Mechanical stimuli can trigger intracellular calcium (Ca2 +) responses in osteocytes and osteoblasts. Successful construction of bone cell networks necessitates more elaborate and systematic analysis for the spatiotemporal properties of Ca2 + signaling in the networks. In the present study, an unsupervised algorithm based on independent component analysis (ICA) was employed to extract the Ca2 + signals of bone cells in the network. We demonstrated that the ICA-based technology could yield higher signal fidelity than the manual region of interest (ROI) method. Second, the spatiotemporal properties of Ca2 + signaling in osteocyte-like MLO-Y4 and osteoblast-like MC3T3-E1 cell networks under laminar and steady fluid flow stimulation were systematically analyzed and compared. MLO-Y4 cells exhibited much more active Ca2 + transients than MC3T3-E1 cells, evidenced by more Ca2 + peaks, less time to the 1st peak and less time between the 1st and 2nd peaks. With respect to temporal properties, MLO-Y4 cells demonstrated higher spike rate and Ca2 + oscillating frequency. The spatial intercellular synchronous activities of Ca2 + signaling in MLO-Y4 cell networks were higher than those in MC3T3-E1 cell networks and also negatively correlated with the intercellular distance, revealing faster Ca2 + wave propagation in MLO-Y4 cell networks. Our findings show that the unsupervised ICA-based technique results in more sensitive and quantitative signal extraction than traditional ROI analysis, with the potential to be widely employed in Ca2 + signaling extraction in the cell networks. The present study also revealed a dramatic spatiotemporal difference in Ca2 + signaling for osteocytic and osteoblastic cell networks in processing the mechanical stimulus. The higher intracellular Ca2 + oscillatory behaviors and intercellular coordination of MLO-Y4 cells provided further evidences that osteocytes may behave as the major mechanical sensor in bone modeling and remodeling processes.