Mechanical Transfer of Black Phosphorus on a Silk Fibroin Substrate: A Viable Method for Photoresponsive and Printable Biomaterials.

Despite the technological importance of semiconductor black phosphorus (BP) in materials science, maintaining the stability of BP crystals in organic media and protecting them from environmental oxidation remains challenging. In this study, we present the synthesis of bulk BP and the exploitation of the viscoelastic properties of a regenerated silk fibroin (SF) film as a biocompatible substrate to transfer BP flakes, thereby preventing oxidation. A model based on the flow of polymers revealed that the applied flow-induced stresses exceed the yield stress of the BP aggregate. Raman spectroscopy was used to investigate the exfoliation efficiency as well as the environmental stability of BP transferred on the SF substrate. Notably, BP flakes transferred to the SF substrate demonstrated improved stability when SF was dissolved in a phosphate-buffered saline medium, and in vitro cancer cell viability experiments demonstrate the tumor ablation efficiency under visible to near-infrared (Vis-nIR) radiation. Moreover, the SF and BP-enriched SF (SF/BP) solution was shown to be processable via extrusion-based three-dimensional (3D) printing. Therefore, this work paves the way for a general method for the transferring of BP on natural biodegradable polymers and processing them via 3D printing toward novel functionalities and complex shapes for biomedical purposes.

Correction to "Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications".

[This corrects the article DOI: 10.1021/acsomega.3c04563.].

4D bioprinted self-folding scaffolds enhance cartilage formation in the engineering of trachea.

Trachea defects that required surgical interventions are increasing in number in the recent years, especially for pediatric patients. However, current gold standards, such as biological grafts and synthetic prothesis, do not represent an effective solution, due to the lack of mimicry and regeneration capability. Bioprinting is a cutting-edge approach for the fabrication of biomimetic scaffold to empower tissue engineering toward trachea replacement. In this study, we developed a self-folding gelatin-based bilayer scaffold for trachea engineering, exploiting the 4D bioprinting approach, namely the fabrication of dynamic scaffolds, able to shape morph in a predefined way after the application of an environmental stimulus. Indeed, starting form a 2D flat position, upon hydration, this scaffold forms a closed tubular structure. An analytical model, based on Timoshenko's beam thermostats, was developed, and validated to predict the radius of curvature of the scaffold according to the material properties and the scaffold geometry. The 4D bioprinted structure was tested with airway fibroblast, lung endothelial cells and ear chondral progenitor cells (eCPCs) toward the development of a tissue engineered trachea. Cells were seeded on the scaffold in its initial flat position, maintained their position after the scaffold actuation and proliferated over or inside it. The ability of eCPCs to differentiate towards mature cartialge was evaluated. Interestingly, real-time PCR revealed that differentiating eCPCs on the 4D bioprinted scaffold promote healthy cartilage formation, if compared with eCPCs cultured on 2D static scaffold. Thus, eCPCs can perceive scaffold folding and its final curvature and to react to it, towards the formation of mature cartilage for the airway.

Electrospun Nanofibrous UV Filters with Bidirectional Actuation Properties Based on Salmon Sperm DNA/Silk Fibroin for Biomedical Applications.

In this study, we dissolved Bombyx mori degummed silk [i.e., silk fibroin (SF)] and salmon sperm deoxyribonucleic acid (DNA) in water and used a bioinspired spinning process to obtain an electrospun nanofibrous SF-based patch (ESF). We investigated the bidirectional macroscale actuation behavior of ESF in response to water vapor and its UV-blocking properties as well as those of ESF/DNA films. Fourier transform infrared (FTIR) results suggest that the formation of ß-sheet-rich structures promotes the actuation effect. ESF/DNA film with high-ordered and ß-sheet-rich structures exhibits higher electrical conductivity and is water-insoluble. Given the intrinsic ability of both SF and DNA to absorb UV radiation, we performed biological experiments on the viability of keratinocyte HaCaT cells after exposure to solar spectrum components. Our findings indicate that the ESF/DNA patch is photoprotective and can increase the cellular viability of keratinocytes after UV exposure. Furthermore, we demonstrated that ESF/DNA patches treated with water vapor can serve as suitable scaffolds for tissue engineering and can improve tissue regeneration when cellularized with HaCaT cells. The 3D shape morphing capability of these patches, along with their potential as UV filters, could offer significant practical advantages in tissue engineering.

