INTRODUCTION

Mechanical force is one of the most important stimuli that the bone receives to regulate bone mass, shape and microarchitecture. The endoskeleton reacts to an increase in load by forming more bone or decreasing its mass in the absence of mechanical stress¹. This is because the stimulation triggers the mechanotransduction process in which osteocytes, considered bone´s key mechanosensory cells, when stimulated, send chemical signals that affect the paracrine regulation of osteoblast and osteoclast behavior²,³. It also has been found to have an anti-apoptotic effect on osteocytes⁴.

With mechanical loading, the expression of sclerostin, which is an inhibitor of the protein signaling pathway Wnt/β-catenin constitutively secreted by osteocytes, decreases thus causing an increase in osteoblastogenesis⁵,⁶. On the other hand, apoptotic osteocytes induce the secretion of the receptor activator for nuclear factor κ B ligand (RANKL), indirectly stimulating osteoclastogenesis ⁷. In addition, some chemokines, a family of chemotactic cytokines, could be involved in bone remodeling when expressed by bone cells and provide key signals to recruit different cellular subpopulations⁸.

Recent studies indicate that the high mobility group box 1 protein (HMGB1), the regulated upon activation, normal T cell expressed, and secreted protein or chemokine (C-C motif) ligand 5 (RANTES or CCL5) and the monocyte chemoattractant protein 1 or chemokine (C-C motif) ligand 2 (MCP1 or CCL2) intervene to recruit mesenchymal stem cells to promote tissue repair⁹,¹⁰.

Based on this evidence, our objectives are focused on recreating an in vitro charge model to generate mechanotransduction in a controlled culture environment¹¹ and to study the effect of conditioned medium secreted by osteocytes after being mechanically stimulated in the promotion of the proliferative and migratory capacity of mesenchymal cells and bone cells as well as the possible protein expression of certain chemotactic factors involved in proliferation and migration processes.

MATERIALS AND METHODS

Cell cultures. For our assays, different cell types were used:

Mechanical stimulation tests by fluid passage (Fluid Flow, FF). This technique generates physiologically relevant mechanical stimulation in bone cells in vitro¹¹. For this, 250,000 MLO-Y4 cells were seeded on Teflon-bound glass slides leaving a space of 15 cm² previously collagenized and incubated for at least 48 hours at 37ºC with 5% CO₂ until they reached the confluence. Subsequently, the cells were subjected to mechanical stimulation or not (static control or SC) with the Flexcell Streamer device of medium cut stress that produces a stress of 10 dynes/cm² for 10 minutes (Flexcell International Corporation, Hillsborough, North Carolina, USA.). The cells were then incubated with α MEM Medium without phenol red (Gibco) with 0.5% CS, 0.5% FBS and 1% penicillin-streptomycin to obtain conditioned media (CM) from the different experimental groups: CM of stimulated cells (FF) collected at 6 hours after the stimulus, CM of SC cells collected at 24 hours after the stimulus and CM of FF cells collected at 24 hours after the stimulus.

Proliferation assay. To carry out the proliferation assay, both mouse pre-osteoblast cells and human pre-adipose cells were seeded at a concentration of 6,000 cells/well in 12-well culture plates (Jet Biofil), one plate per condition with each of the lines Cells, and incubated at 37ºC with 5% CO₂. The following day, the medium was exchanged for 20% of conditioned medium and 80% of its culture medium, adjusting the FBS to 10%. After 24 hours of incubation at 37ºC with 5% CO₂, the cells were raised with Trypsin-EDTA and a cell count was made with Trypan Blue 0.4% in PBS (GE Healthcare, Hyclone, Logan, Utah, USA) in the Neubauer chamber. The process was repeated at 48 and 72 hours, obtaining a proliferation assessment with each of the conditioned media for 3 days and in triplicate.

Migration trial in Transwell. Seed in 4 Transwell 6 well culture plates (Corning, Costar, Life Sciences, New York, USA) 75,000 cells/well with its culture medium on the membrane, and 20% conditioned medium was placed underneath and 80% of your culture medium at 1% FBS. After 24 hours of incubation at 37ºC with 5% CO₂, both media and the upper cell layer of the membrane were removed with the aid of a cotton swab. The cells remaining at the bottom of the membrane were fixed with 4% paraformaldehyde in PBS (Alfa Aesar, Thermo Fisher) for 10 minutes and stained with 0.1% crystal violet in distilled water (MERCK, Kenilworth, New Jersey, USA) for 15 minutes. Finally, the membranes were mounted on slides and observed in the phase contrast microscope (Leica Microsystems DM5500 CTR6000) from which 20 images were obtained at 50 μm per well to analyze the number of cells that had migrated as a function of the conditioned medium used.

