Exploiting light-based 3D-printing for the fabrication of mechanically enhanced, patient-specific aortic grafts.

Despite polyester vascular grafts being routinely used in life-saving aortic aneurysm surgeries, they are less compliant than the healthy, native human aorta. This mismatch in mechanical behaviour has been associated with disruption of haemodynamics contributing to several long-term cardiovascular complications. Moreover, current fabrication approaches mean that opportunities to personalise grafts to the individual anatomical features are limited. Various modifications to graft design have been investigated to overcome such limitations; yet optimal graft functionality remains to be achieved. This study reports on the development and characterisation of an alternative vascular graft material. An alginate:PEGDA (AL:PE) interpenetrating polymer network (IPN) hydrogel has been produced with uniaxial tensile tests revealing similar strength and stiffness (0.39 ± 0.05 MPa and 1.61 ± 0.19 MPa, respectively) to the human aorta. Moreover, AL:PE tubular conduits of similar geometrical dimensions to segments of the aorta were produced, either via conventional moulding methods or stereolithography (SLA) 3D-printing. While both fabrication methods successfully demonstrated AL:PE hydrogel production, SLA 3D-printing was more easily adaptable to the fabrication of complex structures without the need of specific moulds or further post-processing. Additionally, most 3D-printed AL:PE hydrogel tubular conduits sustained, without failure, compression up to 50% their outer diameter and returned to their original shape upon load removal, thereby exhibiting promising behaviour that could withstand pulsatile pressure in vivo. Overall, these results suggest that this AL:PE IPN hydrogel formulation in combination with 3D-printing, has great potential for accelerating progress towards personalised and mechanically-matched aortic grafts.

Understanding the Role of Biofilms in Acute Recurrent Tonsillitis through 3D Bioprinting of a Novel Gelatin-PEGDA Hydrogel.

Acute recurrent tonsillitis is a chronic, biofilm-related infection that is a significant burden to patients and healthcare systems. It is often treated with repeated courses of antibiotics, which contributes to antimicrobial resistance. Studying biofilms is key to understanding this disease. In vitro modelling using 3D bioprinted hydrogels is a promising approach to achieve this. A novel gelatin-PEGDA pseudomonas fluorescens-laden bioink was developed and bioprinted in a 3D hydrogel construct fabricated using computer-aided design to mimic the tonsillar biofilm environment. The bioprinted constructs were cultured at 37 °C in lysogeny broth for 12 days. Bacterial growth was assessed by spectrophotometry. Cellular viability analysis was conducted using optical fluorescence microscopy (FDA/PI staining). A biocompatible 3D-printed bacteria-laden hydrogel construct was successfully fabricated. Bacterial growth was observed using optical fluorescence microscopy. A live/dead cellular-staining protocol demonstrated bacterial viability. Results obtained after the 12-day culture period showed higher bacterial growth in the 1% gelatin concentration construct compared to the 0% control. This study demonstrates the first use of a bacteria-laden gelatin-PEGDA hydrogel for biofabrication of a 3D-printed construct designed to model acute recurrent tonsillitis. Initiating a study with clinically relevant ex vivo tonsil bacteria will be an important next step in improving treatment of this impactful but understudied disease.

Stratified tissue biofabrication by rotational internal flow layer engineering.

The bioassembly of layered tissue that closely mimics human histology presents challenges for tissue engineering. Existing bioprinting technologies lack the resolution and cell densities necessary to form the microscale cell-width layers commonly observed in stratified tissue, particularly when using low-viscosity hydrogels, such as collagen. Here we present rotational internal flow layer engineering (RIFLE), a novel, low-cost biofabrication technology for assembling tuneable, multi-layered tissue-like structures. Using high-speed rotating tubular moulds, small volumes of cell-laden liquids added to the inner surface were transitioned into thin layers and gelled, progressively building macroscale tubes composed of discrete microscale strata with thicknesses a function of rotational speed. Cell encapsulation enabled the patterning of high-density layers (108cells ml-1) into heterogenous constructs. RIFLE versatility was demonstrated through tunica media assembly, encapsulating human smooth muscle cells in cell-width (12.5µm) collagen layers. Such deposition of discrete microscale layers, facilitates the biofabrication of composite structures mimicking the nature of native stratified tissue. This enabling technology has the potential to allow researchers to economically create a range of representative layered tissue.

