Tensiometric estimation of material properties of tissue spheroids

Tissue spheroids have been proposed to use as building blocks in biofabrication and as bioinks in 3D bioprinting technologies. Tissue fusion is an ubiqious phenomenon during embryonic development. Biomimetic tissue spheroid fusion is a fundamental constructional principle of emerging organ printing technology because closely placed tissue spheroids could fuse into tissue and organ-like constructs in fusion permissive bioprintable hydrogel. From physical point of view tissue spheroids could be considered as a visco-elastic-plastic soft matter or complex fluid. We hypothesize that quantitative estimation of material properties of tissue spheroids using tensiometry could predict their tissue spreading and tissue fusion behavior as well as provide a powerful insight about possible speed of post-printed tissue and organ-like constructs compaction and maturation. Tissue spheroids from human fibroblasts, ovine and human chondrocytes and immortalised human keratinocytes have been biofabricated using non-adhesive cell culture plates (Corning, USA). For estimation of material properties of tissue spheroids commercial tensiometer Microsquisher have been emploied (CellScale, Toronto, Canada). Modulus of elasticity of tissue spheroids have been calculated based on peformed tissue compression tests. In order to identify structural determinants of material properties of tissue spheroids standard perturbants of cytoskeleton such as Cytochalasin D (Sigma, USA) for disruption of microfilaments and Nocodazole (Sigma, USA) for disruption of microtubules have been used. Viability of tissue spheroids have been also estimated and their morphology have been analysed using light microscopy, histochemistry, immunohistochemistry, semithin sections stained wih toluidine blue and transmission and scanning electron microscopy. Kinetics of tissue spheroids spreading on electrospun polyurethane matrices have been analysed. Kinetics of two closely placed tissue spheroids fusion have been analysed in hanging drop. Additionally toxic effect of water solution of paramagnetic gadolinium salt (Omniscan®, GE Health Care, USA) on material properties of tissue spheroids have been investigated. It have been demonstrated that material properties of tissue spheroids biofabricated from different cell types have different modulus of elasticity. Even tissue spheroids biofabricated the same cell types but from different species have different material properties. Incubation with Cytochlasin D dramatically reduces estimated material properties of tissue spheroids. Incubation with Nocodazole does not significantly change material properties of tissue spheroids. Material properties of tissue spheroids from chondrocytes (chondrospheres) correlates very well with increasing deposition and accumulation of extracellular matrix (confirmed by expression of collagen type II and glycosoaminoglycans). The incubation with toxic concentration of gadolinium solution dramatically reduces material properties of chondrospheres. There is no any significant correlation between material properties of tissue spheriods and their spreading kinetics. However, there is a certain correction between material properties of tissue spheroids and their tissue fusion kinetics. Our data demonstrate that beside already well established role of cell adhesion receptors such as cadherin and integrins in the realisation of cell cohesion inside tissue spheroids the structural determinants of material properties of tissue spheroids also include components of cytoskeleton such as actin micofilaments and accumulated extracellular matrix. It is possible to predict post-printing tissue fusion behaviour of tissue spheroids based on preliminary estimation of their material properties. Finally, it have been also shown that material properties of tissue spheroids correlate with their viability. Thus, tensiometry is a valuable method for systematic characterization of material properties of tissue spheroids and for prediction of tissue spheroids post-printed tissue fusion behaviour

Commercial articulated collaborative in situ 3D bioprinter for skin wound healing

In situ bioprinting is one of the most clinically relevant techniques in the emerging bioprinting technology because it could be performed directly on the human body in the operating room and it does not require bioreactors for post-printing tissue maturation. However, commercial in situ bioprinters are still not available on the market. In this study, we demonstrated the benefit of the originally developed first commercial articulated collaborative in situ bioprinter for the treatment of full-thickness wounds in rat and porcine models. We used an articulated and collaborative robotic arm from company KUKA and developed original printhead and correspondence software enabling in situ bioprinting on curve and moving surfaces. The results of in vitro and in vivo experiments show that in situ bioprinting of bioink induces a strong hydrogel adhesion and enables printing on curved surfaces of wet tissues with a high level of fidelity. The in situ bioprinter was convenient to use in the operating room. Additional in vitro experiments (in vitro collagen contraction assay and in vitro 3D angiogenesis assay) and histological analyses demonstrated that in situ bioprinting improves the quality of wound healing in rat and porcine skin wounds. The absence of interference with the normal process of wound healing and even certain improvement in the dynamics of this process strongly suggests that in situ bioprinting could be used as a novel therapeutic modality in wound healing.publishersversionPeer reviewe

3D scanning probe nanotomography of tissue spheroid fibroblasts interacting with electrospun polyurethane scaffold

We present a 3D study of nanostructural features of a bioprinted tissue spheroid interacting with polyurethane dual-scale biocompatible scaffold manufactured by three-dimensional printing and electrospinning. Three-dimensional analysis of fibroblasts interacting with electrospun polyurethane fibers was conducted using scanning probe nanotomography with an experimental setup combining ultramicrotome and a scanning probe microscope. Three-dimensional reconstruction demonstrates direct visualization of cell membrane protrusions and coherent cell-fiber interfaces, the formation of which is a prerequisite for an efficient tissue engineered implant. Analysis of obtained 3D data allows for quantitative calculation of the important morphological parameters of adhered cells, scaffolds, and cell-scaffold interfaces. The proposed method may be successfully applied to investigate 3D cell-scaffold constructs at nanoscale

Bioprinting of a functional vascularized mouse thyroid gland construct

Bioprinting can be defined as additive biofabrication of 3D tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. Thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS), as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modelling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, endothelial cells from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of thyroid gland construct, we depleted endogenous EC from thyroid spheroids before bioprinting. EC from allantoic spheroids completely revascularized depleted thyroid tissue. Cultured bioprinted construct was functional as it could normalize blood thyroxin levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents further advance in bioprinting technology exploring self-assembling properties of tissue spheroids

Biofabrication of a Functional Tubular Construct from Tissue Spheroids Using Magnetoacoustic Levitational Directed Assembly

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Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows