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Cell Biology International (2006) 30, 714–720 (Printed in Great Britain)
Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts
Qinghong Zhanga, Xing Lianga*, Baoming Zhua, Qiang Donga, Ling Xua, Lu Xiaa, Jian Hua, Jun Fua and Mengtao Liub
aKey Laboratory of Oral Biomedical Engineering of Ministry of Education, West China College of Stomatology, Sichuan University, Chengdu 610041, China
bDepartment of Prosthetics, The Red Cross Hospital of Kunming, Yunnan 650021, China


Abstract

The present study was designed to determine the effects of fluid shear stress on the mRNA expression of carbonic anhydrase II (CAII) in polarized rat osteoclasts. Cellular morphology of the polarized osteoclasts generated by a mechanical anatomical technique was examined by tartrate-resistant acid phosphatase (TRAP) staining and the osteoclastic resorption of dentine slices. The polarized osteoclasts were then stress-loaded by using a flow shear stress device newly developed by the osteoclast research group (patent number 200420034438; China), at 9dyne/cm2 for various time periods [0 (control group), 15, 30, 60, and 120min], or at various stress levels [0 (control), 0.9, 2.9, 8.7, and 26.3dyne/cm2] for 30min. The mRNA expression of CAII was quantified using real-time fluorescent quantitative PCR (RT-PCR) and the data were analyzed with SPSS 12.0 software. The polarized osteoclasts were larger than regular monocytes (about 30μm diameter) with irregular configuration, and the majority of polarized osteoclasts appeared to be spherical and had approximately 2–20 nuclei. The TRAP positive polarized osteoclasts showed asymmetrical red staining in the cytoplasm, and had many filaments and vacuoles. These cells formed resorptive pits in dentine slices. The levels of CAII mRNA expression were shown to be time-dependent, with the E+5 copy numbers being 7.88±0.09, 11.14±0.12, 15.83±0.18, 1.94±0.02, and 1.37±0.01 in cells treated at 9dyne/cm2 for 0, 15, 30, 60 and 120min, respectively (P<0.05). The levels of CAII mRNA expression (E+5 copy numbers) in cells treated with the stress levels of 0, 0.9, 2.9, 8.7 and 26.3dyne/cm2 were 7.97±0.201, 11.26±0.688, 15.94±0.201, 31.88±1.496, and 45.08±2.639, respectively (P<0.05). These results indicate that there is a relationship between the fluid shear stress and the mRNA expression of CAII in polarized rat osteoclasts.


Keywords: Osteoclast, Fluid shear stress, Carbonic anhydrase II, Morphology.

*Corresponding author. Tel./fax: +86 28 85503570.


1 Introduction

Skeletons are continuously remodeled by stress throughout their entire life span (Buchter et al., 2005; Draenert et al., 2005). Bone tissue has a porous structure (Gregory et al., 2005; Lorc'h-Bukiet et al., 2005; Thomsen et al., 2005) and its development involves the resorption of the older bone through polarized osteoclasts that are functionally mature giant cells, and the formation of the new bone through osteoblasts under stress (Katagiri and Takahashi, 2002). The bone-resorbing osteoclasts have been shown to play a major role in the development and progression of many bone diseases such as osteoporosis and osteopetrosis (Tondravi et al., 1997; Teti et al., 1999; Chen et al., 2004; Balkan et al., 2005).

The process of acid demineralization in osteoclasts is accomplished through H+-ATPase that pumps hydrogen ions formed and released by specific carbonic anhydrase II (CAII) into the extracellular compartment (Mattson et al., 1997; Wood et al., 1998). CAII is a highly active metalloprotease found in polarized osteoclasts of bone marrow. It catalyzes the reaction of CO2 with H2O to synthesize H2CO3 that is then dissociated into H+ and HCO3 (Salo et al., 1997). If the CAII activity is blocked, the acid demineralization of bone tissues and the solution of the matrix will be severely affected (Mahon et al., 2001). Therefore, CAII plays an essential role in polarized osteoclastic resorption (Mostov and Werb, 1997; Grigoriadis et al., 1994; Zelzer and Olsen, 2003).