4D Printing Shape-Morphing Hybrid Biomaterials for Advanced Bioengineering Applications.

Four-dimensional (4D) printing is an innovative additive manufacturing technology used to fabricate structures that can evolve over time when exposed to a predefined environmental stimulus. 4D printed objects are no longer static objects but programmable active structures that accomplish their functions thanks to a change over time in their physical/chemical properties that usually displays macroscopically as a shapeshifting in response to an external stimulus. 4D printing is characterized by several entangled features (e.g., involved material(s), structure geometry, and applied stimulus entities) that need to be carefully coupled to obtain a favorable fabrication and a functioning structure. Overall, the integration of micro-/nanofabrication methods of biomaterials with nanomaterials represents a promising approach for the development of advanced materials. The ability to construct complex and multifunctional triggerable structures capable of being activated allows for the control of biomedical device activity, reducing the need for invasive interventions. Such advancements provide new tools to biomedical engineers and clinicians to design dynamically actuated implantable devices. In this context, the aim of this review is to demonstrate the potential of 4D printing as an enabling manufacturing technology to code the environmentally triggered physical evolution of structures and devices of biomedical interest.

Chitosan and its derivatives in 3D/4D (bio) printing for tissue engineering and drug delivery applications.

Tissue engineering research has undergone to a revolutionary improvement, thanks to technological advancements, such as the introduction of bioprinting technologies. The ability to develop suitable customized biomaterial inks/bioinks, with excellent printability and ability to promote cell proliferation and function, has a deep impact on such improvements. In this context, printing inks based on chitosan and its derivatives have been instrumental. Thus, the current review aims at providing a comprehensive overview on chitosan-based materials as suitable inks for 3D/4D (bio)printing and their applicability in creating advanced drug delivery platforms and tissue engineered constructs. Furthermore, relevant strategies to improve the mechanical and biological performances of this biomaterial are also highlighted.

Biomaterial Inks from Peptide-Functionalized Silk Fibers for 3D Printing of Futuristic Wound-Healing and Sensing Materials.

This study illustrates the sensing and wound healing properties of silk fibroin in combination with peptide patterns, with an emphasis on the printability of multilayered grids, and envisions possible applications of these next-generation silk-based materials. Functionalized silk fibers covalently linked to an arginine-glycine-aspartic acid (RGD) peptide create a platform for preparing a biomaterial ink for 3D printing of grid-like piezoresistors with wound-healing and sensing properties. The culture medium obtained from 3D-printed silk fibroin enriched with RGD peptide improves cell adhesion, accelerating skin repair. Specifically, RGD peptide-modified silk fibroin demonstrated biocompatibility, enhanced cell adhesion, and higher wound closure rates at lower concentration than the neat peptide. It was also shown that the printing of peptide-modified silk fibroin produces a piezoresistive transducer that is the active component of a sensor based on a Schottky diode harmonic transponder encoding information about pressure. We discovered that such biomaterial ink printed in a multilayered grid can be used as a humidity sensor. Furthermore, humidity activates a transition between low and high conductivity states in this medium that is retained unless a negative voltage is applied, paving the way for utilization in non-volatile organic memory devices. Globally, these results pave the way for promising applications, such as monitoring parameters such as human wound care and being integrated in bio-implantable processors.

Biocompatible and Printable Ionotronic Sensing Materials Based on Silk Fibroin and Soluble Plant-Derived Polyphenols.

The emergence of ionotronic materials has been recently exploited for interfacing electronics and biological tissues, improving sensing with the surrounding environment. In this paper, we investigated the synergistic effect of regenerated silk fibroin (RS) with a plant-derived polyphenol (i.e., chestnut tannin) on ionic conductivity and how water molecules play critical roles in regulating ion mobility in these materials. In particular, we observed that adding tannin to RS increases the ionic conductivity, and this phenomenon is accentuated by increasing the hydration. We also demonstrated how silk-based hybrids could be used as building materials for scaffolds where human fibroblast and neural progenitor cells can highly proliferate. Finally, after proving their biocompatibility, RS hybrids demonstrate excellent three-dimensional (3D) printability via extrusion-based 3D printing to fabricate a soft sensor that can detect charged objects by sensing the electric fields that originate from them. These findings pave the way for a viable option for cell culture and novel sensors, with the potential base for tissue engineering and health monitoring.

