Materials Today Bio

AbstractsThe ideal engineered microbial smart-drug should be capable of functioning on demand at specific sites in vivo. However, precise regulation of engineered microorganisms poses challenges in the convoluted and elongated intestines. Despite the promising application potential of optogenetic regulation strategies based on light signals, the poor tissue penetration of light signals limits their application in large experimental animals. Given the rapid development of ingestible electronic capsules in recent years, taking advantage of them as regulatory devices to deliver light signals in situ to engineered bacteria within the intestines has become feasible. In this study, we established an electronic-microorganism signaling system, realized by two Bluetooth-controlled luminescent electronic capsules were designed. The "Manager" capsule is equipped with a photosensor to monitor the distribution of engineered bacteria and to activate the optogenetic function of the bacteria by emitting green light. The other capsule, "Locator", can control the in situ photopolymerization of hydrogels in the intestines via ultraviolet light, aiding in the retention of engineered bacteria at specific sites. These two electronic capsules are expected to work synergistically to regulate the distribution and function of engineered bacteria in vivo, and their application in the treatment of colitis in pigs is currently being investigated, with relevant results to be updated subsequently.

Cellular agriculture, as an emerging food production system, holds potential to address sustainability, food security, and agricultural resilience. Within the cell-based meat supply chain, one of the key steps is scaffolding. In this study, we assessed decellularized banana leaves, various coating materials, and different cell seeding strategies to determine their effects on cell viability, cell growth, cell alignment, and the response of the materials to thermal processing. The efficiency of decellularization was verified through DNA quantification, which decreased from 445 ng/mg in fresh banana leaves to non-detectable levels in the decellularized samples. This was further confirmed by FTIR and PCA modeling. Cell viability exceeded 98% on uncoated, soy-coated, and gelatin-coated samples of the decellularized banana leaves. Alignment of cells on gelatin-coated samples was the highest among the samples, with a dominant orientation of 65.8{degrees}, compared to soy-coated and uncoated samples with dominant orientations of 9.2{degrees} and -6.3{degrees}, respectively. In terms of quality attributes, the kinetics of shrinkage indicated that coating with soy and the presence of cells increased the activation energy due to the higher energy required for protein denaturation. Moreover, the kinetics of area changes in plain scaffolds without cells followed a first-order pattern, while with seeded cells a second-order pattern was followed. In summary, decellularized banana leaves present a sustainable and suitable biomaterial to support cells towards future needs related to meat production.

Developing wound dressings that support healing and allow real-time monitoring is a key priority in modern wound care. In this study, we designed a curcumin-loaded carboxymethyl cellulose (CMC)/polyvinyl alcohol (PVA) composite dressing with integrated pH-responsive colorimetric sensing. The films were made by solution blending and freeze-drying. They formed porous, absorbent structures that quickly absorbed fluid and managed wound exudates effectively. Curcumin served as both a therapeutic agent--delivering antioxidant, anti-inflammatory, and antibacterial effects--and a natural colorimetric indicator through its keto-enol tautomerism, enabling reversible pH-dependent transitions visible to the naked eye. UV-Vis spectroscopy confirmed absorbance shifts under acidic and alkaline conditions. It also showed that curcumin remained [~]80% stable after 14 days in the polymer matrix FTIR and SEM confirmed successful incorporation and uniform distribution of curcumin within the polymer network. Cytotoxicity assays demonstrated excellent biocompatibility, while disc diffusion and MIC assays revealed significant antibacterial activity of the curcumin-loaded films against Pseudomonas aeruginosa, confirming their potential to reduce bacterial growth. Smartphone-based RGB analysis showed a strong correlation with pH (R2 {approx} 0.99), highlighting the feasibility of low-cost digital wound monitoring. Mechanical testing indicated sufficient tensile strength and flexibility for practical wound application. Quantitative antibacterial data (inhibition zone diameter and MIC) supported strong antimicrobial performance. The primary objective of this study was to develop a multifunctional wound dressing capable of both protecting and monitoring wounds in real-time. The proposed system is specifically designed for chronic and infected wounds where pH imbalance delays healing. In addition to antimicrobial activity, the fabricated films demonstrated desirable swelling capacity and sustained curcumin release, further highlighting the practical applicability of the dressing in wound care. Cost- benefit analysis demonstrated clear economic advantages over commercial gauze-based and hydrocolloid dressings. The fabrication method is compatible with industrial scale-up, although process optimization is required. Overall, the curcumin-loaded CMC/PVA dressing provides a multifunctional platform that combines biocompatibility, antibacterial activity, pH-responsive biosensing, and cost-effectiveness for next-generation wound care. Future studies will investigate in vivo performance, long-term stability, and clinical translation potential to validate its effectiveness in real-world conditions. Overall, the curcumin-loaded CMC/PVA dressing provides a multifunctional platform that combines biocompatibility, antibacterial activity, pH-responsive biosensing, mechanical stability, and cost-effectiveness for next-generation wound care. Future studies will investigate in vivo performance, long-term stability, and clinical translation potential.