In recent years, there has been an increasing interest in studying the differentiation and function of osteoclasts (Boyle et al., 2003). The studies have focused on osteoclasts under fluid shear stress, by which osteocytes are mainly affected (Kitano et al., 1997; Sakai et al., 1999; Mcallister and Frangos, 1999). Although several studies have suggested the importance of the CAII expression in osteoclasts under stress in relation to bone metabolism, many questions remain with respect to mechanisms in culture, maturation, migration, apoptosis and gene changes occurring in polarized osteoclasts under stress. Thus, it is imperative to carry out studies on polarized osteoclasts, especially the change of CAII expression in osteoclasts under stress (Alliston and Derynck, 2002; Rodan and Martin, 2000). The present study was designed to determine the strength-dependent effects of fluid shear stress on the mRNA expression of CAII in polarized rat osteoclasts.

2 Materials and methods

2.1 Experimental animals

Six-week-old, male Sprague Dawley (SD) rats (170±10g) were obtained from the Western China Animal Center of Sichuan University. The animal use and care protocol was approved by the Institutional Animal Use and Care Committee of Sichuan University.

2.2 Chemicals, reagents, and equipment

Fetal calf serum and α-MEM culture medium were purchased from Gibco-BRL (USA) and Hyclone (Logan, UT, USA), respectively. Poly-lysine (Sigma, USA) was used for enhancing the adherence between cells and the glass surface. A scanning electron microscope (KYKY-2800, Chinese Academy of Sciences, Beijing, China) was used for taking photographs of osteoclasts excavated onto the dentine slice. The tartrate-resistant acid phosphatase (TRAP) staining solution was generously provided by Dr. Zhu (West China College of Stomatology, Sichuan University, China); it contained acetic buffer solution, 6-NN pararosaniline solution (18ml), naphtol–formamide solution (1ml), and C4O6H4NaK (282mg). An image analytical system (Image-Pro Plus.v 5.0.2, Media Cybernetics Corp., USA) was used for the measurement of the cell diameter, cell number/unit area, and the average distance between neighboring cells. The experimental system for generating the fluid shear stress used in the present study was newly developed by our osteoclast research group (patent number 200420034438; China).

2.3 Pretreatment of glass slides

Glass slides were soaked in sulfuric acid–potassium dichromate solution overnight. They were then cleaned in distilled water and dried. After sterilization, they were coated with poly-lysine for 5min, after which the unbound poly-lysine was removed by suction according to the manufacturer's instructions, and the slides were air-dried.

2.4 Preparation of dentine slices

SD rats were killed by cervical dislocation and were soaked in 75% alcohol for 3min. The entire third molars were dissected along a transverse section with a hard-tissue slicer and the sections were ground to 30μm thickness and 8mm diameter. They were cleaned ultrasonically three times for 30min each, cultured overnight at 4°C in α-MEM medium containing 1000U/ml penicillin and streptomycin, air-dried under aseptic conditions, and finally sterilized for 30min. The dentine slices were used for bone resorption experiments with polarized osteoclasts.

2.5 Culture of polarized osteoclasts

After rapid removal of soft tissues under aseptic conditions, the femora and tibiae were soaked in D-Hanks liquid containing 1000U/ml penicillin and streptomycin at 4°C for 5min. The bones were rapidly cut into pieces with surgical scissors in a small beaker containing 30ml α-MEM culture medium at 4°C. The beaker was vibrated on a mixer for 3×5s with 20s intervals between the vibration episodes; the supernatants were then centrifuged at 3500rpm for 3min. The resultant pellets were diluted to 109/ml for hematometry analysis using α-MEM culture medium containing 20% fetal calf serum and 100U/ml penicillin and streptomycin. The cell suspension (4ml) was inoculated onto a glass slide in a petri dish, with the suspension margin at least 2mm from the edge of the slide. The culture medium was removed from the slide after 30min and 30ml of α-MEM culture medium was added to the petri dish for continuous culture at 37°C in a 5% CO2 constant-temperature incubator. The culture medium was replaced three times, once every 3h. In addition, the cell suspension was inoculated into six wells of a 24-well culture plate containing one dentine slice per well. Another six wells containing the same cell suspension but without the dentine slice were used as controls. The culture medium was replaced after 30min to remove suspended cells, and then replaced three times, once every 3h.

