Development of 3D bioprinting:From printing methods to biomedical applications

Biomanufacturing of tissues/organs in vitro is our big dream,driven by two needs:organ transplantation and accurate tissue models.Over the last decades,3D bioprinting has been widely applied in the construction of many tissues/organs such as skins,vessels,hearts,etc.,which can not only lay a foundation for the grand goal of organ replacement,but also be served as in vitro models committed to pharmacokinetics,drug screening and so on.As organs are so complicated,many bioprinting methods are exploited to figure out the challenges of different applications.So the question is how to choose the suitable bioprinting method?Herein,we systematically review the evolution,process and classification of 3D bioprinting with an emphasis on the fundamental printing principles and commercialized bioprinters.We summarize and classify extrusion-based,dropletbased,and photocuring-based bioprinting methods and give some advices for applications.Among them,coaxial and multi-material bioprinting are highlighted and basic principles of designing bioinks are also discussed.

Triply periodic minimal surface(TPMS)porous structures:from multi-scale design,precise additive manufacturing to multidisciplinary applications

Inspired by natural porous architectures,numerous attempts have been made to generate porous structures.Owing to the smooth surfaces,highly interconnected porous architectures,and mathematical controllable geometry features,triply periodic minimal surface(TPMS)is emerging as an outstanding solution to constructing porous structures in recent years.However,many advantages of TPMS are not fully utilized in current research.Critical problems of the process from design,manufacturing to applications need further systematic and integrated discussions.In this work,a comprehensive overview of TPMS porous structures is provided.In order to generate the digital models of TPMS,the geometry design algorithms and performance control strategies are introduced according to diverse requirements.Based on that,precise additive manufacturing methods are summarized for fabricating physical TPMS products.Furthermore,actual multidisciplinary applications are presented to clarify the advantages and further potential of TPMS porous structures.Eventually,the existing problems and further research outlooks are discussed.

Bioprinting of novel 3D tumor array chip for drug screening

Biomedical field has been seeking a feasible standard drug screening system consisting of 3D tumor model array for drug researching due to providing sufficient samples and simulating actual in vivo tumor growth situation,which is still a challenge to rapidly and uniformly establish though.Here,we propose a novel drug screening system,namely 3D tumor array chip with“layer cake”structure,for drug screening.Accurate gelatin methacryloyl hydrogel droplets(~0.1μL)containing tumor cells can be automatically deposited on demand with electrohydrodynamic 3D printing.Transparent conductive membrane is introduced as a chip basement for preventing charges accumulation during fabricating and convenient observing during screening.Culturing chambers formed by stainless steel and silicon interlayer is convenient to be assembled and recycled.As this chip is compatible with the existing 96-well culturing plate,the drug screening protocols could keep the same as convention.Important properties of this chip,namely printing stability,customizability,accuracy,microenvironment,tumor functionalization,are detailly examined.As a demonstration,it is applied for screening of epirubicin and paclitaxel with breast tumor cells to confirm the compatibility of the proposed screening system with the traditional screening methods.We believe this chip will potentially play a significant role in drug evaluation in the future.

Rapid assembling organ prototypes with controllable cell-laden multi-scale sheets

A native organ has heterogeneous structures, sirength, and cell components. It is a big challenge to fabricate organ prototypes with controllable shapes, strength, and cells. Herein, a hybrid method is developed to fabricate organ prototypes with controlled cell deposition by integrating extrusion-based 3D printing, electrospinning, and 3D bioprinting. Multi-scale sheets were first fabricated by 3D printing and electrospinning;then, all the sheets were assembled into organ prototypes by sol-gel react io n duri ng bioprinting. With this method, macroscale structures fabricated by 3D printing ensure the customized structures and provide mechanical support, nanoscale structures fabricated by electrospinning offer a favorable environment for cell growth, and different types of cells with controllable densities are deposited in accurate locations by bioprinting. The results show that L929 mouse fibroblasts encapsulated in the structures exhibited over 90% survival within 10 days and maintai ned a high proliferation rate. Furthermore, the cells grew in spherical shapes first and then migrated to the nano scale fibers showing stretched morphology. Additionally, a branched vascular structure was successfully fabricated using the presented method. Compared with other methods, this strategy offers an easy way to simultancously realize the shape control, nanolibrous structures, and cell accurate deposition, which will have potemidi applications in tissue cngineering.

Sacrificial microgel‑laden bioink‑enabled 3D bioprinting of mesoscale pore networks

Three-dimensional(3D)bioprinting is a powerful approach that enables the fabrication of 3D tissue constructs that retain complex biological functions.However,the dense hydrogel networks that form after the gelation of bioinks often restrict the migration and proliferation of encapsulated cells.Herein,a sacrificial microgel-laden bioink strategy was designed for directly bioprinting constructs with mesoscale pore networks(MPNs)for enhancing nutrient delivery and cell growth.The sacrificial microgel-laden bioink,which contains cell/gelatin methacryloyl(GelMA)mixture and gelled gelatin microgel,is first thermo-crosslinked to fabricate temporary predesigned cell-laden constructs by extrusion bioprinting onto a cold platform.Then,the construct is permanently stabilized through photo-crosslinking of GelMA.The MPNs inside the printed constructs are formed after subsequent dissolution of the gelatin microgel.These MPNs allowed for effective oxygen/nutrient diffusion,facilitating the generation of bioactive tissues.Specifically,osteoblast and human umbilical vein endothelial cells encapsulated in the bioprinted large-scale constructs(≥1 cm)with MPNs showed enhanced bioactivity during culture.The 3D bioprinting strategy based on the sacrificial microgel-laden bioink provided a facile method to facilitate formation of complex tissue constructs with MPNs and set a foundation for future optimization of MPN-based tissue constructs with applications in diverse areas of tissue engineering.

Balancing the customization and standardization:exploration and layout surrounding the regulation of the growing field of 3D-printed medical devices in China

Medical devices are instruments and other tools that act on the human body to aid clinical diagnosis and disease treatment,playing an indispensable role in modern medicine.Nowadays,the increasing demand for personalized medical devices poses a significant challenge to traditional manufacturing methods.The emerging manufacturing technology of three-dimensional(3D)printing as an alternative has shown exciting applications in the medical field and is an ideal method for manufacturing such personalized medical devices with complex structures.However,the application of this new technology has also brought new risks to medical devices,making 3D-printed devices face severe challenges due to insufficient regulation and the lack of standards to provide guidance to the industry.This review aims to summarize the current regulatory landscape and existing research on the standardization of 3D-printed medical devices in China,and provide ideas to address these challenges.We focus on the aspects concerned by the regulatory authorities in 3D-printed medical devices,highlighting the quality system of such devices,and discuss the guidelines that manufacturers should follow,as well as the current limitations and the feasible path of regulation and standardization work based on this perspective.The key points of the whole process quality control,performance evaluation methods and the concept of whole life cycle management of 3D-printed medical devices are emphasized.Furthermore,the significance of regulation and standardization is pointed out.Finally,aspects worthy of attention and future perspectives in this field are discussed.

Why choose 3D bioprinting? Part I: a brief introduction of 3D bioprinting for the beginners

Graphic abstract Concept of 3D bioprinting 3D printing,also called additive manufacturing,is a layerby-layer manufacturing method,and it has been implemented in a wide variety of areas.3D printing could be treated as the reverse process of potato cutting,automatically assembling the chips or slicing into a potato[1].When this simple idea is met with biomedical engineering,3D bioprinting is born.

Physical understanding of axonal growth patterns on grooved substrates:groove ridge crossing versus longitudinal alignment

Surface topographies such as micrometric edges and grooves have been widely used to improve neuron outgrowth.However,finding the mechanism of neuron–surface interactions on grooved substrates remains a challenge.In this work,PC12 cells and chick forebrain neurons(CFNs)were cultured on grooved and smooth polyacrylonitrile substrates.It was found that CFNs showed a tendency of growing across groove ridges;while PC12 cells were only observed to grow in the longitudinal direction of grooves.To further investigate these observations,a 3D physical model of axonal outgrowth was developed.In this model,axon shafts are simulated as elastic 3D beams,accounting for the axon outgrowth as well as the focal contacts between axons and substrates.Moreover,the bending direction of axon tips during groove ridge crossing is governed by the energy minimization principle.Our physical model predicts that axonal groove ridge crossing is contributed by the bending compliance of axons,caused by lower Young’s modulus and smaller diameters.This work will aid the understanding of the mechanisms involved in axonal alignment and elongation of neurons guided by grooved substrates,and the obtained insights can be used to enhance the design of instructive scaffolds for nerve tissue engineering and regeneration applications.