The combination of synthetic biology and additive manufacturing has driven major changes in production of biomaterials, especially through the use of three-dimensional (3D) bioprinting to create engineered living materials. However, current fabrication methods can be limited by prohibitive hardware costs and the inability to maintain structural fidelity in complex, free-form living architectures. This work demonstrates how to build a low-cost, open-source 3D bioprinting platform that can make complicated bacterial structures with complex geometry and high dimensional accuracy. A commercially available, conventional fused deposition modeling 3D printer was modified to create a bioprinting system that is simple to build. The modified bioprinter, which costs around $450, is less expensive than many commercial bioprinters. This 3D-printing technology uses slurry-based support bath methods featuring low-cost gelatin and agarose microparticles, resulting in structures with a high aspect ratio (>8:1) and feature sizes as small as 260 m. The optimization of critical printing settings, including the ability of the bioink to retract during non-print movements, resulted in a reduction of unwanted bacterial deposition by nearly two orders of magnitude. Long-term viability experiments showed that bacteria in the bioprints could survive for at least 28 days with nutrient supplementation. Additionally, 3D-printed engineered biofilms revealed that incubation conditions and extracellular matrix composition significantly impacted the mechanical properties of printed constructs, with tradeoffs between matrix production and mechanical integrity. This study showcases an accessible 3D bioprinting platform for advanced bioprinting technologies, enabling development of engineered living materials with potential applications in synthetic biology, biotechnology, and tissue engineering.

Hydrogels have emerged as an important field of study in biomedical engineering, biomaterials research and soft tissue modeling due to their versatility and customizable structures. They are three-dimensional networks capable of absorbing significant amounts of water and can be chemically or physically bonded, rendering them insoluble. The unique properties of hydrogels, such as excellent biocompatibility, degradation abilities, and ability to form chemical bonds between macromolecules, make them valuable in a variety of applications. One of the main applications of hydrogels is tissue engineering, where they are used in implantation, surgical procedures, targeted drug delivery and tissue regeneration. Hydrogels can act as scaffolds to mimic the properties of natural tissues, including mechanical strength, degradation properties, gel transformation, and swelling. Crosslinking hydrogels with other polymers, both natural and synthetic, allows their degradation rate to be controlled. Composite hydrogels or biohybrid gels offer advantages such as consistent properties between batches and a high degree of control in the manufacturing process. Using different types of polymers, researchers can fine-tune the chemical and physical properties of the resulting hydrogel, which is crucial for certain biomedical applications such as drug delivery, biocompatibility, and mechanical stability. In this study, our aim is to make hybrid hydrogel scaffolds more suitable for biomedical applications, which can show the healing effect of adding natural polymers to the scaffold structure. Our results revealed that the hybrid hydrogel obtained by chemically linking gelatin polymer chains to PEG polymers during crosslinking via UV light provides more advantageous properties in the final hydrogel structure, such as better durability and pore sizes for future cell placement in tissue engineering applications.

