3D tumor model biofabrication

Animal models have been extensively used in cancer pathology studies and drug discovery.These models,however,fail to reflect the complex human tumor microenvironment and do not allow for high-throughput drug screening in more human-like physiological conditions.Three-dimensional(3D)cancer models present an alternative to automated high-throughput cancer drug discovery and oncology.In this review,we highlight recent technology innovations in building 3D tumor models that simulate the complex human tumor microenvironment and responses of patients to treatment.We discussed various biofabrication technologies,including 3D bioprinting techniques developed for characterizing tumor progression,metastasis,and response to treatment.

Digital biofabrication to realize the potentials of plant roots for product design

Technological and economic opportunities,alongside the apparent ecological benefits,point to biodesign as a new industrial paradigm for the fabrication of products in the twenty-first century.The presented work studies plant roots as a biodesign material in the fabrication of self-supported 3D structures,where the biologically and digitally designed materials provide each other with structural stability.Taking a material-driven design approach,we present our systematic tinkering activities with plant roots to better understand and anticipate their responsive behaviour.These helped us to identify the key design parameters and advance the unique potential of plant roots to bind discrete porous structures.We illustrate this binding potential of plant roots with a hybrid 3D object,for which plant roots connect 600 computationally designed,optimized,and fabricated bioplastic beads into a low stool.

Biofabrication of Ag nanoparticles using Moringa oleifera leaf extract and their antimicrobial activity

Objective:To formulate a simple rapid procedure for bioreduction of silver nanoparticles using aqueous leaves extract of Moringa oleifera(M.oleifera).Methods:10 mL of leaf extract was mixed to 90 mL of 1 mM aqueous of AgNO_3 and was heated at 60-80 ℃ for 20 min.A change from brown to reddish color was observed.Characterization using UV-Vis spectrophotometry, Transmission Electron Microscopy(TEM) was performed.Results:TEM showed the formation of silver nanoparticles with an average size of 57 nm.Conclusions:M.oleifera demonstrates strong potential for synthesis of silver nanoparticles by rapid reduction of silver ions(Ag^+ to Ag^0). Biological methods are good competents for the chemical procedures,which are eco-friendly and convenient.

Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue.Three-dimensional(3D)printing offers a method of fabricating complex anatomical features of clinically relevant sizes.However,the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging.This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions.The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional(2D).The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices,which were arrayed.These 2D slices with each layer of a defined pattern were laser cut,and then successfully assembled with varying thicknesses of 100μm or 200μm.It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions,where the clinically relevant sizes ranging from a simple cube of 20 mm dimension,to a more complex,50 mm-tall human ears were created.In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure.The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice,where a range of hole diameters from 200μm to 500μm were laser cut in an array where cell confluence values of at least 85%were found at three weeks.Cells were also seeded onto a simpler stacked construct,albeit made with micromachined poly fibre mesh,where cells can be found to migrate through the stack better with collagen as bioadhesives.This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future.

Jetting-based bioprinting:process,dispense physics,and applications

Jetting-based bioprinting facilitates contactless drop-on-demand deposition of subnanoliter droplets at well-defined positions to control the spatial arrangement of cells,growth factors,drugs,and biomaterials in a highly automated layer-by-layer fabrication approach.Due to its immense versatility,jetting-based bioprinting has been used for various applications,including tissue engineering and regenerative medicine,wound healing,and drug development.A lack of in-depth understanding exists in the processes that occur during jetting-based bioprinting.This review paper will comprehensively discuss the physical considerations for bioinks and printing conditions used in jetting-based bioprinting.We first present an overview of different jetting-based bioprinting techniques such as inkjet bioprinting,laser-induced forward transfer bioprinting,electrohydrodynamic jet bioprinting,acoustic bioprinting and microvalve bioprinting.Next,we provide an in-depth discussion of various considerations for bioink formulation relating to cell deposition,print chamber design,droplet formation and droplet impact.Finally,we highlight recent accomplishments in jetting-based bioprinting.We present the advantages and challenges of each method,discuss considerations relating to cell viability and protein stability,and conclude by providing insights into future directions of jetting-based bioprinting.

