Low magnitude vibration alleviates age-related bone loss by inhibiting cell senescence of osteogenic cells in naturally senescent rats

Animals

3-month-old male (n=10) and female (n=20) SD rats were selected as the control group and 22-month-old male (n=10) and female (n=30) SD rats were selected as the elderly group. All rats were healthy specific pathogen free (SPF) animals and were purchased from the Experimental Animal Center of Sichuan University. All rats were exposed to a relative humidity of 40%-70% and a temperature of 18-26° C. Animal experiments were approved by ethics committee of Sichuan University.

Isolation, culture and identification of BMSCs, osteoblasts and osteocytes

BMSCs were isolated as References [17] reported, simply described, the bone marrow was flushed out with serum-free medium using a 10 mL syringe, collected in a centrifuge tube at 800 rpm for 5 min, and the supernatant was discarded to retain the cell precipitate. Then cells were incubated at 37° C in 5% CO2 incubator; after 3 hours, the first change of culture medium was made; then the culture medium was changed every three days. Adherent cells were passaged at 80%-90% confluence and the passages 3-4 were used for the subsequent experiments. The surface markers of BMSCs were analyzed by flow cytometry assay to identify BMSCs.

Osteoblasts were isolated as References [18] reported, simply described, the femur and tibia were separated aseptically, and the bone fragments were cut into 0.1 cm × 0.1 cm pieces using bone gnashing forceps, then the bone fragments were sequentially digested by collagenase I at 37° C. The digestion solution was collected, centrifuged and resuspended in a culture flask. The medium was changed every 3 days. The osteoblasts were identified by alizarin red staining and alkaline phosphatase (ALP) staining of P3-P4 generation osteoblast from 3-month-old female SD rats.

Osteocytes were isolated as References [19] reported, simply described, the femur and tibia were separated aseptically into 0.1 cm × 0.1 cm bone fragments, and the bone fragments were digested sequentially at 37° C with collagenase I and EDTA. When the fusion degree of primary osteoblasts reached 80-90%, the osteoblasts were digested and passed to the next generation. The osteocytes were identified by E11/gp38 immunofluorescence staining of P3-P4 generation osteocytes from 3-month-old female SD rats.

Senescence-associated heterochromatic foci (SAHF) and aging-related DNA damage detection

Cells were seeded on coverslip and fixed with 4% paraformaldehyde for 15 min, then permeabilized in 0.1% Triton X-100 for 15 min and blocked with 2% bovine serum albumin for 30 min. Primary antibodies anti-H3K9me3 (CST, USA) or 53BP1 (Abcam, USA) were added at room temperature for 1 h. Then fluorescent secondary antibodies were added for 2 h at room temperature. Next, DAPI was used to counterstain nuclei. SAHF formation was detected by observation of the fluorescence signals of H3K9me3, and fluorescence signals of 53BP1 was observed to detect DNA damage.

SA-β-gal staining

Cells were seeded on coverslip and fixed with 4% paraformaldehyde for 15 min, and rinsed twice with PBS. Then the cells were incubated with SA-β-gal reaction solution (Beyotime, China) at 37° C for 12 h, rinsed twice with double-steaming water, observed and counted under the optical microscope.

CCK-8 detection

Cells from each group were seeded on 96-well plates at a density of 3×10³/well. 10 μL CCK-8 reaction solution (ApexBio, USA) was added to each well after indicated times (1d, 3d, 5d and 7d) of natural growth, and incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader.

Alkaline phosphatase staining

The cells were fixed with 95% ethanol for 10 min, stained with alkaline phosphatase staining solution (Beyotime, China) for 20 min, rinsed three times with ddH2O, observed and photographed under the optical microscope.

Alizarin red staining

Cells were fixed with 95% ethanol for 10 min, stained with 0.2% alizarin red staining solution (Solarbio, China) for 30 min, rinsed three times with ddH2O, observed and photographed under the optical microscope.

Detection of alkaline phosphatase activity

Alkaline phosphatase activity was detected by alkaline phosphatase (ALP) assay kit (Beyotime, China) following the instructions of the manufacturer. Briefly, 50 μL of samples or standard reagents were incubated with a chromogenic substrate for 30 minutes at 37° C, and the OD value in each group was detected at 405 nm.

Oil red o staining

The cells were fixed with 95% ethanol for 10 min, and 0.3% Oil Red O staining solution (Solarbio, China) was added for 15 min. Then the cells were rinsed with ddH2O three times, and the lipid droplets were observed and photographed under the optical microscope.