IntroductionBoth developing and developed nations have made the creation of innovative wound-healing nanomaterials based on natural extracts a top research goal. The objective of this research was to create a gel containing collagen nanoparticles and evaluate its therapeutic potential for skin lesions. MethodsCollagen nanoparticles from fish scales were produced for the first time using desolvation techniques. Using Fourier transform infrared spectroscopy (FTIR), the structure of the isolated collagen and its similarities to collagen type 1 were identified. The surface morphology of the isolated collagen and its reformulation into nanoparticles were examined using transmission and scanning electron microscopy. Human skin fibroblast cells were employed to examine the cytotoxicity of the nanomaterials, and an experimental model was used to evaluate the wound healing capability. ResultsCollagen nanoparticles formulation was confirmed using FTIR, SEM and TEM analysis. Cytotoxicity studies demomstrated that the manufactured nanoparticles have minor toxicity at high concentrations on human skin fibroblast. Histological investigation proved that the fabricated fish scale collagen nanoparticles promoted the healing process in comparison to the saline group. ConclusionThe fabricated product is a highly influential wound healing product that can be applicable for commercial use. The nanoscale size of collagen nanoparticles, make them interesting candidates for biological applications. Key Summary PointsO_LIThe goal of this research was to create natural, effective wound remedies that could lower health-care costs while also providing pain relief and, ultimately, effective scar repair. C_LIO_LICollagen nanoparticles can be synthesized from fish scale utilizing various nanotechnology-based approaches to stimulate skin cell proliferation and promote wound healing. C_LIO_LICollagen nanoparticles have a rough surface, have a negative potential, and can be used for drug delivery and wound healing. C_LIO_LIHistological and macroscopical analysis showed that the synthesized nanoparticles promoted faster wound healing. C_LI

This study presents the development and characterization of a novel thermosensitive injectable hydrogel designed to enhance the biomechanical properties of poloxamer 407 (P407) through the incorporation of a self-assembling peptide. The primary objective was to engineer a formulation that rapidly gels following intra-articular (i.a.) injection, exhibits improved mechanical strength, and enables sustained release of embedded therapeutic cargo. Gelation time assays demonstrated that the P407-peptide formulation solidified more quickly than P407 alone at equivalent concentrations. Rheological analysis revealed a 1.5 kPa increase in storage modulus in the hybrid hydrogel, confirming improved mechanical integrity. In vitro biocompatibility was assessed using human chondrocytes, with MTS assays and LIVE/DEAD staining indicating no cytotoxicity across tested concentrations. To evaluate in vivo applicability, a near-infrared fluorescent (NIRF) dye was incorporated into the hydrogel and injected intra-articularly into an osteoarthritis (OA) mouse model. The labeled formulation allowed for successful tracking and demonstrated localized gelation, supporting its suitability for site-specific, sustained delivery. Overall, the P407-peptide hydrogel offers a promising platform for i.a. therapeutic applications, combining injectability, rapid thermoresponsive gelation, mechanical reinforcement, and controlled release behavior, making it well-suited for regenerative medicine and OA treatment.

Ternary composite systems formed by lactoferrin (LF), sodium alginate (Alg), and Fe(II) were designed to investigate their potential as an iron delivery platform with enhanced protein stability. The ternary LF-Alg-Fe (LAF) composites demonstrated distinct structures depending on the LF to Alg ratio and the Fe(II) concentrations. At an LF to Alg ratio of 8:2 and final Fe concentrations between 20-30 mM, the system formed complexes stabilized by electrostatic interactions. Whereas Alg-rich formulations formed hydrogels stabilized by Alg-Fe(II) egg-box cross-linking. Rheological analysis and swelling behavior indicated a higher mechanical strength in LF-rich complexes and stronger network integrity in Alg-rich hydrogels, while intermediate LF/Alg ratios showed weaker structures overall. Fourier-transform infrared spectroscopy (FTIR) spectra showed no changes in functional groups or polymer structures after composite formation, confirming composite formation via non-covalent interactions. Thermal studies indicated that these ternary systems improved LF stability, evidenced by preserved secondary structure after heating using circular dichroism (CD), and an increased denaturation temperature compared with free LF in differential scanning calorimetry (DSC). In addition, in LF-rich formulations the Fe(II) release in aqueous solution was [~]50% while in Alg-rich formulations it was much lower (< 10%). LF-Alg-Fe composites exhibit distinct structures governed by protein-polysaccharide interactions and iron-mediated cross-linking, providing a potential strategy for protein stabilization and iron fortification in food systems.

