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Background: The lack of healing at the repaired tendon-bone interface is an important cause of failure after rotator cuff repair. While augmentation with growth factors (GFs) has demonstrated promise, the ideal combination must target all 3 tissue types at the tendon-bone interface. Hypothesis: The GF combination of transforming growth factor beta 1, Insulin-like growth factor 1, and parathyroid hormone will promote tenocyte proliferation and differentiation and improve the biomechanical and histological quality of the repaired tendon-bone interface. Study Design: Controlled laboratory study. Methods: In vitro, human tenocytes were cultured in the presence of the GF combination for 72 hours, and cell growth assays and the expression of genes specific to tendon, cartilage, and bone were analyzed. In vivo, adult rats (N = 46) underwent detachment and repair of the left supraspinatus tendon. A PVA-tyramine gel was used to deliver the GF combination to the tendon-bone interface. Histological, biomechanical, and RNA microarray analysis was performed at 6 and 12 weeks after surgery. Immunohistochemistry for type II and X collagen was performed at 12 weeks. Results: When treated with the GF combination in vitro, human tenocytes proliferated 1.5 times more than control ( P = .04). The expression of scleraxis increased 65-fold ( P = .013). The expression of Sox-9 ( P = .011), type I collagen ( P = .021), fibromodulin ( P = .0075), and biglycan ( P = .010) was also significantly increased, while the expression of PPARγ was decreased ( P = .007). At 6 and 12 weeks postoperatively, the quality of healing on histology was significantly higher in the GF group, with the formation of a more mature tendon-bone interface, as confirmed by immunohistochemistry for type II and X collagen. The GF group achieved a load at failure and Young modulus >1.5 times higher at both time points. Microarrays at 6 weeks demonstrated upregulation of genes involved in leukocyte aggregation ( S100A8, S100A9) and tissue mineralization ( Bglap, serglycin, Fam20c). Conclusion: The GF combination promoted protendon and cartilage responses in human tenocytes in vitro; it also improved the histological appearance and mechanical properties of the repair in vivo. Microarrays of the tendon-bone interface identified inflammatory and mineralization pathways affected by the GF combination, providing novel therapeutic targets for further research. Clinical Relevance: The use of this GF combination is translatable to patients and may improve healing after rotator cuff repair.

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.

Vat polymerization is a form of three-dimensional (3D) printing. Historically, it is the oldest additive manufacturing technique, with the development of stereolithography apparatus (SLA) by Charles Hull in 1986. This article outlines the various forms of vat polymerization techniques used for biomedical applications. Due to the complex nature of this printing process, many key print parameters and material properties need to be considered to ensure a successful print. These influential parameters are addressed throughout the article to inform the reader of the considerations that should be taken when using the vat polymerization technique. The article provides information on vat polymerization printer setup, the photo-cross-linking mechanism, and considerations using vat polymerization. In addition, it outlines and discusses the advancements of vat polymerization in the biomedical industry.

The paracrine signaling, immunogenic properties and possible applications of mesenchymal stromal cells (MSCs) for cartilage tissue engineering and regenerative medicine therapies have been investigated through numerous in vitro, animal model and clinical studies. The emerging knowledge largely supports the concept of MSCs as signaling and modulatory cells, exerting their influence through trophic and immune mediation rather than as a cell replacement therapy. The virtues of allogeneic cells as a ready‐to‐use product with well‐defined characteristics of cell surface marker expression, proliferative ability, and differentiation capacity are well established. With clinical applications in mind, a greater focus on allogeneic cell sources is evident, and this review summarizes the latest published and upcoming clinical trials focused on cartilage regeneration adopting allogeneic and autologous cell sources. Moreover, we review the current understanding of immune modulatory mechanisms and the role of trophic factors in articular chondrocyte‐MSC interactions that offer feasible targets for evaluating MSC activity in vivo within the intra‐articular environment. Furthermore, bringing labeling and tracking techniques to the clinical setting, while inherently challenging, will be extremely informative as clinicians and researchers seek to bolster the case for the safety and efficacy of allogeneic MSCs. We therefore review multiple promising approaches for cell tracking and labeling, including both chimerism studies and imaging‐based techniques, that have been widely explored in vitro and in animal models. Understanding the distribution and persistence of transplanted MSCs is necessary to fully realize their potential in cartilage regeneration techniques and tissue engineering applications., We summarize the latest published and upcoming clinical trials focused on cartilage regeneration adopting allogeneic and autologous cell sources. Moreover, we review the current understanding of immune modulatory mechanisms and trophic factors regulating mesenchymal stromal cell (MSC) activity in vivo, as well as multiple approaches for cell tracking and labeling in vitro and in vivo. Understanding the distribution and persistence of MSCs is necessary to fully realize potential cartilage regeneration techniques and tissue engineering applications.