Process,Material,and Regulatory Considerations for 3D Printed Medical Devices and Tissue Constructs

Three-dimensional(3D)printing is a highly automated platform that facilitates material deposition in a layer-by-layer approach to fabricate pre-defined 3D complex structures on demand.It is a highly promising technique for the fabrication of personalized medical devices or even patient-specific tissue constructs.Each type of 3D printing technique has its unique advantages and limitations,and the selection of a suitable 3D printing technique is highly dependent on its intended application.In this review paper,we present and highlight some of the critical processes(printing parameters,build orientation,build location,and support structures),material(batch-to-batch consistency,recycling,protein adsorption,biocompatibility,and degradation properties),and regulatory considerations(sterility and mechanical properties)for 3D printing of personalized medical devices.The goal of this review paper is to provide the readers with a good understanding of the various key considerations(process,material,and regulatory)in 3D printing,which are critical for the fabrication of improved patient-specific 3D printed medical devices and tissue constructs.

Spheroid construction strategies and application in 3D bioprinting

Tissue engineering has been striving toward designing and producing natural and functional human tissues.Cells are the fundamental building blocks of tissues.Compared with traditional two-dimensional cultured cells,cell spheres are threedimensional(3D)structures that can naturally form complex cell–cell and cell–matrix interactions.This structure is close to the natural environment of cells in living organisms.In addition to being used in disease modeling and drug screening,spheroids have significant potential in tissue regeneration.The 3D bioprinting is an advanced biofabrication technique.It accurately deposits bioinks into predesigned 3D shapes to create complex tissue structures.Although 3D bioprinting is efficient,the time required for cells to develop into complex tissue structures can be lengthy.The 3D bioprinting of spheroids significantly reduces the time required for their development into large tissues/organs during later cultivation stages by printing them with high cell density.Combining spheroid fabrication and bioprinting technology should provide a new solution to many problems in regenerative medicine.This paper systematically elaborates and analyzes the spheroid fabrication methods and 3D bioprinting strategies by introducing spheroids as building blocks.Finally,we present the primary challenges faced by spheroid fabrication and 3D bioprinting with future requirements and some recommendations.

Three-dimensional biofabrication of an aragoniteenriched self-hardening bone graft substitute and assessment of its osteogenicity in vitro and in vivo

A self-hardening three-dimensional(3D)-porous composite bone graft consisting of 65 wt%hydroxyapatite(HA)and 35 wt%aragonite was fabricated using a 3D-Bioplotter®.New tetracalcium phosphate and dicalcium phosphate anhydrous/aragonite/gelatine paste formulae were developed to overcome the phase separation of the liquid and solid components.The mechanical properties,porosity,height and width stability of the end products were optimised through a systematic analysis of the fabrication processing parameters including printing pressure,printing speed and distance between strands.The resulting 3D-printed bone graft was confirmed to be a mixture of HA and aragonite by X-ray diffraction,Fourier transform infrared spectroscopy and energy dispersive X-ray spectroscopy.The compression strength of HA/aragonite was between 0.56 and 2.49 MPa.Cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay in vitro.The osteogenicity of HA/aragonite was evaluated in vitro by alkaline phosphatase assay using human umbilical cord matrix mesenchymal stem cells,and in vivo by juxtapositional implantation between the tibia and the anterior tibialis muscle in rats.The results showed that the scaffold was not toxic and supported osteogenic differentiation in vitro.HA/aragonite stimulated new bone formation that bridged host bone and intramuscular implants in vivo.We conclude that HA/aragonite is a biodegradable and conductive bone formation biomaterial that stimulates bone regeneration.Since this material is formed near 37°C,it will have great potential for incorporating bioactive molecules to suit personalised application;however,further study of its biodegradation and osteogenic capacity is warranted.The study was approved by the Animal Ethical Committee at Tongji Medical School,Huazhong University of Science and Technology(IACUC No.738)on October 1,2017.

