CELL CULTURES

The following cellular lines were used for the assays of the present work: mouse mesenchymal cells uncommitted to osteoblastic lineage (C3H/10T1/2 subclone 8, ATTC, CCL-226). These cells were cultured with BME medium (“basal eagle medium,” Thermofisher, 21010046) + 10 % fetal bovine serum (FBS) (Thermofisher, 10500064) + 1 % L-glutamine (Thermofisher, 25030024) + 1 % penicillin/streptomycin (Thermo- fisher, 15140122) at 37 °C with 5 % of CO₂. Mouse preosteoblastic mesenchymal cells (MC3T3-E1 subclone 4, ATTC, CRL-2593). These cells were cultured with MEM alpha medium (“minimum essential medium,” Thermofisher, A1049001) + 10 % FBS + 1 % L-glutamine and 1 % streptomycin/penicillin at 37 °C with 5 % of CO₂. Murine MLO-Y4 osteocytic cells (Kerafast, EKC002). These cells were cultured with MEM alpha medium without ascorbic acid (Thermofisher, 22571038) + 2.5 % FBS + 2.5 % of calf serum (“calf serum” [CS], Thermofisher, 16010159) and 1 % streptomycin/ penicillin at 37 °C with 5 % of CO₂.

MECHANICAL STIMULATION BY FLUID FLOW

A collection of 250 000 MLO-Y4 cells were seeded on Teflon-coated glass slides in a 15 cm² area previously covered with a collagen matrix (Sigma Aldrich, 000010C8919). After 48 hours, the cells unerwent a mechanical stimulus (FF) using the Flexcell Streamer fluid shear stress device that produces a stress of 10 dynes/cm² for 10 minutes (Flexcell International Corporation, Hillsborough, North Carolina, USA). As static control or SC, MLO-Y4 cells were seeded on a Teflon-coated glass slide without undergoing any mechanical stimulus. Afterwards, the cells were incubated for 18 hours, with MEM alpha medium without phenol red (Gibco) supplemented with 0.5 % of CS, 0.5 % of FBS, and 1 % of penicillin-streptomycin to obtain conditioned media (CM) of the different experimental groups: mechanostimulated cells (FF) and control cells.

PROLIFERATION ASSAY

To conduct proliferation assays, both MC3T3-E1 and C3H/10T1/2 cell lines were seeded at a density of 20 000 cells/well in 24-well culture microplates (VWR,734-2325). The following day the medium was changed for a medium with 250 µl of conditioned medium (static control or fluid flow) and 750 µl of its corresponding culture medium. MC3T3-E1 and C3H/10T1/2 cells were used as control incubated with 250 µL of MEM alpha without phenol red (Gibco) supplemented with 0.5 % of CS, 0.5 % of FBS, 1 % of penicillin-streptomycin, and 750 µl of its corresponding culture medium. After 24 h of incubation the cells were detached with Trypsin-EDTA (Thermofisher, 25300062) and cell count started with Trypan blue (Thermofisher, 15250061) in Neubauer chamber. The same process was repeated at 48, 72 and 96 hours and assessment of the proliferation was obtained.

CELL ADHESION

Adhesion of MC3T3-E1 preosteoblastic cells and C3H/10T1/2 mesenchymal cells was evaluated by cell quantification of marked with vital fluorescent dye cytopainter (Abcam, ab176735). To that end, MC3T3-E1 or C3H/10T1/2 cells were seeded in P6 plates for 24 hours. The following day the medium was changed for a medium with 250 µl of the conditioned medium (static control or fluid flow) and 750 µl of its corresponding culture medium. MC3T3-E1 and C3H/10T1/2 cells were used as control of the experiment and incubated with 250 µL of MEM alpha without phenol red (Gibco) supplemented with 0.5 % of CS, 0.5 % of FBS, 1 % of penicillin-streptomycin, and 750 µl of its corresponding culture medium. After 24 hours, they were incubated with cytopainter for 30 minutes and, after incubation, the cells were lifted, counted, and a total of 20 000 cells/cm² were seeded on a new P6 well for 30 minutes. Finally, the supernatant was collected and the cells that were not adhered were counted in a Neubauer chamber using the exclusion method with Trypan blue. On the other hand, images of the cells adhered to the well surface were obtained through a PAULA microscope (“Personal automated lab assistant, Leica Microsystems”). The number of cells marked with fluorescence was counted in 3 different, random fields.

