Correlation of the regenerative potential of dermal fibroblasts in 2D culture with the biological properties of fibroblast-derived tissue spheroids.

In situ 3D bioprinting is a new emerging therapeutic modality for treating human skin diseases. The tissue spheroids have been previously suggested as a powerful tool in rapidly expanding bioprinting technology. It has been demonstrated that the regenerative potential of human dermal fibroblasts could be quantitatively evaluated in 2D cell culture and confirmed after implantation in vivo. However, the development of unbiassed quantitative criteria of the regenerative potential of 3D tissue spheroids in vitro before their in situ bioprinting remains to be investigated. Here it has been demonstrated for the first time that specific correlations exist between the regenerative potential of human dermal fibroblasts cultured in vitro as 2D cell monolayer with biological properties of 3D tissue spheroids fabricated from these fibroblasts. In vitro assessment of biological properties included diameter, spreading and fusion kinetics, and biomechanical properties of 3D tissue spheroids. This comprehensive characterization could be used to predict tissue spheroids' regenerative potential in vivo.

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.

Design, Fabrication, and Application of Mini-Scaffolds for Cell Components in Tissue Engineering.

The concept of "lockyballs" or interlockable mini-scaffolds fabricated by two-photon polymerization from biodegradable polymers for the encagement of tissue spheroids and their delivery into the desired location in the human body has been recently introduced. In order to improve control of delivery, positioning, and assembly of mini-scaffolds with tissue spheroids inside, they must be functionalized. This review describes the design, fabrication, and functionalization of mini-scaffolds as well as perspectives on their application in tissue engineering for precisely controlled cell and mini-tissue delivery and patterning. The development of functionalized mini-scaffolds advances the original concept of "lockyballs" and opens exciting new prospectives for mini-scaffolds' applications in tissue engineering and regenerative medicine and their eventual clinical translation.

Magnetic Patterning of Tissue Spheroids Using Polymer Microcapsules Containing Iron Oxide Nanoparticles.

Magnetic tissue engineering is one of the rapidly emerging and promising directions of tissue engineering and biofabrication where the magnetic field is employed as temporal removal support or scaffold. Iron oxide nanoparticles are used to label living cells and provide the desired magnetic properties. Recently, polymer microcapsules loaded with iron oxide nanoparticles have been proposed as a novel approach to designing magnetic materials with high local concentrations. These microcapsules can be readily internalized and retained intracellularly for a long time in various types of cells. The low cytotoxicity of these microcapsules was previously shown in 2D cell culture. This paper has demonstrated that cells containing these nontoxic nanomaterials can form viable 3D tissue spheroids for the first time. The spheroids retained labeled fluorescent microcapsules with magnetic nanoparticles without a detectable cytotoxic effect. The high concentration of packed nanoparticles inside the microcapsules enables the evident magnetic properties of the labeled spheroids to be maintained. Finally, magnetic spheroids can be effectively used for magnetic patterning and biofabrication of tissue-engineering constructs.

Cytoskeleton systems contribute differently to the functional intrinsic properties of chondrospheres.

Cytoskeleton systems, actin microfilaments, microtubules (MTs) and intermediate filaments (IFs) provide the biomechanical stability and spatial organization in cells. To understand the specific contributions of each cytoskeleton systems to intrinsic properties of spheroids, we've scrutinized the effects of the cytoskeleton perturbants, cytochalasin D (Cyto D), nocodazole (Noc) and withaferin A (WFA) on fusion, spreading on adhesive surface, morphology and biomechanics of chondrospheres (CSs). We confirmed that treatment with Cyto D but not with Noc or WFA severely affected CSs fusion and spreading dynamics and significantly reduced biomechanical properties of cell aggregates. Noc treatment affected spheroids spreading but not the fusion and surprisingly enhanced their stiffness. Vimentin intermediate filaments (VIFs) reorganization affected CSs spreading only. The analysis of all three cytoskeleton systems contribution to spheroids intrinsic properties was performed for the first time.

Extracellular Matrix Determines Biomechanical Properties of Chondrospheres during Their Maturation In Vitro.