A novel wavy non-uniform ligament chiral stent with J-shaped stress-strain behavior to mimic the native trachea

Tracheal stents are an important form of treatment for benign or malignant central airway obstruction.However,the mechanical behavior of current tracheal stents is significantly different from that of the native trachea,which leads to a variety of serious complications.In this study,inspired by the structure of the native trachea,a wavy non-uniform ligament chiral tracheal stent is proposed,in which J-shaped stress-strain behavior and negative Poisson's ratio response are achieved by replacing the tangential ligament of tetrachiral and anti-tetrachiral hybrid structure with a wavy non-uniform ligament.Through the combination of theoretical analysis,finite element analysis and experimental tests,a wide range of desired J-shaped stress-strain curves are explored to mimic the native porcine trachea by tailoring the stent geometry.Besides,the negative Poisson’s ratio and auxetic diameter curves versus axial strain of the stent are also studied in detail,thus contributing to the enhancement of cross-section ventilation and reducing the migration of the stent.This novel tracheal stent with a unique microstructure shows a potential to perfectly match the physiological activities of the native trachea and thereby reduce potential complications.

Why choose 3D bioprinting?Part II:methods and bioprinters

Classification of 3D bioprinting As we mentioned in the last editorial,3D printing,also known as additive manufacturing,could be considered as the reverse process of potato cutting,automatically assembling sliced potato,shredded potato,diced potato to integrity[1].Generally speaking,cell-laden 3D bioprinting can be classified into three types:extrusion-based,droplet-based and photocuring-based bioprinting according to different printing principles.Extrusion-based bioprinting squeezes out continuous hydrogel fibers to set up structures;dropletbased bioprinting generates droplets as the basic unit for biofabrication;and photocuring-based bioprinting utilizes the characteristics of light-sensitive materials,to stack 3D models layer-by-layer.Different bioprinting approaches own diverse characteristics facing various scenarios and have specific requirements for bioinks.

Review of heterogeneous material objects modeling in additive manufacturing

This review investigates the recent developments of heterogeneous objects modeling in additive manufacturing(AM),as well as general problems and widespread solutions to the modeling methods of heterogeneous objects.Prevalent heterogeneous object representations are generally categorized based on the different expression or data structure employed therein,and the state-of-the-art of process planning procedures for AM is reviewed via different vigorous solutions for part orientation,slicing methods,and path planning strategies.Finally,some evident problems and possible future directions of investigation are discussed.

A review of the design methods of complex topology structures for 3D printing

As a matter of fact,most natural structures are complex topology structures with intricate holes or irregular surface morphology.These structures can be used as lightweight infill,porous scaffold,energy absorber or micro-reactor.With the rapid advancement of 3D printing,the complex topology structures can now be efficiently and accurately fabricated by stacking layered materials.The novel manufacturing technology and application background put forward new demands and challenges to the current design methodologies of complex topology structures.In this paper,a brief review on the development of recent complex topology structure design methods was provided;meanwhile,the limitations of existing methods and future work are also discussed in the end.

Additive nanomanufacturing of lab-on-a-chip fluorescent peptide nanoparticle arrays for Alzheimer＇s disease diagnosis