Building Osteogenic Microenvironments with a Double-Network Composite Hydrogel for Bone Repair.

The critical factor determining the in vivo effect of bone repair materials is the microenvironment, which greatly depends on their abilities to promote vascularization and bone formation. However, implant materials are far from ideal candidates for guiding bone regeneration due to their deficient angiogenic and osteogenic microenvironments. Herein, a double-network composite hydrogel combining vascular endothelial growth factor (VEGF)-mimetic peptide with hydroxyapatite (HA) precursor was developed to build an osteogenic microenvironment for bone repair. The hydrogel was prepared by mixing acrylated ß-cyclodextrins and octacalcium phosphate (OCP), an HA precursor, with gelatin solution, followed by ultraviolet photo-crosslinking. To improve the angiogenic potential of the hydrogel, QK, a VEGF-mimicking peptide, was loaded in acrylated ß-cyclodextrins. The QK-loaded hydrogel promoted tube formation of human umbilical vein endothelial cells and upregulated the expression of angiogenesis-related genes, such as Flt1, Kdr, and VEGF, in bone marrow mesenchymal stem cells. Moreover, QK could recruit bone marrow mesenchymal stem cells. Furthermore, OCP in the composite hydrogel could be transformed into HA and release calcium ions facilitating bone regeneration. The double-network composite hydrogel integrated QK and OCP showed obvious osteoinductive activity. The results of animal experiments showed that the composite hydrogel enhanced bone regeneration in skull defects of rats, due to perfect synergistic effects of QK and OCP on vascularized bone regeneration. In summary, improving the angiogenic and osteogenic microenvironments by our double-network composite hydrogel shows promising prospects for bone repair.

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 100 µm or 200 µm. 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 200 µm to 500 µm 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.

Endothelialized microvessels fabricated by microfluidics facilitate osteogenic differentiation and promote bone repair.

In bone tissue engineering, vascularization is one of the critical factors that limit the effect of biomaterials for bone repair. While various approaches have been tried to build vascular networks in bone grafts, lack of endothelialization still constitutes a major technical hurdle. In this study, we have developed a facile technique to fabricate endothelialized biomimetic microvessels (BMVs) from alginate-collagen composite hydrogels within a single step using microfluidic technology. BMVs with different sizes could be readily prepared by adjusting the flow rate of microfluids. All BMVs supported perfusion and outward penetration of substances in the tube. Endothelial cells could adhere and proliferate on the inner wall of tubes. It was also found that the expression of CD31 and secretion of BMP-2 and PDGF-BB were higher in the rat umbilical vein endothelial cells (RUVECs) in BMVs than those cultured on hydrogel. When co-cultured with bone marrow mesenchymal stem cells (BMSCs), endothelialized BMVs promoted the osteogenic differentiation of BMSCs compared to those in acellular BMV group. In vivo, markedly enhanced new bone formation was achieved by endothelialized BMVs in a rat critical-sized calvarial defect model compared to those with non-endothelialized BMVs or without BMVs. Together, findings from both in vitro and in vivo studies have proven that endothelialized BMVs function to facilitate osteogenesis and promote bone regeneration, and therefore might present an effective strategy in bone tissue engineering. STATEMENT OF SIGNIFICANCE: In bone tissue engineering, limited vascularization is one of the critical factors that limit the effect of biomaterials for bone repair. In this study, we developed a facile technique to fabricate endothelialized biomimetic microvessels (BMVs) from alginate-collagen composite hydrogels within a single step using microfluidic technology. Both in vitro and in vivo studies have proven that endothelialized BMVs function to facilitate osteogenesis and promote bone regeneration, and therefore might present an effective strategy in bone tissue engineering.

A Bioprinted Heart-on-a-Chip with Human Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Evaluation.

