Tissue-engineered tendon constructs that replicate the structural and functional properties of natural tendons are crucial in regenerative medicine to improve treatment outcomes after tendon injury. This study aimed to engineer 3-dimensional biomimetic tendon macro-tissues through bioassembly of cell spheroids. Rat tendon fibroblasts seeded at different cell numbers (1 × 104, 5 × 104, 1 × 105, 2 × 105, 3 × 105) were analysed for spheroid formation and development for 28 d. The spheroid diameters decreased over time with a reduction in cell density while synthesising their own collagen fibres. Spheroids were then bioassembled to form fused macro-tissue constructs using pillar array temporary supports. With the presence of ascorbic acid in growth media, the spheroids fused within 6 d after bioassembly, during which the supports were removed, leaving the constructs scaffold-free. The fused spheroids reorganised over time with increased fibrillar collagen content, showing elongated cell morphology in peripheral regions, which were parallelly aligned with collagen fibres resembling natural tendon micro-anatomy. The presence of scleraxis and tenomodulin gene markers also supports the tenogenic nature of tissue constructs. These biomimetic scaffold-free tenogenic macro-tissues have promising applications in tendon repair and regeneration, asin vitromodels and tendon graft substitutes.

The need for repeated administration of vaccines is costly, logistically challenging and leads to reduced compliance. Osmosis-driven swelling and rupture has been proposed as a mechanism for delayed burst release to mimic follow-up (booster) injections without requiring further intervention. The use of microparticles does not guarantee a single bolus release, and previous studies using monolithic implants failed to demonstrate high instant release at the moment of burst. In this study we developed elastic capsules based on a caprolactone-lactide copolymer that released 75 ± 8 % of their contents instantly after up to several weeks of incubation. Capsules swelled up to 5x their original mass through highly reproducible osmosis-induced water uptake. The water uptake rates depended on osmotic strength and could be approximated with a numerical model revealing a water permeability of 3.6 ± 0.2 mm2/(Pa∙day) for all 118 capsules. The moment of burst depended on osmotic strength and capsule stiffness (median times varied from 16 to 37 days) and showed large scatter. The capsule material was non-toxic to a macrophage cell line and degraded through bulk hydrolysis, with an extrapolated completion time of 8 months. The capsules were administered successfully in bovine cadavers with a widely used applicator.

Engineering 3D tissue-like constructs for applications such as regenerative medicine remains a major challenge in biomedical research. Recently, self-healing, viscoplastic fluids have been introduced as suspension media to allow lower viscosity, water-rich bioinks to be printed within them for the fabrication of more biomimetic structures. Here, we present gellan gum granular gels produced through the application of shear during gelation, as a candidate suspension medium. We demonstrate that these granular gels exhibit viscoplasticity over a wide range of temperatures, permitting their use for 3D bioprinting of filaments and droplets at low (4°C) as well as physiological temperatures. These granular gels exhibit very low yield stresses (down to 0.4 Pa) which facilitated printing at print speeds up to 60 mm.s-1. Furthermore, we demonstrate the printing of cell-laden droplets maintained over 7 days to show the potential for multiple days of cell culture, as well as the fabrication of hydrogel features within a crosslinkable version of the suspension medium containing granular gellan gum and gelatine-methacryloyl. The combination of ease of preparation, high printing speed, wide temperature tolerance, and crosslinkability makes this gellan gum sheared through cooling-induced gelation an attractive candidate for suspended biofabrication.

Small‐diameter vascular grafts having biomechanical properties similar to those of native arteries for the treatment of cardiovascular diseases are still elusive. Here, a hybrid extrusion printing and electrospinning technique is presented in which a layer of electrospun nanofibers is deposited over the printed gelatin‐methacryloyl (gelMA) constructs to improve the mechanical performance of gelMA grafts. Various blends of polycaprolactone (PCL) and poly(L‐lactide‐co‐ε‐caprolactone) (PLCL) polymer solutions are used to produce electrospun nanofibers. The variation of gelMA concentration is found to have a negligible role in the overall strength of the graft. It is shown that the burst pressure and tensile strength of the fiber‐reinforced gelMA constructs are comparable to those of native human arteries. Moreover, the compliance of grafts reinforced by 100% PCL and 75/25% PCL/PLCL nanofibers are found to be similar to human muscular arteries and elastic arteries, respectively. The cytocompatibility assessment shows that gelMA presents a bioactive surface for the endothelial cells to survive and grow. Also, PCL/PLCL electrospun nanofibers offer cellular metabolic activity in the same order of magnitude as observed in the control. Therefore, this hybrid technique opens up new possibilities for the fabrication of tubular constructs in tissue engineering.

IntroductionBioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue.MethodsdROb spheroids in control and mineralization media at different seeding densities (1 × 104, 5 × 104, and 1 × 105 cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin® by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects.ResultsSpheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (p < 0.001), and upregulated alkaline phosphatase (p < 0.01).DiscussionWith the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.

