Microfluidic Techniques to perform more Physiologically Relevant Bone Flow Experiments.

摘要

Mechanical stimulation of bone has often been used to prevent and/or treat various bone mass disorders. Osteocytes, as the major bone mechanosensory cell, are critical in regulating these disorders through the expression of factors that control bone homeostasis. The current methodology to study how fluid mechanostimulation of osteocytes regulates other cells in vitro is through the use of parallel plate flow chambers (PPFCs). In these studies, osteocytes are seeded on glass slides and loaded into PPFCs. Flow is applied to the osteocytes, and conditioned medium is collected, which can then be applied to the other cells being investigated. However, this methodology lacks real-time and direct signaling between the cells, and loses low half-life signal interactions. Additionally, these large PPFCs lack dimensional physiological relevance, and are non-viable for primary osteocyte studies. These limitations, however, can be mitigated through the use of microfluidics and/or co-culture.;In this thesis, we present microfluidic techniques to significantly improve the physiological relevance of in vitro osteocyte flow experiments. First, we developed a microfluidic co-culture device to investigate mechanoregulated osteocyte-osteoclast cross-talk. Specifically, we demonstrated that unstimulated osteocytes create an environment that is preferential for osteoclast precursor aggregation and differentiation. Furthermore, we observed increased osteocyte mechanosensitivity in co-culture with osteoclasts. Next, we investigated how platform dimensions and forces applied to the cells, independent of shear stress, can affect osteocyte mechanosensitivity. We determined that this sensitivity was due to differences in flow rates and drag forces applied to the cells. Finally, we fabricated a microfluidic pump that was capable of applying physiologically relevant oscillatory fluid flow and inducing osteocyte intracellular calcium responses within microchannels.;This work highlights the need to translate osteocyte mechanobiology studies to more dimensionally/biochemically relevant platforms using microfluidic technologies. As well, this work has already led to the development of in vitro platforms investigating osteocyte mechanical regulation of bone metastasis.