In this work, we show that valve-based bioprinting induces no measurable detrimental effects on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The aim of the current study was three-fold: first, to assess the response of hiPSC-CMs to several hydrogel formulations by measuring electrophysiological function; second, to customise a new microvalve-based cell printing mechanism in order to deliver hiPSC-CMs suspensions, and third, to compare the traditional manual pipetting cell-culture method and cardiomyocytes dispensed with the bioprinter. To achieve the first and third objectives, iCell2 (Cellular Dynamics International) hiPSC-CMs were used. The effects of well-known drugs were tested on iCell2 cultured by manual pipetting and bioprinting. Despite the results showing that hydrogels and their cross-linkers significantly reduced the electrophysiological performance of the cells compared with those cultured on fibronectin, the bio-ink droplets containing a liquid suspension of live cardiomyocytes proved to be an alternative to standard manual handling and could reduce the number of cells required for drug testing, with no significant differences in drug-sensitivity between both approaches. These results provide a basis for the development of a novel bioprinter with nanolitre resolution to decrease the required number of cells and to automate the cell plating process.

CD271 antibody-functionalized microspheres capable of selective recruitment of reparative endogenous stem cells for in situ bone regeneration.

In the strategy of in situ bone regeneration, it used to be difficult to specifically recruit bone marrow mesenchymal stem cells (BM-MSCs) by a single marker. Recently, CD271 has been considered to be one of the most specific markers to isolate BM-MSCs; however, the effectiveness of CD271 antibodies in recruiting BM-MSCs has not been explored yet. In this study, we developed novel CD271 antibody-functionalized chitosan (CS) microspheres with the aid of polydopamine (PDA) coating to recruit endogenous BM-MSCs for in situ bone regeneration. The CS microspheres were sequentially modified with PDA and CD271 antibody through dopamine self-polymerization and bioconjugation, respectively. In vitro studies showed that the CD271 antibody-functionalized microspheres selectively captured significantly more BM-MSCs from a fluorescently labeled heterotypic cell population than non-functionalized controls. In addition, the PDA coating was critical for supporting stable adhesion and proliferation of the captured BM-MSCs. Effective early recruitment of CD271+ stem cells by the functionalized microspheres at bone defect site of SD rat was observed by the CD271/DAPI immunofluorescence staining, which led to significantly enhanced new bone formation in rat femoral condyle defect over long term. Together, findings from this study have demonstrated, for the first time, that the CD271 antibody-functionalized CS microspheres are promising for in situ bone regeneration.

3D Bioprinting of Complex, Cell-laden Alginate Constructs.

Biofabrication has been receiving a great deal of attention in tissue engineering and regenerative medicine either by manual or automated processes. Different automated biofabrication techniques have been used to produce cell-laden alginate hydrogel structures, especially bioprinting approaches. These approaches have been limited to 2D or simple 3D structures, however. In this chapter, a novel bioprinting technique is disclosed for the production of more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium ion cross-linking for rigidity of the alginate hydrogel immediately after printing, and tertiary barium ion cross-linking for the long-term stability of the alginate hydrogel in the culture medium.

3D biofabrication for soft tissue and cartilage engineering.

Soft tissue injuries (STIs) affect patients of all age groups and represent a common worldwide clinical problem, resulting from conditions including trauma, infection, cancer and burns. Within the spectrum of STIs a mixture of tissues can be injured, ranging from skin to underlying nerves, blood vessels, tendons and cartilaginous tissues. However, significant limitations affect current treatment options and clinical demand for soft tissue and cartilage regenerative therapies continues to rise. Improving the regeneration of soft tissues has therefore become a key area of focus within tissue engineering. As an emerging technology, 3D bioprinting can be used to build complex soft tissue constructs "from the bottom up," by depositing cells, growth factors, extracellular matrices and other biomaterials in a layer-by-layer fashion. In this way, regeneration of cartilage, skin, vasculature, nerves, tendons and other bodily tissues can be performed in a patient specific manner. This review will focus on recent use of 3D bioprinting and other biofabrication strategies in soft tissue repair and regeneration. Biofabrication of a variety of soft tissue types will be reviewed following an overview of available cell sources, bioinks and bioprinting techniques.

The bioprinting roadmap.

