Development of bone cell microarrays in microfluidic chips for studying osteocyte-osteoblast communication under fluid flow mechanical loading

Abstract

Bone tissue remodels throughout life in response to mechanical loads. Impaired activities of bone cells (osteocytes, osteoblasts and osteoclasts) result in a disruption of the bone remodelling cycle, which eventually leads to bone disorders such as osteoporosis. To develop efficient therapeutic strategies against bone disorders, new tools are needed to unravel the bone remodelling cycle at the molecular level. Here, we developed a microfluidic platform, which should allow understanding the bone remodelling cycle in much more detail and ultimately be used to discover new therapeutic compounds. We focused specifically on studying cell-cell communication between osteocytes and osteoblasts cells via connexin 43-gap junctions. Therefore, a new cell printing method was developed to create living cellular bone cell arrays in a microfluidic channel. Several cell printing designs where osteocytes and osteoblasts heterotypically interacted at localized interfaces were evaluated. Physical contacts between the bone cells were characterised at high resolution by correlative atomic force microscopy (AFM) - fluorescence microscopy. We demonstrated that the platform is compatible with single-cell mechanostimulation by AFM nanoindentation and subsequent fluorescent analysis of the mechanoresponse. As a proof of concept, we showed the functionality of the platform by analysing the induced in vivo-like Ca++ wave in the printed osteocyte-osteoblast network upon mechanical stimulation by fluid flow shear stress.

Bone-on-a-Chip: Biomimetic Models Based on Microfluidic Technologies for Biomedical Applications

“…44 Yvanoff et al focused on osteoblast−osteocyte interactions via connexin-43 gap junctions in response to Ca 2+ mechanostimulation in a bone cell microarray. 59 Chemicalgradient generation in a 3D hydrogel in the microfluidic channel is another useful platform to analyze the remodeling of the ECM matrix by osteoblast cells affected by the hydrogel properties and PDGF gradient. 32 Different osteoblast migratory patterns and ECM degradation abilities were observed under different chemical gradients.…”

“…As summarized in Table , different combinations of cell types have been used in BOCs for various research purposes, such as bone remodeling, bone vascularization, and bone metastasis. Immortalized murine bone cells, including MC3T3-E1, MLO-Y4, and Raw264.7, are widely used in BOCs because of their ease of access and culture, despite having inconsistent clinical results. ,,,, Among primary cells, mesenchymal stem cells (MSCs) are the most widely used to simulate the bone microenvironment in BOCs because of their excellent osteogenic differentiation capacity, good agreement with clinical results, and relative ease to obtain cells from humans and animals. , For vessels, human umbilical vein endothelial cells (HUVECs) ,,,,,, are most commonly used, and some reports have used primary cells isolated from cord blood …”

“…Bone is a highly vascularized tissue, and the flow generated by the blood vessels controls osteogenesis . Microfluidic devices can be used to directly stimulate cells with a constant flow, and using scaffolds in combination with devices can reproduce the interstitial flow stress inside the bone. ,, Microfluidic systems have advantages in generating various levels of shear stress. , Bone cells exposed to shear stimuli in the microenvironment showed significantly improved osteogenic differentiation. ,,,,,,, For example, Babaliari et al demonstrated that exposure to constant FSS (0.5 dyn/cm 2 ), at a flow rate of 50 μL/min, enhanced osteoblast proliferation and osteogenic differentiation compared with that in static conditions . In addition, the intermittent FSS induced the osteogenic differentiation of MSCs more strongly than continuous FSS (Figure B) .…”

“…In general, a higher flow rate results in higher osteogenic differentiation; however, exposure to cells at an excessively high flow rate can negatively affect cell adhesion. In another study, interstitial flow-induced shear stimulation was applied after the 3D culture of bone cells by combining microarray and microfluidics . Through interstitial FSS generated in this microfluidic device, osteocytes, which act as mechanical sensors, had a stronger calcium response than osteoblasts.…”

“…Bone remodeling is achieved by the resorption of bone matrix by osteoclasts and the deposition of fresh bone matrix called osteoid by osteoblasts . Therefore, coculture- or triculture-based in vitro bone models have already been explored to clarify the exact roles of osteoblasts, osteocytes, and osteoclasts in bone remodeling. ,,, Park et al suggested a trabecular bone organoid model by coculturing bone cells on a demineralized bone paper (DBP)-based device to study localized bone remodeling (Figure A) . This model showed that the osteoblast-osteoclast interaction actively regulates bone remodeling synergistically via paracrine signaling and cell–cell contact.…”