, tissue engineered organs with active parts developed with biorobotics techniques.     

A brief review of extrusion‐based tissue scaffold bio‐printing

Extrusion‐based bio‐printing has great potential as a technique for manipulating biomaterials and living cells to create three‐dimensional (3D) scaffolds for damaged tissue repair and function restoration. Over the last two decades, advances in both engineering techniques and life sciences have evolved extrusion‐based bio‐printing from a simple technique to one able to create diverse tissue scaffolds from a wide range of biomaterials and cell types. However, the complexities associated with synthesis of materials for bio‐printing and manipulation of multiple materials and cells in bio‐printing pose many challenges for scaffold fabrication. This paper presents an overview of extrusion‐based bio‐printing for scaffold fabrication, focusing on the prior‐printing considerations (such as scaffold design and materials/cell synthesis), working principles, comparison to other techniques, and to‐date achievements. This paper also briefly reviews the recent development of strategies with regard to hydrogel synthesis, multi‐materials/cells manipulation, and process‐induced cell damage in extrusion‐based bio‐printing. The key issue and challenges for extrusion‐based bio‐printing are also identified and discussed along with recommendations for future, aimed at developing novel biomaterials and bio‐printing systems, creating patterned vascular networks within scaffolds, and preserving the cell viability and functions in scaffold bio‐printing. The address of these challenges will significantly enhance the capability of extrusion‐based bio‐printing.     

A Simple,Low-Cost Conductive Composite Material for 3D Printing of Electronic Sensors

3D printing technology can produce complex objects directly from computer aided digital designs. The technology has traditionally been used by large companies to produce fit and form concept prototypes (‘rapid prototyping’) before production. In recent years however there has been a move to adopt the technology as full-scale manufacturing solution. The advent of low-cost, desktop 3D printers such as the RepRap and emoH@baF has meant a wider user base are now able to have access to desktop manufacturing platforms enabling them to produce highly customised products for personal use and sale. This uptake in usage has been coupled with a demand for printing technology and materials able to print functional elements such as electronic sensors. Here we present formulation of a simple conductive thermoplastic composite we term ‘carbomorph’ and demonstrate how it can be used in an unmodified low-cost 3D printer to print electronic sensors able to sense mechanical flexing and capacitance changes. We show how this capability can be used to produce custom sensing devices and user interface devices along with printed objects with embedded sensing capability. This advance in low-cost 3D printing with offer a new paradigm in the 3D printing field with printed sensors and electronics embedded inside 3D printed objects in a single build process without requiring complex or expensive materials incorporating additives such as carbon nanotubes.     

Quantifying Cell-Surface Biomarker Expression in Thick Tissues with Ratiometric Three-Dimensional Microscopy

The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.     

Advances in 3D printing of composite scaffolds for the repairment of bone tissue associated defects

The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.