Ultrasound in Med. & Biol., Vol. 41, No. 6, pp. 1690–1700, 2015 Copyright Ó 2015 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2015.01.014
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Original Contribution LOW-INTENSITY PULSED ULTRASOUND AFFECTS CHONDROCYTE EXTRACELLULAR MATRIX PRODUCTION VIA AN INTEGRIN-MEDIATED P38 MAPK SIGNALING PATHWAY PENG XIA, SHASHA REN, QIANG LIN, KAI CHENG, SHIHAO SHEN, MINGXIA GAO, and XUEPING LI Department of Rehabilitation Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing, China (Received 26 August 2014; revised 26 December 2014; in final form 17 January 2015)
Abstract—Although low-intensity pulsed ultrasound (LIPUS) regulates p38 mitogen-activated protein kinase (MAPK) and promotes cartilage repair in osteoarthritis, the role of integrin-mediated p38 MAPK in the effect of LIPUS on extracellular matrix (ECM) production of normal and OA chondrocytes remains unknown. The aim of this study was to investigate whether LIPUS affects ECM production in normal and OA rabbit chondrocytes through an integrin–p38 signaling pathway. A rabbit model of OA was established by anterior cruciate ligament transection, and chondrocytes were isolated from normal or OA cartilage and cultured in vitro. Chondrocytes were treated with LIPUS and then pre-incubated with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. Expression of type II collagen, MMP-13, integrin b1, p38 and phosphorylated p38 was assessed by Western blot analysis. We found that type II collagen and integrin b1 were upregulated (p , 0.05), whereas MMP-13 was downregulated (p , 0.05) in normal and OA chondrocytes. Furthermore, phosphorylated p38 was upregulated (p , 0.05) in normal chondrocytes, but downregulated (p , 0.05) in OA chondrocytes after LIPUS stimulation. Pre-incubation of chondrocytes with the integrin inhibitor disrupted the effects of LIPUS on normal and OA chondrocytes. Pre-incubation of chrondocytes with the p38 inhibitor reduced the effects of LIPUS on normal chondrocytes, but had no impact on OA chondrocytes. Our findings suggest that the integrin–p38 MAPK signaling pathway plays an important role in LIPUS-mediated ECM production in chondrocytes. (E-mail: [email protected]) Ó 2015 World Federation for Ultrasound in Medicine & Biology. Key Words: Low-intensity pulsed ultrasound, Osteoarthritis, Chondrocytes, Integrin, p38 MAPK.
hydrolysis of cartilage ECM by proteinase is a key step in the pathogenesis of OA (Takaishi et al. 2008). Matrix metalloproteinases (MMPs) are the most important proteolytic enzymes in the degradation of ECM, and MMP-13 is the most effective enzyme for degrading type II collagen (Blain 2007). In pathologic conditions, chondrocytes produce MMP-13 to degrade type II collagen, resulting in damaged or defective cartilage and, ultimately, OA (Bramono et al. 2004; Mitchell et al. 1996). Mechanical stress interactions within the ECM play a dominant role in cell regulation. Cells are able to sense forces to which they are exposed and generate forces on the substrate, which enables a rearrangement of matrix proteins (Guilak et al. 2009). Integrins are a family of cell surface stress receptors mediating cellular interactions with the ECM as well as cell–cell interactions (Hynes 1992; 2002). Integrin b1, the main integrin found on the chondrocyte membrane, regulates the proliferation and differentiation of chondrocytes (Kim et al. 2011). Stimulation of integrin signals by mechanical stress may transduce
INTRODUCTION Osteoarthritis (OA), caused by articular cartilage degeneration (Lohmander et al. 2003), manifests as joint pain, stiffness, dysfunction and different degrees of deformity in the late stage of the disease (Pearle et al. 2005). The degradation of extracellular matrix (ECM) accelerates degeneration in OA cartilage (Sandell and Aigner 2001). The pathologic changes are specifically associated with the chondrocytes. Chondrocytes play a key role in OA and are very important to maintain the anabolic–catabolic balance of matrix maintenance and tissue function (Aigner et al. 2007). Cartilage ECM is composed mainly of collagen and aggrecan. Type II collagen is the most abundant collagen in cartilage and is a typical marker for chondrocytes. The
Address correspondence to: Xueping Li, Department of Rehabilitation Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China. E-mail: [email protected] 1690
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signals across the cell membrane, leading to activation of downstream pathways, such as the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Kim et al. 2011; MacKenna et al. 2000), which play a key role in regulating the production of MMPs in chondrocytes (Chun 2004). The MAPK signaling pathway consists of extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and cJun N-terminal kinase (JNK), all of which are constitutively expressed in most cell types, including chondrocytes (Karsdal et al. 2008). The p38 MAPK signaling pathway mediates chondrocyte–perichondral communication (Stanton and Beier 2007), stabilizes chondrogenic transcription factor SOX 9 mRNA (Tew and Hardingham 2006) and is necessary for MMP expression and activity (Sondergaard et al. 2010). Therefore, the p38 MAPK signaling pathway plays a major role in chondrocyte differentiation (Zhen et al. 2001). Low-intensity pulsed ultrasound (LIPUS) is a form of mechanical energy that is transmitted through and into tissues as pulsed acoustic pressure waves and has been clinically used as a non-invasive supplemental therapy to promote fracture and wound healing (Gebauer et al. 2005). LIPUS has been reported to stimulate chondrocyte proliferation and matrix production in vitro, suggesting an overall anabolic effect on cartilage formation. Therefore, LIPUS can promote the repair of cartilage after damage and can delay cartilage degeneration, leading to OA in the articular cartilage (Huang et al. 1999; Korstjens et al. 2008). Some studies have reported that LIPUS can activate the p38 MAPK signaling pathway (Ikeda et al. 2006; Zhou et al. 2008). Our previous studies also suggested that LIPUS can promote cartilage repair of OA chondrocytes through downregulation of MMP-13 and p38 MAPK (Li et al. 2011) and that the protective effect of LIPUS on chondrocytes may be mediated by the integrin–PI3K/Akt pathway (Cheng et al. 2014). There has, however, been no study on the correlation of integrins and p38 MAPK with the effect of LIPUS on ECM production of normal and OA chondrocytes. In this study, our aim was to investigate whether LIPUS affects ECM production in normal and OA chondrocytes through the integrin–p38 MAPK pathway. METHODS Reagents Phosphate-buffered saline (PBS), 0.25% trypsin, 0.2% type II collagenase, Dulbecco’s modified Eagle medium-high glucose (DMEM-HG), fetal bovine serum (FBS), an immunohistochemical kit, a total protein extraction kit, toluidine blue and other cell isolation- and culture medium-related supplies were purchased from KeyGEN (Nanjing, Jiangsu, China). Mouse anti-rabbit monoclonal