REAL-TIME POLYMERASE CHAIN REACTION (RT-PCR)

A collection of 300 000 cells were seeded in each well in 6-well culture plates in the case of MC3T3-E1 preosteoblastic cells, and 450 000 in the case of C3H/10T1/2 mesenchymal cells. After 24 hours of incubation, the cells were washed with PBS and then PBS was replaced by 250 µl of MEM alpha in the controls and 250 µl in the corresponding conditioned medium in the other wells. In addition, each well was added 750 µl of its corresponding medium. After 24 hours of incubation the culture medium was removed, each medium was collected from the wells in 1.5 mL tubes using trizol and stored at -80 °C.

The following day the samples were defrosted, and kept in ice to maintain a low temperature and then the RNA extraction protocol was performed. Reverse transcription to obtain cDNA was performed with the cDNA reverse transcription kit (“High-capacity RNA-to-cDNA Kit”, Thermofisher, 4387406) following the instructions. After the cDNA was obtained, PCR was performed in a P384 plate using the “Sybergreen” (TB Green, Condalab RR420A) to study the following genes: RUNX2, alkaline phosphatase (ALP), PPARγ, and osteocalcin. In addition, beta actin (cytoskeletal actin) was used as constitutive gene expression. To that end, an ABI PRISM 7500 thermocycler was used (Applied Biosystems).

STATISTICAL ANALYSIS

Data are expressed as means ± standard error. The distribution of the data was analyzed and since they did not adjust to a normal distribution, the differences among the experimental groups were evaluated by non-parametric variance analysis (Kruskal-Wallis). Determination of the possible differences among the experimental groups was conducted using the Dunn or Mann-Whitney tests. To conduct the statistical analysis the software GraphPad Prism was used. p values < 0.05 were considered statistically significant.

EFFECTS OF THE CONDITIONED MEDIUM OF MECHANICALLY STIMULATED —OR NOT— OSTEOCYTES ON THE PROLIFERATION OF MESENCHYMAL CELLS AND PREOSTEOBLASTS

Proliferation study of mouse MC3T3-E1 preosteoblastic cells and mesenchymal cells (C3H/10T1/2) was conducted with 25 % of conditioned medium of MLO-Y4 mouse osteocytic cells mechanically stimulated (FF) or not (SC) (Fig. 1).

Figure 1. Proliferation and cellular death of MC3T3-E1 preosteoblastic cells (A and B, respectively) and C3H/10T1/2 embryonic mesenchymal cells (C and D, respectively) in the presence or absence of conditioned MLO-Y4 osteocyte media after mechanical stimulation (fluid flow: MC FF) or not (static control: MC SC). The controls represent cells without stimulation of osteocyte conditioned media. The values are the mean ± standard error of the 3 experiments conducted in triplicate. *p < 0.05 vs. control; **p < 0.01 vs. control; ^ap < 0.05 vs. MC SC; ^bp < 0.01 vs. MC SC. 

We observed that in both cell lines the osteocyte secretomes show significant effects in proliferation. While the mechanically stimulated (FF) osteocyte secretome promotes greater proliferation, non-stimulated (SC) osteocytes secretome induces less proliferation versus control (Fig. 1 A and C). No differences were observed in cell viability in any of the three experimental conditions in the 4 days analyzed (Fig. 1 B and D).

EFFECTS OF THE CONDITIONED MEDIA OF MECHANICALLY STIMULATED —OR NOT— OSTEOCYTES ON ADHESION OF MESENCHYMAL CELLS AND PREOSTEOBLASTS

Afterwards, a mouse MC3T3-E1 preosteoblastic cell and mesenchymal cell (C3H/10T1/2) adhesion study was conducted in the presence of secretome of osteocytic MLO-Y4 mouse cells after stimulation (FF) or lack (SC) of mechanical stimulus by fluid passage.