This paper proposes an additive nanomanufacturing approach to fabricate a personalized lab-on-a-chip fluorescent peptide nanoparticles （f-PNPs） array for simultaneous multi-biomarker detection that can be used in Alzheimer＇s disease （AD） diagnosis. We will discuss optimization techniques for the additive nanomanufacturing process in terms of reliability, yield and manufacturing efficiency. One contribution of this paper lies in utilization of additive nanomanufacturing techniques to fabricate a patient-specific customize-designed lab-on-a-chip device for personalized AD diagnosis, which remains a major challenge for biomedical engineering. Through the integrated bio-design and bio-manufacturing process, doctor＇s check- up and computer-aided customized design are integrated into the lab-on-a-chip array for patient-specific AD diagnosis. In addition, f-PNPs with targeting moieties for personalized AD biomarkers will be self-assembled onto the customized lab-on-a- chip through the additive nanomanufacturing process, which has not been done before. Another contribution of this research is the personalized lab-on-a-chip f-PNPs array for AD diagnosis utilizing limited human blood. Blood-based AD assessment has been described as ＂the holy grail＂ of early AD detection. This research created the computer-aided design, fabrication through additive nanomanufacturing, and validation of the f-PNPs array for AD diagnosis. This is a highly interdisciplinary research contributing to nanotechnology, biomaterials, and biomedical engineering for neurodegenerative disease. The conceptual work is preliminary with intent to introduce novel techniques to the application. Large-scale manufacturing based on the proposed framework requires extensive validation and optimization.

Molding of polyether ether ketone(PEEK)and its composites:a review

Over the last half-century,polyether ether ketone(PEEK)has emerged as a widely adopted thermoplastic polymer,primarily due to its lower density,exceptional mechanical properties,high-temperature and chemical resistance,and biocompatibility.PEEK and its composites have found extensive applications across various fields,including machinery,aerospace,military equipment,electronics,and biomedicine,positioning themselves as promising substitutes for traditional metal structures.Nevertheless,achieving optimal performance and functional molding of PEEK and its composites presents a formidable challenge,given their inherent characteristics,such as semi-crystallinity,high melting temperature,heightened viscosity,low dielectric coefficient,and hydrophobic properties.In this paper,we present a comprehensive review of the molding methods and processes of PEEK and its composites,including extrusion molding,hot compression molding,injection molding,and 3D printing.We also introduce typical innovative applications within the fields of mechanics,electricity,and biomedicine while elucidating methodologies that leverage the distinctive advantages of PEEK and its composites.Additionally,we summarize research findings related to manipulating the properties of PEEK and its composites through the optimization of machine parameters,process variables,and material structural adjustments.Finally,we contemplate the prevailing development trends and outline prospective avenues for further research in the advancement and molding of PEEK and its composites.

Fabrication of scaffolds in tissue engineering： A review

Tissue engineering （TE） is an integrated discipline that involves engineering and natural science in the development of biological materials to replace, repair, and improve the function of diseased or missing tissues. Traditional medical and surgical treatments have been reported to have side effects on patients caused by organ necrosis and tissue loss. However, engineered tissues and organs provide a new way to cure specific diseases. Scaffold fabrication is an important step in the TE process. This paper summarizes and reviews the widely used scaffold fabrication methods, including conventional methods, electrospinning, three-dimensional printing, and a combination of molding techniques. Furthermore, the differences among the properties of tissues, such as pore size and distribution, porosity, structure, and mechanical properties, are elucidated and critically reviewed. Some studies that combine two or more methods are also reviewed. Finally, this paper provides some guidance and suggestions for the future of scaffold fabrication.

Printability during projection-based 3D bioprinting

Since projection-based 3D bioprinting(PBP)could provide high resolution,it is well suited for printing delicate structures for tissue regeneration.However,the low crosslinking density and low photo-crosslinking rate of photocurable bioink make it difficult to print fine structures.Currently,an in-depth understanding of the is lacking.Here,a research framework is established for the analysis of printability during PBP.The gelatin methacryloyl(GelMA)-based bioink is used as an example,and the printability is systematically investigated.We analyze the photo-crosslinking reactions during the PBP process and summarize the specific requirements of bioinks for PBP.Two standard quantized models are established to evaluate 2D and 3D printing errors.Finally,the better strategies for bioprinting five typical structures,including solid organs,vascular structures,nerve conduits,thin-wall scaffolds,and micro needles,are presented.