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antibodies against type II collagen, MMP-13, integrin b1, p38, phosphorylated p38 (p-p38) or b-actin were purchased from Acris (Herford, NRW, Germany), and goat antimouse (Fab)2 secondary antibody was purchased from Santa Cruz (Dallas, TX, USA). The integrin inhibitor GRGDSP (glycine–arginine–glycine–aspartic acid– serine–proline) was purchased from AnaSpec (Fremont, CA, USA), and the p38 inhibitor SB203580 was purchased from Selleck (Houston, TX, USA). Animal experiments Twenty-two 2.5- to 3.0-kg, 2-mo-old healthy male New Zealand white rabbits were purchased from the Qinglongshan Experimental Animal Center of Jiangsu Province, China. All experimental protocols were approved by the ethics committee of Nanjing Medical University Nanjing Hospital. A rabbit OA model (right knee, n 5 11) was generated by anterior cruciate ligament transection. Normal rabbits served as the control group (n 5 11). Both normal and OA rabbits (4 wk after transection) were euthanized and subjected to histopathologic observation of femoral condylar cartilage. Chondrocytes from knee joints of normal and OA rabbits were isolated from the cartilage and cultured in vitro, after cartilage changes were evaluated using Mankin scores (Mankin et al. 1971; van der Sluijs et al. 1992). Surgical procedures Anterior cruciate ligament transection (ACLT) of the OA rabbit model was performed as described previously (Boulocher et al. 2008; Jean et al. 2008). Briefly, rabbits were intravenously anesthetized with 3% sodium pentobarbital (1 mL/kg). The knee joint skin was disinfected with iodine after the fur was shaved, and a parapatellar skin incision was made on the medial side of the joint. The ACLT was transected using eye scissors, and a positive anterior drawer test was used to ensure complete transection of the ligament. The patella was relocated and the wound closed with a vicryl 4/O braided absorbable sutures. Penicillin and fentanyl were given to prevent bacterial infection and pain, respectively, after the incised skin was closed. The anterior cruciate ligaments of animals in the normal control (NC) group were not transected. Histopathology The femoral condylar articular cartilage collected from knee joints was fixed in neutral formalin, decalcified in ethylenediaminetetraacetic acid (EDTA) for 3 wk, embedded in paraffin and sectioned into 4-mm-thick sections using a microtome. Pathologic changes in knee joint cartilage samples, including surface irregularities and formation of cracks, were examined under a microscope.
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Fibrosis, matrix distribution, cartilage loss and chondrocyte colonization were evaluated in a double-blind fashion by two independent experts using the Mankin score system (Table 1). Chondrocyte isolation and culture Articular cartilage slices obtained from the femoral condyle of knee joints from OA and normal (control) rabbits were cut into smaller pieces, washed with PBS and digested with 0.25% trypsin for 30 min. The resultant chondrocytes were washed again with PBS; incubated for approximately 10 h in DMEM supplemented with 0.2% type II collagenase, 10% heat-inactivated FBS and antibiotics; washed with culture medium; and collected by centrifugation at 1,000 rpm for 10 min. The isolated chondrocytes were cultured in tissue culture dishes with complete DMEM in 5% CO2/95% air at 37 C. The morphology of the chondrocytes was observed under a microscope. Immunocytochemistry Chondrocytes were fixed with 4% paraformaldehyde for 30 min, washed with PBS three times, incubated with 3% H2O2–methanol solution at room temperature for 10 min, washed with PBS three times again and blocked and incubated with goat serum (50–100 mL) at room temperature for 20 min. The cells were then incubated with type II collagen antibodies (50–100 mL, 1:200 dilution) at 37 C for 2 h, washed with PBS three times before addition of 50 mL of an intensifier and incubated at room temperature for 30 min. Then the cells Table 1. Mankin scoring scale* Subgroup 1: Fibrillation 1. Even surface 2. Uneven surface 3. Fibrillated and fissured within superficial zone only 4. Fissures and erosions extending below the surface zone, without extending beyond the radial zone 5. Fissures and erosions extending into the deeper zone Subgroup 2: Matrix distribution 1. Normal staining 2. Moderate loss in staining 3. Severe loss in staining 4. No staining Subgroup 3: Chondrocyte loss 1. Loss extending into superficial zone 2. Loss extending into midzone 3. Loss extending into radial zone Subgroup 4: Chondrocyte cloning 1. No clusters 2. Chondrocyte clusters in superficial zone 3. Chondrocyte clusters in superficial to midzone (fewer than four cells) 4. Chondrocyte clusters of more than four cells located in superficial to midzone, or chondrocyte clusters in deeper zone * Grading was performed separately for the medial femoral condyle, lateral femoral condyle, medial tibial plateau and lateral tibial plateau. The minimum total score was 4, and the maximum total score was 16.
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were washed with PBS three times, incubated with horseradish peroxidase-conjugated anti-mouse-(Fab)2 antibodies (50 mL) at 37 C for 30 min, washed with PBS three times and then subjected to color development using diaminobenzidine and hematoxylin staining. Three culture dishes were read per condition, and the three areas with most positive staining cells were selected. Type II collagen expression was observed under a microscope, and images were acquired. The integrated option density (IOD) was obtained with Image-Pro Plus software (Cheng et al. 2014; Rizzardi et al. 2012). Low-intensity pulsed ultrasound stimulation Normal and OA chondrocytes were subdivided into the control group and the treated group, which was subdivided into four subgroups (1–4) treated with LIPUS (HT2009-1, Ito, Tokyo, Japan) at intensities of 20, 30, 40 and 50 mW/cm2, respectively. A layer of coupling agent (,1 mm thick) was applied between the LIPUS probe and culture dish, and the probe was placed below the tissue culture dish (Fig. 1). Chondrocytes were treated once daily for 6 d at an on–off ratio of 20%, irradiation of 3 MHz, for 20 min in a 37 C incubator under a humidified atmosphere with 5% CO2 in air (Cheng et al. 2014; Korstjens et al. 2008). Application of integrin and p38 MAPK inhibitors To investigate the role of integrin and p38 in the effects of LIPUS on normal and OA chondrocytes, the chondrocytes were pre-treated with a 10, 30 or 50 mM concentration of the integrin inhibitor GRGDSP or a 1, 5 or 10 mM concentration of the p38 inhibitor SB203580, or mock treated with dimethyl sulfoxide for 4 h before the appropriate intensity of LIPUS stimulation. The optimal concentrations of inhibitors were determined using Western blot analysis. Then chondrocytes were incubated with the optimal concentration of inhibitors, 50 mM for GRGDSP or 10 mM for SB203580, and exposed or not exposed to LIPUS for 6 d.
