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Autologous fat grafting offers significant promise for the repair of soft tissue deformities; however, high resorption rates indicate that engineered solutions are required to improve adipose tissue (AT) survival. Advances in material development and biofabrication have laid the foundation for the generation of functional AT constructs; however, a balance needs to be struck between clinically feasible delivery and improved structural integrity of the grafts. A new approach combining the objectives from both the clinical and research communities will assist in developing morphologically and genetically mature AT constructs, with controlled spatial arrangement and increased potential for neovascularization. In a rapidly progressing field, this review addresses research in both the preclinical and bioengineering domains and assesses their ability to resolve functional challenges.

There remains a critical need to develop new technologies and materials that can meet the demands of treating large bone defects. The advancement of 3-dimensional (3D) printing technologies has allowed the creation of personalized and customized bone grafts, with specific control in both macro- and micro-architecture, and desired mechanical properties. Nevertheless, the biomaterials used for the production of these bone grafts often possess poor biological properties. The incorporation of growth factors (GFs), which are the natural orchestrators of the physiological healing process, into 3D printed bone grafts, represents a promising strategy to achieve the bioactivity required to enhance bone regeneration. In this review, the possible strategies used to incorporate GFs to 3D printed constructs are presented with a specific focus on bone regeneration. In particular, the strengths and limitations of different methods, such as physical and chemical cross-linking, which are currently used to incorporate GFs to the engineered constructs are critically reviewed. Different strategies used to present one or more GFs to achieve simultaneous angiogenesis and vasculogenesis for enhanced bone regeneration are also covered in this review. In addition, the possibility of combining several manufacturing approaches to fabricate hybrid constructs, which better mimic the complexity of biological niches, is presented. Finally, the clinical relevance of these approaches and the future steps that should be taken are discussed.

The biophysical properties of biomaterials are key to directing the biological responses and biomaterial integration and function in in situ tissue engineering approaches. We present silk photo-lyogels, a biomaterial format fabricated using a new combinatorial approach involving photo-initiated crosslinking of silk fibroin via di-tyrosine bonds followed by lyophilization to generate 3D, porous lyogels showing physical properties distinct to those of lyophilized silk sponges or silk hydrogels. This fabrication approach allowed introduction of microchannels into 3D constructs via biofabrication approaches involving silk crosslinking around an array of 3D printed photocurable resin pillars to generate parallel channels or around a 3D printed sacrificial thermosensitive gel to generate interconnected channels in a rapid manner and without the need for chemical modification of silk fibroin. The presence of interconnected microchannels significantly improved migration of endothelial cells into 3D photo-lyogels in vitro, and tissue infiltration, photo-lyogel integration, and vascularization when implanted in vivo in a mouse subcutaneous model. Taken together, these findings demonstrate the feasibility and utility of a new combinatorial fabrication approach for generation of silk biomaterials that support cell interactions and implant integration for in situ tissue engineering approaches.

The rise of antibiotic resistance, coupled with increased expectations for mobility in later life, is creating a need for biofilm inhibitors and delivery systems that will reduce surgical implant infection. A limitation of some of these existing delivery approaches is toxicity exhibited toward host cells. Here, we report the application of a novel inhibitor of the enzyme, methylthioadenosine nucleosidase (MTAN), a key enzyme in bacterial metabolic pathways, which include S-adenosylmethionine catabolism and purine nucleotide recycling, in combination with a poly(vinyl alcohol)-tyramine-based (PVA-Tyr) hydrogel delivery system. We demonstrate that a lead MTAN inhibitor, selected from a screened library of 34 candidates, (2S)-2-(4-amino-5H-pyrrolo3,2-dpyrimidin-7-ylmethyl)aminoundecan-1-ol (31), showed a minimum biofilm inhibitory concentration of 2.2 ± 0.4 μM against a clinical staphylococcal species isolated from an infected implant. We observed that extracellular DNA, a key constituent of biofilms, is significantly reduced when treated with 10 μM compound 31, along with a decrease in biofilm thickness. Compound 31 was incorporated into a hydrolytically degradable photo-cross-linked PVA-Tyr hydrogel and the release profile was evaluated by HPLC studies. Compound 31 released from the PVA-hydrogel system significantly reduced biofilm formation (77.2 ± 8.4% biofilm inhibition). Finally, compound 31 released from PVA-Tyr showed no negative impact on human bone marrow stromal cell (MSC) viability, proliferation, or morphology. The results demonstrate the potential utility of MTAN inhibitors in treating infections caused by Gram-positive bacteria, and the development of a nontoxic release system that has potential for tunability for time scale of delivery.

Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses due to their simplistic organization and lack of stimuli. The rise of biofabrication and bioreactor technologies has sought to address this through the development of techniques to spatially organize components of a tissue construct, and devices to supply these tissue constructs with an increasingly in vivo-like environment. Current bioreactors supporting both parenchymal and barrier tissue constructs in interconnected systems for body-on-a-chip platforms have chosen to emphasize study throughput or system/tissue complexity. Here, we report a platform to address this disparity in throughput and both system complexity (by supporting multiple in situ assessment methods) and tissue complexity (by adopting a construct-agnostic format). We introduce an ANSI/SLAS-compliant microplate and docking station fabricated via stereolithography (SLA), or precision machining, to provide up to 96 samples (Ø6 × 10 mm) with two individually-addressable fluid circuits (192 total), loading access, and inspection window for imaging during perfusion. Biofabricated ovarian cancer models were developed to demonstrate the in situ assessment capabilities via microscopy and a perfused resazurin-based metabolic activity assay. In situ microscopy highlighted flexibility of the sample housing to accommodate a range of sample geometries. Utility for drug screening was demonstrated by exposing the ovarian cancer models to an anticancer drug (doxorubicin) and generating the dose-response curve in situ, while achieving an assay quality similar to static wellplate culture. The potential for quantitative analysis of temporal tissue development and screening studies was confirmed by imaging soft- (gelatin) and hard-tissue (calcium chloride) analogs inside the bioreactor via spectral computed tomography (CT) scanning. As a proof-of-concept for particle tracing studies, flowing microparticles were visualized to inform the design of hydrogel constructs. Finally, the ability for mechanistic yet high-throughput screening was demonstrated in a vascular coculture model adopting endothelial and mesenchymal stem cells (HUVEC-MSC), encapsulated in gelatin-norbornene (gel-NOR) hydrogel cast into SLA-printed well inserts. This study illustrates the potential of a scalable dual perfusion bioreactor platform for parenchymal and barrier tissue constructs to support a broad range of multi-organ-on-a-chip applications.

Evolution of metallurgy and biomaterials has progressively shifted the focus of metallic bone-interfacing implant design from adequate mechanical strength and biocompatibility to rapid osseointegration and infection inhibition. The now relatively well-established technology - powder bed additive manufacturing (AM), offers the ability to fabricate porous implants with precise mechanical properties, topological pore architectures and patient-specific design functions, has revolutionized the production of customized multifunctional metallic implants for the individual patient with anatomic-specific requirement. Even though AM titanium and its alloy Ti-6Al-4V have been investigated and adopted for clinical application for decades, the development of porous AM titanium implants is far from complete and further research is required to achieve excellent long-term clinical performance. In this review, we summarize the current status of AM in bone-interfacing implant fabrication, with particular focus on the experimental outcomes of various factors that influence osseointegration, bone and vascular ingrowth as well as hybrid strategies to combat infection, including: pore size, porosity, pore structure, surface modification techniques and incorporation of biological factors. In addition, we also discuss the osteogenic capacity of constructs fabricated through different manufacturing methods and titanium alloys. To this end, we highlight the exciting prospect of AM for bone-interfacing implant manufacture through optimization via material development, implant design, bio-functionalization to clinical evaluation to provide enhanced patient specificity and long-term function.

Microphysiological systems (MPSs) have been proposed as an improved tool to recreate the complex biological features of the native niche with the goal of improving in vitro–in vivo extrapolation. In just over a decade, MPS technologies have progressed from single-tissue chips to multitissue plates with integrated pumps for perfusion. Concurrently, techniques for biofabrication of complex 3D constructs for regenerative medicine and 3D in vitro models have evolved into a diverse toolbox for micrometer-scale deposition of cells and cell-laden bioinks. However, as the complexity of biological models increases, experimental throughput is often compromised. This review discusses the existing disparity between MPS complexity and throughput, then examines an MPS-terminated biofabrication line to identify the hurdles and potential approaches to overcoming this disparity.

