The Effect of Mechanical Loading on the Metabolism of Growth Plate Chondrocytes

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1 Annals of Biomedical Engineering, Vol. 36, No. 5, May 2008 (Ó 2008) pp DOI: /s The Effect of Mechanical Loading on the Metabolism of Growth Plate Chondrocytes MASASHI UEKI, NOBUAKI TANAKA, KOTARO TANIMOTO, CLARICE NISHIO, KOBUN HONDA, YU-YU LIN, YUKI TANNE, SATORU OHKUMA, TAKASHI KAMIYA, EIJI TANAKA, and KAZUO TANNE Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Kasumi, Minami-ku, Hiroshima , Japan (Received 31 October 2007; accepted 5 February 2008; published online 16 February 2008) Abstract It is well known that mechanical loading influences the endochondral bone formation essential for the growth and development of longitudinal bones. The question was, however, asked whether the effect of mechanical loading on the chondrocyte metabolism is dependent on the loading frequency. This study was aimed at evaluating the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. The chondrocytes obtained from rib growth plate cartilage of 4-week-old male Wistar strain rats were cultured by day 4 and day 11 and used as proliferating and matrixforming chondrocytes, respectively. Intermittent tensile stresses with different frequencies were applied to each stage chondrocyte. DNA syntheses were examined by measuring the incorporation of [ 3 H]thymidine into the cells. Furthermore, the rates of collagen and proteoglycan syntheses were determined by measuring the incorporation of [2,3-3 H]proline and [ 35 S]sulfate into the cells, respectively. At the proliferating stage, intermittent tensions with the frequencies of 30 cycles/min and 150 cycles/min significantly (p < 0.05) upregulated the syntheses of DNA, which indicates the promotion of chondrocyte proliferation. At the matrixforming stage, collagen, and proteoglycan syntheses also enhanced with increase of the loading frequency. In particular, the intermittent tension with the frequencies of 30 cycles/min and 150 cycles/min increased significantly (p < 0.05 or p < 0.01) both the collagen and proteoglycan syntheses. These results suggest that the proliferation and differentiation of growth plate chondrocytes are regulated by the mechanical loading and that the chondrocyte metabolism enhanced with increase of loading frequency. These may give more insight into the possible mechanism leading to endochondral bone formation. Keywords Mechanical loading, Loading frequency, Endochondral bone formation, Growth plate chondrocytes. Address correspondence to Eiji Tanaka, Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Kasumi, Minami-ku, Hiroshima , Japan. Electronic mail: etanaka@ hiroshima-u.ac.jp 793 INTRODUCTION Longitudinal bone growth is achieved by endochondral bone formation or ossification. A sequence in the longitudinal bone growth with endochondral bone formation is the modification of immature proliferating chondrocytes into hypertrophic chondrocytes, and the maturation process appears to be the most important determinant for longitudinal growth. Endochondral bone formation is regulated by many factors such as VEGF, CTGF, MMPs, and Cbfa1. 46,47,50,63 In addition, it has been suggested that mechanical loading can influence the rate of chondrocyte maturation. 49,61 For example, the spaceflight experience may result in changes in the composition of extracellular matrix, which could have a negative impact on conversion of growth plate cartilage into normal cancellous bone by endochondral ossification. 49 This implies that immobility and unloading could suppress longitudinal bone growth. Mechanical loading derived from physical exercise influence long bone growth, although these effects vary considerably. Some studies report that physical exercise suppresses bone growth, 2,32 while others obviously reveal enhanced growth with physical training. 55 This controversial may be due to the different magnitude, type, and frequency of the mechanical loading. In fact, mathematical models of endochondral ossification have suggested that tensile stress can accelerate the formation of endochondral bone, 5 while hydrostatic compression preserves the cartilage phenotype. 4 Also, Ohashi et al. 40 showed that longitudinal growth suppression caused by axial loading of the ulna, which is proportional to the magnitude of load, although the largest load (17N) caused morphological changes in the distal growth plate cartilage. 44 In our previous study, 25 the application of an excessive mechanical stress of 17 kpa to cultured articular chondrocytes reduced matrix synthesis and enhanced the gene /08/ /0 Ó 2008 Biomedical Engineering Society

2 794 UEKI et al. expression of matrix metalloproteinases (MMPs). Therefore, it is obvious that mechanical loading can influence longitudinal bone growth. Mechanical loading is classified into sustained and intermittent by its frequency. Many studies have been conducted using cartilage tissue explants and demonstrated that intermittent mechanical stimulation enhanced cartilage metabolism irrespective of the loading type (tension and compression). 15,27,45,56,60,62 In previous studies, mechanical stresses upregulated DNA and glycosaminoglycan syntheses with a significant increase in the intracellular cyclic AMP 54 and PTHrP levels 52 in rat rib growth plate chondrocytes. The oscillatory mechanical stimuli were also reported to affect the metabolism of chondrocytes. Relatively little information, however, is available on the dependency of loading frequency on the growth plate chondrocyte metabolism. Although the detailed mechanism of the effects of the mechanical stimulation on chondrocytes have not been fully understood, it is suggested that the loading frequency may be the most important key for the regulation of cartilage metabolism with mechanical loadings. Therefore, we hypothesized a loading frequency-dependence of chondrocyte metabolism in growth plate. Thus, we applied mechanical tensile stresses with different loading frequencies to the primary growth plate chondrocytes at the proliferating and matrix-forming stages. The aim of this study was to evaluate the effect of tensile loadings with various frequencies on the proliferation of growth plate chondrocytes and extracellular matrix synthesis. MATERIALS AND METHODS Cell Isolation and Culture Rib growth plate chondrocytes were extracted and cultured according to the previously established methods. 48,65 Briefly, rib growth plate cartilage of 4-week-old male Wistar strain rats were resected and digested in sequential trypsin (0.1%)/collagenase (0.05%; Worthington Biochemical, Lakewood, NJ). The resulting cell suspension was filtered through a nylon sieve (pore size = 62 lm) and washed three times with a-minimum essential medium (a-mem; Sanko Junyaku, Tokyo, Japan) containing 50 lg/ml ascorbic acid (Katayamakagaku, Osaka, Japan), 32 units/ml penicillin (Meijiseika, Tokyo, Japan), 60 lg/ml kanamycin (Meijiseika) and 250 ng/ml amphotericin B (ICN Biomedicals, Costa Mesa, CA) (Medium A). The protocol of the experiment was approved by the Animal Care and Use Committee at Hiroshima University. The cells were seeded at per a dish on Bioflex silicone-elastomer multiwell plate (35 mm diameter, Flexcell, McKeesport, PA). Before seeding cells, the experimental and control dishes were coated with 500 ll collagen solution [10 lg/ml bovine type II collagen (acid-soluble, pepsin-resistant; Koken, Osaka, Japan) in phosphate-buffered saline containing 10 mm NaHCO 3 ] to allow the subsequent attachment of cells to the dishes. Chondrocytes were cultured in Medium A containing 10% fetal bovine serum (FBS; Daiichi Kagaku, Tokyo, Japan), and maintained in an atmosphere of 5% CO 2 in a humidified incubator. Through the experimental culture, medium was changed every two days. bfgf (1 ng/ml) was added to the cultures to stimulate proliferation until the cells became confluent. After confluence was achieved, bfgf was replaced by ascorbic acid (50 lg/ml) to promote a sequential differentiation of the cells. 52,54 This method enables us to culture chondrocytes up to prehypertrophic stage. 41 Application of Mechanical Stress Intermittent tensile stresses were applied to the chondrocytes using a computer-driven vacuum operated strain unit (Flexercell strain unit; Flexcell) (Fig. 1). One day before mechanical application, the medium was replaced with a-mem containing 1% FBS without bfgf and ascorbic acid. Furthermore, the medium was changed into a-mem containing 0.5% FBS immediately before mechanical application. Tensile strain was applied with an amplitude of 3%, which resulted in 2 kpa of tensile stress. 16 The oscillation frequencies were 2, 30, and 150 cycles/min, which were defined as low, middle, and high frequencies, respectively. Cultured chondrocytes without mechanical stresses served as the control. Analyses of DNA, Collagen, and Proteoglycan Syntheses To assess DNA synthesis, we measured [ 3 H]thymidine incorporation into chondrocytes. The proliferating FIGURE 1. Schema of the experimental system of tensile stress application. Growth plate chondrocytes on the Bioflex plates were extended by mechanical strain by using a Flexercell strain unit.

3 Effect of Loading on Growth Plate Chondrocytes 795 chondrocytes were exposed to 5 lci/ml [ 3 H]thymidine in 2 ml Medium A containing 0.5% FBS for the final 4 h of 12-h mechanical stress application. After mechanical stimulation, the cell layer was washed three times with PBS and fixed with 10% trichloroacetic acid (TCA). After removal of TCA solution, the cell layer was solubilized with 1 N NaOH and neutirized with 6 N HCl. The radioactivities were measured in the cell layers with a scintillation counter. For proteoglycan and collagen synthesis assay, the matrix-forming chondrocytes were exposed to 2.5 lci/ ml [ 35 S]sulfate and 10 lci/ml [2,3-3 H]proline incorporation into chondrocytes in 2 ml of Medium A containing 0.5% FBS for the final 4 h of 24-h mechanical stress application. After labeling of [ 35 S]sulfate to assess proteoglycan synthesis, the cell layer was washed three times with PBS and solubizied with 2 mg/ml of Pronase E (Kaken Pharmaceutical Company, Tokyo, Japan) in 5 mm CaCl 2 and 0.2 M Tris/HCl at 56 C for 3 h. The precipitates incorporating [ 35 S]sulfate were collected on glass fiber filter papers and washed three times with cetyl pyridinium chloride (CPC; Nacalai Tesque, Kyoto, Japan). The radioactivities of the cells precipitated with CPC were measured in a scintillation counter. With respect to assessment of collagen synthesis, after labeling of [2,3-3 H]proline, the cell layer was washed three times with PBS and homogenized with a polyton in 0.2% Triton-X 100 and 50 mm Tris/HCl. The cell homogenate was digested in the presence or absence of 0.02% collagenase and incubated for 4 h at 37 C. After incubation, the each sample was added the 10% TCA containing 0.5% tannic acid and centrifuged at 10,000 rpm for 10 min and washed three times with 10% TCA/0.5% tannic acid. The precipitates were each solubilized in 50 mm Tris/HCl and the radioactivities were measured in a scintillation counter. The collagen synthesis was substracted the radioactivies in presence of collagenase digestion from the radioactivities in the absence of collagenase digestion. Final data of the radioactivities of [ 35 S]sulfate and [2,3-3 H]proline in the matrix-forming chondrocytes were divided by the DNA content of the each sample. Total DNA Amount Total DNA amount was measured by a Pico- GreenÒ dsdna quantitation kit (Molecular Probes, Leiden, the Netherlands). Chondrocytes were rinsed with PBS and digested overnight at 60 C in a solution of proteinase K (0.1 mg/ml) in solution buffer (150 mm NaCl, 10 mm Tris HCl, 10 mm EDTA, ph 8.0). Nucleic acids in solution buffer were mixed with a diluted PicoGreen reagent (1:200 in TE buffer). The samples were incubated for 10 min at room temperature under a condition protected from light. After the incubation, the fluorescence intensity was measured with a spectrofluorometer (type 850, Hitachi, Tokyo, Japan). Excitation (k ex ) and emission (k em ) wavelengths were 480 and 520 nm, respectively. Statistical Analysis The experiments were repeated three times. Results were expressed as the mean ± standard deviation (SD). All data were tested for normality of distribution (Kolmogorov Smirnov test) and for uniformity (Bartlett s test). Statistical comparisons of the means were performed by one-way analysis of variance with a Bonferroni s modification of Student s t-test as a post hoc test. Probabilities of less than 0.05 were considered to be significant. RESULTS Differentiation Stages of Growth Plate Chondrocytes Cultured chondrocytes exhibited active proliferation with cell division up to day 5 (Fig. 2a). Abundant cartilaginous matrices were synthesized from day 7 to day 14, as noted by the bright white outline in the pericellular region (Fig. 2b). Thus, we determined the cultured cells on day 4 as proliferating, and those on day 11 as matrix-forming chondrocytes in the subsequent experiments described below. Effects of Intermittent Tensile Stresses on the Proliferating and Matrix-Forming Chondrocytes At the proliferating stage, intermittent tensile stresses with high and middle frequencies increased significantly (p < 0.05) the incorporation of [ 3 H]thymidine into the cultured chondrocytes as compared with the controls (Fig. 3). However, intermittent tensile stress with low frequency could not significantly affect the incorporation of [ 3 H]thymidine in the cultured cells. The incorporation rate of [ 3 H]thymidine relative to the control groups upregulated with increase of the loading frequency. Intermittent tensile stress with high frequency enhanced DNA synthesis by about 1.5-fold relative to that in control. At the matrix-forming stage, the incorporation rate of [ 35 S]sulfate into the cultured chondrocytes with three different frequencies was evaluated. Prior to the examination of the proteoglycan synthesis in chondrocytes, we confirmed that the DNA contents in the cells at this stage were not changed substantially by intermittent tensile stresses irrespective of the loading frequency (Fig. 4a). The incorporation rate of [ 35 S]sulfate relative to the controls increased due to

4 796 UEKI et al. FIGURE 2. Cell appearances in cultured growth plate chondrocytes. Photomicrographs of chondrocytes in monolayer culture on day 4 (a) and on day 11 (b). The yellow arrow indicated appearance of cell divisions on day 4 (a), and the red arrow indicated appearance of abundant cartilaginous matrices on day 11 (b). Bars = 30 lm. especially that with high and middle frequencies (p < 0.01 and p < 0.05, respectively). DISCUSSION FIGURE 3. Effects of intermittent tensile stress on DNA synthesis of chondrocytes at the proliferating stage (day 4). The values of DNA synthesis were defined as the total amount of the [ 3 H]thymidine incorporation into chondrocytes. White bars are the values of the controls. Color bars are the values in the chondrocytes applied intermittent tensile stresses with high, middle, and low frequencies. Error bars are standard deviations (for each group n = 10). *p < intermittent tensile stress application. Especially tensile stresses with high and middle frequencies significantly (p < 0.01 and p < 0.05, respectively) enhanced the incorporation of [ 35 S]sulfate into the cultured cells at the matrix-forming stage as compared to the control cells. At high frequency, the proteoglycan synthesis was about 1.5 times of that in the control (Fig. 4b), and the incorporation rate of [ 35 S]sulfate relative to the controls increased with the loading frequency of tensile stress. The incorporation rate of [2,3-3 H]proline was also increased by intermittent tensile stresses (Fig. 4c). The incorporation rate enhanced with increase of the loading frequency, In the present study, the process of the chondrocyte differentiation was represented in in vitro cell culture system. In the BioFlex culture system, the chondrocytes underwent to the proliferating and matrixforming stages with active cell division and matrix synthesis on day 4 6 and day 9 12, respectively. Since the endochondral ossification is regulated by numerous factors such as growth factors and related proteins 1,8,11,29,37,38,58 components of the extracellular matrix 6,14,28,58 proteases, 26,51 and mechanical stimuli, 34 there is a limitation for mimicking the whole process by in vitro culture system. The 2D culture system used in the present study was suitable to examine the effect of mechanical loadings with various loading frequencies on the proliferation of chondrocytes and extracellular matrix formation. A series of the experiments on the rib growth plate chondrocytes was, in the present study, carried out for three frequencies of 2, 30, and 150 cycles/min (0.03, 0.5, and 2.5 Hz, respectively). The frequency of inspiratory motion of the rib ranges from 0.5 to 2.0 Hz at rest or during walking. 12 According to a previous study 57 examining the strain distributions in a single well of a Flexercell plate using a finite element method, the scattering of elongation rate in the siliconeelastomer was relatively small under the condition used in the present study. Therefore, the range examined in the present study can be considered sufficient to cover in vivo habitual and physiological loading although there is a large variation among the species. Our results showed that intermittent tensile stresses with various frequencies enhanced the DNA synthesis in the proliferating, and the collagen and proteoglycan

5 Effect of Loading on Growth Plate Chondrocytes 797 FIGURE 4. Effects of intermittent tensile stress on DNA contents (a), proteoglycan (b), and collagen syntheses (c) of chondrocytes at the matrix-forming stage (day 11). DNA contents were the total amount of DNA. The values of proteoglycan and collagen syntheses were defined as the amounts of the [ 35 S]sulfate and [2,3-3 H]proline incorporation into chondrocytes. White bars are the values of the controls. Color bars are the values in the chondrocytes applied intermittent tensile stresses with high, middle and low frequencies. Error bars are standard deviations (for each group n = 10). **p < 0.01, *p < syntheses in the matrix-forming chondrocytes. These metabolic activities were enhanced by the intermittent tensile stress in a frequency-dependent manner, and this modulation was induced even with relatively lower magnitude than that used in our previous study. 