Fig. 1. LIPUS irradiation of chondrocytes in vitro. A layer of coupling agent (,1 mm thick) was applied between the LIPUS probe and culture dish, and the probe was placed below the tissue culture dish. Normal and osteoarthritis chondrocytes were subdivided a control group that did not receive LIPUS stimulation and a treated group that was subdivided into four subgroups that received LIPUS at intensities of 20, 30, 40 and 50 mW/cm2, respectively. Chondrocytes were treated once a day for 6 d at an on–off ratio of 20%, irradiation of 3 MHz, for 20 min. LIPUS 5 low-intensity pulsed ultrasound.
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Fig. 2. Comparison of femoral condylar cartilage of normal rabbits and rabbits with OA. (a, b) Identification of normal and OA chondrocytes (3400): (a) Normal femoral condylar cartilage has a smooth articular surface with a bright luster with no cracks. (b) The soft articular surface of OA femoral condylar cartilage has crannies (arrows), but lacks the original luster. (c, d) Hematoxylin–eosin (3100): (c) Smooth and evenly stained normal articular cartilage surface with chondrocytes arranged in normal order. (d) Apparent articular cartilage fissures extending deep from the surface with significant loss of staining (arrows) and disorganized and fewer chondrocytes in OA femoral condylar cartilage. (e, f) Immunohistochemical staining of type II collagen: (e) Normal chondrocytes with abundant cytoplasm and round nuclei (arrows). (f) OA chondrocytes with hypertrophic nuclei mixed with some dendrite-like cells (arrows). (g) Type II collagen expression in normal and OA chondrocytes. (h) Mankin scores in normal and OA cartilage. n 5 11. OA 5 osteoarthritis. *p , 0.05.
Western blot analysis After LIPUS stimulation for 6 d, chondrocytes were collected, and expression of type II collagen, MMP-13, integrin b1, p38 and p-p38 was determined by Western blot analysis. Proteins were extracted from the chondrocytes using a total protein extraction kit, and 25 mg/mL was loaded onto sodium dodecyl sulfate–polyacrylamide gel for electrophoresis and electroblotted onto nitrocellulose membranes. The membranes were blocked with skim milk for 2 h and incubated with primary antibodies: anti-type II collagen (1:500 dilution), anti-MMP-13 (1:500 dilution), anti-integrin b1 (1:500 dilution), anti-p38 (1:1000 dilution), anti-p-p38 (1:1,000 dilution) and anti-b-actin (1:500 dilution) at 4 C overnight. Then the membranes were subsequently washed three times with Tween 20 in PBS and incubated with peroxidase-conjugated goat antimouse IgG secondary antibody (1:5,000 dilution) at 37 C for 2 h, followed by another three washes as described above. The membranes were developed with the ECL kit.
Statistical analysis All data were expressed as the mean 6 standard deviation (SD) and analyzed using SPSS 18.0 software (IBM, Armonk, NY, USA). Differences between groups were analyzed by single-factor analysis of variance (ANOVA). Modified Mankin scores were analyzed using the Wilcoxon signed rank test. A p-value , 0.05 was considered to indicate statistical significance. RESULTS General and histopathologic observations Four weeks after ACLT, the articular surface of normal femoral condylar cartilage was smooth, had a bright luster and did not any cracks (Fig. 2a), whereas the articular surface of OA femoral condylar cartilage had become soft, lost its original luster and bore crannies (Fig. 2b). Femoral condylar cartilage from normal and OA rabbits was stained with hematoxylin and eosin and examined under a
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Fig. 3. Expression of type II collagen and MMP-13 in normal chondrocytes and chondrocytes from rabbits with OA after LIPUS stimulation. (a, b) Western blot analysis of type II collagen and MMP-13 in normal and OA chondrocytes with b-actin as a loading control. (c) Quantitative analysis of type II collagen expression in normal chondrocytes. (d) Quantitative analysis of type II collagen expression in OA chondrocytes. (e) Quantitative analysis of MMP-13 expression in normal chondrocytes. (f) Quantitative analysis of MMP-13 expression in OA chondrocytes. The ratio between the target and b-actin was used to normalize the data for comparison. n 5 4. MMP-13 5 matrix metalloproteinase 13; OA 5 osteoarthritis; LIPUS 5 low-intensity pulsed ultrasound. *p , 0.05.
microscope. The surface of articular cartilage from normal rabbits was smooth and evenly stained with the chondrocytes arranged in normal order (Fig. 2c); however, in articular cartilage from rabbits with OA, there were fissures extending deep from the surface with significant loss of staining (Fig. 2d). The chondrocytes from rabbits with OAwere also disorganized and reduced in number (Fig. 2d). Modified Mankin scores Compared with those of the normal control group, the Mankin scores of the OA rabbit model group were
overall significantly higher (p , 0.05). Mankin scores for subgroups 1–4 of the OA rabbit model group were also significantly higher (p , 0.05) than those of the normal control group (Fig. 2h). Identification of normal and OA chondrocytes The normal chondrocytes had abundant cytoplasm and a round nucleus, which grew to full confluence within 4 to 7 d (arrows in Fig. 2e). The OA chondrocytes had hypertrophic nuclei, which were mixed with some dendrite-like cells and grew to full confluence within 9
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Fig. 4. Expression of integrin b1, p38 and p-p38 in normal and osteoarthritis chondrocytes after LIPUS stimulation. (a) Western blot analysis of integrin b1, p38 and p-p38 with b-actin as a loading control. (b) Quantitative analysis of integrin b1. (c) Ratio of p-p38 to p38. The ratio between the target and b-actin was used to normalize the data for comparison. p-p38 5 phosphorylated p38; LIPUS 5 low-intensity pulsed ultrasound; NC 5 normal control group; N 1 L 5 normal 1 LIPUS group; OA 5 osteoarthritis group; O 1 L 5 osteoarthritis 1 LIPUS group. n 5 4. *p , 0.05.