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.

Biofunctionalization of silk biomaterial surfaces with extracellular matrix (ECM) molecules, cell binding peptides, or growth factors is important in a range of applications, including tissue engineering and development of implantable medical devices. Passive adsorption is the most common way to immobilize molecules of interest on preformed silk biomaterials but can lead to random molecular orientations and displacement from the surface, limiting their applications. Herein, we developed techniques for covalent immobilization of biomolecules using enzyme- or photoinitiated formation of dityrosine bonds between the molecule of interest and silk. Using recombinantly expressed domain V of the human basement membrane proteoglycan perlecan (rDV) as a model molecule, we demonstrated that rDV can be covalently immobilized via dityrosine cross-linking without the need to modify rDV or silk biomaterials. Dityrosine-based immobilization resulted in a different molecular orientation to passively absorbed rDV with less C- and N-terminal region exposure on the surface. Dityrosine-based immobilization supported functional rDV immobilization where immobilized rDV supported endothelial cell adhesion, spreading, migration, and proliferation. These results demonstrate the utility of dityrosine-based cross-linking in covalent immobilization of tyrosine-containing molecules on silk biomaterials in the absence of chemical modification, adding a simple and accessible technique to the silk biofunctionalization toolbox.

The aim of this paper is to outline the impact Coronavirus disease (COVID)-19 has had on health research in New Zealand. We used data from the University of Otago, Christchurch, a research intensiv...

This review provides a detailed overview of the rapidly advancing field of biofabrication, particularly with regards to the use of photo-cross-linking (i.e., light-based) techniques. The major emphasis of this review is on the fundamentals of photo-cross-linking and key criteria identified for the successful design and implementation of photo-cross-linked bioinks and bioresins in extrusion-based and lithography-based bioprinting. The general mechanisms associated with photo-cross-linking (e.g., free-radical chain polymerization, thiol-ene, photomediated redox) of natural and synthetic materials are described to inform bioink and bioresin design, which includes the selection of polymers, functional group modifications, photoinitiators, and light sources that enable facile and cytocompatible photo-cross-linking. Depending on material selection and the bioprinting technique of interest, we describe the specific bioink or bioresin properties and criteria that must be achieved to ensure optimal printability and utility. Finally, examples of current state-of-the-art applications of light-based bioprinting for in vitro tissue models, tissue engineering, and regenerative medicine are provided to further motivate future opportunities within the bioprinting landscape that are facilitated with light.

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 translation of growth factors (GFs) into clinical applications is limited by their low stability in physiological environments. Controlled GF delivery through biomaterial vehicles provides protection from proteases, targeted delivery, and longer term release profiles. However, current methods used to incorporate GFs into biomaterials still present limitations. While direct adsorption and encapsulation result in burst release, covalent incorporation provides a tailorable release profile but generally requires more complicated processes and chemical modification of the GFs. Bioaffinity methods provide long-term release profiles but fail in their specificity, resulting in GF-dependent applicability and release profiles. In the present study, we introduce tyraminated poly-vinyl-alcohol (PVA-Tyr) as a GF-delivery vehicle that can covalently incorporate native GFs through a photo-initiated cross-linking process via formation of bi-phenol bonds. Mass loss and release studies revealed that protein-loaded PVA-Tyr hydrogels had highly tailorable degradation times from 7 to 92 days, during which the covalently incorporated proteins were released in a linear fashion. The incorporation of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or brain-derived growth factor (BDNF) resulted in similar incorporation efficiencies and release profiles, demonstrating the low specificity and versatility of the system. Furthermore, functional studies demonstrated that VEGF, bFGF and BDNF released from the PVA-Tyr hydrogels retained the ability to increase the metabolic activity, migration, and 3D vessel formation of endothelial cells and mesenchymal stem cells. Taken together, this demonstrates that PVA-Tyr shows high potential as a highly tailorable GF delivery tool for a range of different regenerative medicine applications.