This bioprinting roadmap features salient advances in selected applications of the technique and highlights the status of current developments and challenges, as well as envisioned advances in science and technology, to address the challenges to the young and evolving technique. The topics covered in this roadmap encompass the broad spectrum of bioprinting; from cell expansion and novel bioink development to cell/stem cell printing, from organoid-based tissue organization to bioprinting of human-scale tissue structures, and from building cell/tissue/organ-on-a-chip to biomanufacturing of multicellular engineered living systems. The emerging application of printing-in-space and an overview of bioprinting technologies are also included in this roadmap. Due to the rapid pace of methodological advancements in bioprinting techniques and wide-ranging applications, the direction in which the field should advance is not immediately clear. This bioprinting roadmap addresses this unmet need by providing a comprehensive summary and recommendations useful to experienced researchers and newcomers to the field.

Rapid antibiotic susceptibility testing using low-cost, commercially available screen-printed electrodes.

Antimicrobial resistance (AMR) is an issue of upmost global importance, with an annually increasing mortality rate and growing economic burden. Poor antimicrobial stewardship has resulted in an abundance and diverse range of antimicrobial resistance mechanisms. To tackle AMR effectively, better diagnostic tests must be developed in order to improve antibiotic stewardship and reduce the emergence of antibiotic resistant organisms. This study employs a low-cost, commercially available screen printed electrode modified with an agarose-based hydrogel deposit to monitor bacterial growth using the techniques of electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) giving rise to a new approach to measuring susceptibility. Susceptible and drug resistant Staphylococcus aureus strains were deposited onto agarose gel modified electrodes which contained clinically important antibiotics to establish growth profiles for each bacterial strain and monitor the influence of the antibiotic on bacterial growth. The results show that S. aureus is able to grow on electrodes modified with gel containing no antibiotic, but is inhibited when the gel modified electrode is seeded with antibiotic. Conversely, methicillin-resistant S. aureus (MRSA; the drug resistant strain) is able to grow on gel modified electrodes containing clinically relevant concentrations of antibiotic. Results show rapid growth profiles, with possible time to results for antibiotic susceptibility <45 min, a significant improvement on the current gold standards of at least 1-2 days.

3D bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing.

The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated 3D bioprinting of thicker biofilms (>4 mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 d in culture. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had denser biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E. coli biofilms had greater resistance to tetracycline than thinner constructs over 7 d of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

Microfluidic Fabrication of Biomimetic Helical Hydrogel Microfibers for Blood-Vessel-on-a-Chip Applications.

Nature has created many perfect helical microstructures, including DNA, collagen fibrils, and helical blood vessels, to achieve unique physiological functions. While previous studies have developed a number of microfabrication strategies, the preparation of complex helical structures and cell-laden helical structures for biomimetic applications remains challenging. In this study, a one-step microfluidics-based methodology is presented for preparing complex helical hydrogel microfibers and cell-laden helical hydrogel microfibers. Several types of complex helical structures, including multilayer helical microfibers and superhelical hollow microfibers with helical channels, are prepared by simply tuning the flow rates or modifying the geometry of microfluidic device. With the decent perfusability, the hollow microfibers may simulate the structural characteristics of helical blood vessels and create swirling blood flow in a blood-vessel-on-chip setup. Such hydrogel-based helical microstructures may potentially be used in areas such as blood vessel tissue engineering, organ-on-chips, drug screening, and biological actuators.

Microfluidics-Based Fabrication of Cell-Laden Hydrogel Microfibers for Potential Applications in Tissue Engineering.

Fibrous hydrogel scaffolds have recently attracted increasing attention for tissue engineering applications. While a number of approaches have been proposed for fabricating microfibers, it remains difficult for current methods to produce materials that meet the essential requirements of being simple, flexible and bio-friendly. It is especially challenging to prepare cell-laden microfibers which have different structures to meet the needs of various applications using a simple device. In this study, we developed a facile two-flow microfluidic system, through which cell-laden hydrogel microfibers with various structures could be easily prepared in one step. Aiming to meet different tissue engineering needs, several types of microfibers with different structures, including single-layer, double-layer and hollow microfibers, have been prepared using an alginate-methacrylated gelatin composite hydrogel by merely changing the inner and outer fluids. Cell-laden single-layer microfibers were obtained by subsequently seeding mouse embryonic osteoblast precursor cells (MC3T3-E1) cells on the surface of the as-prepared microfibers. Cell-laden double-layer and hollow microfibers were prepared by directly encapsulating MC3T3-E1 cells or human umbilical vein endothelial cells (HUVECs) in the cores of microfibers upon their fabrication. Prominent proliferation of cells happened in all cell-laden single-layer, double-layer and hollow microfibers, implying potential applications for them in tissue engineering.