to 12 d (arrows in Fig. 2f). Immunohistochemical staining of type II collagen in normal chondrocytes (Fig. 2e) was much deeper than that in OA chondrocytes (Fig. 2f). The mean density of type II collagen was significantly higher in normal chondrocytes than in OA chondrocytes (p , 0.05) (Fig. 2g). Expression of type II collagen and MMP-13 in normal and OA chondrocytes after 20 min of LIPUS stimulation at four different intensities To determine an appropriate LIPUS intensity for normal and OA rabbit chondrocytes, we examined the expression of type II collagen and MMP-13 in chondrocytes after LIPUS stimulation at four different intensities (20, 30, 40 and 50 mW/cm2). Western blot analysis revealed that in normal and OA rabbit chondrocytes, type II collagen expression was significantly increased (p , 0.05) and MMP-13 expression was significantly decreased (p , 0.05) after LIPUS stimulation at all four intensities. In normal chondrocytes, type II collagen expression was significantly increased (p , 0.05) in the 40 mW/cm2 intensity group compared with all other groups, whereas MMP-13 expression was significantly decreased (p , 0.05) in the 40 mW/cm2 intensity group compared with the
20 mW/cm2 intensity (p , 0.05), 50 mW/cm2 intensity (p , 0.05) and control (p , 0.05) groups. However, no significant difference (p . 0.05) was observed compared with the 30 mW/cm2 intensity group. In OA chondrocytes, type II collagen expression was significantly increased (p , 0.05) in the 30 mW/cm2 intensity group compared with the 40 mW/cm2 intensity group (p , 0.05), 50 mW/cm2 intensity group (p , 0.05) and control group (p , 0.05). However, no significant difference (p . 0.05) was observed compared with the 20 mW/cm2 intensity group. MMP-13 expression was also significantly decreased (p , 0.05) in the 30 mW/cm2 intensity group compared with all other groups (Fig. 3). Integrin b1 expression and p38 phosphorylation in normal and OA rabbit chondrocytes after 20 min of LIPUS stimulation We examined integrin b1 expression and p38 phosphorylation in normal and OA rabbit chondrocytes after LIPUS stimulation. LIPUS at 40 and 30 mW/cm2 was used to stimulate normal and OA rabbit chondrocytes, respectively. Integrin b1 expression significantly increased after LIPUS stimulation (p , 0.05) in both normal and OA chondrocytes, whereas expression in
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Fig. 5. Expression of integrin b1 and p-p38 in normal chondrocytes and OA chondrocytes after treatment with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. (a–d) Western blot analyses of integrin b1 and p38 with b-actin as a loading control in normal and OA chondrocytes. (e) Quantitative analysis of integrin b1 after treatment with 10, 30 or 50 mM GRGDSP in normal and OA chondrocytes. (f) Quantitative analysis of p-p38 after treatment with 1, 5 or 10 mM SB203580 in normal and OA chondrocytes. The ratio between the target and b-actin was used to normalize the data for comparison. n 5 3. OA 5 osteoarthritis. *p , 0.05.
OA chondrocytes was significantly higher than in normal chondrocytes (p , 0.05). The level of p38 phosphorylation increased significantly after LIPUS stimulation in normal chondrocytes (p , 0.05), but significantly decreased after LIPUS stimulation in OA chondrocytes (p , 0.05). The level of p38 phosphorylation was significantly higher in OA chondrocytes than normal chondrocytes (p , 0.05) (Fig. 4). Expression of integrin b1 and p-p38 in normal and OA rabbit chondrocytes after treatment with integrin and p38 inhibitors The expression of integrin b1 and p-p38 in normal and OA chondrocytes was examined after treatment with inhibitors. Integrin b1 expression in normal and OA rabbit chondrocytes significantly decreased (p , 0.05) after treatment with 10, 30 or 50 mM GRGDSP, with the lowest expression in chondrocytes at 50 mM GRGDSP (p , 0.05). Expression of p-p38
in normal and OA rabbit chondrocytes significantly decreased (p , 0.05) after treatment with 1, 5 or 10 mM SB203580, with the lowest expression at 10 mM (p , 0.05) (Fig. 5). Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes after integrin inhibitor treatment Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes was examined after treatment with the integrin inhibitor GRGDSP at 50 mM. After GRGDSP treatment, type II collagen expression significantly increased (p , 0.05), and MMP-13 expression significantly decreased (p , 0.05) in normal and OA chondrocytes. The phosphorylation level of p38 significantly increased (p , 0.05) in normal chondrocytes, but significantly decreased (p , 0.05) in OA chondrocytes. Expression of type II collagen expression significantly increased (p , 0.05) and that
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Fig. 6. Expression of type II collagen, MMP-13, p38 and p-p38 in NC with or without LIPUS stimulation after treatment with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. (a) Western blot analysis of type II collagen, MMP-13, p38 and p-p38 with b-actin as a loading control. (b) Quantitative analysis of type II collagen. (c) Quantitative analysis of MMP-13. (d) Ratio of p-p38 to p38. The ratio between the target and b-actin was used to normalize the data for comparison. MMP-13 5 matrix metalloproteinase 13; NC 5 normal chondrocytes; p-p38 5 phosphorylated p38; LIPUS 5 low-intensity pulsed ultrasound. n 5 4. *p , 0.05.
