Biofabrication最新文献

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.

Recombinant collagen holds immense potential in the development of medical functional materials, yet its widespread application remains hindered by the absence of a suitable self-assembly strategy. In this article, we report the discovery that the bacterial-derived collagen-like protein Scl2 can rapidly self-gelation (~1 minute at pH ~7) due to properties enabled by metal coordination crosslinking. This was achieved by fusing metal ion chelating peptides to both termini of the protein. Our research further reveals the critical role of electrostatic interaction between globular domains (V domains) of recombinant collagen in the self-assembly process. We show that modifying the negative charge load of the N-terminal α-helix of the V domain enables control over the self-assembly time (from 1 min to 30 min) and strength (from 8 kPa to 26 kPa) of the Scl2 hydrogel. By adjusting the molecular weight of the core collagen-like (CL) domain, we have remarkably further enhanced the strength of the Scl2 hydrogel to 78 kPa. Moreover, we innovatively employed electro-oxidized tea polyphenols (EOTP) to enhance the stability of the Scl2 hydrogel, resulting in the formation of a reliable self-assembled metal coordination hydrogel at physiological temperature. This approach not only eliminates the need for toxic chemical crosslinking agents but also confers the material with multiple functionalities, such as adhesion, antibacterial, and antioxidant properties. The novel recombinant Scl2 hydrogel exhibited exceptional in situ self-gelation and injectable properties. This innovative hydrogel not only demonstrates remarkable biological activity but also exhibits remarkable tissue repair-promoting capabilities in full-thickness skin injury models (shorten healing cycle by more than 30%). The convenient and versatile nature of this recombinant collagen hydrogel makes it promising for clinical applications in injury treatment, demonstrating broad applications in the future.

Kidney diseases are among the leading causes of death globally. With the increasing rates of acute kidney injury (AKI) requiring hospitalisation, a better understanding of pathophysiological mechanisms is needed to treat the patients more efficiently. Nephrotoxicity is one of the most common causes of AKI, mainly due to the high availability of over-the-counter drugs and natural supplements, which may interact with prescribed drugs at the level of pharmacokinetics, among other factors. The latter can lead to clinically relevant complications (including AKI), which is even more pronounced given the increasingly ageing population in the Western world and the associated increase in polypharmacy. Drug testing starts at the preclinical level, where a reliable model is needed to predict human response to a tested drug with sufficient accuracy. Recently, in-vitro kidney models of different complexities have been created to study various aspects of kidney diseases. Because the proximal tubule plays a vital role in several mechanisms, many models include proximal tubular epithelial cells (PTECs). Monocultures of PTECs do not represent in-vivo tissue accurately enough. Therefore, more complex models with more cell types are being built. To our knowledge, this is the first review focusing on co-culture models and cell types used alongside PTECs for studying the nephrotoxicity of drugs and other mechanisms of AKI and chronic kidney disease (CKD). .

Bioprinting a resilient yet optically transparent corneal tissue substitute remains a challenge. In this study we introduce an innovative methodology aimed at bolstering the mechanical and optical attributes of silk fibroin (SF) hydrogels, pivotal for the progression of cornea tissue engineering. We devised a unique eosin Y-based photoinitiator system to instigate di-tyrosine linkages within highly concentrated pristine SF solutions under green light exposure. This pioneering technique resulted in SF hydrogels fortified by dityrosine covalent bonds, preserving exceptional transparency and soft elastomeric qualities devoid of spontaneous transitions to stiff, opaque beta-sheet conformations. Furthermore, we synergistically combined SF with decellularized corneal matrix (DCM) hydrogel, leveraging photo-polymerization under green light followed by thermal gelation to establish resilient and stable gel formation. The ensuing dual crosslinked hybrid hydrogels exhibited superior mechanical and thermal resilience in comparison to dual crosslinked DCM hydrogels. The inclusion of SF in DCM further augmented the hydrogel's elasticity and shear recovery, positioning it as an optimal bioink for cornea bioprinting endeavors. During the extrusion printing process, photocrosslinking of the bioink superficially fortified SF and DCM polymer chains via di-tyrosine linkages, furnishing initial stability and mechanical fortitude. Subsequent post-printing thermal gelation further reinforced collagen chains through self-assembly. Notably, the bioprinted cornea constructs, housing human limbal mesenchymal stem cells (hLMSCs), manifested transparency, structural integrity, and optimal functionality, underscored by the expression of keratocyte proteoglycans. In summation, our engineered 3D constructs exhibit promising potential for in vivo applications in cornea tissue engineering, marking a significant stride forward in the field's advancement.

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 this present study, we introduce an innovative hybrid 3D bioprinting methodology that integrates fused deposition modeling (FDM) with top-down digital light processing (DLP) for the fabrication of an artificial trachea. Initially, polycaprolactone (PCL) was incorporated using an FDM 3D printer to provide essential mechanical support, replicating the structure of tracheal cartilage. Subsequently, a chondrocyte-laden glycidyl methacrylated silk fibroin (Sil-MA) hydrogel was introduced via top-down DLP into the PCL scaffold (PCL-Sil scaffold). The mechanical evaluation of PCL-Sil scaffolds showed that they have greater flexibility than PCL scaffolds, with a higher deformation rate (PCL-Sil scaffolds: 140.9±5.37% vs. PCL scaffolds: 124.3±6.25%) and ability to withstand more force before fracturing (3.860±0.140 N for PCL-Sil scaffolds vs. 2.502±0.126 N for PCL scaffolds, ***P < 0.001). Both types of scaffolds showed similar axial compressive strengths (PCL-Sil scaffolds: 4.276±0.127 MPa vs. PCL scaffolds: 4.291±0.135 MPa). Additionally, PCL-Sil scaffolds supported fibroblast proliferation, indicating good biocompatibility. In vivo testing of PCL-Sil scaffolds in a partial tracheal defect rabbit model demonstrated effective tissue regeneration. The scaffolds were pre-cultured in the omentum for two weeks to promote vascularization before transplantation. Eight weeks after transplantation into the animal, bronchoscopy and histological analysis confirmed that the omentum-cultured PCL-Sil scaffolds facilitated rapid tissue regeneration and maintained the luminal diameter at the anastomosis site without signs of stenosis or inflammation. Validation study to assess the feasibility of our hybrid 3D bioprinting technique showed that structures, not only the trachea but also the vertebral bone-disc and trachea-lung complex, were successfully printed. .

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.

The recent occurrence of the Covid-19 pandemic and frequent wildfires have worsened pulmonary diseases and raised the urgent need for investigating host-pathogen interactions and advancing drug and vaccine therapies. Historically, research and experimental studies have relied on two-dimensional cell culture dishes and/or animal models, which suffer from physiological differences from the human lung. More recently, there has been investigation into the use of lung-on-a-chip models and organoids, while the use of bioprinting technologies has also emerged to fabricate three-dimensional constructs or lung models with enhanced physiological relevance. Concurrently, achievements have also been made to develop biomimetic strategies for simulating the in vivo biomechanical conditions induced by lung breathing, though challenges remain with incorporating these strategies with bioprinted models. Bioprinted models combined with advanced biomimetic strategies would represent a promising approach to advance disease discovery and therapeutic development. As inspired, this article briefly reviews the recent progress of both bioprinted in vitro lung models and biomechanical strategies, with a focus on native lung tissue microstructure and biomechanical properties, bioprinted constructs, and biomimetic strategies to mimic the native environment. This article also urges that the integration of bioprinting advances and biomimetic strategies would be essential to achieve synergistic effects for in vitro lung modelling. Key issues and challenges are also identified and discussed along with recommendations for future research.

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.