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Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro
Accepted Manuscript Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro Yong Guo, Yang Wang, Yinqin Liu, Haitao Wang, Chun Guo, Xizheng Zhang, Chaoyong Bei PII:
S1008-1275(15)00045-0
DOI:
10.1016/j.cjtee.2014.09.004
Reference:
CJTEE 31
To appear in:
Chinese Journal of Traumatology
Received Date: 29 July 2014 Revised Date:
24 August 2014
Accepted Date: 11 September 2014
Please cite this article as: Guo Y, Wang Y, Liu Y, Wang H, Guo C, Zhang X, Bei C, Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro, Chinese Journal of Traumatology (2015), doi: 10.1016/j.cjtee.2014.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro Yong Guoa,c, Yang Wanga, Yinqin Liua, Haitao Wanga,c, Chun Guob*, Xizheng
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Zhangc*, Chaoyong Beid
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College of Biotechnology, Guilin Medical University, Guilin 541004, China Department of Medicine, Luohe Medical College, Luohe 462000, China c Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, China d Department of Orthopedic, Affiliated Hospital of Guilin Medical University, Guilin 541000, China
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*Corresponding authors: Tel: 86-13788568212, Email: [email protected] (C. Guo); Tel: 86-13512062555，Email: [email protected] (XZ. Zhang)
Revised 24Aug 2014 Accepted 11Sep 2014
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Fund
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Received 29Jul 2014
This work was financially supported by the National Natural Science Foundation of China (No.11372351, No.31370942, No.81160223), and Scientific Research
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Foundation of Guangxi Higher Education (No.KY2015LX241).
Abstract
Purpose: To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods: Primary osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs, cultured in osteoinductive medium) and RAW264.7 cells cultured in
ACCEPTED MANUSCRIPT osteoclast inductive medium were all subjected to a 1,000 µstrain (µs) at 1 Hz cyclic mechanical stretch for 30 minutes (twice a day). Results: After mechanical stimulation, the alkaline phosphatase (ALP) activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the levels
of
ALP
and
collagen
type
I
increased.
Additionally,
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mRNA
extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant
acid
phosphatase-positive
multinucleated
cells,
matrix
metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium
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solvency all decreased.
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Conclusion: The results demonstrated that 1,000 µs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.
Key words：Mechanical loading；Bones；Osteoblasts；Mesenchymal stem cells；
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RAW264.7 cells；Osteogenesis；Osteoclastogene
ACCEPTED MANUSCRIPT 1. Introduction
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Osteoclasts and osteoblasts are the key differentiated cell types that are responsible for bone resorption, and formation respectively.1 Osteoclasts, a kind of large multinuclear cell, are derived from hematopoietic stem cells in bone marrow, blood and spleen.2-5 Osteoblasts are progeny of resident bone marrow-derived mesenchymal stem cells (BMSCs) or bone marrow stromal cells.1,6
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The balance between bone formation and bone resorption maintains adequate bone mass for each individual's habitual physical activity.7 Mechanical loading has great influence on the skeleton because it can stimulate a series of intra- and intercellular events to inhibit bone resorption or promote bone formation.8 Cyclic mechanical loading, at physiologically-relevant magnitudes, can enhance bone formation significantly.9
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It is well-known that mechanical loading can affect or enhance osteoblastic differentiation of mesenchymal stem cells and osteoblasts’ activity. For example, cyclic mechanical stretching (4,000 µε elongation at 1 Hz frequency) applied on BMSCs and C3H10T1/2 cells, resulted in more osteoblasts.10 Cyclic-stretching at 500-1,500 µε promoted osteoblasts proliferation and increased collagen synthesis.11 Some studies indicate that mechanical loading also influences bone marrow-derived pre-osteoclast-like cell activity, osteoclastogenesis and bone-resorption activity of osteoclasts.12-15 In vivo, osteoblasts (pre-osteoblasts) and osteoclasts (pre-osteoclasts), in the area of bone formation or resorption, are stimulated by nearly the same mechanical loading. Therefore, stimulating osteoblasts (pre-osteoblasts), BMSCs and osteoclasts (pre-osteoclasts) with the same mechanical loading in vitro is an important means to study the effect of mechanical stimulation on bones.
However, the effect of the same mechanical loading on differentiation and activity of osteoblasts, BMSCs, and osteoclasts (pre-osteoclasts) in vitro is not fully understood. Few researchers have applied the same mechanical loading on these cells at the same time and investigated the effects of the same mechanical loading on these cells. In this study, in order to better understand how the same mechanical loading affects osteoblasts, BMSCs and pre-osteoclasts, we investigated the effects of the same mechanical loading on primary osteoblasts activity, osteoblastic differentiation of BMSCs and osteoclastic differentiation of RAW264.7 cells (pre-osteoclast-like cells).
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2. Materials and methods 2.1 Animals, reagents and instruments
2.2 Cells culture
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Mouse monocyte/macrophage cell line RAW264.7 cells were obtained from the cell culture center of Peking Union Medical College, China. C57BL/6 mouse and Wistar rats were obtained from the experimental animal center of Beijing. α-MEM medium, fetal calf serum, streptomycin, penicillin, collagenase I, trypsin were purchased from Invitrogen. Receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech Inc; tartrate-resistant acid phosphatase (TRAP) Staining Kit, ALP Activity Assay Kit, TRAP Activity Assay Kit, BCA Protein Assay Kit and Calcium Assay Kit were from Nanjing Jiancheng Biotechnology Co., Ltd, China; dexamethasone, β-glycerophosphate and ascorbic acid 2-phosphate were from Sigma-Aldrich; PVDF membranes were from Millipore; rabbit anti osteocalcin, goat anti matrix metalloproteinase-9 (MMP-9), RIPA lysis buffer and enhanced chemiluminescence detection kit were from Santa Cruz Biotechnology Co., Ltd; Quant Script RT Kit and SYBR Green I PCR Mix were from Beijing Cowin Biotechnology Co., Ltd, China.