Advanced biofabrication strategies for biomimetic composite scaffolds to regenerate ligament-bone interface

The natural ligament-bone interface features gradient changes in matrix composition,architecture and cell phenotype,which play critical roles in reliable ligament fixation and smooth loading transfer.Mimicking such organisations in artificial composite tissue-engineering scaffolds is important for realising functional fixation between ligament implants and host bones.Here,the authors aim to provide a comprehensive review on the latest strategies to fabricate biomimetic composite scaffolds for the regeneration of ligament-to-bone interface.The biomimetic composite scaffolds are divided into stratified and gradient scaffolds,which are characterised as layer-specific and continuous changes,respectively,in scaffold materials and/or microstructures.Biofabrication strategies for different types of composite scaffolds are summarised.The effects of material/structural changes on cellular morphology,cell differentiation,in vivo osteointegration and multi-tissue interface regeneration are highlighted.Finally,the potential challenges and future perspectives in engineering biomimetic composite scaffolds for ligament-bone interface regeneration are discussed.

Towards the future of plastic surgery:from flaps to microsurgery and regenerative medicine and biofabrication?

Plastic surgery is a specialty that is now worldwide recognized as its own academic discipline within the surgical community. The roots however are as old as 600 BC when in the Sushruta Ayurveda the reconstruction of a nose with a flap from the forehead was described. Plastic surgery is a problem solving discipline that meanwhile is an integral part within modern surgical concepts.

Polyvinylpyrrolidone-based bioink:influence of bioink properties on printing performance and cell proliferation during inkjet-based bioprinting

Among the different bioprinting techniques,the drop-on-demand(DOD)jetting-based bioprinting approach facilitates contactless deposition of pico/nanoliter droplets ofmaterials and cells for optimal cell–matrix and cell–cell interactions.Although bioinks play a critical role in the bioprinting process,there is a poor understanding of the influence of bioink properties on printing performance(such as filament elongation,formation of satellite droplets,and droplet splashing)and cell health(cell viability and proliferation)during the DOD jetting-based bioprinting process.An inert polyvinylpyrrolidone(PVP360,molecular weight=360 kDa)polymerwas used in this study to manipulate the physical properties of the bioinks and investigate the influence of bioink properties on printing performance and cell health.Our experimental results showed that a higher bioink viscoelasticity helps to stabilize droplet filaments before rupturing from the nozzle orifice.The highly stretched droplet filament resulted in the formation of highly aligned“satellite droplets,”which minimized the displacement of the satellite droplets away from the predefined positions.Next,a significant increase in the bioink viscosity facilitated droplet deposition on the wetted substrate surface in the absence of splashing and significantly improved the accuracy of the deposited main droplet.Further analysis showed that cell-laden bioinks with higher viscosity exhibited higher measured average cell viability(%),as the presence of polymer within the printed droplets provides an additional cushioning effect(higher energy dissipation)for the encapsulated cells during droplet impact on the substrate surface,improves the measured average cell viability even at higher droplet impact velocity and retains the proliferation capability of the printed cells.Understanding the influence of bioink properties(e.g.,bioink viscoelasticity and viscosity)on printing performance and cell proliferation is important for the formulation of new bioinks,and we have demonstrated precise DOD deposition of living cells and fabrication of tunable cell spheroids(nL–μL range)using multiple types of cells in a facile manner.

Three-dimensional bioprinting of gelatin methacryloyl (GelMA)

The three-dimensional (3D)bioprinting technology has progressed tremendously over the past decade.By controlling the size, shape,and architecture of the bioprinted constructs,3D bioprinting allows for the fabrication of tissue/organ-like constructs with strong structural-functional similarity with their in vivo counterparts at high fidelity.The bioink,a blend of biomaterials and living cells possessing both high biocompatibility and printability,is a critical component of bioprinting.In particular, gelatin methacryloyl (GelMA)has shown its potential as a viable bioink material due to its suitable biocompatibility and readily tunable physicochemical properties.Current GelMA-based bioinks and relevant bioprinting strategies for GelMA bioprinting are briefly reviewed.

