Bioactive glass nanoparticles (BGNs) are well-recognized multifunctional biomaterials for bone tissue regeneration due to their capability to stimulate various cellular processes through released biologically active ions. Understanding the correlation between BGN composition and cellular responses is key to developing clinically usable BGN-based medical devices. This study investigated the influence of CaO content of binary SiO₂-CaO BGNs (CaO ranging from 0 to 10 mol%) on osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) and in vivo bone regeneration in zebrafish osteoporosis model. The results showed that BGNs could promote osteogenic differentiation of rBMSCs by indirectly releasing active ions or directly interacting with rBMSCs by internalization. In both situations, BGNs of a higher CaO content could promote the osteogenic differentiation of rBMSCs to a greater extent. The internalized BGNs could activate the transcription factors RUNX2 and OSX, leading to the expression of osteogenesis-related genes. The results in the zebrafish osteoporosis model indicated that the presence of BGNs of higher CaO contents could enhance bone regeneration and rescue dexamethasone-induced osteoporosis to a greater extent. These findings demonstrate that BGNs can stimulate osteogenic differentiation of rBMSCs by releasing active ions or internalization. A higher CaO content facilitates osteogenesis and bone regeneration of zebrafish as well as relieving dexamethasone-induced osteoporosis. The zebrafish osteoporosis model can be a potent tool for evaluating the in vivo bone regeneration effects of bioactive materials. Statement of significance: Bioactive glass nanoparticles (BGNs) are increasingly used as fillers of nanocomposites or as delivery platforms of active ions to regenerate bone tissue. Various studies have shown that BGNs can enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by releasing active ions. However, the correlation between BGN composition and cellular responses and in vivo bone regeneration effect has still not been well investigated. Establishment of a suitable in vivo animal model for investigating this correlation is also challenging. The present study reports the influence of CaO content in binary SiO₂-CaO BGNs on osteogenic differentiation of BMSCs extracellularly and intracellularly. This study also demonstrates the suitability of zebrafish osteoporosis model to investigate in vivo bone regeneration effect of BGNs.

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Bisphosphonates (BPs) are commonly used to treat skeletal diseases. However, long-term use of BPs can lead to BP-related jaw bone necrosis (BRONJ), particularly in patients undergoing tooth extraction surgery. The treatment of BRONJ remains challenging due to the local inflammatory microenvironment. To address this issue, we developed catechol-conjugated chitosan-based hydrogels incorporating cerium-doped mesoporous bioactive glass nanoparticles (Ce-MBGNs) and apoptotic extracellular vesicles (ApoEVs). The hydrogels containing 1 wt % Ce-MBGNs demonstrated biodegradability and non-cytotoxicity. The incorporation of Ce-MBGNs did not significantly impact gelation and rheological behavior. By leveraging the effects of Ce-MBGNs on regulating macrophage polarization and ApoEVs on mediating macrophage chemotaxis, hydrogels containing 1 wt % Ce-MBGNs and 1 mg/mL ApoEVs were able to recruit inflammatory macrophages and promote their M2 polarization. The in vivo experiments using a Zometa (ZA)-induced mice BRONJ model revealed that the synergistic effects of Ce-MBGNs and ApoEVs enhanced M2 polarization while reducing M1 polarization. This resulted in improved mucosal healing, bone regeneration, and alleviation of osteonecrosis. Overall, our study offers a new strategy for BRONJ therapy by addressing the inflammatory environment. Catechol-conjugated chitosan-based hydrogels containing Ce-MBGNs and ApoEVs have demonstrated impressive potential in the alleviation of BRONJ.

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Sustainable structural materials with excellent impact-resistance properties are urgently needed but challenging to produce, especially in a scalable fashion and with control over 3D shape. Here, we show that bacterial cellulose (BC) and bacterially precipitated calcium carbonate self-assemble into a layered structure reminiscent of tough biomineralized materials in nature (nacre, bone, dentin). The fabrication method consists of biomineralizing BC to form an organic/inorganic mixed slurry, in which calcium carbonate crystal size is controlled with bacterial poly(γ-glutamic acid) and magnesium ions. This slurry self-assembles into a layered material that combines high toughness and high impact and fire resistance. The rapid fabrication is readily scalable, without involving toxic chemicals. Notably, the biomineralized BC can be repeatedly recycled and molded into any desired 3D shape and size using a simple kitchen blender and sieve. This fully biodegradable composite is well suited for use as a component in daily life, including furniture, helmets, and protective garments.

