Biofabrication最新文献

In this research, carboxymethyl cellulose (CMC)/gelatin (Gel)/graphene oxide (GO)-based scaffolds were produced by using extrusion-based 3D printing for cardiac tissue regeneration. Rheological studies were conducted to evaluate the printability of CMC/Gel/GO inks, which revealed that CMC increased viscosity and enhanced printability. The 3D-printed cardiac patches were crosslinked with N-(3-dimethylaminopropyl)-n'-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) (100:20 mM, 50:10 mM, 25:5 mM) and then characterized by mechanical analysis, electrical conductivity testing, contact angle measurements and degradation studies. Subsequently, cell culture studies were conducted to evaluate the viability of H9C2 cardiomyoblast cells by using the Alamar Blue assay and fluorescence imaging. A high concentration of EDC/NHS (100:20 mM) led to the stability of the patches; however, it drastically reduced the flexibility of the scaffolds. Conversely, a concentration of 25:5 mM resulted in flexible but unstable scaffolds in phosphate buffer saline solution. The suitable EDC/NHS concentration was found to be 50:10 mM, as it produced flexible, stable, and stiff cardiac scaffolds with high ultimate tensile strength. Mechanical characterization revealed that % strain at break of C15/G7.5/GO1 exhibited a remarkable increase of 61.03% compared to C15/G7.5 samples. The improvement of flexibility was attributed to the hydrogen bonding between CMC, Gel and GO. The electrical conductivity of 3D printed CMC/Gel/GO cardiac patches was 7.0 × 10^-3S cm^-1, demonstrating suitability for mimicking the desired electrical conductivity of human myocardium. The incorporation of 1 wt% of GO and addition of CMC concentration from 7.5 wt% to 15 wt% significantly enhanced relative % cell viability. Overall, although this research is at its infancy, CMC/Gel/GO cardiac patches have potential to improve the physiological function of cardiac tissue.

Engineered neural tissue (EngNT) is a stabilised aligned cellular hydrogel that offers a potential alternative to the nerve autograft for the treatment of severe peripheral nerve injury. This work aimed to automate the production of EngNT, to improve the feasibility of scalable manufacture for clinical translation. Endothelial cells were used as the cellular component of the EngNT, with the formation of endothelial cell tube-like structures mimicking the polarised vascular structures formed early on in the natural regenerative process. Gel aspiration-ejection for the production of EngNT was automated by integrating a syringe pump with a robotic positioning system, using software coded in Python to control both devices. Having established the production method and tested mechanical properties, the EngNT containing human umbilical vein endothelial cells (EngNT-HUVEC) was characterised in terms of viability and alignment, compatibility with neurite outgrowth from rat dorsal root ganglion neurons and formation of endothelial cell networksin vitro. EngNT-HUVEC manufactured using the automated system contained viable and aligned endothelial cells, which developed into a network of multinucleated endothelial cell tube-like structures inside the constructs and an outer layer of endothelialisation. The EngNT-HUVEC constructs were made in various sizes within minutes. Constructs provided support and guidance to regenerating neuritesin vitro. This work automated the formation of EngNT, facilitating high throughput manufacture at scale. The formation of endothelial cell tube-like structures within stabilised hydrogels provides an engineered tissue with potential for use in nerve repair.

In the body, capillary beds fulfill the metabolic needs of cells by acting as the sites of diffusive transport for vital gasses and nutrients. In artificial tissues, replicating the scale and complexity of capillaries has proved challenging, especially in a three-dimensional context. In order to better develop thick artificial tissues, it will be necessary to recreate both the form and function of capillaries. Here we demonstrate a top-down method of patterning hydrogels using sacrificial templates formed from thermoresponsive microfibers whose size and architecture approach those of natural capillaries. Within the resulting microchannels, we cultured endothelial monolayers that remain viable for over three weeks and exhibited functional barrier properties. Additionally, we cultured endothelialized microchannels within hydrogels containing fibroblasts and characterized the viability of the co-cultures to demonstrate this approach's potential when applied to cell-laden hydrogels. This method represents a step forward in the evolution of artificial tissues and a path towards producing viable capillary-scale microvasculature for engineered organs.