Decellularisation of tissues, utilising their biochemical cues, poses exciting tissue engineering (TE) opportunities. However, removing DNA from cartilage (dCart) requires harsh treatments due to its dense structure, causing loss of bioactivity and limiting its application as a cartilaginous extra cellular matrix (ECM). In this study, we demonstrate for the first time the successful application of vitreous humor (VH), a highly hydrated tissue closely resembling the glycosaminoglycan (GAG) and collagen composition of cartilage, as an ECM hydrogel to support chondrogenic differentiation. Equine VH was extracted followed by biochemical quantifications, histological examinations, cytotoxicity (human mesenchymal stromal cells, hMSCs and human articular chondrocytes, hACs) and U937 cell proliferation studies. VH was further seeded with hACs or hMSCs and cultured for 3-weeks to study chondrogenesis compared to scaffold-free micro-tissue pellet cultures and collagen-I hydrogels. Viability, metabolic activity, GAG and DNA content, chondrogenic gene expression (aggrecan, collagen I/II mRNA) and mechanical properties were quantified and matrix deposition was visualised using immunohistochemistry (Safranin-O, collagen I/II). VH was successfully extracted, exhibiting negligible amounts of DNA (0.4 ± 0.4 µg/mg dry-weight) and notable preservation of ECM components. VH displayed neither cytotoxic responses nor proliferation of macrophage-like U937 cells, instead enhancing both hMSC and hAC proliferation. Interestingly, encapsulated cells self-assembled the VH-hydrogel into spheroids, resulting in uniform distribution of both GAGs and collagen type II with increased compressive mechanical properties, rendering VH a permissive native ECM source to fabricate cartilaginous hydrogels for potential TE applications. STATEMENT OF SIGNIFICANCE: Fabricating bioactive and cell-instructive cartilage extracellular matrix (ECM) derived biomaterials and hydrogels has over recent years proven to be a challenging task, often limited by poor retention of inherent environmental cues post decellularisation due to the dense and avascular nature of native cartilage. In this study, we present an alternative route to fabricate highly permissive and bioactive ECM hydrogels from vitreous humor (VH) tissue. This paper specifically reports the discovery of optimal VH extraction protocols and cell seeding strategy enabling fabrication of cartilaginous matrix components into a hydrogel support material for promoting chondrogenic differentiation. The work showcases a naturally intact and unmodified hydrogel design that improves cellular responses and may help guide the development of cell instructive and stimuli responsive hybrid biomaterials in a number of TERM applications.

Microchannels are simple, perfusable architectural features engineered into biomaterials to promote mass transport of solutes to cells, effective cell seeding and compartmentalisation for tissue engineering applications, control over spatiotemporal distribution of molecules and ligands, and survival, integration, and vascularisation of engineered tissue analogues in vivo. Advances in biofabrication have led to better control over microchannel fabrication in 3D scaffolds, enabling sophisticated designs that drive the development of complex tissue structures. This review addresses the importance of microchannel structures in biomaterial design and regenerative medicine, and discusses their function, fabrication methods, and proposed mechanisms underlying their effects.

Radical photo-cross-linking of polymers has been at the forefront in the development of biomedical applications to meet many of the biomaterial design criteria needed to address clinical and healthcare challenges, particularly in relation to regenerative and restorative medicine strategies, to treat damaged or diseased tissues and organs. Exciting new hybrid designs, elegantly expanding the range of properties and applications available to individual polymeric materials, are starting to offer detailed customization with complex and dynamic interactions similar to the events occurring within native tissue microenvironments in vivo. Yet the variety in success reported in the literature highlights the many unknown design criteria and parameters affecting functional restoration of damaged tissues and organs. The applied light curing units and radical initiating system, as well as underlying chemical reactions and resultant network structures, all require detailed consideration as means to modulate biological function while further being assessed as sources of toxicity. This is especially important when cells embedded in the polymeric material (or in surrounding tissues) are directly exposed to photo-irradiation. Ultimately, achieving successful clinical translation necessitates the chemical photo-polymerization platforms to be efficacious, safe, and customizable but also convenient for clinical use and cost-effective production. This review thus aims to summarize current and emerging toolkits to photo-polymerize biomedical polymers requiring the direct irradiation of cells and/or mammalian tissues and its associated impact on biological functionality. This specifically includes (1) in vitro photo-polymerization of cell-laden 3D-hydrogels for tissue engineering and regeneration medicine applications, (2) in vivo transdermal photo-polymerization of injectable hydrogels for cell/drug delivery, and (3) in vivo photo-polymerization of cell-free, injectable resin-based composites for load-bearing restorative surgery, all fast-growing and highly competitive fields of modern medicine. We herein summarize both design considerations and biological risk factors associated with selecting suitable light sources, photo-initiators, functional groups, chemical propagation, as well as how subsequent network properties can modulate biological function and ultimately clinical applicability. As more knowledge is continuously accumulated through materials science, matrix biology, and technology, this review provides recommendations for researchers to extend their chemical, biological, and structural characterizations to systematically enrich the paradigm of photo-polymerizable materials for biomedical applications to help ensure efficient and safe radical photo-processing.