of MMP-13 significantly decreased (p , 0.05) after LIPUS stimulation in normal and OA chondrocytes. When GRGDSP was used, there was no significant change in the expression of type II collagen and MMP-13 in normal and OA chondrocytes (p . 0.05) after LIPUS stimulation. The phosphorylation level of p38 did not significantly change (p . 0.05) in normal chondrocytes and OA chondrocytes after LIPUS stimulation (Figs. 6 and 7). Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes after p38 MAPK inhibitor treatment Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes was examined after treatment with the p38 MAPK inhibitor. Type II collagen expression significantly increased in normal and OA chondrocytes (p , 0.05). Type II collagen expression did not change significantly in normal chondrocytes (p . 0.05), but significantly decreased in OA chondrocytes (p , 0.05) after LIPUS stimulation following SB203580 treatment. MMP-13 expression significantly decreased (p , 0.05) in normal and OA chondrocytes after SB203580 treatment. On exposure to LIPUS, MMP-13 expression significantly decreased in OA chondrocytes (p , 0.05), but LIPUS
had no significant effect on normal chondrocytes treated with SB203580 (p . 0.05). The phosphorylation level of p38 significantly decreased (p , 0.05) in normal and OA chondrocytes treated with SB203580. LIPUS stimulation significantly decreased the phosphorylation level of p38 in OA chondrocytes (p , 0.05), but had no significant effect in normal chondrocytes (p . 0.05) after LIPUS stimulation following SB203580 treatment (Figs. 6 and 7). DISCUSSION In this study, we sought to determine whether LIPUS affects ECM production through an integrin–p38 MAPK signaling pathway in normal and OA chondrocytes. We found that LIPUS significantly increases ECM production and decreases MMP-13 expression through an integrin–p38 MAPK signaling pathway. We found that after LIPUS stimulation, type II collagen expression significantly increased (p , 0.05), but MMP-13 expression significantly decreased (p , 0.05). We suggested that the increased levels of MMP-13 degraded type II collagen in the ECM of chondrocytes to cause OA and that LIPUS prevented the type II collagen from being degraded by MMP-13 in both normal and OA chondrocytes.
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Fig. 7. Expression of type II collagen, MMP-13, p38 and p-p38 in OA chondrocytes with or without LIPUS stimulation after treatment with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. (a) Western blot analysis of type II collagen, MMP-13, p38 and p-p38 with b-actin as a loading control. (b) Quantitative analysis of type II collagen. (c) Quantitative analysis of MMP-13. (d) Ratio of p-p38 to p38. The ratio between the target and b-actin was used to normalize the data for comparison. MMP-13 5 matrix metalloproteinase 13; p-p38 5 phosphorylated p38; OA 5 osteoarthritis; LIPUS 5 low-intensity pulsed ultrasound. n 5 4. *p , 0.05.
We further observed that inhibition of integrin by GRGDSP suppressed MMP-13 expression, which may prevent the degradation of type II collagen and decrease the activity of p38 in both normal and OA chondrocytes. Therefore, we speculate that integrin may have a negative effect on chondrocytes without LIPUS stimulation. We also found that integrin b1 expression in both normal and OA chondrocytes significantly increased (p , 0.05) after LIPUS stimulation. In fact, integrin plays an important role in mechanical signal transduction in chondrocytes by participating in the proliferation, differentiation and migration of chondrocytes (Huang et al. 2004; Humphries 2000; Loeser et al. 2000; Luo et al. 2013; Stamenovic and Wang 2000). Fibronectin fragment (Fn-f), on one hand, stimulates MMP production to cause chondrocyte degeneration; on the other hand, integrin binds to Fn-f (Guo et al. 2009; Yasuda 2010). Therefore, we deduced that the increased integrin expression caused by LIPUS in both normal and OA chondrocytes may be ascribed to the binding to Fn-f to eliminate the negative effect of Fn-f on chondrocyte degradation. In this case, integrins might have a beneficial effect on chondrocytes. To sum up, integrins played different roles under different situations. We found that after LIPUS stimulation, phosphorylation levels of p38 increased significantly (p , 0.05) in normal chondrocytes, but decreased significantly
(p , 0.05) in OA chondrocytes. This indicates that when integrin was activated by LIPUS, the downstream p38 MAPK of the integrin–p38 MAPK signaling pathway was activated in normal chondrocytes, but inhibited in OA chondrocytes. In fact, p38 activity is much higher in OA chondrocytes, and inhibition of p38 can curb apoptosis and hypertrophic terminal differentiation of cultured OA chondrocytes (Reilly et al. 2005; Takebe et al. 2011), whereas p38 can be an effector kinase of mechanotransduction in various cell types and is essential to normal articular cartilage (Sondergaard et al. 2010). On the other hand, in adult articular tissues, p38 is known to play an important role in the inflammatory process (Joos et al. 2009), and some evidence suggests that p38 is a major signaling molecule in the induction of MMP-13 and the activity of p38 can have negative effects on cartilage matrix maintenance (Julovi et al. 2011; Mengshol et al. 2000; Pei et al. 2006). In addition, moderate mechanical stress such as knee loading reduces MMP-13 activity through the p38 MAPK signaling pathway in normal articular cartilage (Hamamura et al. 2013). Therefore, we speculated that LIPUS inhibited phosphorylation of p38 in OA chondrocytes, but facilitated phosphorylation of p38 in normal chondrocytes to promote adaptation to a moderate mechanical stress. We found that after application of the integrin inhibitor GRGDSP, levels of type II collagen, MMP-13 and p38 phosphorylation did not significantly change
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in both normal and OA chondrocytes after LIPUS stimulation, suggesting again that p38 MAPK was downstream of integrin and that inhibition of integrin expression disrupted the regulation of p38 and ECM production by LIPUS. We also found that p38 inhibition affected both normal and OA chondrocytes and the effects of LIPUS on normal chondrocytes. Expression of type II collagen significantly increased and expression of MMP-13 significantly decreased in normal and OA chondrocytes treated with the p38 inhibitor SB203580, indicating that p38 may be a negative factor in both normal and OA chondrocytes without LIPUS stimulation. Our results also indicated that expression of type II collagen and MMP-13 did not significantly change in normal chondrocytes after LIPUS stimulation following SB203580 treatment, but in OA chondrocytes, LIPUS stimulation significantly increased type II collagen expression and decreased MMP-13 expression. Thus, we deduced that p38 was mainly a protective factor in normal chondrocytes, but mainly an inflammatory and injurious factor in OA chondrocytes under LIPUS stimulation. We found that inhibition of p38 disrupted the protective effects of LIPUS on ECM production in normal chondrocytes. Because LIPUS had the same inhibitory effect on p38 as SB203580 in OA chondrocytes, the inhibition of p38 by SB203580 had no impact on the effects of LIPUS on ECM production in OA chondrocytes. These results indicate that p38 of normal and OA chondrocytes has different roles under different conditions. For the protective effect of LIPUS, p38 has a more important role in normal chondrocytes than in OA chondrocytes under LIPUS simulation. Of note, the protective effect of LIPUS on chondrocytes is exerted not only through the p38 MAPK pathway, but also through other pathways such as PI3K/Akt pathway, as described in our previous study (Cheng et al. 2014). Therefore, we speculated that the p38 MAPK pathway and PI3K/Akt pathway might have a synergistic effect on ECM production of chondrocytes under LIPUS stimulation, which should be investigated in a further study. CONCLUSIONS Low-intensity pulsed ultrasound was found to have effects on the expression of type II collagen and MMP13 through the integrin–p38 MAPK signaling pathway. Therefore, we deduced that the integrin–p38 MAPK signaling pathway plays an important role in LIPUSmediated ECM production in chondrocytes. Our findings further revealed the mechanism underlying the effects of LIPUS on chondrocytes and provide some basis for the development of OA treatments using LIPUS.