In embedded 3D printing (EMB3D), a nozzle extrudes continuous filaments inside of a viscoelastic support bath. Compared to other extrusion processes, EMB3D enables softer structures and print paths that conform better to the shape of the part, allowing for complex structures such as tissues and organs. However, strategies for high-quality dimensional accuracy and mechanical properties remain undocumented in EMB3D. This work uses computational fluid dynamics simulations in OpenFOAM to probe the underlying physics behind two processes: deformation of the printed part due to nearby nozzle motion and fusion between neighboring filaments during printing. Through simulations, we disentangle yielding from viscous dissipation, and we isolate interfacial tension effects from rheology effects, which are difficult to separate in experiments. Critically, these simulations find that disturbance and fusion are controlled by the flow of support fluid around the nozzle. To avoid part deformation, the nozzle must remain far from existing parts during non-printing moves, moreso when traveling next to the part than above the part and especially when the interfacial tension between the ink and support is non-zero. Additionally, because support can become trapped between filaments at zero interfacial tension, the spacing between filaments must be tight enough to produce over-printing, or printing too much material for the designed space. In non-Newtonian fluids, spacings for vertical walls must be even tighter than spacings for horizontal planes. At these spacings, printing a new filament sometimes creates and sometimes mitigates shape defects in the old filament. While non-zero ink-support interfacial tensions produce better inter-filament fusion than zero interfacial tension, interfacial tension also produces shape defects. Slicing algorithms that consider these unique EMB3D defects are needed to improve mechanical properties and dimensional accuracy of bioprinted constructs.

In vitrobone models are pivotal for understanding tissue behavior and cellular responses, particularly in unravelling certain pathologies' mechanisms and assessing the impact of new therapeutic interventions. A desirablein vitrobone model should incorporate primary human cells within a 3D environment that mimics the mechanical properties characteristics of osteoid and faithfully replicate all stages of osteogenic differentiation from osteoblasts to osteocytes. However, to date, no bio-printed model using primary osteoblasts has demonstrated the expression of osteocytic protein markers. This study aimed to develop bio-printedin vitromodel that accurately captures the differentiation process of human primary osteoblasts into osteocytes. Given the considerable impact of hydrogel stiffness and relaxation behavior on osteoblast activity, we employed three distinct cross-linking solutions to fabricate hydrogels. These hydrogels were designed to exhibit either similar elastic behavior with different elastic moduli, or similar elastic moduli with varying relaxation behavior. These hydrogels, composed of gelatin (5% w/v), alginate (1%w/v) and fibrinogen (2%w/v), were designed to be compatible with micro-extrusion bioprinting and proliferative. The modulation of their biomechanical properties, including stiffness and viscoelastic behavior, was achieved by applying various concentrations of cross-linkers targeting both gelatin covalent bonding (transglutaminase) and alginate chains' ionic cross-linking (calcium). Among the conditions tested, the hydrogel with a low elastic modulus of 8 kPa and a viscoelastic behavior over time exhibited promising outcomes regarding osteoblast-to-osteocyte differentiation. The cessation of cell proliferation coincided with a significant increase in alkaline phosphatase activity, the development of dendrites, and the expression of the osteocyte marker PHEX. Within this hydrogel, cells actively influenced their environment, as evidenced by hydrogel contraction and the secretion of collagen I. This bio-printed model, demonstrating primary human osteoblasts expressing an osteocyte-specific protein, marks a significant achievement. We envision its substantial utility in advancing research on bone pathologies, including osteoporosis and bone tumors.

Bacterial infection is a major challenge to human health. Although various potent antibiotics have emerged in recent decades, current challenges arise from the increasing number of multi-drug-resistant species. Infections associated with implants represent a particular challenge because they are usually diagnosed at an advanced stage and are difficult to treat with antibiotics owing to the formation of protective biofilms. In this study, we designed and explored a synthetic biology-inspired cell-based biosensor/actor for the detection and counteraction of bacterial infections. The system is generic, as it senses diverse types of infections and acts by enhancing the endogenous immune system. This strategy is based on genetically engineered sensor/actor cells that can sense type I interferons (IFNs), which are released by immune cells at the early stages of infection. IFN signalling activates a synthetic circuit to induce reporter genes with a sensitivity of only 5 pg ml^-1of IFN and leads to a therapeutic protein output of 100 ng ml^-1, resulting in theranostic cells that can visualize and fight infections. Robustness and resilience were achieved by implementing a positive feedback loop. We showed that diverse gram-positive and gram-negative implant-associated pathogenic bacteria activate the cascade in co-culture systems in a dose-dependent manner. Finally, we showed that this system can be used to secrete chemoattractants that facilitate the infiltration of immune cells in response to bacterial triggers. Together, the system is not only universal to bacterial infections, but also hypersensitive, allowing the sensing of infections at initial stages.