The field of bioprinting has made significant advancements in recent years and allowed for the precise deposition of biomaterials and cells. However, within this field lies a major challenge, which is developing high resolution constructs, with complex architectures. In an effort to overcome these challenges a biofabrication technique known as vat polymerization is being increasingly investigated due to its high fabrication accuracy and control of resolution (µm scale). Despite the progress made in developing hydrogel precursors for bioprinting techniques, such as extrusion-based bioprinting, there is a major lack in developing hydrogel precursor bioresins for vat polymerization. This is due to the specific unique properties and characteristics required for vat polymerization, from lithography to the latest volumetric printing. This is of major concern as the shortage of bioresins available has a significant impact on progressing this technology and exploring its full potential, including speed, resolution, and scale. Therefore, this review discusses the key requirements that need to be addressed in successfully developing a bioresin. The influence of monomer architecture and bioresin composition on printability is described, along with key fundamental parameters that can be altered to increase printing accuracy. Finally, recent advancements in bioresins are discussed together with future directions.

The integration of light-driven technologies into biofabrication has revolutionized the field of tissue engineering and regenerative medicine, with numerous breakthroughs in the last few years. Light-based bioprinting approaches (lithography, multiphoton and volumetric bioprinting) have shown the potential to fabricate large scale tissue engineering constructs of high resolution, with great flexibility and control over the cellular organization. Given the unprecedented degree of freedom in fabricating convoluted structures, key challenges in regenerative medicine, such as introducing complex channels and pre-vascular networks in 3D constructs have also been addressed. Light has also been proven as a powerful tool, leading to novel photo-chemistry in designing bioinks, but also able to impart spatial-temporal control over cellular functions through photo-responsive chemistry. For instance, smart constructs able to undergo remotely controlled shape changes, stiffening, softening and degradation can be produced. The non-invasive nature of light stimulation also enables to trigger such responses post-fabrication, during the maturation phase of a construct. Such unique ability can be used to mimic the dynamic processes occurring in tissue regeneration, as well as in disease progression and degenerative processes in vivo. Bringing together these novel multidisciplinary expertise, the present Special Issue aims to discuss the most recent trends, strategies and novel light-based technologies in the field of biofabrication. These include: 1) using light-based bioprinting to develop in vitro models for drug screening, developmental biology models, disease models, and also functional tissues for implantation; 2) novel light-based biofabrication technologies; 3) development of new photo-responsive bioinks or biomaterial inks.

Bone regeneration of critical-sized bone defects, bone fractures or joint replacements remains a significant clinical challenge. Although there has been rapid advancement in both the fields of bone tissue engineering and additive manufacturing, functional bone implants with rapid vascularization capacity to ensure osseointegration and long-term biological fixation in large bone defects remains limited in clinics. In this study, we developed an in vitro vascularized bone implant by combining cell-laden hydrogels with direct metal printed (DMP) porous titanium alloys (Ti–6Al–4V). A 5 wt% allylated gelatin (GelAGE), was utilized to co-encapsulate human mesenchymal stromal cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) to investigate concurrent osteogenic and vasculogenic performance. DMP macro-porous Ti–6Al–4V scaffolds were subsequently infused/enriched with cell-laden GelAGE to examine the feasibility to deliver cells and engineer vascular-like networks in the hybrid implant. Furthermore, as a proof of concept, a full-scale porous Ti–6Al–4V acetabular cup was impregnated with cell-laden hydrogel to validate the clinical potential of this strategy. The vasculogenic potential was evaluated by examining micro-capillary formation coupled with capillary network maturation and stabilization. Osteogenic differentiation was assessed via alkaline phosphatase activity as well as osteocalcin and osteopontin expression. Our results suggested that GelAGE supported HUVECs spreading and vascular-like network formation, along with osteogenesis of hMSCs. Titanium hybrid constructs with cell-laden hydrogel demonstrated enhanced osteogenesis with similar vasculogenic capability compared to the cell-laden hydrogel alone constructs. The full-scale implant with cell-laden hydrogel coating similarly showed cell distribution and spreading, implying the potential for further clinical application. Our study presents the feasibility of integrating bio-functional hydrogels with porous titanium implants to fabricate a vascularized hybrid construct with both mechanical support and preferable biological functionality (osteogenesis/vasculogenesis), which paves the way for improved strategies to enhance bone regeneration in complex large bone defects achieving long-term bone-implant fixation.

