Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell‐based organoids are promising as functional, self‐organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self‐organizing co‐culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration).

Recent studies have shown that mechanical stimulation, in the form of fluid perfusion and mechanical compression, can enhance osteogenic differentiation of mesenchymal stem cells and bone cells within tissue engineering scaffolds in vitro. The precise nature of mechanical stimulation within tissue engineering scaffolds is not only dictated by the exogenously applied loading regime, but also depends on the geometric features of the scaffold, in particular architecture, pore size and porosity. However, the precise contribution of each geometric feature towards the resulting mechanical stimulation within a scaffold is difficult to characterise due to the wide range of interacting parameters. In this study, we have applied a fluid–structure interaction model to investigate the role of scaffold geometry (architecture, pore size and porosity) on pore wall shear stress (WSS) under a range of different loading scenarios: fluid perfusion, mechanical compression and a combination of perfusion and compression. It is found that scaffold geometry (spherical and cubical pores), in particular the pore size, has a significant influence on the stimulation within scaffolds. Furthermore, we observed an amplified WSS within scaffolds under a combination of fluid perfusion and mechanical compression, which exceeded that caused by individual fluid perfusion or mechanical compression approximately threefold. By conducting this comprehensive parametric variation study, an expression was generated to allow the design and optimisation of 3D TE scaffolds and inform experimental loading regimes so that a desired level of mechanical stimulation, in terms of WSS is generated within the scaffold.

Recent studies have shown that mechanical stimulation, by means of flow perfusion and mechanical compression (or stretching), enhances osteogenic differentiation of mesenchymal stem cells and bone cells within biomaterial scaffolds in vitro. However, the precise mechanisms by which such stimulation enhances bone regeneration is not yet fully understood. Previous computational studies have sought to characterise the mechanical stimulation on cells within biomaterial scaffolds using either computational fluid dynamics or finite element (FE) approaches. However, the physical environment within a scaffold under perfusion is extremely complex and requires a multiscale and multiphysics approach to study the mechanical stimulation of cells. In this study, we seek to determine the mechanical stimulation of osteoblasts seeded in a biomaterial scaffold under flow perfusion and mechanical compression using multiscale modelling by two-way fluid–structure interaction and FE approaches. The mechanical stimulation, in terms of wall shear stress (WSS) and strain in osteoblasts, is quantified at different locations within the scaffold for cells of different attachment morphologies (attached, bridged). The results show that 75.4 % of scaffold surface has a WSS of 0.1–10 mPa, which indicates the likelihood of bone cell differentiation at these locations. For attached and bridged osteoblasts, the maximum strains are 397 and 177,200 \(\upmu \) \(\upvarepsilon \) , respectively. Additionally, the results from mechanical compression show that attached cells are more stimulated ( \(\mathrm{maximum\,strain}=22,600\,\upmu \) \(\upvarepsilon \) ) than bridged cells ( \(\mathrm{maximum\,strain}=10,000\,\upmu \) \(\upvarepsilon \) ). Such information is important for understanding the biological response of osteoblasts under in vitro stimulation. Finally, a combination of perfusion and compression of a tissue engineering scaffold is suggested for osteogenic differentiation.

Generating 3D bone cell networks in vitro that mimic the dynamic process during early bone formation remains challenging. Here, we report a synthetic biodegradable microporous hydrogel for efficient formation of 3D networks from human primary cells, analysis of cell-secreted extracellular matrix (ECM) and microfluidic integration. Using polymerization-induced phase separation, we demonstrate dynamic in situ formation of microporosity (5–20 µm) within matrix metalloproteinase-degradable polyethylene glycol hydrogels in the presence of living cells. Pore formation is triggered by thiol-Michael-addition crosslinking of a viscous precursor solution supplemented with hyaluronic acid and dextran. The resulting microporous architecture can be fine-tuned by adjusting the concentration and molecular weight of dextran. After encapsulation in microporous hydrogels, human mesenchymal stromal cells and osteoblasts spread rapidly and form 3D networks within 24 hours. We demonstrate that matrix degradability controls cell-matrix remodeling, osteogenic differentiation, and deposition of ECM proteins such as collagen. Finally, we report microfluidic integration and proof-of-concept osteogenic differentiation of 3D cell networks under perfusion on chip. Altogether, this work introduces a synthetic microporous hydrogel to efficiently differentiate 3D human bone cell networks, facilitating future in vitro studies on early bone development.

In bone tissue engineering experiments, fluid-induced shear stress is able to stimulate cells to produce mineralised extracellular matrix (ECM). The application of shear stress on seeded cells can for example be achieved through bioreactors that perfuse medium through porous scaffolds. The generated mechanical environment (i.e. wall shear stress: WSS) within the scaffolds is complex due to the complexity of scaffold geometry. This complexity has so far prevented setting an optimal loading (i.e. flow rate) of the bioreactor to achieve an optimal distribution of WSS for stimulating cells to produce mineralised ECM. In this study, we demonstrate an approach combining computational fluid dynamics (CFD) and mechano-regulation theory to optimise flow rates of a perfusion bioreactor and various scaffold geometries (i.e. pore shape, porosity and pore diameter) in order to maximise shear stress induced mineralisation. The optimal flow rates, under which the highest fraction of scaffold surface area is subjected to a wall shear stress that induces mineralisation, are mainly dependent on the scaffold geometries. Nevertheless, the variation range of such optimal flow rates are within 0.5-5 mL/min (or in terms of fluid velocity: 0.166-1.66 mm/s), among different scaffolds. This approach can facilitate the determination of scaffold-dependent flow rates for bone tissue engineering experiments in vitro, avoiding performing a series of trial and error experiments.

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.