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Primary osteoblasts were isolated from neonatal C57BL/6 mice calvarial bone by a sequential enzyme digestion based on methods previously described.16,17 Briefly, after the Wistar rats calvarial bone was minced with scissors and rinsed with PBS, the bone fragments were digested in 0.25% trypsin for 10 minutes one time, and in 0.1% collagenase I for 30 minutes 3 times. The first digestate was discarded, and the cell suspension was centrifuged twice. Then the collected cells were grown in α-MEM medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin and 100 U/ml penicillin at 37 ˚C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every three days and passage was conducted when the cells reached confluence, and the third passage cells were used for the following experiments. BMSCs were isolated and cultured in vitro using the previously reported method.10 Briefly, the femur and tibia of the two-month-old male Wistar rats were isolated. After rinsing three times with PBS, each end of femur and tibia was removed and douched with α-MEM medium. The flushed mixture was filtered through a 100 µm mesh, then centrifuged for 5 minutes at 800 g. The collected cells were re-suspended in medium
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and a 4-fold volume of red blood cell lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3, and 10 µmol/L EDTA) was added. After 10-minute incubation and 5 minute centrifugation at 800 g, the cells were re-suspended in medium and centrifuged twice more. Then, the re-suspended cells were seeded to 25 cm2 plastic flasks at a density of 2×105-3×105 cells per flask. Two weeks later, cell clones had formed, the cells were digested with 0.25% trypsin and passaged to new flasks. After the first passage, the medium was changed every three days and passage was performed when cells reached confluence, and BMSCs’ third passage was used for the following experiments.
2.3 Application of mechanical loading
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RAW264.7 cells can differentiate into osteoclast-like cells in the presence of 40 ng/mL RANKL and 40 ng/mL M-CSF, as was confirmed by bone resorption assay and TRAP staining in our lab. The cells were cultured in α-MEM medium mentioned above in flasks for the experiments.
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The application of mechanical loading on the cells was conducted with a specially designed four-point bending device described previously.18,19 In this instrument, the cells on the polystyrene cell carrier were subjected to homogeneously distributed uniaxial bending stimuli (Figure 1).
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Bending stimuli was measured in strain. Strain is the difference in length of the surface with and without application of bending stimuli divided by the length without the stimuli (1,000 µstrain equal an elongation of 0.1%). The primary osteoblasts and the BMSCs cultured in osteoinductive medium (α-MEM medium mentioned above supplemented with 100 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate, 250 µmol/L ascorbic acid 2-phosphate), and the RAW264.7 cells cultured in osteoclast-inductive medium (α-MEM mentioned above supplemented with 40 ng/mL M-CSF and 40 ng/mL RANKL) were seeded to the cell carriers coated with osteoblasts extracellular matrix (ECM) prepared by our lab (data not show). The application of mechanical loading at the frequency of 1 Hz for 30 minutes twice a day (the applied values were 1,000 µs) started after the cells were seeded onto the cell carriers. Control cultures were maintained under identical culture conditions without mechanical loading.
2.4 ALP and secreted TRAP activity assay The primary osteoblasts and BMSCs were digested with 0.2% trypsin, and centrifuged. Then cells were lysed by brief sonication on ice in a lysis buffer (250 mmol/L sucrose, 5 mmol/L Tris-HCl, 0.1% TritonX-100, pH 7.5). According to the procedure provided
ACCEPTED MANUSCRIPT by the manufacturer, the protein concentration of the cell lysates was measured with BCA Protein Assay Kit. ALP activity of the cell lysates was assayed with ALP Activity Assay Kit according to manufacturer’s protocol. One unit (U) of ALP activity represents 1 µmol of p-nitrophenyl phosphate hydrolyzed to p-nitrophenol per minute, so that the ALP activity in the proteins was expressed in U/g protein.
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Secreted TRAP activity of the RAW264.7 cells medium was detected with TRAP Activity Assay Kit according to manufacturer’s protocol. One unit (U) of TRAP activity was defined as the amount of enzyme required to hydrolyze 1 µmol of p-nitrophenyl phosphate per minute at 37 °C; the TRAP activity of the medium was expressed in U/L.
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2.5 TRAP staining
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The RAW264.7 cells cultured for a given period were washed with PBS and fixed with 10% neutral formalin. They were then washed with distilled water and stained with TRAP cytochemical staining kit. After washing, TRAP-positive cells with more than two nuclei were considered to be osteoclast-like cells. The number of osteoclast-like cells was counted under Olympus BX51 microscope (Olympus, Japan), and the photographs were taken.
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2.6 Extracellular-deposited calcium assay
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The cells on the cell carriers were removed into PBS containing 0.5% Triton X-100 and 25 mmol/L NH4OH, rinsed with distilled deionized water gently. The extracellular-deposited calcium content of the cell carrier was measured by the calcium assay kit according to the instructions of the manufacturer.
