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Three-dimensional (3D) printing technology enables the design of personalized scaffolds with tunable pore size and composition. Combining decellularization and 3D printing techniques provides the opportunity to fabricate scaffolds with high potential to mimic native tissue. The aim of this study is to produce novel decellularized bone extracellular matrix (dbECM)-reinforced composite-scaffold that can be used as a biomaterial for bone tissue engineering. Decellularized bone particles (dbPTs, ∼100 ​μm diameter) were obtained from rabbit femur and used as a reinforcement agent by mixing with gelatin (GEL) in different concentrations. 3D scaffolds were fabricated by using an extrusion-based bioprinter and crosslinking with microbial transglutaminase (mTG) enzyme, followed by freeze-drying to obtain porous structures. Fabricated 3D scaffolds were characterized morphologically, mechanically, and chemically. Furthermore, MC3T3-E1 mouse pre-osteoblast cells were seeded on the dbPTs reinforced GEL scaffolds (GEL/dbPTs) and cultured for 21 days to assess cytocompatibility and cell attachment. We demonstrate the 3D-printability of dbPTs-reinforced GEL hydrogels and the achievement of homogenous distribution of the dbPTs in the whole scaffold structure, as well as bioactivity and cytocompatibility of GEL/dbPTs scaffolds. It was shown that Young's modulus and degradation rate of scaffolds were enhanced with increasing dbPTs content. Multiphoton microscopy imaging displayed the interaction of cells with dbPTs, indicating attachment and proliferation of cells around the particles as well as into the GEL-particle hydrogels. Our results demonstrate that GEL/dbPTs hydrogel formulations have potential for bone tissue engineering.

Biofabrication can be a tool to three-dimensionally (3D) print muscle cells embedded inside hydrogel biomaterials, ultimately aiming to mimic the complexity of the native muscle tissue and to create in-vitro muscle analogues for advanced repair therapies and drug testing. However, to 3D print muscle analogues of high cell alignment and synchronous contraction, the effect of biofabrication process parameters on myoblast growth has to be understood. A suitable biomaterial matrix is required to provide 3D printability as well as matrix degradation to create space for cell proliferation, matrix remodelling capacity, and cell differentiation. We demonstrate that by the proper selection of nozzle size and extrusion pressure, the shear stress during extrusion-bioprinting of mouse myoblast cells (C2C12) can achieve cell orientation when using oxidized alginate-gelatin (ADA-GEL) hydrogel bionk. The cells grow in the direction of printing, migrate to the hydrogel surface over time, and differentiate into ordered myotube segments in areas of high cell density. Together, our results show that ADA-GEL hydrogel can be a simple and cost-efficient biodegradable bioink that allows the successful 3D bioprinting and cultivation of C2C12 cells in-vitro to study muscle engineering.

We present an enhanced version of our previously engineered MyoRobot system for reliable, versatile and automated investigations of skeletal muscle or linear polymer material (bio)mechanics. That previous version already replaced strenuous manual protocols to characterize muscle biomechanics properties and offered automated data analysis. Here, the system was further improved for precise control over experimental temperature and muscle single fiber sarcomere length. Moreover, it also now features the calculation of fiber cross-sectional area via on-the-fly optical diameter measurements using custom-engineered microscope optics. With this optical systems integration, the MyoRobot 2.0 allows to tailor a wealth of recordings for relevant physiological parameters to be sequentially executed in living single myofibers. Research questions include assessing temperature-dependent performance of active or passive biomechanics, or automated control over length-tension or length-velocity relations. The automatically obtained passive stress-strain relationships and elasticity modules are important parameters in (bio)material science. From the plethora of possible applications, we validated the improved MyoRobot 2.0 by assessing temperature-dependent myofibrillar Ca2+ sensitivity, passive axial compliance and Young's modulus. We report a Ca2+ desensitization and a narrowed dynamic range at higher temperatures in murine M. extensor digitorum longus single fibers. In addition, an increased axial mechanical compliance in single muscle fibers with Young's moduli between 40 - 60 kPa was found, compatible with reported physiological ranges. These applications demonstrate the robustness of our MyoRobot 2.0 for facilitated single muscle fiber biomechanics assessment.

Many different biofabrication approaches as well as a variety of bioinks have been developed by researchers working in the field of tissue engineering. A main challenge for bioinks often remains the difficulty to achieve shape fidelity after printing. In order to overcome this issue, a homogeneous pre-crosslinking technique, which is universally applicable to all alginate-based materials, was developed. In this study, the Young’s Modulus after post-crosslinking of selected hydrogels, as well as the chemical characterization of alginate in terms of M/G ratio and molecular weight, were determined. With our technique it was possible to markedly enhance the printability of a 2% (w/v) alginate solution, without using a higher polymer content, fillers or support structures. 3D porous scaffolds with a height of around 5 mm were printed. Furthermore, the rheological behavior of different pre-crosslinking degrees was studied. Shear forces on cells as well as the flow profile of the bioink inside the printing nozzle during the process were estimated. A high cell viability of printed NIH/3T3 cells embedded in the novel bioink of more than 85% over a time period of two weeks could be observed.

High-content screening microscopy relies on automation infrastructure that is typically proprietary, non-customizable, costly and requires a high level of skill to use and maintain. The increasing availability of rapid prototyping technology makes it possible to quickly engineer alternatives to conventional automation infrastructure that are low-cost and user-friendly. Here, we describe a 3D printed inexpensive open source and scalable motorized positioning stage for automated high-content screening microscopy and provide detailed step-by-step instructions to re-building the device, including a comprehensive parts list, 3D design files in STEP (Standard for the Exchange of Product model data) and STL (Standard Tessellation Language) format, electronic circuits and wiring diagrams as well as software code. System assembly including 3D printing requires approx. 30h. The fully assembled device is light-weight (1.1kg), small (33×20×8cm) and extremely low-cost (approx. EUR 250). We describe positioning characteristics of the stage, including spatial resolution, accuracy and repeatability, compare imaging data generated with our device to data obtained using a commercially available microplate reader, demonstrate its suitability to high-content microscopy in 96-well high-throughput screening format and validate its applicability to automated functional Cl−- and Ca2+-imaging with recombinant HEK293 cells as a model system. A time-lapse video of the stage during operation and as part of a custom assembled screening robot can be found at https://vimeo.com/158813199.