Neurodegenerative diseases (NDs) are a broad class of pathologies characterized by the progressive loss of neurons in the central nervous system. The main problem in the study of NDs is the lack of an adequate realistic experimental model to study the pathogenic mechanisms. Induced pluripotent stem cells (iPSCs) partially overcome the problem, with their capability to differentiate into almost every cell types; even so, these cells alone are not sufficient to unveil the mechanisms underlying NDs. 3D bioprinting allows to control the distribution of cells such as neurons, leading to the creation of a realistic in vitro model. In this work, we analyzed two biomaterials: sodium alginate and gelatin, and three different cell types: a neuroblastoma cell line (SH-SY5Y), iPSCs, and neural stem cells. All cells were encapsulated inside the bioink, printed and cultivated for at least seven days; they all presented good viability. We also evaluated the maintenance of the printed shape, opening the possibility to obtain a reliable in vitro neural tissue combining 3D bioprinting and iPSCs technology, optimizing the study of the degenerative processes that are still widely unknown.

Three-dimensional printing of poly(ε-caprolactone) (PCL) is a consolidated scaffold manufacturing technique for bone regenerative medicine. Simultaneously, the mesenchymal stem/stromal cell (MSC) secretome is osteoinductive, promoting scaffold colonization by cells, proliferation, and differentiation. The present paper combines 3D-printed PCL scaffolds with lyosecretome, a freeze-dried formulation of MSC secretome, containing proteins and extracellular vesicles (EVs). We designed a lyosecretome 3D-printed scaffold by two loading strategies: (i) MSC secretome adsorption on 3D-printed scaffold and (ii) coprinting of PCL with an alginate-based hydrogel containing MSC secretome (at two alginate concentrations, i.e., 6% or 10% w/v). A fast release of proteins and EVs (a burst of 75% after 30 min) was observed from scaffolds obtained by absorption loading, while coprinting of PCL and hydrogel, encapsulating lyosecretome, allowed a homogeneous loading of protein and EVs and a controlled slow release. For both loading modes, protein and EV release was governed by diffusion as revealed by the kinetic release study. The secretome’s diffusion is influenced by alginate, its concentration, or its cross-linking modes with protamine due to the higher steric hindrance of the polymer chains. Moreover, it is possible to further slow down protein and EV release by changing the scaffold shape from parallelepiped to cylindrical. In conclusion, it is possible to control the release kinetics of proteins and EVs by changing the composition of the alginate hydrogel, the scaffold’s shape, and hydrogel cross-linking. Such scaffold prototypes for bone regenerative medicine are now available for further testing of safety and efficacy.

Alginate-based hydrogels are extensively used to create bioinks for bioprinting, due to their biocompatibility, low toxicity, low costs, and slight gelling. Modeling of bioprinting process can boost experimental design reducing trial-and-error tests. To this aim, the cross-linking kinetics for the chemical gelation of sodium alginate hydrogels via calcium chloride diffusion is analyzed. Experimental measurements on the absorbed volume of calcium chloride in the hydrogel are obtained at different times. Moreover, a reaction-diffusion model is developed, accounting for the dependence of diffusive properties on the gelation degree. The coupled chemical system is solved using finite element discretizations which include the inhomogeneous evolution of hydrogel state in time and space. Experimental results are fitted within the proposed modeling framework, which is thereby calibrated and validated. Moreover, the importance of accounting for cross-linking-dependent diffusive properties is highlighted, showing that, if a constant diffusivity property is employed, the model does not properly capture the experimental evidence. Since the analyzed mechanisms highly affect the evolution of the front of the solidified gel in the final bioprinted structure, the present study is a step towards the development of reliable computational tools for the in silico optimization of protocols and post-printing treatments for bioprinting applications.

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The study aims to investigate a novel bioink made from Chitosan (Cs)/ poly(gamma‐glutamic acid) (Gamma‐PGA) hydrogel that takes advantage of the two biodegradable and biocompatible polymers meeting most of the requirements for biomedical applications. The bioink could be an alternative to other materials commonly used in 3D‐bioprinting such as gelatin or alginate. Cs/ Gamma‐PGA hydrogel was prepared by double extrusion of Gamma‐PGA and Cs solutions, where 2 × 105 human adult fibroblasts per ml Cs solution had been loaded, through Cellink 3D‐Bioprinter at 37°C. A computer aided design model was used to get 3D‐bioprinting of a four layers grid hydrogel construct with 70% infill. Hydrogel characterization involved rheology, FTIR analysis, stability study (mass loss [ML], fluid uptake [FU]), and cell retaining ability into hydrogel. 3D‐bioprinted hydrogel gelation time resulted to be <60 s, hydrogel structure was maintained up to 36.79 Pa shear stress, FTIR analysis demonstrated Gamma‐PGA/Cs interpolyelectrolyte complex formation. The 3D‐bioprinted hydrogel was stable for 35 days (35% ML) in cell culture medium, with increasing FU. Cell loaded 3D‐bioprinted Cs 6% hydrogel was able to retain 70% of cells which survived to printing process and cell viability was maintained during 14 days incubation.