2.6 Staining of polarized osteoclasts

After 24h culture, the medium in the plates was replaced and the plates were incubated at 37°C for 5min. The cells were fixed with glutaraldehyde for 10min, rinsed and then air-dried. TRAP staining fluid was added (3ml per well) and the plates were incubated at 37°C for an additional 60min. After the removal of the TRAP solution, the plates were washed three times with distilled water and air-dried. The slices were sealed with balata and polarized osteoclasts were observed under a microscope for morphological examinations including cellular configuration, size, nuclei and vesicles.

2.7 Bone resorption

After 3 day culture with osteoclasts, the dentine slices were removed and cleaned ultrasonically three times with 30min each in a 0.25M NH4OH solution to remove adherent cells. They were then rinsed with phosphate buffer solution (PBS), fixed in 2.5% (v/v) glutaraldehyde, post-fixed in 1% osmium tetroxide, and dehydrated with an alcohol series. Finally, the dentine samples were subjected to CO2 critical-point drying and gold staining and the resorptive lacunae were observed by scanning electronic microscopy (SEM).

2.8 Treatment with flow shear stress

A flow shear stress device was developed by the Osteoclast Research Group (patent number: 200420034438, China). When stress was loaded, a clear eyeshot was randomly selected from the cell creeping slice to ensure a clear observation of the configuration of osteoclasts. The magnification, light strength and position for observing cells remained stable during the stress loading process. Finally, Image-Pro Plus analytical software was used to measure the cell diameter, cell number/unit area, and the average distance between neighboring cells. To carry out the time-dependent studies, experimental groups were subjected to stress at 2.9dyne/cm2 for the following times: 0 (control group), 15, 30, 60, and 120min. To investigate the effects at various stress levels for a fixed time (30min), experimental groups were subjected to the following flow stresses: 0 (control), 0.9, 2.9, 8.7, and 26.3dyne/cm2.

2.9 Real-time fluorescent quantitative PCR

Primers and probes for GAPDH and CAII mRNA analyses in polarized osteoclasts were designed and synthesized according to the complete GenBank sequences (Table 1). The real-time fluorescent quantitative PCR procedures included RNA extraction, 1% agarose gel electrophoresis, reverse transcription, routine PCR, RT-PCR, and the measurement of copy numbers for specific genes. Amplification conditions for the CAII gene were as follows: 94°C 2min, 94°C 20s, 55°C 30s and 60°C 40s (45 cycles).


Table 1.

Primer and probe sequences for real-time fluorescent quantitative PCR

PrimerSequence
CAII
 Forward5′-CCAGTTTCACTTTCACTG-3′
 Reverse5′-AGGCAGGTCCAATCTTCAA-3′
 Taqman probe5′-CACTTGGTTCACTGGAACACC-3′

GAPDH
 Forward5′-TGGGTGTGAACCACGAGAA-3′
 Reverse5′-GGCATGGACTTGGTCATGA-3′
 Taqman probe5′-CTGCACCACAACTGCTTAGC-3′


2.10 Statistical analysis

Data are expressed as mean±standard deviation (SD), and statistical analysis was performed with ANOVA (SPSSD 12.0, Chicago, USA). A P value of less than 0.05 was considered statistically significant.

3 Results

After three changes of culture medium, the number and density of the cells decreased significantly, as observed under light microscopy. Polarized osteoclasts were mainly observed along with few osteocytes. The polarized osteoclasts displayed various morphologies (elliptical, kettle-shaped or irregular) but most were spherical, ranging from about 30 to 80μm in diameter. There were irregular cytoplasmic vesicles and occasional pseudopodia. Darkly-stained nuclei with unclear boundaries were frequently observed. In the course of stress loading, the cell density was about 106/cm2, and the average distance between neighboring cells was about 50μm.

TRAP staining clearly identified the polarized osteoclasts scattered among the monocytes. They were larger than monocytes, often elliptical in configuration, and had unclear boundaries. Spherical nuclei were dispersed throughout the cytoplasm and some vesicles were visible. Abundant filaments occupied the cellular periphery. In contrast, the monocytes were smaller and negatively stained, and their nuclei were punctate (Fig. 1).