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Acknowledgments—We thank Medjaden Bioscience Limited for editing and proofreading this article. We also thank the National Natural Science Foundation of China (Grant 81272151) for financial support.
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Volume 41, Number 6, 2015 Reilly GC, Golden EB, Grasso-Knight G, Leboy PS. Differential effects of ERK and p38 signaling in BMP-2 stimulated hypertrophy of cultured chick sternal chondrocytes. Cell Commun Signal 2005;3:3. Rizzardi AE, Johnson AT, Vogel RI, Pambuccian SE, Henriksen J, Skubitz AP, Metzger GJ, Schmechel SC. Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring. Diagn Pathol 2012;7:42. Sandell LJ, Aigner T. Articular cartilage and changes in arthritis, an introduction: Cell biology of osteoarthritis. Arthritis Res 2001;3: 107–113. Sondergaard BC, Schultz N, Madsen SH, Bay-Jensen AC, Kassem M, Karsdal MA. MAPKs are essential upstream signaling pathways in proteolytic cartilage degradation divergence in pathways leading to aggrecanase and MMP-mediated articular cartilage degradation. Osteoarthritis Cartilage 2010;18:279–288. Stamenovic D, Wang N. Invited review: Engineering approaches to cytoskeletal mechanics. J Appl Physiol 2000;89:2085–2090. Stanton LA, Beier F. Inhibition of p38 MAPK signaling in chondrocyte cultures results in enhanced osteogenic differentiation of perichondral cells. Exp Cell Res 2007;313:146–155. Takaishi H, Kimura T, Dalal S, Okada Y, D’Armiento J. Joint diseases and matrix metalloproteinases: A role for MMP-13. Curr Pharm Biotechnol 2008;9:47–54. Takebe K, Nishiyama T, Hayashi S, Hashimoto S, Fujishiro T, Kanzaki N, Kawakita K, Iwasa K, Kuroda R, Kurosaka M. Regulation of p38 MAPK phosphorylation inhibits chondrocyte apoptosis in response to heat stress or mechanical stress. Int J Mol Med 2011;27:329–335. Tew SR, Hardingham TE. Regulation of SOX9 mRNA in human articular chondrocytes involving p38 MAPK activation and mRNA stabilization. J Biol Chem 2006;281:39471–39479. van der Sluijs JA, Geesink RG, van der Linden AJ, Bulstra SK, Kuyer R, Drukker J. The reliability of the Mankin score for osteoarthritis. J Orthop Res 1992;10:58–61. Yasuda T. Comparison of hyaluronan effects among normal, osteoarthritis, and rheumatoid arthritis cartilages stimulated with fibronectin fragment. Biomed Res 2010;31:63–69. Zhen X, Wei L, Wu Q, Zhang Y, Chen Q. Mitogen-activated protein kinase p38 mediates regulation of chondrocyte differentiation by parathyroid hormone. J Biol Chem 2001;276:4879–4885. Zhou S, Bachem MG, Seufferlein T, Li Y, Gross HJ, Schmelz A. Low intensity pulsed ultrasound accelerates macrophage phagocytosis by a pathway that requires actin polymerization, Rho, and Src/MAPKs activity. Cell Signal 2008;20:695–704.
http://dx.doi.org/10.1016/j.ultrasmedbio.2015.01.014
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Original Contribution LOW-INTENSITY PULSED ULTRASOUND AFFECTS CHONDROCYTE EXTRACELLULAR MATRIX PRODUCTION VIA AN INTEGRIN-MEDIATED P38 MAPK SIGNALING PATHWAY PENG XIA, SHASHA REN, QIANG LIN, KAI CHENG, SHIHAO SHEN, MINGXIA GAO, and XUEPING LI Department of Rehabilitation Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing, China (Received 26 August 2014; revised 26 December 2014; in final form 17 January 2015)
Abstract—Although low-intensity pulsed ultrasound (LIPUS) regulates p38 mitogen-activated protein kinase (MAPK) and promotes cartilage repair in osteoarthritis, the role of integrin-mediated p38 MAPK in the effect of LIPUS on extracellular matrix (ECM) production of normal and OA chondrocytes remains unknown. The aim of this study was to investigate whether LIPUS affects ECM production in normal and OA rabbit chondrocytes through an integrin–p38 signaling pathway. A rabbit model of OA was established by anterior cruciate ligament transection, and chondrocytes were isolated from normal or OA cartilage and cultured in vitro. Chondrocytes were treated with LIPUS and then pre-incubated with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. Expression of type II collagen, MMP-13, integrin b1, p38 and phosphorylated p38 was assessed by Western blot analysis. We found that type II collagen and integrin b1 were upregulated (p , 0.05), whereas MMP-13 was downregulated (p , 0.05) in normal and OA chondrocytes. Furthermore, phosphorylated p38 was upregulated (p , 0.05) in normal chondrocytes, but downregulated (p , 0.05) in OA chondrocytes after LIPUS stimulation. Pre-incubation of chondrocytes with the integrin inhibitor disrupted the effects of LIPUS on normal and OA chondrocytes. Pre-incubation of chrondocytes with the p38 inhibitor reduced the effects of LIPUS on normal chondrocytes, but had no impact on OA chondrocytes. Our findings suggest that the integrin–p38 MAPK signaling pathway plays an important role in LIPUS-mediated ECM production in chondrocytes. (E-mail: [email protected]) Ó 2015 World Federation for Ultrasound in Medicine & Biology. Key Words: Low-intensity pulsed ultrasound, Osteoarthritis, Chondrocytes, Integrin, p38 MAPK.