Western Blot. The cells were prepared to extract the total protein with RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitors and phosphatases (Calbiochem). On the other hand, the conditioned medium was lyophilized and the pellet was resuspended in MiliQ water. To quantify the amount of protein in each of the samples, both lyophilized conditioned medium and cell lysate, the Varioskan Flash Multimode Reader (Thermo Scientific) with a Comassie template were used. Once quantified, they were separated in 15% acrylamide gels and transferred to nitrocellulose membranes. The membrane was then blocked with 5% bovine serum albumin (BSA) dissolved in TBS with Tween 20 (Sigma-Aldrich) for one hour at room temperature and incubated overnight at 4ºC with polyclonal antibodies. of rabbit: anti-HMGB1, anti-MCP1 and anti-RANTES (Abcam, Cambridge, UK). As a control, the anti-α tubulin mouse monoclonal antibody was used. It was then incubated for one hour at room temperature with the corresponding IgG coupled to peroxidase and the membrane was revealed in the transilluminator (Syngene DYV 6-E) with the ECL system (Electro-chemo-luminescence, GE-Amersham, Pittsburgh, USA). The intensities of the bands were quantified by densitometry.

Statistic analysis. In the statistical analysis of the results, the data are expressed as mean ± standard deviation of at least two experiments carried out in triplicate. It was performed using the GraphPad Prism V 7.0 software (GraphPad software, La Jolla, California, USA), using a non-parametric study using a two-tailed t-test or U-Mann-Whitney test for two-to-two comparisons, and the Kuskal-Wallis test for group comparisons. Outliers were detected and excluded using the GraphPad QuickCalcs^©2018 program that uses the Grubb test, and values of p<0.05 were considered as significant results.

RESULTS

Effect of conditioned media of osteocytes mechanically stimulated in the proliferation of pre-osteoblasts and pre-adipose mesenchymal cells

A proliferation study of mouse pre-osteoblastic cells MC3T3-E1 and pre-adipose mesenchymal cells was performed with 20% conditioned media of mouse osteocytic cells MLO-Y4 in the presence (FF 6 hours and FF 24 hours) and absence (SC or control static) of mechanical stimulus by fluid passage.

As shown in figure 1A, there is no significant difference in the proliferation of the cell line MC3T3-E1 after 24 hours or after 72 hours in the presence of conditioned media of 6 and 24 hours. In the case of the pre-adipose mesenchymal cells, the results also showed no significant differences after 24 hours or after 72 hours in the presence of the same conditioned media (Figure 1B).

Figure 1. Cell proliferation of MC3T3-E1 (A) and ASC (B) (mesenchymal cells of adipose origin) in the presence and absence of conditioned media of 6 and 24 hours after mechanical stimulation. The values are the mean ± standard deviation of 3 independent experiments in triplicate. Results presented as number of cells vs. control 

Effect of conditioned media of osteocytes mechanically stimulated in the migration of pre-osteoblasts, preadipose mesenchymal cells and macrophages

The study of migration of pre-osteoblastic cells MC3T3-E1, mesenchymal pre-adipose and macrophage cells RAW 264.7 was performed with culture medium specific to each cell line (control) and conditioned media of osteocytic MLO-Y4 cells in the presence (FF 6 hours and FF 24 hours) and absence (SC) of mechanical stimulation.

The pre-osteoblastic cells doubled and tripled their migration in the presence of the conditioned media of the osteocytes collected after 6 and 24 hours of being subjected to stimuli by fluid passage, respectively (Figure 2).

Figure 2. Migration of MC3T3-E1 cells. Representative images corresponding to the migration of MC3T3-E1 in each of the study conditions (A-C). Number of cells per field of MC3T3 migration in the absence and presence of conditioned study media (D). The values are the mean ± standard deviation of 2 independent experiments in triplicate. **p<0.001 vs. static control 

In the same way, the pre-adipose mesenchymal cells also duplicated their migration in the presence of that media (Figure 3).

Figure 3. Migration of ASC cells (mesenchymal cells of adipose origin). Representative images corresponding to the migration of ASC in each of the study conditions (A-C). Number of cells per field of ASC migration in the absence and presence of conditioned study media (D). The values are the mean ± standard deviation of 2 independent experiments in triplicate. **p<0.001 vs. static control 

In the case of the RAW 264.7 mouse macrophage line, our results indicate a three-fold decrease in their migration in the presence of conditioned media collected after 24 hours of performing the Fluid Flow (Figure 4).

Figure 4. Migration of RAW 264.7 cells. Representative images corresponding to the migration of RAW 264.7 in each of the study conditions (A-C). Number of cells per field of RAW migration 264.7 in the absence and presence of conditioned study media (D). The values are the mean ± standard deviation of 2 independent experiments in triplicate. **p<0.001 vs. static control 

Analysis of chemokine expression and secretion after mechanically stimulating osteocytes

In order to corroborate the results obtained previously, the analysis of chemoattractant protein expression was carried out using the Western Blot technique. For this, the lysates of osteocytic cells MLO-Y4 were obtained in the presence and absence of mechanical stimulation by the passage of fluid and, on the other hand, the lyophilisation of their respective conditioned media, as described in the section on materials and methods.

