Abstract— Osteoporosis is a common bone and metabolic disease that is characterized by bone density loss and microstructural degeneration. Human bone marrow-derived mesenchymal stem cells have great potential for bone tissue engineering and cell-based therapy due to their excellent multipotency, especially osteogenic differentiation. Although low fluid shear force plays an important role in bone osteogenic differentiation, the cellular and molecular mechanisms underlying this effect remain poorly understood due to a lack of effective tools to detect gene expression at the single-cell level. Here, we presented a double-stranded nucleic acid biosensor to examine the regulatory role of Notch signaling during osteogenic differentiation. The effects of orbital shear stress on hMSC proliferation, morphology change, osteogenic differentiation and Notch1-Dll4 signaling were examined. Osteogenic differentiation was studied by characterizing alkaline phosphatase (ALP) activity. We further investigated how orbital shear modulates Notch1-Dll4 signaling during osteogenic differentiation. Our results showed Notch1-Dll4 signaling is involved in orbital shear- regulated osteogenic differentiation. Inhibition of Notch signaling will mediate the effects of shear stress on human osteogenic differentiation. Keywords: osteogenic differentiation, mesenchymal stem cells, Notch signaling, shear stress I. INTRODUCTION Osteoporosis is a systemic metabolic bone disease with bone mass loss and microstructural degeneration. In recent years, the cost for treating osteoporosis is increasing due to the increased aged population and space travel, causing challenges to public health care. In space, the reasons for developing osteoporosis is mainly related to low (micro- to zero-) gravity conditions, with possible contributions of cosmic ray radiation.[1] For example, bone density loss occurs in the weightless environment of space due to the lack of gravity force. Thus, the bone no longer needs to support the body against gravity. Astronauts lose about 1% - 2% of their bone mineral density every month during space travel. Osteoporosis is one of the major consequences of long- duration spaceflights in astronauts, seriously undermining their heath.[2] The current treatment of osteoporosis is to stimulate osteogenesis or inhibit bone resorption through drug-based agents, i.e., bisphosphonates.[3] However, drug- based agents are limited due to their side effects and lack of This work is supported by NASA CT Space Grant and Research Grant from Taglitela College of Engineering, University of New Haven. 1 Department of Chemistry, Chemical and Biomedical Engineering, University of New Haven, West Haven, CT, 06516, USA. 2 Department of Biomedical Engineering, Lehigh University, Bethlehem, PA, 18015, USA. 3 Department of Biomedical Engineering, Duke University, Durham, NC, 27708, USA. capability of regaining the lost bone density. Thus, there is an urgent need for alternative therapeutic approaches for osteoporosis, especially therapies that are able to counteract bone mass bone, which is crucial for prolonged Space missions. Human bone marrow-derived mesenchymal stem cells are ideal candidates for cell-based therapies for bone tissue engineering and regenerative medicine due to their multipotency. Under mechanical or chemical stimulation, hMSCs can be induced to differentiate into different lineages, including osteoblasts (bone), neuroblasts (neural tissue), adipoblasts (fat), myoblasts (muscle), and chondroblasts (cartilage)[4]. Osteogenic differentiation is a dynamic process and involves several significant signaling pathways, including YAP/TAZ signaling, Notch signaling, and RhoA signaling [5, 6]. Although it has been shown that low fluid shear force, including that encountered in microgravity models, regulates in vitro osteogenic differentiation,[7-10] the fundamental mechanisms that are underlying this effect remains poorly characterized due to a lack of effective tools to detect gene expression profiles at the single-cell level. Here, we present a double-stranded locked nucleic acid biosensor to investigate the effects of low orbital fluid shear stress on hMSCs proliferation and osteogenic differentiation. The phenotypic behaviors, including cell morphology, proliferation, and differentiation, were compared and characterized. We further tracked Notch 1 ligand Delta-like 4 (Dll4) gene expression by incorporating this biosensor with hMSCs imaging during osteogenic differentiation. Finally, we examined how Notch1-Dll4 signaling regulates osteogenic differentiation of hMSCs that is under orbital shear stress. Pharmacological administration is applied to disrupt Notch1- Dll4 signaling to investigate the molecular mechanisms that govern osteogenic differentiation. Our results provide convincing evidence that orbital shear stress induces osteogenesis through Notch1-Dll4 signaling. II. MATERIAL AND METHODS A. Cell Culture and Reagents Human mesenchymal stem cells (hMSCs) were acquired from Lonza and maintained in mesenchymal stem cell basal medium (MSCBM) with GA-1000, L-glutamine, and 4 Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA *Corresponding Author: Dr. Kagya Amoako (kamoako@newhaven.edu) and Dr. Shue Wang (swang@newhaven.edu) are with the University of New Haven, West Haven, CT, 06516, USA. Low Fluid Shear Stress Regulates Osteogenic Differentiation of Human Mesenchymal Stem Cells through Notch1-Dll4 Signaling Yuwen Zhao 1,2 , Kiarra Richardson 1,3 , Rui Yang 1,4 , Zoe Bousraou 1 , Yoo Kyoung Lee 1 , Dr. Kagya Amoako 1,* , Dr. Shue Wang 1,* , Member, IEEE