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2.7 Western blot analysis
After trypsinization and centrifugation, the cells were lysed with RIPA lysis buffer containing protease inhibitors. Protein content of the cell lysates was quantified by BCA Protein Assay Kit. Equal amount of protein from each sample was separated in 15% SDS-polyacrylamide gels by electrophoresis, and then the protein was transferred to PVDF membranes. After blocking with 5% BSA, the membranes were incubated overnight with the primary antibody, then incubated with secondary antibody conjugated with horseradish peroxidase. The immunoreactive bands were visualized using an enhanced chemiluminescence detection kit. The optical density of the protein bands was determined with Gel Doc 2000 (Bio-Rad, USA). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control
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2.8 qRT-PCR reaction
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Total RNA was extracted with Trizol reagent (Invitrogen), and then cDNA was synthesized using the Quant Script RT Kit. qPCR was performed to detect mRNA levels of ALP, Col I and GAPDH (internal control reference) using SYBR Green I PCR Mix on a Real-Time PCR System (7900; Applied Biosystems, USA) according to the manufacturer’s instructions. Primer sequences are listed in Table 1. The amplification reaction included a denaturation step at 94°C for 3 minutes followed by 40 cycles of 94°C for 20 s, and annealing and extension at each annealing temperature for 35 s. Using the relative quantitative method (2-∆∆Ct), the levels of the PCR products of interest were calculated relative to those control groups.
Table 1. Primers used for qPCR analysis of mRNA
ALP
Primer sequence (5’-3’)
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Gene
F:CGGGACTGGTACTCGGATAA
Products size Annealing
(bps)
temperature (℃)
156
58
130
58
159
58
R:ATTCCACGTCGGTTCTGTTC F:GGTATGCTTGATCTGTATCTG
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Col Ⅰ
R:TCTTCTGAGTTTGGTGATACG GAPDH
F: ACCCATCACCATCTTCCAGGAG
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R: GAAGGGGCGGAGATGATGAC
2.9 Statistical analysis
Results were presented as mean ±SD. Data were analyzed by Students’ t test. A value of p <0.05 was considered statistically significant.
3. Results After osteoblasts were stimulated with the 1,000 µs mechanical loading for 4 days and 7
ACCEPTED MANUSCRIPT days respectively, the activity of ALP in the cells enhanced remarkably compared with control group, the results of BMSCs stimulated with the mechanical loading were similar to the results of osteoblasts. After stimulation for 7 days, the extracellular-deposited calcium contents of the osteoblasts or BMSCs increased. These data showed that the 1,000 µs mechanical loading enhanced osteoblast activity and promoted osteoblastic differentiation (Figure 2).
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After RAW264.7 cells were mechanically stimulated for 4 days and 7 days respectively, the activity of secreted TRAP decreased remarkably compared with control group (Figure 2), and the number of TRAP-positive multinucleated cells also decreased compared with control group (Figures 3 and 4). TRAP-positive multinucleated cells were designated osteoclast-like cells, so the mechanical loading suppressed osteoclastic differentiation of RAW264.7 cells.
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After mechanical stimulation for 7 days, the MMP-9 protein level of Raw264.7 decreased (Figure 5), and the extracellular-deposited calcium contents of Raw264.7 cells seeded on cell carrier increased (Figure 2D), which showed extracellular calcium solvency of the cells was reduced. These results also testified the mechanical loading suppressed osteoclastic differentiation.
4. Discussion
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In addition, after 7 days of mechanically stimulating, the osteocalcin protein level of both osteoblasts and BMSCs increased (Figure 6). The mRNA expression levels of ALP and collagen type I (Col I) in these cells increased, too (Figure 7). The results confirmed that the mechanical loading enhanced osteoblast activity, and promoted osteoblasic differentiation.
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Many studies have explored the effect of mechanical loading on BMSCs and osteoblasts. Some studies showed that mechanical loading can stimulate mesenchymal stem cells early osteo-chondrogenic response20, and cyclic mechanical strain (5% or 2,000 µs, 1 Hz) could promote or induce osteogenic differentiation of MSCs in vitro.21,22 Mechanical loading (2,000 µs, 200 cycles per day, 1 Hz) loaded on MSCs, could enhance collagen type I and osteonectin expressions of MSCs, and the calcium content of the cells may also increase.23 When human BMSCs were stimulated by a daily cyclic stretch of either 2% or 8% elongation at 1 Hz in vitro, osteogenic differentiation of the BMSCs was initiated.24 Cyclic tension applied to human osteoblasts led to an increase of osteoprotegerin synthesis and a reduction of soluble RANKL release and RANKL mRNA expression.25 After mechanical stretching (1,000 µs, 0.5 Hz), the mRNA levels of c-fos, bone morphogenetic proteins, bone morphogenetic proteins’ receptor, and production of macrophage colony-stimulating factor of human osteoblast in vitro all increased.26
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RAW264.7 cells are preosteoclasts, which can differentiate into osteoclast-like cells in vitro. The cells are also sensitive to mechanical loading; after stimulated by mechanical loading (applied by flexcell tension system, 10% elongation at 0.5 Hz ), the osteoclastic differentiation decreased with mechanical loading and increased after mechanical loading was removed.14 Exposed for six hours to fluid shear stress of 16 dynes/cm2, bone marrow-derived preosteoclast-like cells released more prostaglandin and nitric oxide.12 The 1,100 µs mechanical stimulation (5 minutes/day at 0.1 Hz, 5 days ) induced a significant increase of ALP’s activity in osteoblast, and the mechanical stimulation for 1 minute/day at 0.3 Hz (over 5 days) decreased the fusion and resorption activity of the osteoclast-like cells in vitro.27
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Those studies demonstrated the mechanical loading influence of these cells activity, and osteoblastic or osteoclastic differentiation evidently. However, few researchers stimulated two or more kinds of cells with the same mechanical loading at the same time respectively. Therefore, in this study, the primary osteoblasts, BMSCs and RAW264.7 cells were stimulated with the same mechanical loading respectively at the same time, which can stimulate osteoblast, BMSCs and osteoclast (preosteoclasts) in bone tissue subjected to mechanics in vivo partially. In vivo studies had shown that bone strains in or above the range of 1,500-3,000 µs resulted in bone mass increases.28-30 In those studies, the magnitude, duration and frequency of mechanical loading applied to the cells were different, but the effects were similar. Besides characteristics of the loadings being different, bone cells exhibited diverse responses in vitro to mechanical stimulation depending on the type of material where they adhere31, so that these results were acceptable. Our previous study indicated that mechanical loading of 1,000-2,000 µs at 1 Hz could enhance osteoblasts, activity and promote osteoblastic differentiation of BMSCs in vitro (data not published). Hence, the magnitude of mechanical loading was 1,000 µs in this study.
