3D Co-Printing and Substrate Geometry Influence the Differentiation of C2C12 Skeletal Myoblasts.

Cells are influenced by several biomechanical aspects of their microenvironment, such as substrate geometry. According to the literature, substrate geometry influences the behavior of muscle cells; in particular, the curvature feature improves cell proliferation. However, the effect of substrate geometry on the myogenic differentiation process is not clear and needs to be further investigated. Here, we show that the 3D co-printing technique allows the realization of substrates. To test the influence of the co-printing technique on cellular behavior, we realized linear polycaprolactone substrates with channels in which a fibrinogen-based hydrogel loaded with C2C12 cells was deposited. Cell viability and differentiation were investigated up to 21 days in culture. The results suggest that this technology significantly improves the differentiation at 14 days. Therefore, we investigate the substrate geometry influence by comparing three different co-printed geometries-linear, circular, and hybrid structures (linear and circular features combined). Based on our results, all structures exhibit optimal cell viability (>94%), but the linear pattern allows to increase the in vitro cell differentiation, in particular after 14 days of culture. This study proposes an endorsed approach for creating artificial muscles for future skeletal muscle tissue engineering applications.

3D bioprinted osteosarcoma model for experimental boron neutron capture therapy (BNCT) applications: Preliminary assessment.

Osteosarcoma is the most frequently primary malignant bone tumor characterized by infiltrative growth responsible for relapses and metastases. Treatment options are limited, and a new therapeutic option is required. Boron neutron capture therapy (BNCT) is an experimental alternative radiotherapy able to kill infiltrative tumor cells spearing surrounding healthy tissues. BNCT studies are performed on 2D in vitro models that are not able to reproduce pathological tumor tissue organization or on in vivo animal models that are expensive, time-consuming and must follow the 3R's principles. A 3D in vitro model is a solution to better recapitulate the complexity of solid tumors meanwhile limiting the animal's use. Objective of this study is to optimize the technical assessment for developing a 3D in vitro osteosarcoma model as a platform for BNCT studies: printing protocol, biomaterial selection, cell density, and crosslinking process. The best parameters that allow a fully colonized 3D bioprinted construct by rat osteosarcoma cell line UMR-106 are 6 × 106 cells/ml of hydrogel and 1% CaCl2 as a crosslinking agent. The proposed model could be an alternative or a parallel approach to 2D in vitro culture and in vivo animal models for BNCT experimental study.

Characterization of a Bioink Combining Extracellular Matrix-like Hydrogel with Osteosarcoma Cells: Preliminary Results.

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.

Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities.

The intertwined adoption of synthetic biology and 3D bioprinting has the potential to improve different application fields by fabricating engineered living materials (ELMs) with unnatural genetically-encoded sense & response capabilities. However, efforts are still needed to streamline the fabrication of sensing ELMs compatible with field use and improving their functional complexity. To investigate these two unmet needs, we adopted a workflow to reproducibly construct bacterial ELMs with synthetic biosensing circuits that provide red pigmentation as visible readout in response to different proof-of-concept chemical inducers. We first fabricated single-input/single-output ELMs and we demonstrated their robust performance in terms of longevity (cell viability and evolutionary stability >15 days, and long-term storage >1 month), sensing in harsh, non-sterile or nutrient-free conditions compatible with field use (soil, water, and clinical samples, including real samples from Pseudomonas aeruginosa infected patients). Then, we fabricated ELMs including multiple spatially-separated biosensor strains to engineer: level-bar materials detecting molecule concentration ranges, multi-input/multi-output devices with multiplexed sensing and information processing capabilities, and materials with cell-cell communication enabling on-demand pattern formation. Overall, we showed successful field use and multiplexed functioning of reproducibly fabricated ELMs, paving the way to a future automation of the prototyping process and boosting applications of such devices as in-situ monitoring tools or easy-to-use sensing kits.

Computer-aided engineering and additive manufacturing for bioreactors in tissue engineering: State of the art and perspectives.

Two main challenges are currently present in the healthcare world, i.e., the limitations given by transplantation and the need to have available 3D in vitro models. In this context, bioreactors are devices that have been introduced in tissue engineering as a support for facing the mentioned challenges by mimicking the cellular native microenvironment through the application of physical stimuli. Bioreactors can be divided into two groups based on their final application: macro- and micro-bioreactors, which address the first and second challenge, respectively. The bioreactor design is a crucial step as it determines the way in which physical stimuli are provided to cells. It strongly depends on the manufacturing techniques chosen for the realization. In particular, in bioreactor prototyping, additive manufacturing techniques are widely used nowadays as they allow the fabrication of customized shapes, guaranteeing more degrees of freedom. To support the bioreactor design, a powerful tool is represented by computational simulations that allow to avoid useless approaches of trial-and-error. In the present review, we aim to discuss the general workflow that must be carried out to develop an optimal macro- and micro-bioreactor. Accordingly, we organize the discussion by addressing the following topics: general and stimulus-specific (i.e., perfusion, mechanical, and electrical) requirements that must be considered during the design phase based on the tissue target; computational models as support in designing bioreactors based on the provided stimulus; manufacturing techniques, with a special focus on additive manufacturing techniques; and finally, current applications and new trends in which bioreactors are involved.