Implantations of degradable biomaterials for drug delivery or restoration and regenerative medicine cause fibrosis and acute inflammation which may lead to chronic side effects. Additionally, the fibrous encapsulation can itself inhibit the local cells from proliferating and forming new healthy tissue. Current drug delivery models require a new method to decrease the adverse effects following implantation of biomaterials in patients and inhibit the activation signaling sent to quiescent fibroblasts. This research details the novel chemical and physical properties of reduced keratin (KTN) hydrogels extracted from residual human hair with sheared cuticle layers, obtained from barbershops and beauty salons. KTN hydrogels with and without calcium ions conjugated with atorvastatin (Ator), which is thought to affect TGF-{beta} signaling pathways directly by competitively inhibiting key signal transmitters and cellular responses, were investigated via electron microscopy, absorbance spectroscopy, and computational modeling and simulation to observe the proliferative inhibition of fibroblastic cells in comparison to the alginate hydrogel controls. Hydrogel extracts were tested for non-cytotoxic effects via L929 adipose fibroblast cell culture following ISO 10993-5 standard for safety of medical device biomaterials. Two cell models were exposed to increasing serial concentrations of Ator to evaluate the half maximal effective concentration (EC50). For quiescent fibroblasts, Ators EC50 was observed at 368 M in PBS. For mesenchymal stem cells (MSCs), Ators EC50 was observed at 209 M in PBS. The mass of drug was normalized in all groups after calculating their timed absorption and release, and KTN hydrogels conjugated with calcium ions and Ator (ATCK) completely absorbed the drug < 0.5 h while KTN hydrogels with Ator alone (ATKR) completely absorbed the drug at 1h. After 7d in PBS, ATCK released Ator at 2.2 {+/-} 0.7% while Ator in calcium alginate spheres (ATLG) was released at 7.8 {+/-} 1.1% and ATKR released 11.2 {+/-} 4.3% of drug. On-going research focuses on other active fibroblastic cell types and in vivo experimentation to observe foreign body response in subcutaneous mouse tissue.

Ischemia-reperfusion injury (IRI) is a significant complication in kidney transplantation, often affecting the viability and function of organs. Normothermic machine perfusion (NMP) is a technique used to improve the condition of organs prior to transplantation. In this study, we show that incorporating antioxidant poly(propylene sulfide) nanoparticles (PPS-NPs) during cold-storage and NMP significantly enhances its efficacy in reducing IRI upon porcine kidney transplantation. We found that by scavenging reactive oxygen species, PPS-NPs reduced oxidative stress and inflammation that occurs during ischemia-reperfusion with oxidized DNA reduced 5.3x and both TNF- and complement activation approximately halved. Our studies show that this approach led to significantly improved hemodynamics, better renal function, and tissue health compared to NMP alone. The results suggest that incorporating PPS-NPs into transplantation protocols may expand the pool of kidneys suitable for transplantation and enhance overall transplantation success rates. The broader impact of this work could extend to other organ transplants, suggesting a wider application for nanoantioxidant technologies in organ preservation.

