Calcium response in bone cells at different osteogenic stages under unidirectional or oscillatory flow

Abstract

It was found that preosteoblast MC3T3-E1 cells were less responsive in calcium signaling than mature osteocyte MLO-Y4 cells when a steady fluid flow was exerted on a micropatterned cell network. However, the effect of fluid flow on the calcium response in preosteocyte MLO-A5 was seldom investigated. In the present study, MLO-A5 as well as MC3T3-E1 and MLO-Y4 cells were cultured on a regular substrate with high or low density under unidirectional or oscillatory fluid flow. The results showed that calcium oscillation in the cells during late osteogenesis was significantly stronger than during early osteogenesis regardless of the fluid flow type or the presence of a physical cell-cell connection. Calcium oscillation produced by the oscillatory flow in the three types of cells was stronger than that produced by the unidirectional flow, but MC3T3-E1 and MLO-A5 cells exhibited limited potential for calcium oscillation compared with MLO-Y4 cells. After suramin was used to block the binding of extracellular adenosine triphosphate (ATP) to the membrane P2 receptor, the calcium oscillation in the three types of bone cells with or without physical connections was significantly suppressed as a single responsive peak under unidirectional flow. For the ATP-blocking group of low-density cells under oscillatory flow, the number of oscillation peaks in three types of cells was still more than two. It indicates that besides the ATP pathway, other mechanosensitive calcium pathways may exist under oscillatory flow. The present study provided further evidence for the osteogenic stage-dependent calcium response of bone cells under unidirectional or oscillatory fluid flow.

FIG. 1.

Experimental design and definition of parameters. (a) Schematic of the experimental setup. (b) Schematic of the parallel-plate flow chamber. (c) Schematic of cells with high and low density. (d) The profile of unidirectional and oscillatory flow. (e) The characteristic parameters of flow-induced calcium response. t₁ denotes the time from flow onset to the first response peak; t₂ is the time from the first peak to 50% relaxation; t₃, t₄, t₅, t_6, t₇, and t₈ are the time intervals between different peaks. m₁ represents the normalized magnitude of the first peak.

FIG. 2.

Cell culture with different densities. Light microscopy images of (a) MC3T3-E1, (c) MLO-A5, and (e) MLO-Y4 cells cultured on slides with high or low density. Scale bar, 50 μm. Box charts showing the density distribution of (b) MC3T3-E1, (d) MLO-A5, and (f) MLO-Y4 cells, in which the ends of whiskers represent the 5th and 95th percentiles, and the square denotes the mean value. The middle line in each box plot represents the median and the square denotes the mean; the top and bottom margins of the box represent the 75th and 25th percentiles; the whiskers extend to the 95th and 5th percentiles.

FIG. 3.

Effects of cell density and ATP pathway on the flow-induced calcium response in bone cells under unidirectional flow. (a) Time-lapsed pseudocolor fluorescent images and (b)–(e) typical calcium responsive curves of MC3T3-E1, MLO-A5, and MLO-Y4 cells with different cell density and with suramin treatment or not. The digits on the cells indicate the number of calcium responsive peaks during the 9 min of flow stimulation. Scale bar, 50 μm.

FIG. 4.

Effects of cell density and ATP pathway on the flow-induced calcium response in bone cells under oscillatory flow. (a) Time-lapsed pseudocolor fluorescent images and (b)–(e) typical calcium responsive curves of MC3T3-E1, MLO-A5, and MLO-Y4 cells with different cell density and with suramin treatment or not. The digits on the cells indicate the number of calcium responsive peaks during the 9 min flow stimulation. Scale bar, 50 μm.

FIG. 5.

Property of flow-induced calcium oscillation in bone cells with high and low density. (a) and (b) The percentage of responsive cells; (c) and (d) the average number of responsive peaks in MC3T3-E1, MLO-A5, and MLO-Y4 cells with high and low density under unidirectional (U) and oscillatory (O) flow. * represents the significant difference between the two connected groups and # is with the corresponding high density group. + means significant difference with the corresponding MC3T3-E1 group, ‡ is between MC3T3-E1 and MLO-A5 groups, and $\mp$ is with the corresponding MLO-A5 group but not the MC3T3-E1 group.

FIG. 6.

Sensitivity of the flow-induced calcium response in bone cells with high and low density. (a) and (b) Time to the first peak; (c) and (d) time to the 50% relaxation of the first peak; (e) and (f) magnitude of the first peak in MC3T3-E1, MLO-A5, and MLO-Y4 cells with high and low density under unidirectional (U) and oscillatory (O) flow. * represents the significant difference between the two connected groups and # is with the corresponding high density group. + means significant difference with corresponding MC3T3-E1 group, ‡ is between MC3T3-E1 and MLO-A5 groups, and $\mp$ is with the corresponding MLO-A5 group but not theMC3T3-E1 group.

FIG. 7.

Schematic of calcium response and intercellular calcium transfer among bone cells at different osteogenic stages.