hydrolysis of cartilage ECM by proteinase is a key step in the pathogenesis of OA (Takaishi et al. 2008). Matrix metalloproteinases (MMPs) are the most important proteolytic enzymes in the degradation of ECM, and MMP-13 is the most effective enzyme for degrading type II collagen (Blain 2007). In pathologic conditions, chondrocytes produce MMP-13 to degrade type II collagen, resulting in damaged or defective cartilage and, ultimately, OA (Bramono et al. 2004; Mitchell et al. 1996). Mechanical stress interactions within the ECM play a dominant role in cell regulation. Cells are able to sense forces to which they are exposed and generate forces on the substrate, which enables a rearrangement of matrix proteins (Guilak et al. 2009). Integrins are a family of cell surface stress receptors mediating cellular interactions with the ECM as well as cell–cell interactions (Hynes 1992; 2002). Integrin b1, the main integrin found on the chondrocyte membrane, regulates the proliferation and differentiation of chondrocytes (Kim et al. 2011). Stimulation of integrin signals by mechanical stress may transduce
INTRODUCTION Osteoarthritis (OA), caused by articular cartilage degeneration (Lohmander et al. 2003), manifests as joint pain, stiffness, dysfunction and different degrees of deformity in the late stage of the disease (Pearle et al. 2005). The degradation of extracellular matrix (ECM) accelerates degeneration in OA cartilage (Sandell and Aigner 2001). The pathologic changes are specifically associated with the chondrocytes. Chondrocytes play a key role in OA and are very important to maintain the anabolic–catabolic balance of matrix maintenance and tissue function (Aigner et al. 2007). Cartilage ECM is composed mainly of collagen and aggrecan. Type II collagen is the most abundant collagen in cartilage and is a typical marker for chondrocytes. The
Address correspondence to: Xueping Li, Department of Rehabilitation Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China. E-mail: [email protected] 1690
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signals across the cell membrane, leading to activation of downstream pathways, such as the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathway (Kim et al. 2011; MacKenna et al. 2000), which play a key role in regulating the production of MMPs in chondrocytes (Chun 2004). The MAPK signaling pathway consists of extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and cJun N-terminal kinase (JNK), all of which are constitutively expressed in most cell types, including chondrocytes (Karsdal et al. 2008). The p38 MAPK signaling pathway mediates chondrocyte–perichondral communication (Stanton and Beier 2007), stabilizes chondrogenic transcription factor SOX 9 mRNA (Tew and Hardingham 2006) and is necessary for MMP expression and activity (Sondergaard et al. 2010). Therefore, the p38 MAPK signaling pathway plays a major role in chondrocyte differentiation (Zhen et al. 2001). Low-intensity pulsed ultrasound (LIPUS) is a form of mechanical energy that is transmitted through and into tissues as pulsed acoustic pressure waves and has been clinically used as a non-invasive supplemental therapy to promote fracture and wound healing (Gebauer et al. 2005). LIPUS has been reported to stimulate chondrocyte proliferation and matrix production in vitro, suggesting an overall anabolic effect on cartilage formation. Therefore, LIPUS can promote the repair of cartilage after damage and can delay cartilage degeneration, leading to OA in the articular cartilage (Huang et al. 1999; Korstjens et al. 2008). Some studies have reported that LIPUS can activate the p38 MAPK signaling pathway (Ikeda et al. 2006; Zhou et al. 2008). Our previous studies also suggested that LIPUS can promote cartilage repair of OA chondrocytes through downregulation of MMP-13 and p38 MAPK (Li et al. 2011) and that the protective effect of LIPUS on chondrocytes may be mediated by the integrin–PI3K/Akt pathway (Cheng et al. 2014). There has, however, been no study on the correlation of integrins and p38 MAPK with the effect of LIPUS on ECM production of normal and OA chondrocytes. In this study, our aim was to investigate whether LIPUS affects ECM production in normal and OA chondrocytes through the integrin–p38 MAPK pathway. METHODS Reagents Phosphate-buffered saline (PBS), 0.25% trypsin, 0.2% type II collagenase, Dulbecco’s modified Eagle medium-high glucose (DMEM-HG), fetal bovine serum (FBS), an immunohistochemical kit, a total protein extraction kit, toluidine blue and other cell isolation- and culture medium-related supplies were purchased from KeyGEN (Nanjing, Jiangsu, China). Mouse anti-rabbit monoclonal
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antibodies against type II collagen, MMP-13, integrin b1, p38, phosphorylated p38 (p-p38) or b-actin were purchased from Acris (Herford, NRW, Germany), and goat antimouse (Fab)2 secondary antibody was purchased from Santa Cruz (Dallas, TX, USA). The integrin inhibitor GRGDSP (glycine–arginine–glycine–aspartic acid– serine–proline) was purchased from AnaSpec (Fremont, CA, USA), and the p38 inhibitor SB203580 was purchased from Selleck (Houston, TX, USA). Animal experiments Twenty-two 2.5- to 3.0-kg, 2-mo-old healthy male New Zealand white rabbits were purchased from the Qinglongshan Experimental Animal Center of Jiangsu Province, China. All experimental protocols were approved by the ethics committee of Nanjing Medical University Nanjing Hospital. A rabbit OA model (right knee, n 5 11) was generated by anterior cruciate ligament transection. Normal rabbits served as the control group (n 5 11). Both normal and OA rabbits (4 wk after transection) were euthanized and subjected to histopathologic observation of femoral condylar cartilage. Chondrocytes from knee joints of normal and OA rabbits were isolated from the cartilage and cultured in vitro, after cartilage changes were evaluated using Mankin scores (Mankin et al. 1971; van der Sluijs et al. 1992). Surgical procedures Anterior cruciate ligament transection (ACLT) of the OA rabbit model was performed as described previously (Boulocher et al. 2008; Jean et al. 2008). Briefly, rabbits were intravenously anesthetized with 3% sodium pentobarbital (1 mL/kg). The knee joint skin was disinfected with iodine after the fur was shaved, and a parapatellar skin incision was made on the medial side of the joint. The ACLT was transected using eye scissors, and a positive anterior drawer test was used to ensure complete transection of the ligament. The patella was relocated and the wound closed with a vicryl 4/O braided absorbable sutures. Penicillin and fentanyl were given to prevent bacterial infection and pain, respectively, after the incised skin was closed. The anterior cruciate ligaments of animals in the normal control (NC) group were not transected. Histopathology The femoral condylar articular cartilage collected from knee joints was fixed in neutral formalin, decalcified in ethylenediaminetetraacetic acid (EDTA) for 3 wk, embedded in paraffin and sectioned into 4-mm-thick sections using a microtome. Pathologic changes in knee joint cartilage samples, including surface irregularities and formation of cracks, were examined under a microscope.
