Introduction: Bone remodeling is the combined process of bone resorption by osteoclasts and bone formation by osteoblasts. This process is regulated by mechanosensing osteocytes. It is the most fundamental physiological process that defines living bone. An imbalance in this process can cause metabolic bone diseases such as osteoporosis. Currently, no complete in vitro bone remodeling model is available. Such models have the potential to increase our knowledge on the physiological and pathological processes underlying bone remodeling and could potentially improve drug development processes. Bone-on-a-chip technology has the great potential to advance bone research, allowing for the study of low cell numbers in high temporal and/or spatial resolution. In this study, microfluidic chip technology is used to create a bone remodeling model. Currently, the study focusses on three-dimensional (3D) bone formation inside the microfluidic chip by osteogenic differentiation of human bone marrow derived mesenchymal stromal cells (MSCs) into osteoblasts. Next, the aim is to achieve bone-remodeling-on-a-chip by facilitating interaction between osteoblasts, osteocytes and osteoclasts.
Methods: A bone-on-a-chip microfluidic device that facilitates 3D in vitro bone-like tissue formation was developed. A polydimethylsiloxane (PDMS) microfluidic device was fabricated by means of photo- and soft-lithography. The device contained rectangular-shaped cell culture channels that were coated with fibronectin and seeded with MSCs. The MSCs were dynamically cultured for a period of 21 days by applying medium flow, resulting in shear stresses of around 2.3 mPa acting on the cells. Osteogenic medium was used to differentiate the MSCs along the osteogenic lineage.
Results: Time-lapse brightfield imaging revealed self-assembly into 3D constructs within the channel. At the end of the 21-day culture period, deposition of calcium (Alizarin Red staining) and collagen (Picrosirius Red staining) in the extracellular matrix produced by the cells was visible. Confocal microscopy revealed the formation of 3D bone-like struts through self-assembly. Immunohistochemical staining confirmed the formation of collagen type 1 and revealed the expression of osteopontin and DMP-1, confirming the differentiation of the MSCs into the osteogenic lineage.
Conclusion: Overall, the results revealed mineralized bone-like struts. With this, the developed bone-on-a-chip microfluidic device showed the first step towards a 3D in vitro bone remodeling model, exhibiting 3D bone-like tissue formation. In future research, osteoclasts will be added to the model to facilitate the bone resorption process.