Three-dimensional (3D) bioprinting allows the production of artificial 3D cellular microenvironments thanks to the controlled spatial deposition of bioinks. Proper bioink characterization is required to achieve the essential characteristics of printability and biocompatibility for 3D bioprinting. In this work, a protocol to standardize the experimental characterization of a new bioink is proposed. A functionalized hydrogel based on gelatin and chitosan was used. The protocol was divided into three steps: pre-printing, 3D bioprinting, and post-printing. For the pre-printing step, the hydrogel formulation and its repeatability were evaluated. For the 3D-bioprinting step, the hydrogel-printability performance was assessed through qualitative and quantitative tests. Finally, for the post-printing step, the hydrogel biocompatibility was investigated using UMR-106 cells. The hydrogel was suitable for printing grids with good resolution from 4 h after the cross-linker addition. To guarantee a constant printing pressure, it was necessary to set the extruder to 37 °C. Furthermore, the hydrogel was shown to be a valid biomaterial for the UMR-106 cells’ growth. However, fragmentation of the constructs appeared after 14 days, probably due to the negative osteosarcoma-cell interference. The protocol that we describe here denotes a strong approach to bioink characterization to improve standardization for future biomaterial screening and development.

Polycaprolactone (PCL) and hydroxyapatite (HA) composite are widely used in tissue engineering (TE). They are fit to being processed with three-dimensional (3D) printing technique to create scaffolds with verifiable porosity. The current challenge is to guarantee the reliability and reproducibility of 3D printed scaffolds and to create sterile scaffolds which can be used for in vitro cell cultures. In this context it is important for successful cell culture, to have a protocol in order to evaluate the sterility of the printed scaffolds. We proposed a systematic approach to sterilise 90%PCL-10%HA pellets using a 3D bioprinter before starting the printing process. We evaluated the printability of PCL-HA composite and the shape fidelity of scaffolds printed with and without sterilised pellets varying infill pattern, and the sterility of 3D printed scaffolds following the method established by the United States Pharmacopoeia. Finally, the thermal analyses supported by the Fourier Transform Infrared Spectroscopy were useful to verify the stability of the sterilisation process in the PCL solid state with and without HA. The results show that the use of the 3D printer, according to the proposed protocol, allows to obtain sterile 3D PCL-HA scaffolds suitable for TE applications such as bone or cartilage repair.

Skeletal muscle regeneration is one of the major areas of interest in sport medicine as well as trauma centers. Three-dimensional (3D) bioprinting (BioP) is nowadays widely adopted to manufacture 3D constructs for regenerative medicine but a comparison between the available biomaterial-based inks (bioinks) is missing. The present study aims to assess the impact of different hydrogels on the viability, proliferation, and differentiation of murine myoblasts (C2C12) encapsulated in 3D bioprinted constructs aided to muscle regeneration. We tested three different commercially available hydrogels bioinks based on: (1) gelatin methacrylate and alginate crosslinked by UV light; (2) gelatin methacrylate, xanthan gum, and alginate-fibrinogen; (3) nanofibrillated cellulose (NFC)/alginate-fibrinogen crosslinked with calcium chloride and thrombin. Constructs embedding the cells were manufactured by extrusion-based BioP and C2C12 viability, proliferation, and differentiation were assessed after 24 h, 7, 14, 21, and 28 days in culture. Although viability, proliferation, and differentiation were observed in all the constructs, among the investigated bioinks, the best results were obtained by using NFC/alginatefibrinogen-based hydrogel from 7 to 14 days in culture, when the embedded myoblasts started fusing, forming at day 21 and day 28 multinucleated myotubes within the 3D bioprinted structures. The results revealed an extensive myotube alignment all over the linear structure of the hydrogel, demonstrating cell maturation, and enhanced myogenesis. The bioprinting strategies that we describe here denote a strong and endorsed approach for the creation of in vitro artificial muscle to improve skeletal muscle tissue engineering for future therapeutic applications.