3D bioprinting:current status and trends-a guide to the literature and industrial practice

The multidisciplinary research field of bioprinting combines additive manufacturing,biology and material sciences to cre-ate bioconstructs with three-dimensional architectures mimicking natural living tissues.The high interest in the possibility of reproducing biological tissues and organs is further boosted by the ever-increasing need for personalized medicine,thus allowing bioprinting to establish itself in the field of biomedical research,and attracting extensive research efforts from companies,universities,and research institutes alike.In this context,this paper proposes a scientometric analysis and critical review of the current literature and the industrial landscape of bioprinting to provide a clear overview of its fast-changing and complex position.The scientific literature and patenting results for 2000-2020 are reviewed and critically analyzed by retrieving 9314 scientific papers and 309 international patents in order to draw a picture of the scientific and industrial landscape in terms of top research countries,institutions,journals,authors and topics,and identifying the technology hubs worldwide.This review paper thus offers a guide to researchers interested in this field or to those who simply want to under-stand the emerging trends in additive manufacturing and 3D bioprinting.

Leading Approaches to Vascularize Kidney Constructs in Tissue Engineering

There is an unprecedented need for new treatments for renal failure,as the incidence of this disease is increasing disproportionately to advancements in therapies.Current treatments are limited by the availability of viable organs,for which there is a worldwide lack.These treatment modalities also require a substantial amount of infrastructure,significantly limiting the access to care in most countries.Kidney tissue engineering approaches promise to develop alternative solutions that address many of the inadequacies in current care.Although many advancements have been made—primarily in the past decade—in biofabrication and whole-organ tissue engineering,many challenges remain.One major hindrance to the progress of current tissue engineering approaches is establishing successful vascularization of developed engineered tissue constructs.This review focuses on the recent advancements that address the vascular challenge,including the biofabrication of vasculature,whole-organ engineering through decellularization and recellularization approaches,microscale organogenesis,and vascularization using organoids in the context of kidney tissue engineering.We also highlight the specific challenges that remain in developing successful strategies capable of clinical translation.

Microfluidic bubble-generator enables digital light processing 3D printing of porous structures

Three-dimensional(3D)printing is an emerging technique that has shown promising success in engineering human tissues in recent years.Further development of vatphotopolymerization printing modalities has significantly enhanced the complexity level for 3D printing of various functional structures and components.Similarly,the development of microfluidic chip systems is an emerging research sector with promising medical applications.This work demonstrates the coupling of a digital light processing(DLP)printing procedure with a microfluidic chip system to produce size-tunable,3D-printable porosities with narrow pore size distributions within a gelatin methacryloyl(GelMA)hydrogel matrix.It is found that the generation of size-tunable gas bubbles trapped within an aqueous GelMA hydrogel-precursor can be controlled with high precision.Furthermore,the porosities are printed in two-dimensional(2D)as well as in 3D using the DLP printer.In addition,the cytocompatibility of the printed porous scaffolds is investigated using fibroblasts,where high cell viabilities as well as cell proliferation,spreading,and migration are confirmed.It is anticipated that the strategy is widely applicable in a range of application areas such as tissue engineering and regenerative medicine,among others.

Building biological foundries for next-generation synthetic biology

Synthetic biology is an interdisciplinary field that takes top-down approaches to understand and engineer biological systems through design-build-test cycles. A number of advances in this relatively young field have greatly accelerated such engineering cycles. Specifically, various innovative tools were developed for in silico biosystems design, DNA de novo synthesis and assembly, construct verification, as well as metabolite analysis, which have laid a solid foundation for building biological foundries for rapid prototyping of improved or novel biosystems. This review summarizes the state-of-the-art technologies for synthetic biology and discusses the challenges to establish such biological foundries.