We studied three possible proteins involved in the migration of mesenchymal cells after a mechanical stimulus, two of them belonging to the C-C chemokine family: MCP1 and RANTES, and the high mobility protein group 1 (HMGB1). Tubulin was used to normalize the cell lysate samples.

As seen in figure 5, in the cell lysates of mechanically stimulated MLO-Y4 there was a two-fold decrease in the expression of the chemotactic protein MCP1 (Figures 5A-5B). In the samples of lyophilized conditioned media it was observed that the secretion of MCP1 also decreased under the conditions of mechanical stimulation, in this case it decreased three times (Figures 5C-5D).

Figure 5. Secretion and expression of chemotactic proteins measured by Western Blot (A-C). Expression of the MCP1 protein in MLO-Y4 cell lysate in the absence of mechanical stimulation (SC) and after 6 and 24 hours of performing FF for 10 minutes (FF6h and FF24h) (C-D). Secretion of the MCP1 protein in conditioned MLO-Y4 media in the absence and presence of mechanical stimulus (E-F). Expression of HMGB1 in cell lysate of MLO-Y4 in the absence and presence of mechanical stimulus (G-H). Expression of RANTES in cell lysate of MLO-Y4 in the absence and presenceof mechanical stimulation. The relative densitometric values are the mean ± standard deviation of 2 independent experiments in triplicate. *p<0.05 vs. static control 

In mechanically stimulated MLO-Y4 cell lysates, a two-fold decrease in the expression of the HMGB1 chemotactic protein was observed in the FF condition at 24 hours (Figures 5E-5F).

Similarly, in mechanically stimulated MLO-Y4 cell lysates, a threefold decrease in the expression of the RANTES chemotactic protein was observed under FF conditions at 6 and 24 hours (Figures 5G-5H).

DISCUSSION

Aging, loss of sex steroids, excess glucocorticoids and certain bone diseases such as osteoporosis, cause a decoupling in bone remodeling and a loss of bone quality due to the accumulation of apoptotic osteocytes that precede the recruitment of precursors osteoclasts and their differentiation to carry out the process of directed bone resorption¹⁴,¹⁵.

However, physiological levels of mechanical stimulation such as physical exercise maintains the viability of osteocytes and, furthermore, as demonstrated in this work, acts on their behavior by modifying the production of certain chemokines and regulating the migration of different cell types.

In our results, we found that the exposure of MC3T3-E1 pre-osteoblastic cells and human pre-adipose mesenchymal cells to conditioned media of mechanically stimulated mouse MLO-Y4 osteocitic cells does not affect their proliferation, but increases their migratory capacity. Previous studies have already shown that the conditioned medium of mechanically stimulated osteocytes is able to recruit osteoprogenitors (mesenchymal cells and osteoblasts) and promote the commitment of the osteogenic lineage of these cells to replenish depleted osteoblasts, improve bone formation and strengthen tissue¹⁶,¹⁷.

On the other hand, our results indicate a decrease in the migration of RAW 264.7 macrophages in the presence of conditioned media of MLO-Y4 cells collected after 24 hours of performing the Fluid Flow. This corroborates what has been observed by other authors who indicate that this conditioned medium is also capable of inhibiting osteoclastogenesis¹⁸.

All this suggests a negative feedback mechanism mediated by paracrine factors that would regulate the bone formation and resorption process. Therefore, we check whether certain selected chemokines are involved in this process through Western Blot assays of both the mechanically stimulated osteocytic cells and the conditio- ned media. According to our results, although there is a significant decrease in the monocyte chemotactic protein type 1 (MCP-1), the high mobility protein group 1 (HMGB1) and the RANTES chemotactic protein in the cell lysates of MLO- Y4, mechanically stimulated, do not seem to be directly associated with the migration of bone forming and repopulating cells.

However, contrary to what occurs in our study, there are previous studies that indicate that HMGB1 is released in the extracellular environment through the active secretion of stimulated cells¹⁹ and promotes osteogenic migration and differentiation of MSCs⁹,²⁰. In the case of MCP-1, it has been observed that mesenchymal stem cells from the bone marrow migrate in response to this chemokine²¹. And there are findings that indicate that RANTES is capable of causing the migration of different cell types, including mesenchymal stem cells from bone marrow, through the induction of autophagy²²-²⁴.

For future research, carrying out a proteomic study of the osteocyte conditioned media would be required, both without stimulation and with mechanical stimulation, to deepen the communication processes of the osteocytes with their environment.