Western blot

The proteins were lysed and collected by cell lysis solution, and the concentration of proteins was detected by BCA kit. Total protein was separated by SDS-PAGE electrophoresis and then transferred onto 0.22 μm PVDF membranes. After that, membranes were blocked and then incubated with primary antibodies overnight at 4° C. After incubated with a secondary antibody for 1 hour at room temperature, the blots were visualized using an ECL kit (Pierce, USA) and quantified with Image J software.

Application of LMV to osteogenic cells and rats

To analyze the effect of LMV on osteogenic cells, the osteogenic cells from old female rats were divided into static group and vibration groups (cells were vibrated at 0.3 g and 90 Hz). For the vibration groups, osteogenic cells were seeded in six-well plates, then the plates were fixed in a box and placed on the vibration platform of the vibration device (ZD-50T, Guangzhou Meiyifeng Test Equipment CO., Ltd, China), and vibrated for 30 minutes, 5 days. To test the effect of vibration at 0.3 g and 90 Hz on the bones of aged rats, 20 of 22-month-old female rats were randomly divided into the static group (n = 10) and vibration group (n = 10). For rats, a vertical whole-body vibration at 0.3 g and 90 Hz was performed for 30 minutes, once daily, 5 days a week until 12 weeks for subsequent analysis.

μCT

The left femur was dissected from the rats, then placed into the scanner Viva CT 80 for scanning. The scanning parameters were as follows: a voltage of 70 kV and a current of 114 μA with a resolution of 12 μm per pixel. The images were analyzed with the μCT Tomography software, and the region of interest (ROI) selected for analysis was from 1.5 to 4.5 mm below the growth plate to determine trabecular bone volume per tissue volume (Tb. BV/TV), trabecular number (Tb. N) and trabecular separation (Tb. Sp).

Morphological staining of bone tissue

The bone tissue section of the proximal femur was selected, and the morphology of bone tissue was observed by HE staining and Masson staining, and the percentage of trabecular bone area was analyzed by Image J software.

Analysis of serum biomarkers

Alkaline phosphatase activity and tartrate acid phosphatase activity was detected by alkaline phosphatase assay kit and tartaric acid-resistant acid phosphatase assay kit following the instructions of the manufacturer (Beyotime, China).

Statistical analysis

Replicated animals were used as replication. Data were analyzed by SPSS 20.0 software and presented as mean ± SD. Comparisons were analyzed using Student's t test for experiments with two groups, or one-way ANOVA for multiple groups. Differences were considered significant at P < 0.05.

Isolation and identification of osteogenic cells from young rats

As shown in Figure 1A, the CD29 positive rate of isolated BMSCs reached 94.8%, the CD45 negative rate reached 93.62%, and the CD11b negative rate reached 92.68%, indicating the purity of the isolated BMSCs. As shown in Figure 1B, the osteoblasts isolated and cultured were positive for Alizarin Red staining and the ALP staining, indicating the purity of the osteoblasts. As shown in Figure 1C, the isolated osteocytes highly expressed E11/gp38, indicating the purity of the osteocytes.

Figure 1. Identification of BMSCs, osteoblasts, and osteocytes from normal female rats. (A) Identification of surface antigens in BMSCs from normal female rats; (B) Alizarin red staining and ALP staining of osteoblasts from normal female rats; (C) E11/gp38 immunofluorescence staining of osteocytes from normal female rats.

Senescence detection of BMSCs in aged rats and young rats

As shown in Figure 2A, 2B, green immunofluorescence showed the higher expression levels of 53BP1 and H3K9Me3 in aged rat BMSCs, indicating that aged rat BMSCs had DNA damage and senescence-related heterochromatin aggregation. As shown in Figure 2C, BMSCs from aged rats highly expressed senescence-related marker proteins p16^INK4a and p21. As shown in Figure 2D, the cells stained blue were β-galactosidase staining positive cells, and the β-galactosidase staining-positive BMSCs in aged rats were significantly higher than those in young rats.

Figure 2. Senescence identification of BMSCs in aged and young rats. (A) 53BP1 immunofluorescence staining of aged and young rat BMSCs; (B) H3K9Me3 immunofluorescence staining of aged and young rat BMSCs; (C) Senescence marker protein assay and relative protein expression statistics of aged and young rat BMSCs $(\overline{x}\pm s,\text{n}=3)$; (D) β-Galactosidase staining plots and statistical plots of the percentage of positive β-galactosidase staining cells $(\overline{x}\pm s,\text{n}=3)$; *P < 0.05 vs. young female rats, **P < 0.01 vs. young female rats, ^#P < 0.05 vs. young male rats, ^##P < 0.01 vs. young male rats.