Fig. 1

TRAP staining of a polarized osteoclast (×400). The cell was larger and configuration appeared elliptical. Round nuclei were dispersed in the cytoplasm, and vesicles were also visible. Many filaments were visible on the cellular periphery.


After a 3-day culture of polarized osteoclasts on the dentine slices, resorptive lacunae were observed under SEM. These lacunae were round, elliptical or irregular. In addition, they varied in size from 5 to 10μm in length. Clear boundaries and demineralized traces were visible, and, in contrast to the dentine tubules, the bases of the lacunae were rough (Fig. 2).


Fig. 2

Resorptive lacuna of a polarized osteoclast (×4000). Lacunae were obvious and appeared elliptical, about 5μm in length. Clear boundaries and demineralized traces were visible, and the base of each resorptive lacuna was rough and different from dentine tubules.


Real-time fluorescent quantitative PCR was carried out after the high purity of the total RNA was confirmed (Fig. 3) and the primer design and probe synthesis were validated (Fig. 4). The Ct values of the samples were determined by comparing with a standard curve (Figs. 5–8) and the copy numbers were subjected to statistical analysis (Tables 2 and 3).


Fig. 3

Electropherogram of total RNA extract. Bands at 28S, 18S and 5S are observed without contamination, indicating that the extraction of total RNA was successful.


Fig. 4

Electropherogram of PCR amplified products of CAII. Bands of CAII amplified products are observed clearly between 200bp and 100bp, indicating that primers and probes for this messenger were correct.


Fig. 5

Kinetic curves of RT-PCR for the GAPDH standard. X-axis=Ct value, Y-axis=DRn.


Fig. 6

Regression line of the GAPDH standard sample. X-axis=logCt, and Y-axis=Ct value.


Fig. 7

Kinetic curves of RT-PCR for the CAII standard (action time groups). Deep green, black, red, blue and light green curves indicate stress at 2.9dyne/cm2 for 0 (the control), 15, 30, 60 and 120min groups, respectively. X-axis=Ct value, Y-axis=DRn.


Fig. 8

Kinetic curves of RT-PCR for the CAII standard (strength groups). Deep green, black, red, blue and light green curves indicate stress strengths of 0 (the control), 0.9, 2.9, 8.7 and 26.3dyne/cm2, respectively, for 30min. X-axis=Ct value, Y-axis=DRn.


Table 2.

Time-dependent analysis of the expression of CAII mRNA in polarized osteoclasts


Table 3.

Expression of CAII mRNA in polarized osteoclasts after treatment with various levels of stress for 30 min

Strength (dyne/cm2)Copy numbers (E+5) (mean ± SD)
0 (Control)7.97 ± 0.201
0.911.26 ± 0.688
2.915.94 ± 0.201
8.731.88 ± 1.496
26.345.08 ± 2.639


Action time (min)Copy numbers (E+5) (mean ± SD)
0 (Control)7.88 ± 0.09
1511.14 ± 0.12
3015.83 ± 0.18
601.94 ± 0.02
1201.37 ± 0.01








4 Discussion

Osteoclasts, a group of multinucleate giant cells in bone tissue, are derived from the hematopoietic system in the bone marrow and differentiate along the mononuclear/macrophage line (Mostov and Werb, 1997; Boyle et al., 2003; Sabokbar et al., 1997). Bone resorption is largely carried out by polarized osteoclasts (Wood et al., 1997; Teitelbaum, 2000) that are known as activated osteoclasts or mature multinucleate giant cells formed by the fusion of osteoclastic precursors (Nakamura et al., 2003).

As hypermetabolic and terminally differentiated cells (Bai et al., 2005), osteoclasts have short survival time and low cell numbers without passage and appear as immature cell strains, which limits the utility of these cells in the field of bone metabolism. Although in vitro culture of osteoclasts was introduced as early as in 1982 (Chambers and Magnus, 1982), the methodologies remain diverse and difficult to apply (James et al., 1996). Common methods include the mechanical anatomical technique, the cranium digested method, bone marrow culture, spleen tissue revulsion and the giant cell tumor method (Scheven et al., 1998; Suda et al., 2003). A large number of osteoclast-like cells may be obtained by the giant cell tumor method, but these cells are derived from tumor tissue, which cannot be equated to osteoclasts in normal bone marrow (Tse et al., 2004; Roodman, 2001). Bone marrow culture or spleen tissue revulsion method can also generate a large number of osteoclasts, but these cells belong to the osteoclastic precursors (Chen and Olson, 2005). The amount of osteoclasts obtained by digesting young animal cranium is poor, and collagenase and trypsinization used in the procedures may cause great damage to osteoclasts (Schwab et al., 2005).