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ALP is extensively used as a marker of osteoblast or osteogenic differentiation, which increases enzymatic activity to an osteoblastic phenotype.32,33 Osteocalcin, a bone matrix protein, is indicative of mature osteoblasts.34 Extracellular-deposited calcium is also indicative of mineralized matrix production of osteoblasts. Therefore, ALP activity, osteocalcin protein and extracellular-deposited calcium were all analysed. TRAP is often used as one of the markers of osteoclast or osteoclastic differentiation35, MMP-9 is osteoclast-specific gene which rates to bone resorption.15,36 Hence, MMP-9 protein, soluble TRAP activity and TRAP staining were all detected. In this study, RAW264.7 cells were seeded on cell carrier coated with osteoblasts ECM containing extracellular-deposited calcium. Once RAW264.7 cells differentiated to osteoclast-like cells, the extracellular-deposited calcium was dissolved, so extracellular calcium solvency of the cells may be an indication of osteoclast function.
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In this study, with the same cyclic mechanical loading of 1,000 µs, the primary osteoblast, BMSCs and RAW264.7 cells were stimulated continuously respectively. These results from this study showed that the mechanical loading resulted in enhancement of the ALP activity, mRNA levels of ALP and Col I, osteocalcin protein level and extracellular-deposited calcium of osteoblasts and BMSCs, led to a decrease in TRAP activity, number of TRAP-positive multinucleated cells, MMP-9 protein level and extracellular calcium solvency of RAW264.7 cells. The three kinds of cells were stimulated mechanically respectively in this experiment. In the future, we hope to investigate the effect of the same mechanical stimulation parameter on the co-culture of two or three cell types.
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In conclusion, the 1,000 µs cyclic mechanical loading enhanced osteoblasts, activity, promoted osteogenic differentiation of BMSCs and restrained osteoclastic differentiation of RAW264.7 cells in vitro. Therefore, the same mechanical loading enhanced osteogenesis and suppressed osteoclastogenesis in vitro.
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Figure legends
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Figure 1. A：Four-point bending device for application of mechanical loading on cells; B: Schematic chart of the device. When loaded, cells were stimulated by mechanical stretch.
Figure 2. After 4 days and 7 days of mechanical loading (1,000 µs, 1Hz), ALP activity of
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osteoblasts and BMSCs, secreted TRAP activity of Raw264.7 cells and extracellular-deposited calcium of cell carrier seeded by these cells were shown. ALP activities of osteoblasts and BMSCs were all enhanced (A, B); TRAP activities of Raw264.7 cells both decreased (C); extracellular-deposited calcium of these cells all increased after 7 days (D), RAW264.7 cells were seeded on cell carrier coated with osteoblasts ECM, extracellular-deposited calcium content in each well of cell carrier was (4.126±0.798) µg, increased extracellular-deposited calcium of RAW264.7 cells seeded cell carrier meant that RAW264.7 cells, extracellular calcium solvency decreased. *p<0.01, versus control, **p<0.01, versus control, n=8.
Figure 3. After 4 days and 7 days of mechanical loading (1,000 µs, 1 Hz), in the TRAP cytochemical staining of RAW264.7, the white arrows denote TRAP-positive multinucleated cells (osteoclasts). Figure. 4 Numbers of TRAP-positive multinucleated cells. After 4 days and 7 days of mechanical loading, the numbers of TRAP-positive multinucleated cells both decreased. *P<0.01, **p<0.01, versus control, n=8.
ACCEPTED MANUSCRIPT Figure 5. After 7 days of 1,000 µs mechanical loading, western blot analysis of MMP-9 in Raw264.7 cells. The relative protein expression level of MMP-9 (normalized to GAPDH) in 1,000 µs group was higher than the control group. *p<0.05, versus control, n=7.
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Figure 6. After 7 days of 1,000 µs mechanical loading, western blot analysis of osteocalcin in osteoblasts and BMSCs, the relative protein expression levels of osteocalcin (normalized to GAPDH) in 1,000 µs group were higher than control group. **p<0.01, *p<0.05, versus control, n=6.
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Figure 7. After 7 days of 1,000 µs mechanical loading, qRT-PCR analysis of ALP and Col I mRNAs in osteoblasts or BMSCs. The relative mRNA expression level of ALP or Col I (normalized to control) in 1,000 µs group was higher than the control group. **p<0.01, versus control, n=7.