Oxygen inhibition is a phenomenon that directly impacts the print fidelity of 3D biofabricated and photopolymerized hydrogel constructs. It typically results in the undesirable physical collapse of fabricated constructs due to impaired cross-linking, and is an issue that generally remains unreported in the literature. In this study, we describe a systematic approach to minimizing oxygen inhibition in photopolymerized gelatin-methacryloyl (Gel-MA)-based hydrogel constructs, by comparing a new visible-light initiating system, Vis + ruthenium (Ru)/sodium persulfate (SPS) to more conventionally adopted ultraviolet (UV) + Irgacure 2959 system. For both systems, increasing photoinitiator concentration and light irradiation intensity successfully reduced oxygen inhibition. However, the UV + I2959 system was detrimental to cells at both high I2959 concentrations and UV light irradiation intensities. The Vis + Ru/SPS system yielded better cell cyto-compatibility, where encapsulated cells remained85% viable even at high Ru/SPS concentrations and visible-light irradiation intensities for up to 21 days, further highlighting the potential of this system to biofabricate cell-laden constructs with high shape fidelity, cell viability, and metabolic activity.

3D bioprinting of constructs for tissue engineering and regenerative medicine has steadily gained attention due to its potential to fabricate anatomically-precise living constructs, localise specific cell types and enable the regeneration of functional tissues in a clinical setting. However, the limited availability of bioinks that can be successfully 3D bioprinted with high fidelity and simultaneously provide encapsulated cells with a tailored, low-stiffness microenvironment supporting functional tissue formation remains an unmet challenge. To address both the physical and biological limitations of available bioinks, this study aimed to develop a nanocomposite bioink (Sr-GelMA) comprising of strontium-carbonate (Sr) nanoparticles and low concentration (5 w/v%) gelatin-methacryloyl (GelMA) hydrogel for extrusion-based 3D bioprinting of low-stiffness cell-laden scaffolds with high shape fidelity and bone-specific cell signalling factors. We systematically investigated the effect of Sr incorporation on hydrogel physico-chemical properties, print fidelity, scaffold shape retention, as well as cell viability, osteogenic differentiation and in vitro bone formation. Nanocomposite Sr-GelMA hydrogels retained their physical and mechanical properties, while rheological studies revealed a significant increase in viscosity profiles that led to notably enhanced printability compared to GelMA alone. Moreover, bioprinted Sr-GelMA scaffolds exhibited excellent shape fidelity evidenced by a defined pore geometry on the x-y-z axis, resulting in an interconnected bioink filament and pore network that was maintained even after long-term culture and osteogenic differentiation (28 days) of human mesenchymal stromal cells (hMSCs). The presence of clustered Sr nanoparticles in the cell-laden bioink allowed high quality bioprinting combined with high hMSC viability (>95%) post-fabrication. Furthermore, Sr addition resulted in enhanced osteogenic differentiation of hMSCs as revealed by higher alkaline phosphatase (ALP) levels, osteocalcin (OCN) and collagen type-I (Col I) expression, with mineralised nodule formation distributed homogenously throughout the bioprinted construct. This study demonstrated that strontium-based nanocomposite bioinks optimised for extrusion-based 3D bioprinting of osteoconductive scaffolds support long-term shape retention with promising potential for bone tissue regeneration.

Growth factors (GFs) are often a key component in tissue engineering and regenerative medicine approaches. In order to fully exploit the therapeutic potential of GFs, GF delivery vehicles have to meet a number of key design criteria such as providing localized delivery and mimicking the dynamic native GF expression levels and patterns. The use of biomaterials as delivery systems is the most successful strategy for controlled delivery and has been translated into different commercially available systems. However, the risk of side effects remains an issue, which is mainly attributed to insufficient control over the release profile. This book chapter reviews the current strategies, chemistries, materials and delivery vehicles employed to overcome the current limitations associated with GF therapies.