Recently developed modular bioassembly techniques hold tremendous potential in tissue engineering and regenerative medicine, due to their ability to recreate the complex microarchitecture of native tissue. Here, we developed a novel approach to fabricate hybrid tissue-engineered constructs adopting high-throughput microfluidic and 3D bioassembly strategies. Osteochondral tissue fabrication was adopted as an example in this study, because of the challenges in fabricating load bearing osteochondral tissue constructs with phenotypically distinct zonal architecture. By developing cell-instructive chondrogenic and osteogenic bioink microsphere modules in high-throughput, together with precise manipulation of the 3D bioassembly process, we successfully fabricated hybrid engineered osteochondral tissue in vitro with integrated but distinct cartilage and bone layers. Furthermore, by encapsulating allogeneic umbilical cord blood-derived mesenchymal stromal cells, and demonstrating chondrogenic and osteogenic differentiation, the hybrid biofabrication of hydrogel microspheres in this 3D bioassembly model offers potential for an off-the-shelf, single-surgery strategy for osteochondral tissue repair.

While decades of research have enriched the knowledge of how to grow cells into mature tissues, little is yet known about the next phase: fusing of these engineered tissues into larger functional structures. The specific effect of multicellular interfaces on tissue fusion remains largely unexplored. Here, a facile 3D‐bioassembly platform is introduced to primarily study fusion of cartilage–cartilage interfaces using spheroids formed from human mesenchymal stromal cells (hMSCs) and articular chondrocytes (hACs). 3D‐bioassembly of two adjacent hMSCs spheroids displays coordinated migration and noteworthy matrix deposition while the interface between two hAC tissues lacks both cells and type‐II collagen. Cocultures contribute to increased phenotypic stability in the fusion region while close initial contact between hMSCs and hACs (mixed) yields superior hyaline differentiation over more distant, indirect cocultures. This reduced ability of potent hMSCs to fuse with mature hAC tissue further underlines the major clinical challenge that is integration. Together, this data offer the first proof of an in vitro 3D‐model to reliably study lateral fusion mechanisms between multicellular spheroids and mature cartilage tissues. Ultimately, this high‐throughput 3D‐bioassembly model provides a bridge between understanding cellular differentiation and tissue fusion and offers the potential to probe fundamental biological mechanisms that underpin organogenesis., This facile bioassembly platform to study cartilage–cartilage interfaces offers a powerful route to probe multicellular fusion mechanisms in developmental stages of tissue growth and regenerative repair strategies. This hybrid 3D‐bioassembly model provides a bridge between understanding, tissue fusion and cellular differentiation by recapitulating detailed biological mechanisms in vitro—systematically unlocking the potential of tissue growth and subsequent organ engineering.

Three-dimensional (3D) printing has emerged as an enabling approach in a variety of different fields. However, the bulk volume of printing systems limits the expansion of their applications. In this study, a portable 3D digital light processing (DLP) printer is built based on a smartphone-powered projector and a custom-written smartphone-operated App. Constructs with detailed surface architectures, porous features, or hollow structures, as well as sophisticated tissue analogues, are successfully printed using this platform, by utilizing commercial resins as well as a range of hydrogel-based inks, including poly(ethylene glycol)-diacrylate (PEGDA), gelatin methacryloyl (GelMA), or allylated gelatin (GelAGE). Moreover, due to the portability of our unique DLP printer, medical implants can be fabricated for point-of-care usage, and cell-laden tissues can be produced in situ, achieving a new milestone for mobile-health technologies. Additionally, the all-in-one printing system described herein enables the integration of the 3D scanning smartphone App to obtain object-derived 3D digital models for subsequent printing. Along with further developments, we anticipate this portable, modular, and easy-to-use smartphone-enabled DLP printer to secure exciting opportunities for applications in resource-limited and point-of-care settings not only in biomedicine but also for home and educational purposes.

As emerging therapeutic factors, extracellular vesicles (EVs) offer significant potential for myocardial infarction (MI) treatment. Current delivery approaches for EVs involve either intra-myocardial or intravenous injection, where both have inherent limitations for downstream clinical applications such as secondary tissue injury and low delivery efficiency. Herein, an injection-free approach for delivering EVs onto the heart surface to treat MI is proposed. By spraying a mixture of EVs, gelatin methacryloyl (GelMA) precursors, and photoinitiators followed by visible light irradiation for 30 s, EVs are physically entrapped within the GelMA hydrogel network covering the surface of the heart, resulting in an enhanced retention rate. Moreover, EVs are gradually released from the hydrogel network through a combination of diffusion and/or enzymatic degradation of the hydrogel, and they are effectively taken up by the sprayed tissue area. More importantly, the released EVs further migrate deep into myocardium tissue, which exerts an improved therapeutic effect. In an MI-induced mice model, the group treated with EVs-laden GelMA hydrogels shows significant recovery in cardiac function after 4 weeks. The work demonstrates a new strategy for delivering EVs into cardiac tissues for MI treatment in a localized manner with high retention.