Engineering Auxetic Cylinders and Intestine to Improve Longitudinal Intestinal Lengthening and Tailoring Procedure.

Auxetic materials can be exploited for coupling different types of tissues. Herein, we designed a material where the microorganism metabolic activity yields the formation of buckled/collapsed bubbles within gelling silicone cylinders thus providing auxetic properties. The finite element model of such hollow auxetic cylinders demonstrated the tubular structure to promote worm-like peristalsis. In this scenario, the described hybrid auxetic structures may be applied to the longitudinal intestinal lengthening and tailoring procedure to promote enteral autonomy in short bowel syndrome. The presented material and analytical design synergistic approach offer a pioneering step for the clinical translation of hybrid auxetic materials.

3D Printing Silk-Based Bioresorbable Piezoelectric Self-Adhesive Holey Structures for In Vivo Monitoring on Soft Tissues.

Flexible and biocompatible adhesives with sensing capabilities can be integrated onto human body and organ surfaces, characterized by complex geometries, thus having the potential to sense their physiological stimuli offering monitoring and diagnosis of a wide spectrum of diseases. The challenges in this innovative field are the following: (i) the coupling method between the smart adhesive and the soft human substrates, (ii) the bioresorbable behavior of the material, and (iii) the electrical exchange with the substrate. Here, we introduce a multifunctional composite by mixing silk fibroin, featuring piezoelectric properties, with a soluble plant-derived polyphenol (i.e., chestnut tannin) modified with graphene nanoplatelets. This material behaves as a glue on different substrates and gives rise to high elongation at break, conformability, and adhesive performances to gastrointestinal tissues in a rat model and favors the printability via extrusion-based 3D printing. Exploiting these properties, we designed a bioresorbable 3D printed flexible and self-adhesive piezoelectric device that senses the motility once applied onto a phantom intestine and the hand gesture by signal translation. Experimental results also include the biocompatibility study using gastrointestinal cells. These findings could have applicability in animal model studies, and, thanks to the bioresorbable behavior of the materials, such an adhesive device could be used for monitoring the motility of the gastrointestinal tract and for the diagnosis of motility disorders.

A Mesoscale 3D Culture System for Native and Engineered Biphasic Tissues: Application to the Osteochondral Unit.

Interface tissues are functionally graded tissues characterized by a complex layered structure, which therefore present a great challenge to be reproduced and cultured in vitro. Here, we describe the design and operation of a 3D printed dual-chamber bioreactor as a culturing system for biphasic native or engineered osteochondral tissues. The bioreactor is designed to potentially accommodate a variety of interface tissues and enables the precise study of tissue crosstalk by creating two separate microenvironments while maintaining the tissue compartments in direct contact.

4D printing in biomedical applications: emerging trends and technologies.

Nature's material systems during evolution have developed the ability to respond and adapt to environmental stimuli through the generation of complex structures capable of varying their functions across direction, distances and time. 3D printing technologies can recapitulate structural motifs present in natural materials, and efforts are currently being made on the technological side to improve printing resolution, shape fidelity, and printing speed. However, an intrinsic limitation of this technology is that printed objects are static and thus inadequate to dynamically reshape when subjected to external stimuli. In recent years, this issue has been addressed with the design and precise deployment of smart materials that can undergo a programmed morphing in response to a stimulus. The term 4D printing was coined to indicate the combined use of additive manufacturing, smart materials, and careful design of appropriate geometries. In this review, we report the recent progress in the design and development of smart materials that are actuated by different stimuli and their exploitation within additive manufacturing to produce biomimetic structures with important repercussions in different but interrelated biomedical areas.

Pectin as Rheology Modifier of a Gelatin-Based Biomaterial Ink.

Gelatin is a natural biopolymer extensively used for tissue engineering applications due to its similarities to the native extracellular matrix. However, the rheological properties of gelatin formulations are not ideal for extrusion-based bioprinting. In this work, we present an approach to improve gelatin bioprinting performances by using pectin as a rheology modifier of gelatin and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a gelatin-pectin crosslinking agent. The preparation of gelatin-pectin formulations is initially optimized to obtain homogenous gelatin-pectin gels. Since the use of GPTMS requires a drying step to induce the completion of the crosslinking reaction, microporous gelatin-pectin-GPTMS sponges are produced through freeze-drying, and the intrinsic properties of gelatin-pectin-GPTMS networks (e.g., porosity, pore size, degree of swelling, compressive modulus, and cell adhesion) are investigated. Subsequently, rheological investigations together with bioprinting assessments demonstrate the key role of pectin in increasing the viscosity and the yield stress of low viscous gelatin solutions. Water stable, three-dimensional, and self-supporting gelatin-pectin-GPTMS scaffolds with interconnected micro- and macroporosity are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The proposed biofabrication approach does not require any additional temperature controller to further modulate the rheological properties of gelatin solutions and it could furthermore be extended to improve the bioprintability of other biopolymers.

Physicochemical Characterization of Pectin-Gelatin Biomaterial Formulations for 3D Bioprinting.

Developing biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion-based 3D bioprinting. Self-supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue-like scaffolds for tissue regeneration.

Carbon Nanotubes/Regenerated Silk Composite as a Three-Dimensional Printable Bio-Adhesive Ink with Self-Powering Properties.

In this study, regenerated silk (RS) obtained from Bombyx Mori cocoons is compounded with carboxyl-functionalized carbon nanotubes (f-CNTs) in an aqueous environment for the fabrication of functional bio-adhesives. Molecular interactions between RS and carboxyl groups of CNTs result in structural increase of the ß-sheet formation, obtaining a resistant adhesive suitable for a wet biological substrate. Moreover, the functionalization of CNTs promotes their dispersion in RS, thus enabling the production of films with controlled electrical conductivity. The practical utility of such a property is demonstrated through the fabrication of a piezoelectric device implanted in a rat to monitor the breathing in vivo and to be used as a self-powered system. Finally, RS/f-CNTs were used as a printable biomaterial ink to three dimensionally print bilayer hollow tubular structures composed of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and RS. Initial tests carried out by seeding and growing human skin fibroblasts demonstrated that the 3D printed bilayer hollow cylindrical structures offer a suitable surface for the seeded cells to attach and proliferate. In general, the herein proposed RS/f-CNT composite serves as a versatile material for solvent-free dispersion processing and 3D printing, thus paving a new approach to prepare multifunctional materials with potential applications of great interest in sealing biological substrates and implantable devices for regenerative medicine.

Recent advances in bioprinting technologies for engineering different cartilage-based tissues.

Inadequate self-repair and regenerative efficiency of the cartilage tissues has motivated the researchers to devise advanced and effective strategies to resolve this issue. Introduction of bioprinting to tissue engineering has paved the way for fabricating complex biomimetic engineered constructs. In this context, the current review gears off with the discussion of standard and advanced 3D/4D printing technologies and their implications for the repair of different cartilage tissues, namely, articular, meniscal, nasoseptal, auricular, costal, and tracheal cartilage. The review is then directed towards highlighting the current stem cell opportunities. On a concluding note, associated critical issues and prospects for future developments, particularly in this sphere of personalized medicines have been discussed.

Endothelial cells support osteogenesis in an in vitro vascularized bone model developed by 3D bioprinting.