Three-dimensional (3D) printing incorporated with controlled delivery is an effective tool for complex tissue regeneration. Here, we explored a new strategy for spatiotemporal delivery of bioactive cues by establishing a precise-controlled micro-thin coating of hydrogel carriers on 3D-printed scaffolds. We optimized the printing parameters for three hydrogel carriers, fibrin cross-linked with genipin, methacrylate hyaluronic acid, and multidomain peptides, resulting in homogenous micro-coating on desired locations in 3D printed polycaprolactone microfibers at each layer. Using the optimized multi-head printing technique, we successfully established spatial-controlled micro-thin coating of hydrogel layers containing profibrogenic small molecules (SMs), Oxotremorine M and PPBP maleate, and a chondrogenic cue, Kartogenin. The delivered SMs showed sustained releases up to 28 d and guided regional differentiation of mesenchymal stem cells, thus leading to fibrous and cartilaginous tissue matrix formation at designated scaffold regionsin vitroandin vivo. Our micro-coating of hydrogel carriers may serve as an efficient approach to achieve spatiotemporal delivery of various bioactive cues through 3D printed scaffolds for engineering complex tissues.

This study introduces a novelin vitromodel for intractable temporal lobe epilepsy (TLE) utilizing 3D bioprinting technology, aiming to replicate the complex neurobiological characteristics of TLE more accurately. Primary neural cell constructs were fabricated and subjected to epileptiform-inducing conditions, fostering synaptic proliferation and neuronal loss. Systematically electrophysiological and immunofluorescent analyses indicated that significant synaptic connectivity and sustained epileptiform activities within the constructs akin to those observed in human epilepsy models. Notably, the model responded to treatments with phenytoin and tetrodotoxin, illustrating its potential utility in drug response kinetics studies. Furthermore, we performed drug permeability simulations using COMSOL Multiphysics to analyze the diffusion characteristics of these drugs within the constructs. These results confirm that our 3D bioprinted neural model provides a physiologically relevant and ethically sustainable platform, which is beneficial for studying TLE mechanisms and developing therapeutic strategies with high accuracy and clinical relevance.

Three-dimensional (3D) organotypic skin in vitro has attracted increasing attention for drug development, cosmetics evaluation, and even clinical applications. However, the severe contraction of these models restricts their application, especially in the analyses based on barrier functions such as percutaneous penetration. For the full-thickness skin equivalents, the mechanical properties of the dermis scaffold plays an important role in the contraction resistance. In this investigation, we optimized a hydrogel composed of gelatine methacrylamide (GelMA), hyaluronic acid methacrylate (HAMA), and type I collagen (Col I), adjusted the elastic moduli to 2.27±0.08 kPa to fit the skin cells growth and resist contraction as well. This optimized hydrogel exhibited a swelling ratio of 23.25 ± 0.94% and demonstrated satisfactory cell viability in fibroblasts cultures. Then, we mixed this hydrogel with fibroblasts of liquid-liquid culture to construct the dermis, on which seeded keratinocytes were seeded for another 14 days of air-liquid culture to form cornified epidermis, and a commercialized hydrogel Ava-FT-Skin was used as control. This optimized skin model could maintained its integrity for a prolonged period of 28 days. Differentiated epidermis presented basal, spinous, granular, and cornified layers, meanwhile, epidermis markers like keratin-10, keratin-14, involucrin, loricrin, filaggrin, and dermis markers vimentin were expressed distinctly in the right distribution. Furthermore, penetration of a 607 Da Cascade blue-labelled dextran was calculated and compared to the Avatarget skin model, both of which could prevent more than 99% of the fluorescent molecule. We consider that this full-thickness skin model could be widely used in pharmaceutical and cosmetic industries, especially in penetration detection, contributing to the excellent contraction resistance.

Emergency wounds are often accompanied by bacterial infection, oxidative stress, and excessive inflammation due to the inability to quickly close and stop bleeding, resulting in chronic wounds that are difficult to heal. Clinically, surgical suturing is the fastest method for wound closure, but it is only suitable for wounds with small bleeding volumes and causes unsightly scar formation. Consequently, there is a critical need for hemostatic dressings versatile enough to address a spectrum of diverse and intricate wounds, especially in emergency scenarios. In this study, we constructed a unique versatile natural gelatin-based hydrogel with hemostasis, antibacterial, and anti-inflammation properties. The hydrogel was composed of 4-(4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy) butyrylethylenediamine-modified methacrylated gelatin (GelMA-NB) and epigallocatechin gallate-grafted polylysine (EPL-EGCG), which imparts adhesion, antibacterial and antioxidant properties to the hydrogel. Simultaneously, the hydrogel was loaded with GelMA microspheres encapsulating natural resveratrol (RES@GM). This combination not only exhibited outstanding hemostatic capabilities but also preserved the anti-inflammatory potential of RES. In different animal models, the hydrogel exhibited outstanding hemostatic and wound healing effects, down-regulated the expression of IL-1βto promote inflammatory regulation and potential for angiogenesis and anti-scar. In conclusion, unique versatile natural gelatin-based hydrogel suitable for various complex wounds provides a promising strategy for emergency wound dressing applications.