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S1008-1275(15)00045-0
DOI:
10.1016/j.cjtee.2014.09.004
Reference:
CJTEE 31
To appear in:
Chinese Journal of Traumatology
Received Date: 29 July 2014 Revised Date:
24 August 2014
Accepted Date: 11 September 2014
Please cite this article as: Guo Y, Wang Y, Liu Y, Wang H, Guo C, Zhang X, Bei C, Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro, Chinese Journal of Traumatology (2015), doi: 10.1016/j.cjtee.2014.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effect of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro Yong Guoa,c, Yang Wanga, Yinqin Liua, Haitao Wanga,c, Chun Guob*, Xizheng
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Zhangc*, Chaoyong Beid
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College of Biotechnology, Guilin Medical University, Guilin 541004, China Department of Medicine, Luohe Medical College, Luohe 462000, China c Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, China d Department of Orthopedic, Affiliated Hospital of Guilin Medical University, Guilin 541000, China
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*Corresponding authors: Tel: 86-13788568212, Email: [email protected] (C. Guo); Tel: 86-13512062555，Email: [email protected] (XZ. Zhang)
Revised 24Aug 2014 Accepted 11Sep 2014
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Fund
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Received 29Jul 2014
This work was financially supported by the National Natural Science Foundation of China (No.11372351, No.31370942, No.81160223), and Scientific Research
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Foundation of Guangxi Higher Education (No.KY2015LX241).
Abstract
Purpose: To investigate the influence of the same mechanical loading on osteogenesis and osteoclastogenesis in vitro. Methods: Primary osteoblasts, bone marrow-derived mesenchymal stem cells (BMSCs, cultured in osteoinductive medium) and RAW264.7 cells cultured in
ACCEPTED MANUSCRIPT osteoclast inductive medium were all subjected to a 1,000 µstrain (µs) at 1 Hz cyclic mechanical stretch for 30 minutes (twice a day). Results: After mechanical stimulation, the alkaline phosphatase (ALP) activity, osteocalcin protein level of the osteoblasts and BMSCs were all enhanced, and the levels
of
ALP
and
collagen
type
I
increased.
Additionally,
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mRNA
extracellular-deposited calcium of both osteoblasts and BMSCs increased. At the same time, the activity of secreted tartrate-resistant acid phosphatase, the number of tartrate-resistant
acid
phosphatase-positive
multinucleated
cells,
matrix
metalloproteinase-9 protein levels of RAW264.7 cells and the extracellular calcium
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solvency all decreased.
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Conclusion: The results demonstrated that 1,000 µs cyclic mechanical loading enhanced osteoblasts activity, promoted osteoblastic differentiation of BMSCs and restrained osteoclastogenesis of RAW264.7 cells in vitro.
Key words：Mechanical loading；Bones；Osteoblasts；Mesenchymal stem cells；
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RAW264.7 cells；Osteogenesis；Osteoclastogene
ACCEPTED MANUSCRIPT 1. Introduction
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Osteoclasts and osteoblasts are the key differentiated cell types that are responsible for bone resorption, and formation respectively.1 Osteoclasts, a kind of large multinuclear cell, are derived from hematopoietic stem cells in bone marrow, blood and spleen.2-5 Osteoblasts are progeny of resident bone marrow-derived mesenchymal stem cells (BMSCs) or bone marrow stromal cells.1,6
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The balance between bone formation and bone resorption maintains adequate bone mass for each individual's habitual physical activity.7 Mechanical loading has great influence on the skeleton because it can stimulate a series of intra- and intercellular events to inhibit bone resorption or promote bone formation.8 Cyclic mechanical loading, at physiologically-relevant magnitudes, can enhance bone formation significantly.9
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It is well-known that mechanical loading can affect or enhance osteoblastic differentiation of mesenchymal stem cells and osteoblasts’ activity. For example, cyclic mechanical stretching (4,000 µε elongation at 1 Hz frequency) applied on BMSCs and C3H10T1/2 cells, resulted in more osteoblasts.10 Cyclic-stretching at 500-1,500 µε promoted osteoblasts proliferation and increased collagen synthesis.11 Some studies indicate that mechanical loading also influences bone marrow-derived pre-osteoclast-like cell activity, osteoclastogenesis and bone-resorption activity of osteoclasts.12-15 In vivo, osteoblasts (pre-osteoblasts) and osteoclasts (pre-osteoclasts), in the area of bone formation or resorption, are stimulated by nearly the same mechanical loading. Therefore, stimulating osteoblasts (pre-osteoblasts), BMSCs and osteoclasts (pre-osteoclasts) with the same mechanical loading in vitro is an important means to study the effect of mechanical stimulation on bones.
However, the effect of the same mechanical loading on differentiation and activity of osteoblasts, BMSCs, and osteoclasts (pre-osteoclasts) in vitro is not fully understood. Few researchers have applied the same mechanical loading on these cells at the same time and investigated the effects of the same mechanical loading on these cells. In this study, in order to better understand how the same mechanical loading affects osteoblasts, BMSCs and pre-osteoclasts, we investigated the effects of the same mechanical loading on primary osteoblasts activity, osteoblastic differentiation of BMSCs and osteoclastic differentiation of RAW264.7 cells (pre-osteoclast-like cells).