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Catastrophic breakage of a material might bring severe accidents in aerospace engineering, construction, and transportation field. Therefore, engineering material with high toughness values is very important for these special applications. Many biological materials in nature, such as nacre, silk, and wood, possess high toughness values because of their highly organized micro- and nanostructure. Inspired by these natural materials, many scientists tried to build tough materials by improving their orientation of the micro- and nanostructure. However, most of the current fabrication methods are either energy-consuming or labor-intensive, the mild and scalable production of engineering tough materials remains challenging. @en

Biofilms are three-dimensional (3D) bacterial communities that exhibit a highly self-organized nature in terms of their composition and complex architecture. Bacteria in biofilms display emergent biological properties, such as resistance to antimicrobials and disinfectants that the individual planktonic cells lack. Bacterial biofilms possess specialized architectural features including unique extracellular matrix compositions and a distinct spatially patterned arrangement of cells and matrix components within the biofilm. It is unclear which of these architectural elements of bacterial biofilms lead to the development of their emergent biological properties. Here, we report a 3D printing-based technique for studying the emergent resistance behaviors of Escherichia coli biofilms as a function of their architecture. Cellulose and curli are the major extracellular-matrix components in E. coli biofilms. We show that 3D-printed biofilms expressing either curli alone or both curli and cellulose in their extracellular matrices show higher resistance to exposure against disinfectants than 3D prints expressing either cellulose alone or no biofilm-matrix components. The 3D-printed biofilms expressing cellulose and/or curli also show thicker anaerobic zones than nonbiofilm-forming E. coli 3D prints. Thus, the matrix composition plays a crucial role in the emergent spatial patterning and biological endurance of 3D-printed biofilms. In contrast, initial spatial distribution of bacterial density or curli-producing cells does not have an effect on biofilm resistance phenotypes. Further, these 3D-printed biofilms could be reversibly attached to different surfaces (bacterial cellulose, glass, and polystyrene) and display resistance to physical distortions by retaining their shape and structure. This physical robustness highlights their potential in applications including bioremediation, protective coatings against pathogens on medical devices, or wastewater treatment, among many others. This new understanding of the emergent behavior of bacterial biofilms could aid in the development of novel engineered living materials using synthetic biology and materials science approaches.

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Living materials, which are fabricated by encapsulating living biological cells within a non-living matrix, have gained increasing attention in recent years. Their fabrication in spatially defined patterns that are mechanically robust is essential for their optimal functional performance but is difficult to achieve. Here, a bioprinting technique employing environmentally friendly chemistry to encapsulate microalgae within an alginate hydrogel matrix is reported. The bioprinted photosynthetic structures adopt pre-designed geometries at millimeter-scale resolution. A bacterial cellulose substrate confers exceptional advantages to this living material, including strength, toughness, flexibility, robustness, and retention of physical integrity against extreme physical distortions. The bioprinted materials possess sufficient mechanical strength to be self-standing, and can be detached and reattached onto different surfaces. Bioprinted materials can survive stably for a period of at least 3 days without nutrients, and their life can be further extended by transferring them to a fresh source of nutrients within this timeframe. These bioprints are regenerative, that is, they can be reused and expanded to print additional living materials. The fabrication of the bioprinted living materials can be readily up-scaled (up to ≥70 cm × 20 cm), highlighting their potential product applications including artificial leaves, photosynthetic bio-garments, and adhesive labels.