Diabetes Mellitus (DM) is one of the most concerning conditions, and its chronic complications are nearly synonymous with inflammation, oxidative stress, and infections. In the acute inflammatory phase of diabetic wound healing (DWH), reducing excessive reactive oxygen species (ROS) and inflammatory response of the wound is a necessary treatment. The current work used a mix of emulsification and lyophilization approaches to investigate the effects of resveratrol microparticles (RES-GMS) loaded chitosan-collagen (CS-CLG) scaffold with doxycycline (DOX) on DWH. Resveratrol (RES) is a powerful antioxidant that promotes cell proliferation in the dermis by improving fibroblast function and enhancing CLG production. DOX can potentially shift the balance away from the chronic wounds pro-inflammatory, proteolytic status toward an environment that promotes vascular ingrowth and, eventually, epithelial development. Cross-linked scaffolds had optimal porosity, reduced matrix degradation, and prolonged drug release when compared to non-cross-linked scaffolds, according to the results of composite scaffold characterization. Cell proliferation assay employing mouse fibroblasts was used to study the kinetics and bioactivity of growth factors produced from the scaffold. The RES-DOX-CS-CLG scaffold was biocompatible and promoted cell development compared to the control and CS-CLG scaffolds in in vitro experiments. DOX-loaded CS-CLG scaffold loaded with R-GMS delivers a prolonged release of RES, according to in vitro tests.

Bone morphogenetic proteins (BMPs) and transforming growth factors (TGF-{beta}) are members of the transforming growth factors superfamily, known for their role in several physiological and pathological processes. These factors are known to bind in vivo to BMP and TGF-{beta} receptors respectively, which induces the phosphorylation of the Smad (pSmad) transcription factors. This pathway is generally studied with western blot and luciferase bioluminescence assay, which present some limitations. In our work, we developed and optimized a high-content immunofluorescence assay to study the pSmad pathway on glass as well as on biomaterials by overcoming the technical challenges raised by image acquisition and analysis. Furthermore, with this assay, we present here a proof-of concept for drug testing on glass and on biomimetic films using drug inhibitors of the BMP receptor and of the TGF-{beta} receptors. Altogether, our results open perspectives for future drug testing on biomimetic films that present various growth factors and extracellular matrix proteins or peptides.

This paper focuses on the design, fabrication, and characterization of a platform aiming to dynamically culture cells under mechanical stimulation. The platform, made of polymethylmethacrylate (PMMA), allows real-time mechanical stimuli, providing valuable insights for living tissue models. Through mechanical testing and dynamic microfluidic tests, the chip functionality was assessed. The experimental results validation showcases the potential of the device in mimicking physiological conditions, offering a promising avenue for pharmaceutical testing advancement.

It can be found from the results that nano hydroxyapatite- silver -3.0 wt% carageenan (nHA-Ag-CG3.0) improved the mechanical properties of the as-formed hydrogel scaffold after incorporation of higher CG concentration. The Youngs modulus of hydroxyapatite- silver - 1.5wt% carageenan (nHA-Ag-CG1.5) was found to be 0.36 {+/-} 0.07 MPa that increased in case of nHA-Ag-CG3.0 demonstrating better interfacial compatibility of their matrix with respect to the reinforcement. This increase in reinforcement concentration resulted in higher stiffness that dissipated energy. The higher swelling ratio is envisaged to induce better cell adhesion and proliferation. The biodegradability test was performed in phosphate buffered saline at body temperature for 3 weeks. The biodegradability rate of nHA-Ag-CG1.5 was found to be equivalent to nHA-Ag-CG3.0 hydrogels at day 7 while it increased faster in nHA-Ag-CG3.0 on days 14 and 21 that may be ascribed to the possible interaction of nHA and Ag with their CG matrix. The bacterial cell viability of Staphylococcus aureus (S. aureus) was performed after 10 h, 20 h and 30 h of culture. The nHA-Ag-CG1.5 exhibited restrained growth of S. aureus as compared to nHA-Ag-CG3.0 and these results were validated by CLSM analysis. Hence, nHA-Ag-CG3.0 may be considered to have more cytocompatibility than nHA-Ag-CG 1.5.

In response to clinical demands for advanced wound dressings, a "smart bandage" was developed by combining polycaprolactone (PCL) nanofibers and the thermoresponsive polymer poly(N-isopropylacrylamide) (PNIPAAm). This bandage incorporates curcumin, an anti-inflammatory and antioxidant agent, to enhance wound healing. The LCST of PNIPAAm (32{degrees}C) enables an "on-off" drug delivery system, transitioning from a hydrophilic coil to a hydrophobic state. The nanofiber matrix was characterized using FTIR spectroscopy, SEM, water contact angle, and dynamic mechanical analysis. Curcumin release was assessed both above and below the LCST. The bandage is comfortable, easy to apply and remove, and promotes rapid wound healing. In vitro studies confirmed its non-toxicity to human dermal fibroblast cells. This "smart bandage" represents a significant advancement in wound care, with the potential to meet clinical requirements and enhance patient compliance, offering controlled drug delivery combined with the therapeutic benefits of curcumin.