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2. Materials and methods 2.1 Animals, reagents and instruments
2.2 Cells culture
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Mouse monocyte/macrophage cell line RAW264.7 cells were obtained from the cell culture center of Peking Union Medical College, China. C57BL/6 mouse and Wistar rats were obtained from the experimental animal center of Beijing. α-MEM medium, fetal calf serum, streptomycin, penicillin, collagenase I, trypsin were purchased from Invitrogen. Receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech Inc; tartrate-resistant acid phosphatase (TRAP) Staining Kit, ALP Activity Assay Kit, TRAP Activity Assay Kit, BCA Protein Assay Kit and Calcium Assay Kit were from Nanjing Jiancheng Biotechnology Co., Ltd, China; dexamethasone, β-glycerophosphate and ascorbic acid 2-phosphate were from Sigma-Aldrich; PVDF membranes were from Millipore; rabbit anti osteocalcin, goat anti matrix metalloproteinase-9 (MMP-9), RIPA lysis buffer and enhanced chemiluminescence detection kit were from Santa Cruz Biotechnology Co., Ltd; Quant Script RT Kit and SYBR Green I PCR Mix were from Beijing Cowin Biotechnology Co., Ltd, China.
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Primary osteoblasts were isolated from neonatal C57BL/6 mice calvarial bone by a sequential enzyme digestion based on methods previously described.16,17 Briefly, after the Wistar rats calvarial bone was minced with scissors and rinsed with PBS, the bone fragments were digested in 0.25% trypsin for 10 minutes one time, and in 0.1% collagenase I for 30 minutes 3 times. The first digestate was discarded, and the cell suspension was centrifuged twice. Then the collected cells were grown in α-MEM medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin and 100 U/ml penicillin at 37 ˚C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every three days and passage was conducted when the cells reached confluence, and the third passage cells were used for the following experiments. BMSCs were isolated and cultured in vitro using the previously reported method.10 Briefly, the femur and tibia of the two-month-old male Wistar rats were isolated. After rinsing three times with PBS, each end of femur and tibia was removed and douched with α-MEM medium. The flushed mixture was filtered through a 100 µm mesh, then centrifuged for 5 minutes at 800 g. The collected cells were re-suspended in medium
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and a 4-fold volume of red blood cell lysis buffer (0.15 mol/L NH4Cl, 10 mmol/L KHCO3, and 10 µmol/L EDTA) was added. After 10-minute incubation and 5 minute centrifugation at 800 g, the cells were re-suspended in medium and centrifuged twice more. Then, the re-suspended cells were seeded to 25 cm2 plastic flasks at a density of 2×105-3×105 cells per flask. Two weeks later, cell clones had formed, the cells were digested with 0.25% trypsin and passaged to new flasks. After the first passage, the medium was changed every three days and passage was performed when cells reached confluence, and BMSCs’ third passage was used for the following experiments.
2.3 Application of mechanical loading
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RAW264.7 cells can differentiate into osteoclast-like cells in the presence of 40 ng/mL RANKL and 40 ng/mL M-CSF, as was confirmed by bone resorption assay and TRAP staining in our lab. The cells were cultured in α-MEM medium mentioned above in flasks for the experiments.
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The application of mechanical loading on the cells was conducted with a specially designed four-point bending device described previously.18,19 In this instrument, the cells on the polystyrene cell carrier were subjected to homogeneously distributed uniaxial bending stimuli (Figure 1).
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Bending stimuli was measured in strain. Strain is the difference in length of the surface with and without application of bending stimuli divided by the length without the stimuli (1,000 µstrain equal an elongation of 0.1%). The primary osteoblasts and the BMSCs cultured in osteoinductive medium (α-MEM medium mentioned above supplemented with 100 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate, 250 µmol/L ascorbic acid 2-phosphate), and the RAW264.7 cells cultured in osteoclast-inductive medium (α-MEM mentioned above supplemented with 40 ng/mL M-CSF and 40 ng/mL RANKL) were seeded to the cell carriers coated with osteoblasts extracellular matrix (ECM) prepared by our lab (data not show). The application of mechanical loading at the frequency of 1 Hz for 30 minutes twice a day (the applied values were 1,000 µs) started after the cells were seeded onto the cell carriers. Control cultures were maintained under identical culture conditions without mechanical loading.
2.4 ALP and secreted TRAP activity assay The primary osteoblasts and BMSCs were digested with 0.2% trypsin, and centrifuged. Then cells were lysed by brief sonication on ice in a lysis buffer (250 mmol/L sucrose, 5 mmol/L Tris-HCl, 0.1% TritonX-100, pH 7.5). According to the procedure provided
ACCEPTED MANUSCRIPT by the manufacturer, the protein concentration of the cell lysates was measured with BCA Protein Assay Kit. ALP activity of the cell lysates was assayed with ALP Activity Assay Kit according to manufacturer’s protocol. One unit (U) of ALP activity represents 1 µmol of p-nitrophenyl phosphate hydrolyzed to p-nitrophenol per minute, so that the ALP activity in the proteins was expressed in U/g protein.
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Secreted TRAP activity of the RAW264.7 cells medium was detected with TRAP Activity Assay Kit according to manufacturer’s protocol. One unit (U) of TRAP activity was defined as the amount of enzyme required to hydrolyze 1 µmol of p-nitrophenyl phosphate per minute at 37 °C; the TRAP activity of the medium was expressed in U/L.
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2.5 TRAP staining
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The RAW264.7 cells cultured for a given period were washed with PBS and fixed with 10% neutral formalin. They were then washed with distilled water and stained with TRAP cytochemical staining kit. After washing, TRAP-positive cells with more than two nuclei were considered to be osteoclast-like cells. The number of osteoclast-like cells was counted under Olympus BX51 microscope (Olympus, Japan), and the photographs were taken.
