Osteocytes are stellate cells that are encased within bone tissue. Their cell processes connect to processes on neighboring osteocytes by gap junctions, forming a complex functional network capable of coordinated biological efforts. The spaces where the osteocyte cell body and cell processes reside are called lacunae and canaliculi, respectively;​ the space between the cells and the bony walls of the lacunae and canaliculi, called the lacuno-canalicular system (LCS), is filled by proteoglycans and fluid. Movement of the fluid surrounding osteocytes in the LCS is responsible for both delivering circulating factors to osteocytes, as well as transmitting tissue level mechanical loads into a local stimulus that is detected at the cellular level the osteocytes. Emerging data point to osteocytes as the central regulator of bone biology due to their mechanical sensitivity, central location, and ability to modulate the activities of both bone building and bone resorbing cells (osteoblasts and osteoclasts respectively) via paracrine and endocrine signaling. These cells play a role in guiding accrual of bone during growth, maintenance of bone during adulthood and controlling bone loss in osteoporosis and aging.

The last two decades have seen an explosion in osteocyte research, driven substantially by development of isolated osteocyte culture systems for in vitro studies and advances in immunohistochemistry for in situ studies. However, a major stumbling block in the study of osteocytes has been the inability to observe these cells while alive and within their native environment in bone. This is particularly critical for mechanotransduction studies, where the correct fluid mechanical environment that osteocytes experience in their three-dimensional LCS environment cannot be modeled in cell culture systems. Moreover, the emerging consensus suggests that the response of osteocytes to mechanical loading is a major integration point for bone health;​ systemic influences on bone such as hormones act on osteocytes and influence the same molecular signaling cascades as mechanical loading.

The research undertaken in this thesis tackles the difficult but essential issue of how to study responses of osteocytes in living bone to mechanical loading and hormonal challenges. We developed an approach to visualize osteocyte Ca²⁺+ signaling in vivo;​ Ca²⁺+ is a ubiquitous primary messenger in cells and increases in cytosolic Ca²⁺+ levels lead to downstream pathway modulation and changes in gene transcription. 1) First we created a genetically modified mouse strain with a genetically encoded calcium indicator (GCaMP3) expressed in osteocytes;​ this marker fluoresces with increases in cytosolic Ca²⁺+and provides a robust system for measuring acute osteocyte responses. 2) We then developed a combined multiphoton microscopy and precision mechanical loading system to allow us to determine how authentic osteocytes in vivo respond to mechanical challenge. Using these technologies, we performed the first studies of how osteocytes, both individually and as a population, respond to mechanical loading in vivo, and how these Ca²⁺+ signaling responses may encode mechanical strain information into biologically relevant signals. Our final series of studies investigated the long-held but never tested hypothesis that estrogen and parathyroid hormone function in vivo as gain modulators, altering the mechanical sensitivity of osteocytes to mechanical stimuli. This work is a large step forward in the field of bone research, generating a new investigative tool and deeper understanding about osteocytes, the central regulator of bone biology