Dyslipidemia, characterized by a high level of lipids (cholesterol, triglycerides, or both), can increase the risk of developing and progressing atherosclerosis. As atherosclerosis progresses, the number and severity of aterial plagues increases with greater risk of myocardial infarction, a major contributor to cardiovascular mortality. Atherosclerosis progresses in four phases, namely endothelial dysfunction, fatty streak formation, lesion progression and plaque rupture, and eventually thrombosis and arterial obstruction. With greater understanding of the pathological processes underlying atherosclerosis, researchers have identified that lipoproteins play a significant role in the development of atherosclerosis. In particular, apolipoprotein B (apoB)-containing lipoproteins have been shown to associate with atherosclerosis. Oxidized low-density lipoproteins (ox-LDLs) also contribute to the progression of atherosclerosis whereas high-density lipoproteins (HDL) contribute to the removal of cholesterol from macrophages thereby inhibiting the formation of foam cells. Given these known associations, lipoproteins may have potential as biomarkers for predicting risk associated with atherosclerotic plaques or may be targets as novel therapeutic agents. As such, the rapid development of drugs targeting lipoprotein metabolism may lead to novel treatments for atherosclerosis. A comprehensive review of lipoprotein function and their role in atherosclerosis, along with the latest development of lipoprotein targeted treatment, is timely. This review focuses on the functions of different lipoproteins and their involvement in atherosclerosis. Further, diagnostic and therapeutic potential are highlighted giving insight into novel lipoprotein-targetted approaches to treat atherosclerosis.

Recent advances highlight the potential of photopolymerizable allylated gelatin (GelAGE) as a versatile hydrogel with highly tailorable properties. It is, however, unknown how different photoinitiating system affects the stability, gelation kinetics and curing depth of GelAGE. In this study, sol fraction, mass swelling ratio, mechanical properties, rheological properties, and curing depth were evaluated as a function of time with three photo-initiating systems: Irgacure 2959 (Ig2959, 320–500 nm), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 320–500 nm), and ruthenium/sodium persulfate (Ru/SPS, 400–500 nm). Results demonstrated that GelAGE precursory solutions mixed with either Ig2959 or LAP remained stable over time while the Ru/SPS system enabled the onset of controllable redox polymerization without irradiation during pre-incubation. Photo-polymerization using the Ru/SPS system was significantly faster (&lt, 5 s) compared to both Ig2959 (70 s) and LAP (50 s). Plus, The Ru/SPS system was capable of polymerizing a thick construct (8.88 ± 0.94 mm), while Ig2959 (1.62 ± 0.49 mm) initiated hydrogels displayed poor penetration depth with LAP (7.38 ± 2.13 mm) in between. These results thus support the use of the visible light based Ru/SPS photo-initiator for constructs requiring rapid gelation and a good curing depth while Ig2959 or LAP can be applied for photo-polymerization of GelAGE materials requiring long-term incubation prior to application if UV is not a concern.

To understand the typical presentation, risk factors, location and size, treatment, neurological recovery and survival of spontaneous spinal epidural haematomas (SSEH) in children. A systematic review of the English literature from 1 January 1960 to 1 March 2018 was performed on children aged 18 years and younger. Individual patient data were extracted and collated. Outcome measures were mode of presentation, risk factors, initial neurological findings, initial presumed diagnosis, diagnostic investigations, site and size of the SSEH, treatment, neurological recovery and survival. Thirty-one publications and 36 patients were reviewed. All age groups were affected. 83% of patients did not have a known risk factor. Back pain was reported in 61% and neurological dysfunction in 97% of patients, although not all articles defined these parameters. Initially 28% of patients were suspected of having an alternative diagnosis. All patients had an MRI and/or CT scan confirming the diagnosis. The cervical-thoracic region was most commonly affected, and the average haematoma size extended across 6.3 vertebral levels. Surgical decompression was performed in 72% of patients. Neurological function improved in 83% of patients. Two patients died as a consequence of their SSEH. SSEHs affect all paediatric age groups and typically present with neurological dysfunction and/or back pain. The initial diagnosis is incorrect in up to 28% of cases, but cross-sectional spinal imaging is diagnostic. Most SSEHs are located in the cervico-thoracic region and affect multiple spinal levels. The treatment depends on whether the patient has a bleeding disorder and their neurological status. These slides can be retrieved under Electronic Supplementary Material.