In this study, a mechanical anatomical technique was used to obtain polarized osteoclasts from the large bones of SD rats. This method may be considered a classic one, because several advantages are appealing. First, the osteoclasts obtained by the mechanical anatomical technique are derived from resorbing bone tissue, namely, polarized osteoclasts. These cells from animal bone marrow are most likely to resemble the characteristics of osteocytes in the physiological condition. Second, this method produces sufficient osteoclasts for the experiments. As shown in the present study, the polarized osteoclasts obtained through this procedure were larger than monocytes. In addition, they had more nuclei than monocytes with an abundant cytoplasm and were more active than those generated by other methods.

In the present study, several methods were used to ensure satisfactory identification and characterization of the polarized osteoclasts: morphological observation, TRAP staining and dentine resorption in culture. The mature osteoclasts were shown as giant multinucleate cells about 20–100μm in diameter, with a single osteoclast containing possibly 2 to over 20 nuclei. In this study, the polarized osteoclasts displayed various morphological appearances: most had spherical configurations; others were ovoid, kettle-shaped or edge-shaped. On average, they were about 30μm in diameter. When considering species differences, rat osteoclasts are smaller than other species in size, such as rabbit, but they have similar functions (Goto et al., 1996). TRAP staining of the osteoclasts revealed uneven red deposits in the cytoplasm of these large cells as TRAP can hydrolyze Naphtol AS-BI phosphate to Naphtol AS-BI, which then combines with 6-NN pararosaniline to form unique staining (Hayman et al., 1996). In addition, some vacuoles were observed and the nuclei were found to be dark and spherical. In addition, the activity of these cells was observed in the resorption experiment. It was revealed that the resorptive lacunae excavated by the polarized osteoclasts were irregular with clear boundaries. Demineralized traces were apparent and the base of each resorptive lacuna was rough, in contrast to that of the dentine tubules.

The fluid shear stress device used in this experiment was developed by the osteoclast research group using the parallel plate flow chamber (patent number 200420034438) (Liang et al., 2004). The device has two main parts: the uniform rectangular parallel plate flow chamber and the liquid irrigation system. The parallel plate flow chamber has dimensions of 104mm×26.2mm×0.25mm. A glass slide can be inserted directly into the chamber; and the cells cultured on it can be subjected to various stable fluid shear stresses. The irrigation system comprises upper and lower liquid pools, a constant flow peristaltic pump, and silica gel tubules connecting the lower storage pools with the upper storage pools. The upper pool can be kept constant since any liquid above this surface would flow into the lower storage pool through the silica gel tubule. The whole device is connected to a chamber with a constant temperature of 37°C.

The shear stress can be calculated from the formula σ=6μQ/H2W, where σ is fluid shear stress (dyne/cm2), μ is the coefficient of viscosity (mPas), Q is volume flow through the parallel plate chamber (ml/min) and H refers to the vertical height between the inner top surface of the parallel plate chamber and the upper surface of the glass slide. α-MEM culture medium was used as the liquid in this stress experiment and the coefficient of viscosity was 0.7522mPas. Thus, in this study, experimental stress strength could be varied by adjusting the height and the velocity of flow, and all the stress experiments could be conducted under sterile conditions.