Additive-lathe 3D bioprinting of bilayered nerve conduits incorporated with supportive cells

Nerve conduits have been identified as one of the most promising treatments for peripheral nerve injuries,yet it remains unsolved how to develop ideal nerve conduits with both appropriate biological and mechanical properties.Existing nerve conduits must make trade-offs between mechanical strength and biocompatibility.Here,we propose a multi-nozzle additive-lathe 3D bioprinting technology to fabricate a bilayered nerve conduit.The materials for printing consisted of gelatin methacrylate(GelMA)-based inner layer,which was cellularized with bone marrow mesenchymal stem cells(BMSCs)and GelMA/poly(ethylene glycol)diacrylate(PEGDA)-based outer layer.The high viability and extensive morphological spreading of BMSCs encapsulated in the inner layer was achieved by adjusting the degree of methacryloyl substitution and the concentration of GelMA.Strong mechanical performance of the outer layer was obtained by the addition of PEGDA.The performance of the bilayered nerve conduits was assessed using in vitro culture of PC12 cells.The cell density of PC12 cells attached to cellularized bilayered nerve conduits was more than 4 times of that on acellular bilayered nerve conduits.The proliferation rate of PC12 cells attached to cellularized bilayered nerve conduits was over 9 times higher than that on acellular bilayered nerve conduits.These results demonstrate the additive-lathe 3D bioprinting of BMSCs embedded bilayered nerve conduits holds great potential in facilitating peripheral nerve repair.

In-situ density measurement for plastic injection molding via ultrasonic technology

Density variation during the injection molding process directly reflects the state of plastic melt and contains valuable information for process monitoring and optimization.Therefore,in-situ density measurement is of great interest and has significant application value.The existing methods,such as pressure−volume−temperature(PVT)method,have the shortages of time-delay and high cost of sensors.This study is the first to propose an in-situ density measurement method using ultrasonic technology.The analyses of the time-domain and frequency-domain signals are combined in the proposed method.The ultrasonic velocity is obtained from the time-domain signals,and the acoustic impedance is computed through a full-spectral analysis of the frequency-domain signals.Experiments with different process conditions are conducted,including different melt temperature,injection speed,material,and mold structure.Results show that the proposed method has good agreement with the PVT method.The proposed method has the advantages of in-situ measurement,non-destructive,high accuracy,low cost,and is of great application value for the injection molding industry.

Ultrasonic measurement of tie-bar stress for die-casting machine

In die casting,the real-time measurement of the stress of the tie-bar helps ensure product quality and protect the machine itself.However,the traditional magnetic-attached strain gauge is installed in the mold and product operating area,which hinders the loading and unloading of the mold and the collection of die castings.In this paper,a method for real-time measurement of stress using ultrasonic technology is proposed.The stress variation of the tie-bar is analyzed,and a mathematical model between ultrasonic signal and stress based on acoustoelastic theory is established.Verification experiments show that the proposed method agrees with the strain gauge,and the maximum of the difference square is only 1.5678(MPa)2.Furthermore,single-factor experiments are conducted.A higher ultrasonic frequency produces a better measurement accuracy,and the mean of difference squares at 2.5 and 5 MHz are 2.3234 and 0.6733(MPa)_(2),respectively.Measurement accuracy is insensitive to probe location and tonnage of the die-casting machine.Moreover,the ultrasonic measurement method can be used to monitor clamping health status and inspect the dynamic pulling force of the tie-bar.This approach has the advantages of high precision,high repeatability,easy installation,and noninterference,which helps guide the production in die casting.

Single-electromagnet levitation for density measurement and defect detection

This paper presents a single-electromagnet levitation device to measure the densities and detect the internal defects of antimagnetic materials.The experimental device has an electromagnet in its lower part and a pure iron core in the upper part.When the electromagnet is activated,samples can be levitated stably in a paramagnetic solution.Compared with traditional magnetic levitation devices,the single-electromagnet levitation device is adjustable.Different currents,electromagnet shapes,and distances between the electromagnet and iron core are used in the experiment depending on the type of samples.The magnetic field formed by the electromagnet is strong.When the concentration of the MnCl aqueous solution is 3 mol/L,the measuring range of the single-electromagnet levitation device ranges from 1.301 to 2.308 g/cm.However,with the same concentration of MnCl aqueous solution(3 mol/L),the measuring range of a magnetic levitation device built with permanent magnets is only from 1.15 to 1.50 g/cm.The single-electromagnet levitation device has a large measuring range and can realize accurate density measurement and defect detection of high-density materials,such as glass and aluminum alloy.