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.

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.

Cardiovascular diseases (CVDs) are a major health problem worldwide, and health professionals are still actively seeking new and effective approaches for CVDs treatment. Presently, extracellular vesicles, particularly exosomes, have gained its popularity for CVDs treatment because of their function as messengers for inter‐ and extra‐cellular communications to promote cellular functions in cardiovascular system. However, as a newly developed field, researchers are still trying to fully understand the role of exosomes, and their mechanism in mediating cardiac repair process. Therefore, a comprehensive review of this topic can be timely and favourable. In this review, we summarized the basic biogenesis and characterization of exosomes and then further extended the focus on the circulating exosomes in cellular communication and stem cell‐derived exosomes in cardiac disease treatment. In addition, we covered interactions between the heart and other organs through exosomes, leading to the diagnostic characteristics of exosomes in CVDs. Future perspectives and limitations of exosomes in CVDs were also discussed with a special focus on exploring the potential delivery routes, targeting the injured tissue and engineering novel exosomes, as well as its potential as one novel target in the metabolism‐related puzzle.

Tissue engineering approaches for bone repair have rapidly evolved due to the development of novel biofabrication technologies, providing an opportunity to fabricate anatomically-accurate living implants with precise placement of specific cell types. However, limited availability of biomaterial inks, that can be 3D-printed with high resolution, while providing high structural support and the potential to direct cell differentiation and maturation towards the osteogenic phenotype, remains an ongoing challenge. Aiming towards a multifunctional biomaterial ink with high physical stability and biological functionality, this work describes the development of a nanocomposite biomaterial ink (Mg-PCL) comprising of magnesium hydroxide nanoparticles (Mg) and polycaprolactone (PCL) thermoplastic for 3D printing of strong and bioactive bone regenerative scaffolds. We characterised the Mg nanoparticle system and systematically investigated the cytotoxic and osteogenic effects of Mg supplementation to human mesenchymal stromal cells (hMSCs) 2D-cultures. Next, we prepared Mg-PCL biomaterial ink using a solvent casting method, and studied the effect of Mg over mechanical properties, printability and scaffold degradation. Furthermore, we delivered MSCs within Mg-PCL scaffolds using a gelatin-methacryloyl (GelMA) matrix, and evaluated the effect of Mg over cell viability and osteogenic differentiation. Nanocomposite Mg-PCL could be printed with high fidelity at 20 wt% of Mg content, and generated a mechanical reinforcement between 30%–400% depending on the construct internal geometry. We show that Mg-PCL degrades faster than standard PCL in an accelerated-degradation assay, which has positive implications towards in vivo implant degradation and bone regeneration. Mg-PCL did not affect MSCs viability, but enhanced osteogenic differentiation and bone-specific matrix deposition, as demonstrated by higher ALP/DNA levels and Alizarin Red calcium staining. Finally, we present proof of concept of Mg-PCL being utilised in combination with a bone-specific bioink (Sr-GelMA) in a coordinated-extrusion bioprinting strategy for fabrication of hybrid constructs with high stability and synergistic biological functionality. Mg-PCL further enhanced the osteogenic differentiation of encapsulated MSCs and supported bone ECM deposition within the bioink component of the hybrid construct, evidenced by mineralised nodule formation, osteocalcin (OCN) and collagen type-I (Col I) expression within the bioink filaments. This study demonstrated that magnesium-based nanocomposite bioink material optimised for extrusion-based 3D printing of bone regenerative scaffolds provide enhanced mechanical stability and bone-related bioactivity with promising potential for skeletal tissue regeneration.