OBJECTIVE: Chondrospheres represent a variant of tissue spheroids biofabricated from chondrocytes. They are already being used in clinical trials for cartilage repair; however, their biomechanical properties have not been systematically investigated yet. The aim of our study was to characterize chondrospheres in long-term in vitro culture conditions for morphometric changes, biomechanical integrity, and their fusion and spreading kinetics. RESULTS: It has been demonstrated that the increase in chondrospheres secant modulus of elasticity is strongly associated with the synthesis and accumulation of extracellular matrix. Additionally, significant interplay has been found between biomechanical properties of tissue spheroids and their fusion kinetics in contrast to their spreading kinetics. CONCLUSIONS: Extracellular matrix is one of the main structural determinants of chondrospheres biomechanical properties during chondrogenic maturation in vitro. The estimation of tissue spheroids' physical behavior in vitro prior to operative treatment can be used to predict and potentially control fusogenic self-assembly process after implantation in vivo.

Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly.

Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering.

Cardiovascular tissue engineering I. Perfusion bioreactors: a review.

Tissue engineering is a fast-evolving field of biomedical science and technology with future promise to manufacture living tissues and organs for replacement, repair, and regeneration of diseased organs. Owing to the specific role of hemodynamics in the development, maintenance, and functioning of the cardiovascular system, bioreactors are a fundamental of cardiovascular tissue engineering. The development of perfusion bioreactor technology for cardiovascular tissue engineering is a direct sequence of previous historic successes in extracorporeal circulation techniques. Bioreactors provide a fluidic environment for tissue engineered tissue and organs, and guarantee their viability, maturation, biomonitoring, testing, storage, and transportation. There are different types of bioreactors and they vary greatly in their size, complexity, and functional capabilities. Although progress in design and functional properties of perfusion bioreactors for tissue engineered blood vessels, heart valves, and myocardial patches is obvious, there are some challenges and insufficiently addressed issues, and room for bioreactor design improvement and performance optimization. These challenges include creating a triple perfusion bioreactor for vascularized tubular tissue engineered cardiac construct; designing and manufacturing fluidics-based perfused minibioreactors; incorporation of systematic mathematical modeling and computer simulation based on computational fluid dynamics into the bioreactor designing process; and development of automatic systems of hydrodynamic regime control. Designing and engineering of built-in noninvasive biomonitoring systems is another important challenge. The optimal and most efficient perfusion and conditioning regime, which accelerates tissue maturation of tissue-engineered constructs also remains to be determined. This is a first article in a series of reviews on critical elements of cardiovascular tissue engineering technology describing the current status, unsolved problems, and challenges of bioreactor technology in cardiovascular tissue engineering and outlining future trends and developments.

Rapid biofabrication of tubular tissue constructs by centrifugal casting in a decellularized natural scaffold with laser-machined micropores.

Centrifugal casting allows rapid biofabrication of tubular tissue constructs by suspending living cells in an in situ cross-linkable hydrogel. We hypothesize that introduction of laser-machined micropores into a decellularized natural scaffold will facilitate cell seeding by centrifugal casting and increase hydrogel retention, without compromising the biomechanical properties of the scaffold. Micropores with diameters of 50, 100, and 200 mum were machined at different linear densities in decellularized small intestine submucosa (SIS) planar sheets and tubular SIS scaffolds using an argon laser. The ultimate stress and ultimate strain values for SIS sheets with laser-machined micropores with diameter 50 mum and distance between holes as low as 714 mum were not significantly different from unmachined control SIS specimens. Centrifugal casting of GFP-labeled cells suspended in an in situ cross-linkable hyaluronan-based hydrogel resulted in scaffold recellularization with a high density of viable cells inside the laser-machined micropores. Perfusion tests demonstrated the retention of the cells encapsulated within the HA hydrogel in the microholes. Thus, an SIS scaffold with appropriately sized microholes can be loaded with hydrogel encapsulated cells by centrifugal casting to give a mechanically robust construct that retains the cell-seeded hydrogel, permitting rapid biofabrication of tubular tissue construct in a "bioreactor-free" fashion.