52 Although the mechanism in signal transduction from mechanical stimuli to chemical signaling in cells has not been fully understood, several mechanisms such as phosphorylation of platelet-derived growth factor (PDGF) receptor, 33 integrin-mediated adhesion 7,59 stretch-activated cation channels, 11,17,24 and G proteins 31 have been suggested as the mechanosensors of cells. It may be possible to explain the detection of cyclic tensile stresses in chondrocytes by the changes in an intracellular calcium ions ([Ca 2+ ]i) level caused by the fluid flow. 64 The fluid flow of the culture medium can be generated during the application of intermittent tensile stress to the cells using the BioFlex system due to the expansion of the flexible plate, and the cells are subjected to fluid flow pressure. 3 Therefore, the fluid flow rate can be affected by the frequency of the stress. The [Ca 2+ ]i level in cultured chondrocytes was enhanced when the cells were submitted to the fluid flow with a high rate. 13,21 Our previous study 52 revealed that the mobilization of Ca 2+ had a crucial role in the upregulation of PTHrP expression by the mechanical stress. From these results, it was speculated that the fluid flow might be a factor in modulation of the DNA and extracellular matrix syntheses in chondrocytes by intermittent tensile stress. On the other hand, the sustained mechanical stress induced the reduction of DNA and proteoglycan syntheses in proliferating chondrocytes and matrixforming chondrocytes, respectively. 18,20,40,43,53,60 When the sustained stress was applied to chondrocytes, the volume of chondrocytes was decreased in proportion to an increase in extracellular mechanical stress. 19,21 In response to higher-osmotic pressure, the most cells attempt to adjust their volume to the physiological status by modulating the uptake and/or expulsion of various solutes. 39 Chondrocytes respond to volume

6 798 UEKI et al. decrease by the activation of membrane transporters such as osmolyte channels and the Na + K + 2Cl - cotransporter. 22,23 The [Ca 2+ ]i is also reported to play a role in regulatory volume control, 36 and has been shown to increase in certain cells in response to higher osmotic pressure. 9,10,30,35 Furthermore, under sustained stress, a lack of oxide reperfusion may occur within the cultured cells. Pufe et al. 42 reported that after mechanical continuous overload chondrocytes were strongly immunopositive for hypoxia-induced transcription factor-1 (HIF-1). Consequently, mechanical sustained overload induces HIF-1, and the subsequently generated VEGF activates the chondrocytes autocrinally for producing MMP-1, -3, and -13. Tissue inhibitors of metalloproteinase (TIMP-1 and -2) are then reduced by mechanical overload. 25 These findings indicate that sustained loading probably facilitates hypoxia in chondrocytes to mediate the destructive processes associated with OA as an autocrine factor. From these considerations, chondrocyte likely senses and responds strictly to the cell deformation and alteration of extracellular circumstances. Taken together with the results in the present study, the application of intermittent mechanical loading by exercise or some therapeutic appliances such as ultrasound therapy would be essential for the cartilage development, resulting in longitudinal bone formation or bone repair. In conclusion, the metabolism of growth plate chondrocytes was significantly enhanced by the intermittent tensile stress even with relatively lower magnitude, suggesting that the frequency of the mechanical loading may be a crucial factor for modulating the maturation of growth plate chondrocytes as well as the magnitude of the stress. ACKNOWLEDGMENTS This study was supported by a Grant-in-aid (# , # , # , # , and # ) for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan. This work was also carried out by the courtesy of the Research Center for Molecular Medicine, Hiroshima University. REFERENCES 1 Bitgood, M. J., and A. P. McMahon. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell cell interaction in the mouse embryo. Dev. Biol. 172: , Bourrin, S., C. Genty, S. Palle, C. Gharib, and C. Alexandre. Adverse effects of strenuous exercise: a densitometric and histomorphometric study in the rat. J. Appl. Physiol. 76: , Brown, T. 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