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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.

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.

Poultry feathers are among the most abundant and polluting keratin-rich waste biomasses. In this work, we developed a one-pot microwave-assisted process for eco-friendly keratin extraction from poultry feathers followed by a direct electrospinning (ES) of the raw extract, without further purification, to obtain keratin-based bioplastics. This microwave-assisted keratin extraction (MAE) was conducted in acetic acid 70% v/v. The effects of extraction time, solvent/feathers ratio, and heating mode (MAE vs. conventional heating) on the extraction yield were investigated. The highest keratin yield (26 ± 1% w/w with respect to initial feathers) was obtained after 5 h of MAE. Waste-derived keratin were blended with gelatin to fabricate keratin-based biodegradable and biocompatible bioplastics via ES, using 3-(Glycidyloxypropyl)trimethoxysilane (GPTMS) as a cross-linking agent. A full characterization of their thermal, mechanical, and barrier properties was performed by differential scanning calorimetry, thermogravimetric analysis, uniaxial tensile tests, and water permeability measurements. Their morphology and protein structure were investigated using scanning electron microscopy and attenuated total reflection-infrared spectroscopy. All these characterizations highlighted that the properties of the keratin-based bioplastics can be modulated by changing keratin and GPTMS concentrations. These bioplastics could be applied in areas such as bio-packaging and filtration/purification membranes.

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 modelled both the extrusion process of the SAPHs as well as the stability over time of a 3D bioprinted wood-pile scaffold. FE modelling revealed that the simulated SAPHs and printing set-ups led to a successful extrusion, within a range of shear stresses that are not detrimental for cells. Finally, we successfully 3D bioprinted a 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.

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.

Neural tissue engineering has recently emerged as an alternative strategy to repair nerve damage and promote nerve regeneration. It involves the fabrication of scaffolds with properties mimicking those of the natural extracellular matrix for guiding a three-dimensional (3D) neural regeneration. These engineered constructs, in addition to mechanical support, should be also capable of providing proper chemical and electrical stimuli for adhesion, migration, and proliferation of the neural cells. In this study, we developed conductive composite hydrogel films based on gelatin and carbon black (CB) as scaffolds for neural tissue engineering applications. The presented hydrogel constructs were fabricated in the form of films using the solvent casting method after dispersing several concentrations of CB in a 5 % (w/v) gelatin solution along with (3-glycidoxypropyl)trimethoxysilane (GPTMS) as the crosslinking agent at a concentration of 1.84 % (v/v). The CB concentrations of 0.3 %, 0.5 %, 0.7 %, and 0.9 % (w/w) with respect to the gelatin amount were chosen. The morphological, compositional, swelling, dissolution, electrical, mechanical, and wettability properties together were characterized as function of CB content and compared with those of pure gelatin-based hydrogel. Results demonstrated that the incorporation of different quantities of CB relatively reduced the water uptake capability of the films and increased the stability in water of the gelatin matrix. Findings from the mechanical tests revealed that composite hydrogels have a lower elastic modulus with respect to the pure gelatin matrix. Moreover, it was found that the incorporation of incremental CB concentrations kept the wettability surface property unchanged while the electrical characterization of the proposed structures showed a reduction of the electrical impedance. Overall, the study suggests that the composite structures could be used as a potential candidate for fabrication of scaffolds for neural regeneration with tunable electrical and mechanical properties by varying the CB concentration in a finite range.

The majority of strategies used in tissue engineering (TE) employ a scaffold, which is used to guide .the proliferation, the migration and the adhesione of cell in 3D to pruduce an engineered tissue. A new trend in scaffolds’s fabrication is represented by the hybrid Rapid Prototyping technologies. This is a new multimaterial and multiscale fabrication approach which combine the common RP technologies with other micro/nanofabrication techiques to fabricate scaffold that mimick the hetereogenty and hierarchical structure typical of the native extracellular matrix. In this new contest our work present: 1) an innovative device for the fabrication of multi material scaffolds based on an open source FDM 3D printer suitably modified to integrate a multi nozzle deposition tool 2) a design proposal for a multi material and multi scale machine to allow a full control over the modulation of the building materials and of the topography in a scaffold 3) and lastly a CAD workflow to guide the fabrication of RP patient specific scaffolds. Multifunctional hydrogel-based scaffold are fabricated as a demonstration of the validity of the proposed devices. Starting from a clinical case we print a patient-specific scaffold with the aim to recover bone defects at mandibular level as a validation of the proposed CAD process.

A parallel manipulator with three translational degrees of freedom for microfabrication of biomaterials was designed for a given workspace. The device was opportunely designed to mount the following biofabrication tools on its mobile platform: a pressure-driven extrusion tool, a piston-driven extrusion tool, and an electrospinning tool. Taking advantage of the modularity of the system, it was possible to combine these different techniques to obtain multimaterial and multiscale scaffolds with structural and mechanical characteristics more similar to biological tissues. In this work, hydrogel-based composite scaffolds were fabricated as a demonstration.

Tissue engineering (TE) aims at producing patient-specific biological substitutes in an attempt to repair, replace and regenerate damaged tissues or organs in order to improve the current state of clinical treatments. A three-dimensional substrate, the scaffold, is a key aspect to promote cell organization to form a tissue. Recently, rapid prototyping (RP) technologies have been successfully used to fabricate complex scaffolds, thanks to the ability to create highly reproducible architecture and compositional variation across the entire structure, due to their precise controlled computer driven fabrication. The drawback of most of the fabrication principles applied in the RP processes is the requirement of particular conditions (e.g., pressure or temperature) that limit the material choice. Natural polymers, such as collagen, have underlined their superiority for TE solutions but they are challenging to be processed with RP techniques. As alternative, scaffold made of natural biomaterial can be produced by indirect fabrication techniques, casting a biomaterial into sacrificial mold realized by RP processes. So far, the indirect rapid prototyping (iRP) has emerged in a number of different approaches with promising results. The present chapter is focused on iRP multistep methods, highlighting strength and weakness and indicating possible future perspectives.