3D biofabrication for tubular tissue engineering.

The therapeutic replacement of diseased tubular tissue is hindered by the availability and suitability of current donor, autologous and synthetically derived protheses. Artificially created, tissue engineered, constructs have the potential to alleviate these concerns with reduced autoimmune response, high anatomical accuracy, long-term patency and growth potential. The advent of 3D bioprinting technology has further supplemented the technological toolbox, opening up new biofabrication research opportunities and expanding the therapeutic potential of the field. In this review, we highlight the challenges facing those seeking to create artificial tubular tissue with its associated complex macro- and microscopic architecture. Current biofabrication approaches, including 3D printing techniques, are reviewed and future directions suggested.

3D bioactive composite scaffolds for bone tissue engineering.

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. Developing bioactive three-dimensional (3D) scaffolds to support bone regeneration has therefore become a key area of focus within bone tissue engineering (BTE). A variety of materials and manufacturing methods including 3D printing have been used to create novel alternatives to traditional bone grafts. However, individual groups of materials including polymers, ceramics and hydrogels have been unable to fully replicate the properties of bone when used alone. Favourable material properties can be combined and bioactivity improved when groups of materials are used together in composite 3D scaffolds. This review will therefore consider the ideal properties of bioactive composite 3D scaffolds and examine recent use of polymers, hydrogels, metals, ceramics and bio-glasses in BTE. Scaffold fabrication methodology, mechanical performance, biocompatibility, bioactivity, and potential clinical translations will be discussed.

Three-dimensional bioprinting of stem-cell derived tissues for human regenerative medicine.

Stem cell technology in regenerative medicine has the potential to provide an unlimited supply of cells for drug testing, medical transplantation and academic research. In order to engineer a realistic tissue model using stem cells as an alternative to human tissue, it is essential to create artificial stem cell microenvironment or niches. Three-dimensional (3D) bioprinting is a promising tissue engineering field that offers new opportunities to precisely place stem cells within their niches layer-by-layer. This review covers bioprinting technologies, the current development of 'bio-inks' and how bioprinting has already been applied to stem-cell culture, as well as their applications for human regenerative medicine. The key considerations for bioink properties such as stiffness, stability and biodegradation, biocompatibility and printability are highlighted. Bioprinting of both adult and pluriopotent stem cells for various types of artificial tissues from liver to brain has been reviewed. 3D bioprinting of stem-cell derived tissues for human regenerative medicine is an exciting emerging area that represents opportunities for new research, industries and products as well as future challenges in clinical translation.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Biofabrication: A Guide to Technology and Terminology.

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.

Additive Manufacturing: Unlocking the Evolution of Energy Materials.

The global energy infrastructure is undergoing a drastic transformation towards renewable energy, posing huge challenges on the energy materials research, development and manufacturing. Additive manufacturing has shown its promise to change the way how future energy system can be designed and delivered. It offers capability in manufacturing complex 3D structures, with near-complete design freedom and high sustainability due to minimal use of materials and toxic chemicals. Recent literatures have reported that additive manufacturing could unlock the evolution of energy materials and chemistries with unprecedented performance in the way that could never be achieved by conventional manufacturing techniques. This comprehensive review will fill the gap in communicating on recent breakthroughs in additive manufacturing for energy material and device applications. It will underpin the discoveries on what 3D functional energy structures can be created without design constraints, which bespoke energy materials could be additively manufactured with customised solutions, and how the additively manufactured devices could be integrated into energy systems. This review will also highlight emerging and important applications in energy additive manufacturing, including fuel cells, batteries, hydrogen, solar cell as well as carbon capture and storage.