AbstractObjectivesThe enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production.MethodsCell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×104, 5×104, 1×105, 2×105 in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope.3D mini-tissue constructs15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix.ResultsRTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×105 RTFs and 3×105 BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage.ConclusionsThis study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model.Declaration of Interest(a) fully declare any financial or other potential conflict of interest

Vat polymerization allows for the accurate and fast fabrication of personalized implants and devices. While the technology advances rapidly and more materials become available, the fabrication of flexible yet tough resorbable materials for biomedical applications remains a challenge. Here, a formulation that can be 3D printed with high accuracy using vat polymerization, yielding materials that are tough, degradable, and non‐toxic is presented. This unique combination of properties is obtained by combining a long‐chain polycaprolactone macromonomer with a small molecule cross‐linker. A wide range of properties is achieved by tuning the ratio of these components. The use of benzyl alcohol as a non‐volatile, benign solvent enables fabrication on a low‐cost desktop 3D printer, with an exposure time of 8 s per 50‐micron layer. The 3D‐printed networks are tough and elastic with a tensile strength of 11 MPa at 116% elongation at break. Cells attach and proliferate on the networks with a viability of >91%. The networks are fully degradable to soluble products. This new 3D printable material opens up a range of opportunities in biomedical engineering and personalized medicine.

Abstract AIMS To succeed in clinical trials for glioblastoma we need in vitro models capable of more faithfully replicating dis- ease biology and more accurately predicting patient drug responses. To this end, new bioprinting technologies have the potential to biofabricate clinically relevant biomimetic tissues which can accelerate drug discovery and additionally serve as a platform for personalized medicine. METHOD We evaluated the effect of individual biomaterials and combinations of biomaterials, including decellularised pig brain extracellular matrix (dECM), fibrin, gelatin-methacryloyl (GelMA), hyaluronic acid-methacrylate (HAMA), Matrigel and alginate, on the proliferation and invasion of aggressive brain cancer cells (U87) in vitro. Cell viability was assessed using propidium iodide. Invasiveness was studied employing confocal microscopy. Data generated from Z-stacks was analysed using ImageJ to determine the size and circularity of cells. RESULTS Although Matrigel supports rapid cell proliferation and invasion, it has mechanical properties unsuited to bio- printing. In contrast, HAMA displays a pronounced shear-thinning behaviour and rapid controllable photo- crosslinking. Combinations of HAMA-fibrin provided results comparable to those seen with Matrigel or HAMA- Matrigel. However, high levels of crosslinking affected these biomaterial mixtures, resulting in a decreased ability of cells to grow and spread. CONCLUSIONS Initial results indicate that the addition of fibrin to HAMA promoted the growth and spreading of U87 cells. In further work, we aim to improve our printed constructs by including porcine brain dECM, microglia and recently established cell lines from paediatric patients. The project will test whether these bioprinted models can provide drug testing data with closer results to human disease than current, simpler alternatives.

Single-administration vaccine delivery systems are intended to improve the efficiency and efficacy of immunisation programs in both human and veterinary medicine. In this work, an osmotically triggered delayed delivery device was developed that was able to release a payload after a delay of approximately 21 days, in a consistent and reproducible manner. The device was constructed out of a flexible poly(ε-caprolactone) photo-cured network fabricated into a hollow tubular shape, which expelled approximately 10% of its total payload within 2 days after bursting. Characterisation of the factors that control the delay of release demonstrated that it was advantageous to adjust material permeability and device wall thickness over manipulation of the osmogent concentration in order to maintain reproducibility in burst delay times. The photo-cured poly(ε-caprolactone) network was shown to be fully degradable in vitro, and there was no evidence of cytotoxicity after 11 days of direct contact with primary dermal fibroblasts. This study provides strong evidence to support further development of flexible biomaterials with the aim of continuing improvement of the device burst characteristics in order to provide the greatest chance of the devices succeeding with in vivo vaccine booster delivery.

Gelatin methacryloyl (GelMA) hydrogels have gained significant attention due to their biocompatibility and tunable properties. Here, a new approach to engineer GelMA-based matrices to mimic the osteoid matrix is provided. Two cross-linking methods were employed to mimic the tissue stiffness: standard cross-linking (SC) based on visible light exposure (VL) and dual cross-linking (DC) involving physical gelation, followed by VL. It was demonstrated that by reducing the GelMA concentration from 10% (G10) to 5% (G5), the dual-cross-linked G5 achieved a compressive modulus of ∼17 kPa and showed the ability to support bone formation, as evidenced by alkaline phosphatase detection over 3 weeks of incubation in osteogenic medium. Moreover, incorporating poly(ethylene) oxide (PEO) into the G5 and G10 samples was found to hinder the fabrication of highly porous hydrogels, leading to compromised cell survival and reduced osteogenic differentiation, as a consequence of incomplete PEO removal.