Senescence detection of osteoblasts in aged rats and young rats

As shown in Figure 3A, 3B, green immunofluorescence showed the higher expression levels of 53BP1 and H3K9Me3 in aged rat osteoblasts, indicating that aged rat osteoblasts had DNA damage and senescence-related heterochromatin aggregation. As shown in Figure 3C, osteoblasts from aged rats highly expressed senescence-related marker proteins p16^INK4a and p21. As shown in Figure 3D, the β-galactosidase staining-positive osteoblasts in aged rats were significantly higher than those in young rats.

Figure 3. Senescence identification of osteoblasts in aged and young rats. (A) 53BP1 immunofluorescence staining of aged rat and young rat osteoblasts; (B) H3K9Me3 immunofluorescence staining of aged rat and young rat osteoblasts; (C) Senescence marker protein assay and protein relative expression statistics of aged rat and young rat osteoblasts $(\overline{x}\pm s,\text{n}=3)$; (D) β-Galactosidase staining plots and statistical plots of the percentage of positive β-galactosidase staining cells $(\overline{x}\pm s,\text{n}=3)$; *P < 0.05 vs. young female rats, **P < 0.01 vs. young female rats, ^#P < 0.05 vs. young male rats, ^##P < 0.01 vs. young male rats.

Senescence detection of osteocytes in aged rats and young rats

As shown in Figure 4A, 4B, 53BP1 and H3K9Me3 with green fluorescence were expressed in the nucleus of aged rat osteocytes, indicating that aged rat osteocytes had DNA damage and senescence-related heterochromatin aggregation. As shown in Figure 4C, aged rat osteocytes expressed senescence-related marker proteins p16^INK4a and p21. As shown in Figure 4D, the β-galactosidase staining-positive osteocytes in aged rats were significantly higher than those in young rats.

Figure 4. Senescence identification of osteocytes in aged and young rats. (A) 53BP1 immunofluorescence staining of aged rat and young rat osteocytes; (B) H3K9Me3 immunofluorescence staining of aged rat and young rat osteocytes; (C) Senescence marker protein assay and protein relative expression statistics of aged rat and young rat osteocytes $(\overline{x}\pm s,\text{n}=3)$; (D) β-Galactosidase staining of aged rat and young rat osteocytes and statistical plots of the percentage of positive β-galactosidase staining cells $(\overline{x}\pm s,\text{n}=3)$; *P < 0.05 vs. young female rats, **P < 0.01 vs. young female rats, ^#P < 0.05 vs. young male rats, ^##P < 0.01 vs. young male rats.

Comparison of the proliferation activity of osteogenic cells between aged rats and young rats

As shown in Figure 5, the OD values of BMSCs, osteoblasts and osteocytes of aged rats were significantly lower than those of young rats at each time point after culture, and the proliferation curves of BMSCs, osteoblasts and osteocytes of aged rats were lower than those of young rats, indicating that the proliferation ability of BMSCs, osteoblasts and osteocytes of aged rats was significantly decreased. It is noteworthy that at 7 days after inoculation, the OD value of osteocytes from 22-month-old female rats decreased by 43.6% compared with those of 3-month-old female rats and the OD value of osteocytes from 22-month-old male rats decreased by 37.1% compared with those from 3-month-old male rats, indicating osteocytes had the most obvious decrease in proliferation.

Figure 5. Growth curves of BMSCs, osteoblasts and osteocytes in aged rats and young rats. (A) Growth curves of BMSCs in aged rats and young rats; (B) Growth curves of osteoblasts in aged rats and young rats; (C) Growth curves of osteocytes in aged rats and young rats.

Comparison of the functional activity of osteogenic cells between aged rats and young rats

As shown in Figure 6A, after osteogenic induced differentiation, the calcium nodule formation was significantly reduced in aged rat BMSCs compared with young rat BMSCs. However, after adipogenic induced differentiation, the lipid droplet formation was significantly more in aged rat BMSCs than in young rat BMSCs, indicating that the adipogenic differentiation potential of aged rat BMSCs was higher than osteogenic differentiation potential. Western blot was used to detect the expression of COL-I, ALP, Runx2, OCN in the osteoblasts of elderly rats and young rats. As shown in Figure 6B, the expression of osteogenic proteins in the osteoblasts from aged rats was significantly decreased compared with those of young rats. In addition, as shown in Figure 6C, 6D, the number of calcified nodules, the staining depth of alkaline phosphatase staining, and the activity of alkaline phosphatase in the osteoblasts from aged rats were all lower than those in young rats, indicating that the osteogenic activity of aged rat osteoblasts was reduced. Western blot was used to detect the expression of sclerosteosis protein in osteocytes of elderly rats and young rats. As shown in Figure 6E, the osteocytes from aged rats highly expressed sclerosteosis, a negative regulator of bone formation, indicating the functional activity of aged rat osteocytes was significantly lower than that of young rats.