AO_SCPLOWBSTRACTC_SCPLOWPolymeric implants for local drug delivery offer significant advantages for treating various medical conditions by enabling the temporal and spatial control of drug release, improving efficacy, and reducing systemic side effects. In this context, {micro}MESH, a 20 m thin, dual-compartmentalized film comprising a poly(lactic-co-glycolic acid) (PLGA) micronetwork intercalated with a polyvinyl alcohol (PVA) microlayer, represents an interesting opportunity as its geometry can be systematically and accurately micropatterned during the fabrication process, enabling the systematic analysis of the effect of geometry on biodegradation rates mechanisms. In this study, four different {micro}MESH films were realized with different surface area-to-volume ratios (Sa/V), ranging from 0.67 to 1.7 {micro}m-1. After characterizing the {micro}MESH geometry via fluorescent and scanning electron microscopy, biodegradations studies were performed up to 60 days in different media to assess the mass loss of PLGA, the reduction in PLGA molecular weight, and the formation of macroscopic defects - pores, holes and crack - within the PLGA micronetwork. By comparing the four {micro}MESH films among themselves and to a flat, continuous PLGA slab (FLAT), it was confirmed the importance of the surface-to-volume ratio and demonstrated that {micro}MESH with higher Sa/V ratios exhibited slower degradation rates compared to FLAT. Scanning electron microscopy images of the PLGA micronetworks revealed morphological changes indicative of bulk erosion, including surface roughening and pore formation, in FLAT and {micro}MESH configurations with low Sa/V ratios. These findings confirm that film micropatterning significantly influences degradation kinetics.

The severe, long-lasting harm caused by plastic pollution to marine ecosystems and coastal economies has led to the development of biodegradable plastics; however, their limited decomposition in cold, dark marine environments remains a challenge. Here, we present our newly developed technologies for creating 3D-bioprinted living materials for bioplastic degradation with specific use in marine environments. Our approach integrates halotolerant bioplastic-degrading bacterium Bacillus sp. NRRL B-14911 into alginate-based bio-ink to print an engineered living material (ELM) termed a "bio-sticker." Quantification of bacteria viability reveals that bioprinted marine bacteria survive within bio-stickers for more than three weeks. The rate at which the bio-stickers degrade the bioplastic polyhydroxybutyrate (PHB) can be tuned by altering bio-sticker biomass concentration, bioplastic concentration, or incubation temperature. Bio-stickers that are transferred to a new PHB sample still retain high biodegradation activity, demonstrating their durability. Strain sweep oscillatory tests demonstrate viscoelastic behavior of the bio-stickers. Monotonic tensile tests indicate that the elastic modulus and the adhesion of the bio-stickers are not negatively impacted by bacteria growth or incubation temperature. Our work paves the way for development of ELMs to facilitate the inclusion of bioplastics within the blue economy, promoting the emergence of more sustainable and eco-friendly materials.

Systemic inflammation presents a significant challenge to the long-term function of biohybrid implants. While endothelialisation of biohybrid implants has been shown to improve device hemocompatibility, its feasibility under the influence of patients inflammatory status remains largely unexplored. To investigate this, we developed a controlled in vitro model which allows to study endothelial dysfunction under inflammatory stress. Endothelial cells were cultured on polydimethylsiloxane under physiological shear stress and exposed to lipopolysaccharide (LPS)-activated peripheral blood mononuclear cells (PBMCs), simulating inflammatory conditions. Endothelial morphology and confluence was assessed using immunohistochemistry and scanning electron microscopy. Leukocyte adhesion was evaluated directly as well as indirectly, using flow cytometry to analyse cell adhesion molecules. Quantitative PCR was used for gene expression analysis of inflammatory mediators. Notably, neither LPS nor PBMCs alone induced endothelial disruption, whereas their combination significantly impaired endothelial confluence: Inflammatory activation led to substantial loss of endothelial confluence, increased leukocyte adhesion, and elevated expression of adhesion molecules ICAM-1, VCAM-1, and E-selectin. Gene expression analysis highlights the upregulation of inflammatory mediators, such as IL-6, IL-8, IL-10, and MCP-1. This study underscores the challenges of implementing endothelialisation in biohybrid devices, particularly in patients with systemic inflammation. By considering translational hurdles, this work contributes to the development of clinically viable biohybrid constructs and highlights the importance of considering inflammatory dynamics when designing next-generation implants.