It is believed that osteoclasts are terminally differentiated cells with a short survival time in vitro (Akiyama et al., 2005). Chambers et al. (1984) reported that the survival time of osteoclasts in vitro was only about 24h. With the continuous improvement of culture techniques, the survival time of osteoclasts can be extended to as long as 144h (Fenton et al., 1993). Weinbaum et al. (1994) indicated that the flow shear stress caused by mechanical stresses in bone marrow was less than 30dyne/cm2. Smalt et al. (1997) later found that osteoclasts showed reaction even when the flow shear stress was only 1.0dyne/cm2. In addition, our preliminary tests demonstrated that most of the osteoclasts could be washed away when the stress strength was 30dyne/cm2 for 3min, and that the osteoclast bases adhering to the glass surface could be destroyed when the strength was 8.7dyne/cm2 over 60min. As a result, during each experiment, we selected the time and the stress loading strength according to the cell responses, ensuring that the loaded stress did not destroy the cell bases adhering to the glass slide surface and that the cells would not appear to have died abnormally when the stress loading ended. After repeated preliminary experiments to establish conditions meeting these criteria, the flow stress values were set at 0 (control), 0.9, 2.9, 8.7 and 26.3dyne/cm2, respectively, with a stress loading time of 30min. The stress loading time of 30min was determined as it was found that this was when the strength of flow shear stress was constant.

As osteoclasts play a crucial role in bone remodeling in response to stress, they are implicated in many bone diseases (Bonewald, 2002; Huiskes et al., 2000). Therefore, it is interesting to investigate the mRNA expression and enzyme levels in osteoclasts and other biological functions under stress (Klein-Nulend et al., 2005; Garat et al., 2005). For example, Wichert et al. (2003) attributed an important role in force transduction to integrins, reasoning that the cellular reaction (including enzyme activation) to an applied force was triggered by kinetic interaction between integrins and the extracellular matrix. Stevens and Frangos (2003) suggested that the cytomembrane might be an incipient receptor of fluid shear stress, because such stress might enhance membrane fluidity. Rubin et al. (1999) was the first to observe the reduced osteoclastic activity in response to strain, and shortly afterwards noted that the culture medium itself had no inhibitory effect, suggesting that strain exerted a direct depressive effect on the cells.

However, Kurata et al. (2001) found increased mRNA expression by TRAP and indicated that tensile stress enhanced osteoclastic resorption. McAllister et al. (2000) observed increased concentrations of NO and PGE2 after osteoclasts were exposed to a fluid sheer stress of 16dyne/cm2. Smalt et al. (1997) applied the flow shear stresses at 1.0, 4.6 and 21.5dyne/cm2 to osteoclasts and observed that the quantity of NO was released and that the flow shear stress at 1.0dyne/cm2 resulted in an apparent cell reaction. From another perspective, Weinbaum et al. (1994) reasoned that the stress forced liquid to flow through the bone marrow under physiological conditions, and speculated that the flow shear stress required for a cell response might be less than 30dyne/cm2. Kazaki et al. (2002) co-cultured periodontal fibrocytes with peripheral blood mononuclear cells and applied stress on these cells. It was found that the number of osteoclasts and the expression of ODF mRNA were increased, indicating that liquid in bone lacunae would exert shear stress on the vicinal cells under mechanical stress or vascular pressure and that osteocytes not only react to shear stress but also exert autocrine effects on their own function.

In this study, α-MEM culture medium was used to exert flow shear stress on polarized osteoclasts, and an exposure time and a range of stress strengths were employed that allowed cell-substrate adhesion while keeping the cytoskeleton intact. Analysis of the CAII gene expression by real-time fluorescent quantitative PCR indicated that the mRNA expression of CAII had a tendency to increase until it reached a peak value at 30min, after which it tended to decrease. We speculate that polarized osteoclasts might become adaptive to a prolonged exposure to stress. In addition, it was found that the levels of CAII mRNA expression were increased proportionally with the increase in flow shear stress (P<0.05). We speculate that these changes in gene expression might be associated with several cellular events caused by the shear stress such as cytomorphosis, stretches in cell membrane, deformation of the cytoskeleton (Nath, 2003; Taylor, 2001) and stress signaling pathways (Berenbaum, 2004; Malemud, 2004; Deschner and Hofman, 2003). The activation of a series of biological effects and the release of signaling molecules may modulate various cellular functions (Falany et al., 2001; Ye et al., 2002), including changes in the mRNA expression of multiple genes (Nulend et al., 2005; Rubin et al., 2006). Nevertheless, the in vitro findings from the present study and the previous reports by others should be further investigated and confirmed in in vivo experiments.

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Received 28 October 2005/18 March 2006; accepted 3 May 2006

doi:10.1016/j.cellbi.2006.05.002


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)