Current research progress of photopolymerized hydrogels in tissue engineering

Owing to the special fo rmation of photopolymerized hydrogels,they can effectively control the formation of hydrogels in space and time.Moreover,the photopolymerized hydrogels have mild formation conditions and biocompatibility;therefore,they can be widely used in tissue engineering.With the development and application of manufacturing technology,photopolymerized hydrogels can be widely used in cell encapsulation,scaffold materials,and other tissue engineering fields through more elaborate manufacturing methods.This review covers the types of photoinitiators,manu facturing technologies for photopolymerized hydrogels as well as the materials used,and a summary of the applications of photopolymerized hydrogels in tissue engineering.

Biomaterials for stem cell engineering and biomanufacturing

Recent years have witnessed the expansion of tissue failures and diseases.The uprising of regenerative medicine converges the sight onto stem cell-biomaterial based therapy.Tissue engineering and regenerative medicine proposes the strategy of constructing spatially,mechanically,chemically and biologically designed biomaterials for stem cells to grow and differentiate.Therefore,this paper summarized the basic properties of embryonic stem cells(ESCs),induced pluripotent stem cells(iPSCs)and adult stem cells.The properties of frequently used biomaterials were also described in terms of natural and synthetic origins.Particularly,the combination of stem cells and biomaterials for tissue repair applications was reviewed in terms of nervous,cardiovascular,pancreatic,hematopoietic and musculoskeletal system.Finally,stem-cell-related biomanufacturing was envisioned and the novel biofabrication technologies were discussed,enlightening a promising route for the future advancement of large-scale stem cell-biomaterial based therapeutic manufacturing.

Effect of cyclic mechanical loading on immunoinflammatory microenvironment in biofabricating hydroxyapatite scaffold for bone regeneration

It has been proven that the mechanical microenvironment can impact the differentiation of mesenchymal stem cells(MSCs).However,the effect of mechanical stimuli in biofabricating hydroxyapatite scaffolds on the inflammatory response of MSCs remains unclear.This study aimed to investigate the effect of mechanical loading on the inflammatory response of MSCs seeded on scaffolds.Cyclic mechanical loading was applied to biofabricate the cell-scaffold composite for 15 min/day over 7,14,or 21 days.At the predetermined time points,culture supernatant was collected for inflammatory mediator detection,and gene expression was analyzed by qRT-PCR.The results showed that the expression of inflammatory mediators(IL1B and IL8)was downregulated(p<0.05)and the expression of ALP(p<0.01)and COL1A1(p<0.05)was upregulated under mechanical loading.The cell-scaffold composites biofabricated with or without mechanical loading were freeze-dried to prepare extracellular matrix-based scaffolds(ECM-based scaffolds).Murine macrophages were seeded on the ECM-based scaffolds to evaluate their polarization.The ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying enhanced the expression of M2 polarization-related biomarkers(Arginase 1 and Mrc1,p<0.05)of macrophages in vitro and increased bone volume/total volume ratio in vivo.Overall,these findings demonstrated that mechanical loading could dually modulate the inflammatory responses and osteogenic differentiation of MSCs.Besides,the ECM-based scaffolds that were biofabricated with mechanical loading before freeze-drying facilitated the M2 polarization of macrophages in vitro and bone regeneration in vivo.Mechanical loading may be a promising biofabrication strategy for bone biomaterials.

Photo-crosslinkable hydrogel and its biological applications

In the past few years,photo-crosslinkable hydrogels have drawn a great attention in tissue engineering applications due to their high biocompatibility and extracellular matrix(ECM)-like structure.They can be easily biofabricated through exposure of a photosensitive system composed of photo-crosslinkable hydrogels,photo-initiators and other compounds such as cells and therapeutic molecules,to ultraviolet or visible light.With the development ofbiofabrication methods,many researchers studied the biological applications of photo-crosslinkable hydrogels in tissue engineering,such as vascular,wound dressing and bone engineering.This review highlights the biomaterials for photo-crosslinkable hydrogels,biofabrication techniques and their biological applications in tissue engineering.Meanwhile,the challenges and prospects of photo-crosslinkable hydrogels are discussed as well.