Figure 6. Functional activities of BMSCs, osteoblasts and osteocytes in aged rats compared to young rats. (A) Comparison of osteogenic and adipogenic differentiation ability of BMSCs between aged rats and young rats. BMSCs were cultured in osteogenic induction medium for 21 days, and alizarin red staining was performed. BMSCs were stained with oil red O after 14 days of culture in adipogenic induction medium, and the red drops in the figure were lipid droplets; (B) Determination of bone formation protein and statistics of protein relative expression in osteoblasts of aged rats and young rats $(\overline{x}\pm s,\text{n}=3)$; (C) Alizarin red staining and alkaline phosphatase staining of osteoblasts from aged rats and young rats; (D) The activity of alkaline phosphatase (ALP) in osteoblasts from old rats and young rats was measured $(\overline{x}\pm s,\text{n}=6)$; (E) The determination of sclerosteosis and the statistics of relative protein expression in osteocytes from old rats and young rats $(\overline{x}\pm s,\text{n}=3)$; *P < 0.05 vs. young female rats, **P < 0.01 vs. young female rats, ^#P < 0.05 vs. young male rats, ^##P < 0.01 vs. young male rats.

Low magnitude vibration inhibited osteogenic cells senescence

Because the previous results proved that there was no significant difference in senescent cells between male and female rats, only female rats were used to verify the effect of low magnitude vibration. As shown in the β-gal staining, there were more β-gal positive osteogenic cells in the old female rats group compared with the young group. And the number of β-gal positive osteogenic cells was decreased significantly after vibration treatment (Figure 7A). Quantitative analysis of β-gal positive osteogenic cells also revealed the same result (Figure 7B).

Figure 7. Low magnitude vibration inhibited osteogenic cells senescence. (A) β-Galactosidase staining of osteogenic cells. Osteogenic cells from aged rats were treated with LMV, thenβ-Galactosidase staining was performed to detect senescent cells. (B) Statistical plots of the percentage of positive β-galactosidase staining cells $(\overline{x}\pm s,\text{n}=3)$; *P < 0.05 vs. old female rats.

Changes in the SIRT1/p53/p21 axis in osteogenic cells after LMV

The SIRT1/p53/p21 axis is a typical senescence regulatory pathway. As a well-known anti-senescence protein, SIRT1 was downregulated significantly in senescent osteogenic cells, accompanied by significant upregulation of p53 and p21. However, after LMV treatment, SIRT1 was significantly upregulated with downregulation of p53 and p21 (Figure 8). These data suggested that the SIRT1/p53/p21 pathway was involved in regulating LMV-mediated anti-senescence process of osteogenic cells.

Figure 8. Changes in the SIRT1/p53/p21 axis in osteogenic cells after LMV. (A) BMSCs were treated with LMV and were harvested to detect the protein levels of SIRT1, p53, and p21 using western blotting $(\overline{x}\pm s,\text{n}=3)$; (B) Osteoblasts were treated with LMV and were harvested to detect the protein levels of SIRT1, p53, and p21 using western blotting $(\overline{x}\pm s,\text{n}=3)$; (C) Osteocytes were treated with LMV and were harvested to detect the protein levels of SIRT1, p53, and p21 using western blotting $(\overline{x}\pm s,\text{n}=3)$. *P < 0.05 vs. old female rats.

LMV promotes bone formation of aged rats

As shown in Figure 9A, 9D, 9G, the femoral bone density, trabecular volume fraction and trabecular number per unit volume were significantly decreased in aged rats compared with young rats, but partly restored after vibration. Besides, the trabecular separation was significantly decreased in the aged rats after vibration. As shown in Figure 9B, 9C, 9H, after vibration, the trabecular area fraction of femur was significantly increased in aged rats. The analysis of serum biomarkers showed that the ALP activity in the vibration group was increased compared with the static group, while the serum TRAP activity in the vibration group was decreased (Figure 9I, 9J). The above results indicated that aged rats had significant osteoporotic bone structure changes and significant bone loss, and LMV promoted the bone formation of aged rats.

Figure 9. LMV promoted bone formation of aged rats. (A) uCT of femurs from rats treated with LMV; (B) HE staining of femurs from rats treated with LMV; (C) Masson staining of femurs from rats treated with LMV; (D–G) Morphometric parameters of femurs obtained by uCT analysis, including BMD, Tb. BV/TV, Tb. N, Tb. Sp $(\overline{x}\pm s,\text{n}=6)$; (H) Tb. Ar obtained by bone histomorphometric staining analysis $(\overline{x}\pm s,\text{n}=6)$; (I, J) Serum ALP activity and TRAP activity detection. *P < 0.05 vs. old female rats.