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Cell Biology International (2006) 30, 714720 (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
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 9
Keywords: Osteoclast, Fluid shear stress, Carbonic anhydrase II, Morphology.
*Corresponding author. Tel./fax: +86 28 85503570.
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 CO
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
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-N
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 5
2.4 Preparation of dentine slices
SD rats were killed by cervical dislocation and were soaked in 75% alcohol for 3
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 1000
2.6 Staining of polarized osteoclasts
2.7 Bone resorption
After 3 day culture with osteoclasts, the dentine slices were removed and cleaned ultrasonically three times with 30
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 2.9
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
Primer and probe sequences for real-time fluorescent quantitative PCR
2.10 Statistical analysis
Data are expressed as mean
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 80
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).
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 10
Resorptive lacuna of a polarized osteoclast (×4000). Lacunae were obvious and appeared elliptical, about 5
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 C
Electropherogram of total RNA extract. Bands at 28S, 18S and 5S are observed without contamination, indicating that the extraction of total RNA was successful.
Electropherogram of PCR amplified products of CAII. Bands of CAII amplified products are observed clearly between 200
Kinetic curves of RT-PCR for the GAPDH standard. X-axis
Regression line of the GAPDH standard sample. X-axis
Kinetic curves of RT-PCR for the CAII standard (action time groups). Deep green, black, red, blue and light green curves indicate stress at 2.9
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 26.3
Time-dependent analysis of the expression of CAII mRNA in polarized osteoclasts
Expression of CAII mRNA in polarized osteoclasts after treatment with various levels of stress for 30 min
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 20–100
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 104
The shear stress can be calculated from the formula σ
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 24
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 PGE
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 30
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Received 28 October 2005/18 March 2006; accepted 3 May 2006doi:10.1016/j.cellbi.2006.05.002