The electronic industry has room for improvement in adopting cleaner strategies, both in production processes (often energy-intensive and polluting) and in waste management. Many small components like security tags are routinely disposed of via general waste, which could be reduced adopting biodegradable polymers. In this work, a method for selective deposition of metallic micro-tracks on polycaprolactone (PCL) for circuitry integration is presented. The polymer is biodegradable, flexible, suitable for 3D printing, and can be obtained from sustainable sources. Photoreduction of Ag ions was used to generate seeds for subsequent selective electroless copper (Cu) plating in a process that avoids common but undesirable compounds such as cyanides and palladium. Two different photopatterning methods were successfully used to achieve selective Cu plating: flood exposure with a 460 nm light-emitting diode (LED) and direct laser writing (DLW) using a 405 nm laser, achieving 47 ± 11 μ m wide tracks. The deposition of uniform Cu layers on PCL substrates is demonstrated, with thicknesses of up to 14 μ m and electrical conductivities of up to 2.06 × 10 7 S m −1 , which is near the conductivity of bulk Cu (5.89 × 10 7 S m −1 ). Cu-plated interconnects were demonstrated to be fully functional for powering a 5 SMD LEDs circuit. Furthermore, DLW enabled the interconnect manufacturing on an uneven substrate. This method is flexible, selective, low-cost, vacuum-free and of minimized environmental impact, and it provides a new route towards the manufacturing of biodegradable electronics.

The lack of in vitro tissue and organ models capable of mimicking human physiology severely hinders the development and clinical translation of therapies and drugs with higher in vivo efficacy. Bioprinting allow us to fill this gap and generate 3D tissue analogues with complex functional and structural organization through the precise spatial positioning of multiple materials and cells. In this review, we report the latest developments in terms of bioprinting technologies for the manufacturing of cellular constructs with particular emphasis on material extrusion, jetting, and vat photopolymerization. We then describe the different base polymers employed in the formulation of bioinks for bioprinting and examine the strategies used to tailor their properties according to both processability and tissue maturation requirements. By relating function to organization in human development, we examine the potential of pluripotent stem cells in the context of bioprinting toward a new generation of tissue models for personalized medicine. We also highlight the most relevant attempts to engineer artificial models for the study of human organogenesis, disease, and drug screening. Finally, we discuss the most pressing challenges, opportunities, and future prospects in the field of bioprinting for tissue engineering (TE) and regenerative medicine (RM).

Shape memory polymers are materials that are able to retain a deformed state until an external stimulus, most typically heat, triggers recovery to the original geometry. Whereas typically, shape memory polymers are required to recover fast (seconds to minutes), many applications, particularly in the medical field, would benefit from a slow recovery (days to weeks). In this work, we exploit the broad glass transition range of photo-cured poly(D,L-lactide) dimethacrylate networks to obtain recovery times of up to 2 weeks, at 11 °C below the peak glass transition temperature of 58 °C. Recovery times decreased considerably for higher recovery temperatures, down to ∼10 min at 55 °C. A large spread in glass transition values (53.3–61.0 °C) was observed between samples, indicating poor reproducibility in sample preparation and, hence, in predicting shape recovery kinetics for individual samples. Furthermore, a staged recovery was observed with different parts of the samples recovering at different times. The ability to prepare complex structures using digital light processing stereolithography 3D printing from these polymers was confirmed. To the best of our knowledge, this work provides the first experimental evidence of prolonged recovery of shape memory polymers.

The interest in bioprinting of sustainable biomaterials is rapidly growing, and lignocellulosic biomaterials have a unique role in this development. Lignocellulosic materials are biocompatible and possess tunable mechanical properties, and therefore promising for use in the field of 3D-printed biomaterials. This review aims to spotlight the recent progress on the application of different lignocellulosic materials (cellulose, hemicellulose, and lignin) from various sources (wood, bacteria, and fungi) in different forms (including nanocrystals and nanofibers in 3D bioprinting). Their crystallinity, leading to water insolubility and the presence of suspended nanostructures, makes these polymers stand out among hydrogel-forming biomaterials. These unique structures give rise to favorable properties such as high ink viscosity and strength and toughness of the final hydrogel, even when used at low concentrations. In this review, the application of lignocellulosic polymers with other components in inks is reported for 3D bioprinting and identified supercritical CO2 as a potential sterilization method for 3D-printed cellulosic materials. This review also focuses on the areas of potential development by highlighting the opportunities and unmet challenges such as the need for standardization of the production, biocompatibility, and biodegradability of the cellulosic materials that underscore the direction of future research into the 3D biofabrication of cellulose-based biomaterials.

5 years ago, we described the emergence of 3D printing in medicine. It was about 3D printing of anatomical structures, patient-specific drilling guides, cutting templates and implants and printing of living cells, growth factors and biomaterials ('bioprinting'). Surgeons are increasingly making use of 3D printing possibilities in preparation of surgeries on patients with complicated anatomies. Using tangible 3D models, it is easier for surgeons to prepare for surgeries and discussions with patients. They can also use 3D models as a tool to help with the training of young surgeons. Permanent titanium implants are increasingly being printed. Bioprinting is still in its infancy and there are no direct clinical applications yet. As we already predicted 5 years ago, many hurdles still have to be taken before broad clinical application of bioprinted products will become a reality.