Bone is a highly vascularized tissue, in which vascularization and mineralization are concurrent processes during skeletal development. Indeed, both components should be included in any reliable and adherent in vitro model platform for the study of bone physiology and pathogenesis of skeletal disorders. To this end, we developed an in vitro vascularized bone model, using a gelatin-nanohydroxyapatite (gel-nHA) three-dimensional (3D) bioprinted scaffold. First, we seeded human mesenchymal stem cells (hMSCs) on the scaffold, which underwent osteogenic differentiation for 2 weeks. Then, we included lentiviral-GFP transfected human umbilical vein endothelial cells (HUVECs) within the 3D bioprinted scaffold macropores to form a capillary-like network during 2 more weeks of culture. We tested three experimental conditions: condition 1, bone constructs with HUVECs cultured in 1:1 osteogenic medium (OM): endothelial medium (EM); condition 2, bone constructs without HUVECs cultured in 1:1 OM:EM; condition 3: bone construct with HUVECs cultured in 1:1 growth medium:EM. All samples resulted in engineered bone matrix. In conditions 1 and 3, HUVECs formed tubular structures within the bone constructs, with the assembly of a complex capillary-like network visible by fluorescence microscopy in the live tissue and histology. CD31 immunostaining confirmed significant vascular lumen formation. Quantitative real-time PCR was used to quantify osteogenic differentiation and endothelial response. Alkaline phosphatase and runt-related transcription factor 2 upregulation confirmed early osteogenic commitment of hMSCs. Even when OM was removed under condition 3, we observed clear osteogenesis, which was notably accompanied by upregulation of osteopontin, vascular endothelial growth factor, and collagen type I. These findings indicate that we have successfully realized a bone model with robust vascularization in just 4 weeks of culture and we highlighted how the inclusion of endothelial cells more realistically supports osteogenesis. The approach reported here resulted in a biologically inspired in vitro model of bone vascularization, simulating de novo morphogenesis of capillary vessels occurring during tissue development.

Modeling the Three-Dimensional Bioprinting Process of ß-Sheet Self-Assembling Peptide Hydrogel Scaffolds.

Extrusion-based three-dimensional (3D) bioprinting is nowadays the most efficient additive manufacturing technology to fabricate well-defined and clinical-scale relevant 3D scaffolds, exploiting soft biomaterials. However, trial and error approaches are usually employed to achieve the desired structures, thus leading to a waste of time and material. In this work, we show the potential of finite element (FE) simulation in predicting the printability of a biomaterial, in terms of extrudability and scaffold mechanical stability over time. To this end, we firstly rheologically characterized a newly developed self-assembling peptide hydrogel (SAPH). Subsequently, we modeled both the extrusion process of the SAPHs and the stability over time of a 3D-bioprinted wood-pile scaffold. FE modeling revealed that the simulated SAPHs and printing setups led to a successful extrusion, within a range of shear stresses that are not detrimental for cells. Finally, we successfully 3D bioprinted human ear-shaped scaffolds with in vivo dimensions and several protrusion planes by bioplotting the SAPH into a poly(vinyl alcohol)-poly(vinyl pyrrolidone) copolymer, which was identified as a suitable bioprinting strategy by mechanical FE simulation.

Pectin-GPTMS-Based Biomaterial: toward a Sustainable Bioprinting of 3D scaffolds for Tissue Engineering Application.

Developing green and nontoxic biomaterials, derived from renewable sources and processable through 3D bioprinting technologies, is an emerging challenge of sustainable tissue engineering. Here, pectin from citrus peels was cross-linked for the first time with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) through a one-pot procedure. Freeze-dried porous pectin sponges, with tunable properties in terms of porosity, water uptake, and compressive modulus, were obtained by controlling GPTMS content. Cell experiments showed that GPTMS did not affect the cytocompatibility of pectin. The addition of GPTMS improved the printability of pectin due to an increase of viscosity and yield stress. Three-dimensional woodpile and complex anatomical-shaped scaffolds with interconnected micro- and macropores were, therefore, bioprinted without the use of any additional support material. These results show the great potential of using pectin cross-linked with GPTMS as biomaterial ink to fabricate patient-specific scaffolds, which could be used to promote tissue regeneration in vivo.

Ultrasonic mixing chamber as an effective tool for the biofabrication of fully graded scaffolds for interface tissue engineering.

One of the main challenges of the interface-tissue engineering is the regeneration of diseased or damaged interfacial native tissues that are heterogeneous both in composition and in structure. In order to achieve this objective, innovative fabrication techniques have to be investigated. This work describes the design, fabrication, and validation of a novel mixing system to be integrated into a double-extruder bioprinter, based on an ultrasonic probe included into a mixing chamber. To validate the quality and the influence of mixing time, different nanohydroxyapatite-gelatin samples were printed. Mechanical characterization, micro-computed tomography, and thermogravimetric analysis were carried out. Samples obtained from three-dimensional bioprinting using the mixing chamber were compared to samples obtained by deposition of the same final solution obtained by manually operated ultrasound probe, showing no statistical differences. Results obtained from samples characterization allow to consider the proposed mixing system as a promising tool for the fabrication of graduated structures which are increasingly being used in interface-tissue engineering.