No significant differences were seen in the adhesion of MC3T3-E1 cells in the different experimental conditions (Fig. 2). Regarding the number of unattached cells, we observed that stimulation with conditioned media (FF and SC) hardly decreased adhesion compared to control conditions (Fig. 2).

Figure 2. Adhesion of MC3T3-E1 preosteoblastic cells in the presence or absence of conditioned MLO-Y4 osteocyte media after mechanical stimulation (fluid flow: MC FF) or not (static control: MC SC). The controls represent cells without stimulation of osteocyte conditioned media. The values are the mean ± standard error of 3 experiments conducted in triplicate. Representative images are shown after being obtained through bright field microscopy (upper panel) or epifluorescence microscopy (lower panel). Results are expressed as the number of attached cells/field (lower left) and unattached cells/mL (lower right). The values are the mean ± standard error of 3 experiments conducted in triplicate. 

However, we did observe a clear difference among different experimental conditions in C3H/10T1/2 cell adhesion. The medium conditioned by mechanically stimulated osteocytes increased adhesion of this cell line (Fig. 3).

Figure 3. Adhesion of C3H/10T1/2 embryonic mesenchymal cells in the presence or absence of conditioned MLO-Y4 osteocyte media after mechanical stimulation (fluid flow: MC FF) or not (static control: MC SC). The controls represent cells without stimulation of osteocyte conditioned media. The values are the mean ± standard error of 3 experiments conducted in triplicate. Representative images are shown after being obtained through bright field microscopy (upper panel) or epifluorescence microscopy (lower panel). The results are expressed as the number of attached cells/field (lower left) and unattached cells /mL (lower right). The values are the mean ± standard error of 3 experiments conducted in triplicate. *p < 0.05 vs. control. 

ANALYSIS OF GENE EXPRESSION IN MESENCHYMAL AND PREOSTEOBLASTIC CELLS TREATED WITH CONDITIONED MEDIA OF OSTEOCYTES WHETHER STIMULATED MECHANICALLY OR NOT

We conducted a study of differentiation markers and bone formation (Runx2, osteocalcin and alkaline phosphatase) and adipogenic differentiation (PPARᵞ) in mouse MC3T3-E1 preosteoblastic cells and mesenchymal cells (C3H/10T1/2) stimulated with 25 % of conditioned media of mouse MLO-Y4 osteocytic cells in the presence (FF) and absence (SC) of a mechanical stimulus by fluid flow. Figures 4 and 5 show no significant changes on the expression of Runx2, osteocalcin, alkaline phosphatase or any of the two lines studied.

Figure 4. Gene expression analysis of bone differentiation and formation markers (Runx2, osteocalcin, and alkaline phosphatase) and adipogenic differentiation (PPARᵞ) in MC3T3-E1 preosteoblastic cells in the presence or absence of conditioned MLO-Y4 osteocyte media after mechanical stimulation (fluid flow: MC FF) or not (static control: MC SC). The controls represent cells without stimulation of osteocyte conditioned media. The values are the mean ± standard error of 3 experiments conducted in triplicate. *p < 0.05 vs. control; **p < 0.01 vs. control. 

Figure 5. Gene expression analysis of bone differentiation and formation markers (Runx2, osteocalcin, and alkaline phosphatase) and adipogenic differentiation (PPARᵞ) in C3H/10T1/2 preosteoblastic cells in the presence or absence of conditioned MLO-Y4 osteocyte media after mechanical stimulation (fluid flow: MC FF) or not (static control: MC SC). The controls represent cells without stimulus of osteocyte conditioned media. The values are the mean ± standard error of 3 experiments conducted in triplicate. 

However, the PPARγ expression increased after stimulation with conditioned media of osteocytes SC and FF in MC3T3-E1 preosteoblastic cells (Fig. 4) while in C3H/10T1/2 cells, the PPARγ expression levels remained practically unchanged in all the experimental conditions (Fig. 5).