Multicomponent gelatin-methacryloyl (GelMA) hydrogels are regularly adopted for cartilage tissue engineering (TE) applications, where optimizing chemical modifications for preserving biofunctionality is often overlooked. This study investigates the biological effect of two different modification methods, methacrylation and thiolation, to copolymerize GelMA and heparin. The native bioactivity of methacrylated heparin (HepMA) and thiolated heparin (HepSH) is evaluated via thromboplastin time and heparan sulfate-deficient myeloid cell-line proliferation assay, demonstrating that thiolation is superior for preserving anticoagulation and growth factor signaling capacity. Furthermore, incorporating either HepMA or HepSH in chondrocyte-laden GelMA hydrogels, cultured for 5 weeks under chondrogenic conditions, promotes cell viability and chondrocyte phenotype. However, only GelMA-HepSH hydrogels yield significantly greater differentiation and matrix deposition in vitro compared to GelMA. This study demonstrates that thiol-ene chemistry offers a favorable strategy for incorporating bioactives into gelatin hydrogels as compared to methacrylation while furthermore highlighting GelMA-HepSH hydrogels as candidates for cartilage TE applications.

Biofabrication techniques offer the potential to produce living three-dimensional (3D) tissue constructs to repair or replace damaged or diseased human tissues and organs. Using these advanced Biofabrication techniques, constructs exhibiting spatial variation of cells, cellular building blocks, growth factors, and mechanical properties along multiple axes with high geometric complexity can be obtained. The level of control offered by these technologies to develop complex biofabricated tissues will allow tissue engineers to better study factors that modulate tissue formation and function, and provide a valuable tool to study graft performance as well as for high throughput screening of drug candidates in biofabricated tissue organoid models.

Bioprinting of living cells is rapidly developing as an advanced biofabrication approach to engineer tissues. Bioinks can be extruded in three-dimensions (3D) to fabricate complex and hierarchical constructs for implantation. However, lack of functionality can often be attributed to poor bioink properties. Indeed, advanced bioinks encapsulating living cells should: (i) present optimal rheological properties and retain 3D structure post-fabrication, (ii) promote cell viability and support cell differentiation, (iii) localise proteins of interest (e.g. vascular endothelial growth factor (VEGF)) to stimulate encapsulated cell activity and tissue ingrowth upon implantation. In this study, we present the results of the inclusion of a synthetic nanoclay, Laponite (LPN) together with a gelatin methacryloyl (GelMA) bioink and the development of a functional cell-instructive bioink. A nanocomposite bioink displaying enhanced shape fidelity retention and interconnected porosity within extrusion-bioprinted fibres was observed. Human bone marrow stromal cell (HBMSC) viability within the nanocomposite showed no significant changes over 21 days of culture in LPN-GelMA (85.60 ± 10.27 %), compared to a significant decrease in GelMA from 7 (95.88 ± 2.90 %) to 21 days (55.54 ± 14.72 %) (p

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth-)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross-linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol-ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth-)acryloyl chemistry, and without additional nondegradable components. An in-depth analysis of GelAGE synthesis is conducted, and standard UV-initiation is further compared with a recently described visible-light-initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography-based (digital light processing) 3D printing and extrusion-based 3D bioprinting, the latter supporting long-term viability postprinting of encapsulated chondrocytes.

Background Dityrosine crosslinking in proteins is a bioinspired method of forming hydrogels. This study compares oxidative enzyme initiators for their relative crosslinking efficiency and cytocompatibility using the same phenol group and the same material platform. Four common enzyme and enzyme-like oxidative initiators were probed for resulting material properties and cell viability post-encapsulation. Results All four initiators can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. Horseradish peroxidase (HRP) or hematin with hydrogen peroxide led to a more rapid poly (vinyl alcohol)-tyramine (PVA-Tyr) polymerization (10–60 min) because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and therefore took longer to gel (2.5+ hours). The use of hydrogen peroxide as an oxidant reduced cell viability immediately post-encapsulation. Laccase- and tyrosinase-mediated encapsulation of cells resulted in higher cell viability immediately post-encapsulation and significantly higher cell proliferation after one week of culture. Conclusions Overall this study demonstrates that HRP/H2O2, hematin/H2O2, laccase, and tyrosinase can create injectable, in situ phenol-crosslinked hydrogels, however oxidant type and concentration are critical parameters to assess when phenol crosslinking hydrogels for cell-based applications. Electronic supplementary material The online version of this article (doi:10.1186/s40824-016-0077-z) contains supplementary material, which is available to authorized users.