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2.6 Extracellular-deposited calcium assay
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The cells on the cell carriers were removed into PBS containing 0.5% Triton X-100 and 25 mmol/L NH4OH, rinsed with distilled deionized water gently. The extracellular-deposited calcium content of the cell carrier was measured by the calcium assay kit according to the instructions of the manufacturer.
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2.7 Western blot analysis
After trypsinization and centrifugation, the cells were lysed with RIPA lysis buffer containing protease inhibitors. Protein content of the cell lysates was quantified by BCA Protein Assay Kit. Equal amount of protein from each sample was separated in 15% SDS-polyacrylamide gels by electrophoresis, and then the protein was transferred to PVDF membranes. After blocking with 5% BSA, the membranes were incubated overnight with the primary antibody, then incubated with secondary antibody conjugated with horseradish peroxidase. The immunoreactive bands were visualized using an enhanced chemiluminescence detection kit. The optical density of the protein bands was determined with Gel Doc 2000 (Bio-Rad, USA). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control
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2.8 qRT-PCR reaction
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Total RNA was extracted with Trizol reagent (Invitrogen), and then cDNA was synthesized using the Quant Script RT Kit. qPCR was performed to detect mRNA levels of ALP, Col I and GAPDH (internal control reference) using SYBR Green I PCR Mix on a Real-Time PCR System (7900; Applied Biosystems, USA) according to the manufacturer’s instructions. Primer sequences are listed in Table 1. The amplification reaction included a denaturation step at 94°C for 3 minutes followed by 40 cycles of 94°C for 20 s, and annealing and extension at each annealing temperature for 35 s. Using the relative quantitative method (2-∆∆Ct), the levels of the PCR products of interest were calculated relative to those control groups.
Table 1. Primers used for qPCR analysis of mRNA
ALP
Primer sequence (5’-3’)
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Gene
F:CGGGACTGGTACTCGGATAA
Products size Annealing
(bps)
temperature (℃)
156
58
130
58
159
58
R:ATTCCACGTCGGTTCTGTTC F:GGTATGCTTGATCTGTATCTG
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Col Ⅰ
R:TCTTCTGAGTTTGGTGATACG GAPDH
F: ACCCATCACCATCTTCCAGGAG
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R: GAAGGGGCGGAGATGATGAC
2.9 Statistical analysis
Results were presented as mean ±SD. Data were analyzed by Students’ t test. A value of p <0.05 was considered statistically significant.
3. Results After osteoblasts were stimulated with the 1,000 µs mechanical loading for 4 days and 7
ACCEPTED MANUSCRIPT days respectively, the activity of ALP in the cells enhanced remarkably compared with control group, the results of BMSCs stimulated with the mechanical loading were similar to the results of osteoblasts. After stimulation for 7 days, the extracellular-deposited calcium contents of the osteoblasts or BMSCs increased. These data showed that the 1,000 µs mechanical loading enhanced osteoblast activity and promoted osteoblastic differentiation (Figure 2).
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After RAW264.7 cells were mechanically stimulated for 4 days and 7 days respectively, the activity of secreted TRAP decreased remarkably compared with control group (Figure 2), and the number of TRAP-positive multinucleated cells also decreased compared with control group (Figures 3 and 4). TRAP-positive multinucleated cells were designated osteoclast-like cells, so the mechanical loading suppressed osteoclastic differentiation of RAW264.7 cells.
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After mechanical stimulation for 7 days, the MMP-9 protein level of Raw264.7 decreased (Figure 5), and the extracellular-deposited calcium contents of Raw264.7 cells seeded on cell carrier increased (Figure 2D), which showed extracellular calcium solvency of the cells was reduced. These results also testified the mechanical loading suppressed osteoclastic differentiation.
4. Discussion
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In addition, after 7 days of mechanically stimulating, the osteocalcin protein level of both osteoblasts and BMSCs increased (Figure 6). The mRNA expression levels of ALP and collagen type I (Col I) in these cells increased, too (Figure 7). The results confirmed that the mechanical loading enhanced osteoblast activity, and promoted osteoblasic differentiation.
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Many studies have explored the effect of mechanical loading on BMSCs and osteoblasts. Some studies showed that mechanical loading can stimulate mesenchymal stem cells early osteo-chondrogenic response20, and cyclic mechanical strain (5% or 2,000 µs, 1 Hz) could promote or induce osteogenic differentiation of MSCs in vitro.21,22 Mechanical loading (2,000 µs, 200 cycles per day, 1 Hz) loaded on MSCs, could enhance collagen type I and osteonectin expressions of MSCs, and the calcium content of the cells may also increase.23 When human BMSCs were stimulated by a daily cyclic stretch of either 2% or 8% elongation at 1 Hz in vitro, osteogenic differentiation of the BMSCs was initiated.24 Cyclic tension applied to human osteoblasts led to an increase of osteoprotegerin synthesis and a reduction of soluble RANKL release and RANKL mRNA expression.25 After mechanical stretching (1,000 µs, 0.5 Hz), the mRNA levels of c-fos, bone morphogenetic proteins, bone morphogenetic proteins’ receptor, and production of macrophage colony-stimulating factor of human osteoblast in vitro all increased.26
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RAW264.7 cells are preosteoclasts, which can differentiate into osteoclast-like cells in vitro. The cells are also sensitive to mechanical loading; after stimulated by mechanical loading (applied by flexcell tension system, 10% elongation at 0.5 Hz ), the osteoclastic differentiation decreased with mechanical loading and increased after mechanical loading was removed.14 Exposed for six hours to fluid shear stress of 16 dynes/cm2, bone marrow-derived preosteoclast-like cells released more prostaglandin and nitric oxide.12 The 1,100 µs mechanical stimulation (5 minutes/day at 0.1 Hz, 5 days ) induced a significant increase of ALP’s activity in osteoblast, and the mechanical stimulation for 1 minute/day at 0.3 Hz (over 5 days) decreased the fusion and resorption activity of the osteoclast-like cells in vitro.27
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Those studies demonstrated the mechanical loading influence of these cells activity, and osteoblastic or osteoclastic differentiation evidently. However, few researchers stimulated two or more kinds of cells with the same mechanical loading at the same time respectively. Therefore, in this study, the primary osteoblasts, BMSCs and RAW264.7 cells were stimulated with the same mechanical loading respectively at the same time, which can stimulate osteoblast, BMSCs and osteoclast (preosteoclasts) in bone tissue subjected to mechanics in vivo partially. In vivo studies had shown that bone strains in or above the range of 1,500-3,000 µs resulted in bone mass increases.28-30 In those studies, the magnitude, duration and frequency of mechanical loading applied to the cells were different, but the effects were similar. Besides characteristics of the loadings being different, bone cells exhibited diverse responses in vitro to mechanical stimulation depending on the type of material where they adhere31, so that these results were acceptable. Our previous study indicated that mechanical loading of 1,000-2,000 µs at 1 Hz could enhance osteoblasts, activity and promote osteoblastic differentiation of BMSCs in vitro (data not published). Hence, the magnitude of mechanical loading was 1,000 µs in this study.