Aims The intra-articular administration of tranexamic acid (TXA) has been shown to be effective in reducing blood loss in unicompartmental knee arthroplasty and anterior cruciate reconstruction. The effects on human articular cartilage, however, remains unknown. Our aim, in this study, was to investigate any detrimental effect of TXA on chondrocytes, and to establish if there was a safe dose for its use in clinical practice. The hypothesis was that TXA would cause a dose-dependent damage to human articular cartilage. Materials and Methods The cellular morphology, adhesion, metabolic activity, and viability of human chondrocytes when increasing the concentration (0 mg/ml to 40 mg/ml) and length of exposure to TXA (0 to 12 hours) were analyzed in a 2D model. This was then repeated, excluding cellular adhesion, in a 3D model and confirmed in viable samples of articular cartilage. Results Increasing concentrations above 20 mg/ml resulted in atypical morphology, reduced cellular adhesion and metabolic activity associated with increased chondrocyte death. However, the cell matrix was not affected by the concentration of TXA or the length of exposure, and offered cellular protection for concentrations below 20 mg/ml. Conclusion These results show that when in vitro chondrocytes are exposed to higher concentrations of TXA, such as that expected following recommended intra-articular administration, cytotoxicity is observed. This effect is dose-dependent, such that a tissue concentration of 10 mg/ml to 20 mg/ml could be expected to be safe. Cite this article: Bone Joint J 2018;100-B:404–12.

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.

Human bone marrow stromal cells (hBMSCs) have been extensively utilised for bone tissue engineering applications. However, they are associated with limitations that hinder their clinical utility for bone regeneration. Cell fate can be modulated via altering their epigenetic functionality. Inhibiting histone deacetylase (HDAC) enzymes have been reported to promote osteogenic differentiation, with HDAC3 activity shown to be causatively associated with osteogenesis. Therefore, this study aimed to investigate the potential of using an HDAC2 & 3 selective inhibitor - MI192 to induce epigenetic reprogramming of hBMSCs and enhance its therapeutic efficacy for bone formation. Treatment with MI192 caused a time-dose dependant reduction in hBMSCs viability. MI192 was also found to substantially alter hBMSCs epigenetic function through reduced HDAC activity and increased histone acetylation. hBMSCs were pre-treated with MI192 (50 μM) for 48 h prior to osteogenic induction. MI192 pre-treatment significantly upregulated osteoblast-related gene/protein expression (Runx2, ALP, Col1a and OCN) and enhanced alkaline phosphatase specific activity (ALPSA) (1.43-fold) (P ≤ 0.001). Moreover, MI192 substantially increased hBMSCs extracellular matrix calcium deposition (1.4-fold) (P ≤ 0.001) and mineralisation when compared to the untreated control. In 3D microtissue culture, MI192 significantly promoted hBMSCs osteoblast-related gene expression and ALPSA (> 2.41-fold) (P ≤ 0.001). Importantly, MI192 substantially enhanced extracellular matrix deposition (ALP, Col1a, OCN) and mineralisation (1.67-fold) (P ≤ 0.001) within the bioassembled-microtissue (BMT) construct. Following 8-week intraperitoneal implantation within nude mice, MI192 treated hBMSCs exhibited enhanced extracellular matrix deposition and mineralisation (2.39-fold) (P ≤ 0.001) within the BMT when compared to the untreated BMT construct. Taken together, these results demonstrate that MI192 effectively altered hBMSCs epigenetic functionality and is capable of promoting hBMSCs osteogenic differentiation in vitro and in vivo, indicating the potential of using epigenetic reprogramming to enhance the therapeutic efficacy of hBMSCs for bone augmentation strategies.

The principle challenge for engineering viable, cell-laden hydrogel constructs of clinically-relevant size, is rapid vascularization, in order to moderate the finite capacity of passive nutrient diffusion. A multiscale vascular approach, with large open channels and bulk microcapillaries may be an admissible approach to accelerate this process, promoting overall pre-vascularization for long-term viability of constructs. However, the limited availability of bioinks that possess suitable characteristics that support both fabrication of complex architectures and formation of microcapillaries, remains a barrier to advancement in this space. In this study, gelatin-norbornene (Gel-NOR) is investigated as a vascular bioink with tailorable physico-mechanical properties, which promoted the self-assembly of human stromal and endothelial cells into microcapillaries, as well as being compatible with extrusion and lithography-based biofabrication modalities. Gel-NOR constructs containing self-assembled microcapillaries are successfully biofabricated with varying physical architecture (fiber diameter, spacing, and orientation). Both channel sizes and cell types affect the overall structural changes of the printed constructs, where cross-signaling between both human stromal and endothelial cells may be responsible for the reduction in open channel lumen observed over time. Overall, this work highlights an exciting three-way interplay between bioink formulation, construct design, and cell-mediated response that can be exploited towards engineering vascular tissues.