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Bioinspired bacterial cellulose (BC) composites are next-generation renewable materials that exhibit promising industrial applications. However, large-scale production of inorganic/organic BC composites by in situ fermentation remains difficult. The methods based on BC mechanical disintegration impair the mechanical property of dried BC films, while the static in situ fermentation methods fail to incorporate inorganic particles within the BC network because of the limited diffusion ability. Furthermore, the addition of other components in the fermentation medium significantly interferes with the production of BC. Here, a tough BC composite with a layered structure reminiscent of the tough materials found in nature (e.g., nacre, dentin, and bone) is prepared using a semistatic in situ fermentation method. The bacterially produced biopolymer γ-poly(glutamic acid) (PGA), together with graphene oxide (GO), is introduced into the BC fermentation medium. The resulting dried BC-GO-PGA composite film shows high toughness (36 MJ m-3), which makes it one of the toughest BC composite film reported. In traditional in situ fermentation methods, the addition of a second component significantly reduces the wet thickness of the final composites. However, in this report, we show that addition of both PGA and GO to the fermentation medium shows a synergistic effect in increasing the wet thickness of the final BC composites. By gently agitating the solution, GO particles get entrapped into the BC network, as the formed pellicles can move below the liquid level and the GO particles suspended in the liquid can be entrapped into the BC network. Compared to other methods, this method achieves high toughness while using a mild and easily scalable fabrication procedure. These bacterially produced composites could be employed in the next generation of biodegradable structural high-performance materials, construction materials, and tissue engineering scaffolds (tendon, ligament, and skin) that require high toughness.

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Biodegradable porous magnesium (Mg) scaffolds are promising for application in the regeneration of critical-sized bone defects. Although additive manufacturing (AM) carries the promise of offering unique opportunities to fabricate porous Mg scaffolds, current attempts to apply the AM approach to fabricating Mg scaffolds have encountered some crucial issues, such as those related to safety in operation and to the difficulties in composition control. In this paper, we present a room-temperature extrusion-based AM method for the fabrication of topologically ordered porous Mg scaffolds. It is composed of three steps, namely (i) preparing a Mg powder loaded ink with desired rheological properties, (ii) solvent-cast 3D printing (SC-3DP) of the ink to form scaffolds with 0 °/ 90 °/ 0 ° layers, and (iii) debinding and sintering to remove the binder in the ink and then get Mg powder particles bonded by applying a liquid-phase sintering strategy. A rheological analysis of the prepared inks with 54, 58 and 62 vol% Mg powder loading was performed to reveal their viscoelastic properties. Thermal-gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), carbon/sulfur analysis and scanning electron microscopy (SEM) indicated the possibilities of debinding and sintering at one single step for fabricating pure Mg scaffolds with high fidelity and densification. The resulting scaffolds with high porosity contained hierarchical and interconnected pores. This study, for the first time, demonstrated that the SC-3DP technique presents unprecedented possibilities to fabricate Mg-based porous scaffolds that have the potential to be used as a bone-substituting material. Statement of Significance: Biodegradable porous magnesium scaffolds are promising for application in the regeneration of critical-sized bone defects. Although additive manufacturing (AM) carries the promise of offering unique opportunities to fabricate porous magnesium scaffolds, current attempts to apply the AM approach to fabricating magnesium scaffolds still have some crucial limitations. This study demonstrated that the solvent-cast 3D printing technique presents unprecedented possibilities to fabricate Mg-based porous scaffolds. The judicious chosen of formulated binder system allowed for the negligible binder residue after debinding and the short-time liquid-phase sintering strategy led to a great success in sintering pure magnesium scaffolds. The resulting scaffolds with hierarchical and interconnected pores have great potential to be used as a bone-substituting material.

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Natural materials, such as nacre and silk, exhibit both high strength and toughness due to their hierarchical structures highly organized at the nano-, micro-, and macroscales. Bacterial cellulose (BC) presents a hierarchical fibril structure at the nanoscale. At the microscale, however, BC nanofibers are distributed randomly. Here, BC self-Assembles into a highly organized spiral honeycomb microstructure giving rise to a high tensile strength (315 MPa) and a high toughness value (17.8 MJ m-3), with pull-out and de-spiral morphologies observed during failure. Both experiments and finite-element simulations indicate improved mechanical properties resulting from the honeycomb structure. The mild fabrication process consists of an in situ fermentation step utilizing poly(vinyl alcohol), followed by a post-Treatment including freezing-Thawing and boiling. This simple self-Assembly production process is highly scalable, does not require any toxic chemicals, and enables the fabrication of light, strong, and tough hierarchical composite materials with tunable shape and size. @en

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

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