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ALP is extensively used as a marker of osteoblast or osteogenic differentiation, which increases enzymatic activity to an osteoblastic phenotype.32,33 Osteocalcin, a bone matrix protein, is indicative of mature osteoblasts.34 Extracellular-deposited calcium is also indicative of mineralized matrix production of osteoblasts. Therefore, ALP activity, osteocalcin protein and extracellular-deposited calcium were all analysed. TRAP is often used as one of the markers of osteoclast or osteoclastic differentiation35, MMP-9 is osteoclast-specific gene which rates to bone resorption.15,36 Hence, MMP-9 protein, soluble TRAP activity and TRAP staining were all detected. In this study, RAW264.7 cells were seeded on cell carrier coated with osteoblasts ECM containing extracellular-deposited calcium. Once RAW264.7 cells differentiated to osteoclast-like cells, the extracellular-deposited calcium was dissolved, so extracellular calcium solvency of the cells may be an indication of osteoclast function.
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In this study, with the same cyclic mechanical loading of 1,000 µs, the primary osteoblast, BMSCs and RAW264.7 cells were stimulated continuously respectively. These results from this study showed that the mechanical loading resulted in enhancement of the ALP activity, mRNA levels of ALP and Col I, osteocalcin protein level and extracellular-deposited calcium of osteoblasts and BMSCs, led to a decrease in TRAP activity, number of TRAP-positive multinucleated cells, MMP-9 protein level and extracellular calcium solvency of RAW264.7 cells. The three kinds of cells were stimulated mechanically respectively in this experiment. In the future, we hope to investigate the effect of the same mechanical stimulation parameter on the co-culture of two or three cell types.
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In conclusion, the 1,000 µs cyclic mechanical loading enhanced osteoblasts, activity, promoted osteogenic differentiation of BMSCs and restrained osteoclastic differentiation of RAW264.7 cells in vitro. Therefore, the same mechanical loading enhanced osteogenesis and suppressed osteoclastogenesis in vitro.
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Figure legends
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Figure 1. A：Four-point bending device for application of mechanical loading on cells; B: Schematic chart of the device. When loaded, cells were stimulated by mechanical stretch.
Figure 2. After 4 days and 7 days of mechanical loading (1,000 µs, 1Hz), ALP activity of
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osteoblasts and BMSCs, secreted TRAP activity of Raw264.7 cells and extracellular-deposited calcium of cell carrier seeded by these cells were shown. ALP activities of osteoblasts and BMSCs were all enhanced (A, B); TRAP activities of Raw264.7 cells both decreased (C); extracellular-deposited calcium of these cells all increased after 7 days (D), RAW264.7 cells were seeded on cell carrier coated with osteoblasts ECM, extracellular-deposited calcium content in each well of cell carrier was (4.126±0.798) µg, increased extracellular-deposited calcium of RAW264.7 cells seeded cell carrier meant that RAW264.7 cells, extracellular calcium solvency decreased. *p<0.01, versus control, **p<0.01, versus control, n=8.
Figure 3. After 4 days and 7 days of mechanical loading (1,000 µs, 1 Hz), in the TRAP cytochemical staining of RAW264.7, the white arrows denote TRAP-positive multinucleated cells (osteoclasts). Figure. 4 Numbers of TRAP-positive multinucleated cells. After 4 days and 7 days of mechanical loading, the numbers of TRAP-positive multinucleated cells both decreased. *P<0.01, **p<0.01, versus control, n=8.
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Figure 6. After 7 days of 1,000 µs mechanical loading, western blot analysis of osteocalcin in osteoblasts and BMSCs, the relative protein expression levels of osteocalcin (normalized to GAPDH) in 1,000 µs group were higher than control group. **p<0.01, *p<0.05, versus control, n=6.
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Figure 7. After 7 days of 1,000 µs mechanical loading, qRT-PCR analysis of ALP and Col I mRNAs in osteoblasts or BMSCs. The relative mRNA expression level of ALP or Col I (normalized to control) in 1,000 µs group was higher than the control group. **p<0.01, versus control, n=7.
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