Bone tissue loss can occur due to disease, trauma or following surgery, in each case treatment involving the use of bone grafts or biomaterials is usually required. Recent development of three-dimensional (3D) bioprinting (3DBP) has enabled the printing of customized bone substitutes. Bioinks used for bone 3DBP employ various particulate phases such as ceramic and bioactive glass particles embedded in the bioink creating a composite. When composite bioinks are used for 3DBP based on extrusion, particles are heterogeneously distributed causing damage to cells due to stresses created during flow in the matrix of the composite. Therefore, the objective of this study was to develop cell-friendly osteopromotive bioink mitigating the risk of cell damage due to the flow of particles. Towards this end, we have linked organic and inorganic components, gelatin methacryloyl (GelMA) and Ag-doped bioactive glass (Ag-BaG), to produce a hybrid material, GelMA-Ag-BaG (GAB). The distribution of the elements present in the Ag-BaG in the resulting hybrid GAB structure was examined. Rheological properties of the resulting hydrogel and its printability, as well as the degree of swelling and degradation over time, were also evaluated. GAB was compared to GelMA alone and GelMA-Ag-BaG nanocomposites. Results showed the superiority of the hybrid GAB bioink in terms of homogenous distribution of the elements in the structure, rheological properties, printability, and degradation profiles. Accordingly, this new bioink represents a major advance for bone 3DBP.

Critical-sized bone defects represent a challenge in bone tissue engineering, due to insufficient vascularization that results in implant failure. Scaffold pre-vascularization is a promising strategy to create a functional microvascular network that integrates with host vasculature. In this study, we present a hybrid 3D construct comprising a hyaluronic acid-based hydrogel and a 3D printed polycaprolactone/{beta}-tricalcium phosphate scaffold, to support vascular network formation and osteogenic differentiation. Peptide-functionalized (i.e. RGD, YIGSR, IKVAV, QK) hydrogels were obtained via thiol-ene chemistry, using two crosslinkers (PEG-diSH or MMP-diSH). Preliminary biological experiments assessed human mesenchymal stromal cells (hMSCs), endothelial cells (hUVECs), and their co-culture, on different gel formulations. All cell conditions displayed enhanced spreading and metabolic activity on gel formulations comprising RGD; thus these (i.e. RGD only and a combination of RGD/YIGSR) were selected for further studies. Cells were then mixed with the hydrogel precursor solutions, which were injected to embed the scaffolds and crosslinked using a UV lamp. After 7 days, tubule formation was observed only in co-culture conditions, highlighting the importance of cellular crosstalk for the formation of a vascular network. Significant differences were found across the tested formulations. In the RGD-PEG constructs, hUVECs formed tubule-like structures, surrounded by hMSCs, exhibiting pericyte-like behavior, supported by the upregulation of SMA gene. Conversely, in the RGD/YIGSR-MMP conditions, hMSCs were mostly located on the scaffold fibers, and showed the highest expression of early osteogenic markers (RUNX2 and ALP). Overall, we demonstrated that the hybrid system with tailored hydrogel chemistry can support simultaneous microvascular organization and osteogenic commitment, offering a promising platform for bone tissue engineering applications. However, further studies involving longer culture periods will aim at clarifying the complex interplay between material composition, cell crosstalk and spatial organization and their influence on the maturation and stability of the vascular network.