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Fibrosis, matrix distribution, cartilage loss and chondrocyte colonization were evaluated in a double-blind fashion by two independent experts using the Mankin score system (Table 1). Chondrocyte isolation and culture Articular cartilage slices obtained from the femoral condyle of knee joints from OA and normal (control) rabbits were cut into smaller pieces, washed with PBS and digested with 0.25% trypsin for 30 min. The resultant chondrocytes were washed again with PBS; incubated for approximately 10 h in DMEM supplemented with 0.2% type II collagenase, 10% heat-inactivated FBS and antibiotics; washed with culture medium; and collected by centrifugation at 1,000 rpm for 10 min. The isolated chondrocytes were cultured in tissue culture dishes with complete DMEM in 5% CO2/95% air at 37 C. The morphology of the chondrocytes was observed under a microscope. Immunocytochemistry Chondrocytes were fixed with 4% paraformaldehyde for 30 min, washed with PBS three times, incubated with 3% H2O2–methanol solution at room temperature for 10 min, washed with PBS three times again and blocked and incubated with goat serum (50–100 mL) at room temperature for 20 min. The cells were then incubated with type II collagen antibodies (50–100 mL, 1:200 dilution) at 37 C for 2 h, washed with PBS three times before addition of 50 mL of an intensifier and incubated at room temperature for 30 min. Then the cells Table 1. Mankin scoring scale* Subgroup 1: Fibrillation 1. Even surface 2. Uneven surface 3. Fibrillated and fissured within superficial zone only 4. Fissures and erosions extending below the surface zone, without extending beyond the radial zone 5. Fissures and erosions extending into the deeper zone Subgroup 2: Matrix distribution 1. Normal staining 2. Moderate loss in staining 3. Severe loss in staining 4. No staining Subgroup 3: Chondrocyte loss 1. Loss extending into superficial zone 2. Loss extending into midzone 3. Loss extending into radial zone Subgroup 4: Chondrocyte cloning 1. No clusters 2. Chondrocyte clusters in superficial zone 3. Chondrocyte clusters in superficial to midzone (fewer than four cells) 4. Chondrocyte clusters of more than four cells located in superficial to midzone, or chondrocyte clusters in deeper zone * Grading was performed separately for the medial femoral condyle, lateral femoral condyle, medial tibial plateau and lateral tibial plateau. The minimum total score was 4, and the maximum total score was 16.
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were washed with PBS three times, incubated with horseradish peroxidase-conjugated anti-mouse-(Fab)2 antibodies (50 mL) at 37 C for 30 min, washed with PBS three times and then subjected to color development using diaminobenzidine and hematoxylin staining. Three culture dishes were read per condition, and the three areas with most positive staining cells were selected. Type II collagen expression was observed under a microscope, and images were acquired. The integrated option density (IOD) was obtained with Image-Pro Plus software (Cheng et al. 2014; Rizzardi et al. 2012). Low-intensity pulsed ultrasound stimulation Normal and OA chondrocytes were subdivided into the control group and the treated group, which was subdivided into four subgroups (1–4) treated with LIPUS (HT2009-1, Ito, Tokyo, Japan) at intensities of 20, 30, 40 and 50 mW/cm2, respectively. A layer of coupling agent (,1 mm thick) was applied between the LIPUS probe and culture dish, and the probe was placed below the tissue culture dish (Fig. 1). Chondrocytes were treated once daily for 6 d at an on–off ratio of 20%, irradiation of 3 MHz, for 20 min in a 37 C incubator under a humidified atmosphere with 5% CO2 in air (Cheng et al. 2014; Korstjens et al. 2008). Application of integrin and p38 MAPK inhibitors To investigate the role of integrin and p38 in the effects of LIPUS on normal and OA chondrocytes, the chondrocytes were pre-treated with a 10, 30 or 50 mM concentration of the integrin inhibitor GRGDSP or a 1, 5 or 10 mM concentration of the p38 inhibitor SB203580, or mock treated with dimethyl sulfoxide for 4 h before the appropriate intensity of LIPUS stimulation. The optimal concentrations of inhibitors were determined using Western blot analysis. Then chondrocytes were incubated with the optimal concentration of inhibitors, 50 mM for GRGDSP or 10 mM for SB203580, and exposed or not exposed to LIPUS for 6 d.
Fig. 1. LIPUS irradiation of chondrocytes in vitro. A layer of coupling agent (,1 mm thick) was applied between the LIPUS probe and culture dish, and the probe was placed below the tissue culture dish. Normal and osteoarthritis chondrocytes were subdivided a control group that did not receive LIPUS stimulation and a treated group that was subdivided into four subgroups that received LIPUS at intensities of 20, 30, 40 and 50 mW/cm2, respectively. Chondrocytes were treated once a day for 6 d at an on–off ratio of 20%, irradiation of 3 MHz, for 20 min. LIPUS 5 low-intensity pulsed ultrasound.
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Fig. 2. Comparison of femoral condylar cartilage of normal rabbits and rabbits with OA. (a, b) Identification of normal and OA chondrocytes (3400): (a) Normal femoral condylar cartilage has a smooth articular surface with a bright luster with no cracks. (b) The soft articular surface of OA femoral condylar cartilage has crannies (arrows), but lacks the original luster. (c, d) Hematoxylin–eosin (3100): (c) Smooth and evenly stained normal articular cartilage surface with chondrocytes arranged in normal order. (d) Apparent articular cartilage fissures extending deep from the surface with significant loss of staining (arrows) and disorganized and fewer chondrocytes in OA femoral condylar cartilage. (e, f) Immunohistochemical staining of type II collagen: (e) Normal chondrocytes with abundant cytoplasm and round nuclei (arrows). (f) OA chondrocytes with hypertrophic nuclei mixed with some dendrite-like cells (arrows). (g) Type II collagen expression in normal and OA chondrocytes. (h) Mankin scores in normal and OA cartilage. n 5 11. OA 5 osteoarthritis. *p , 0.05.
Western blot analysis After LIPUS stimulation for 6 d, chondrocytes were collected, and expression of type II collagen, MMP-13, integrin b1, p38 and p-p38 was determined by Western blot analysis. Proteins were extracted from the chondrocytes using a total protein extraction kit, and 25 mg/mL was loaded onto sodium dodecyl sulfate–polyacrylamide gel for electrophoresis and electroblotted onto nitrocellulose membranes. The membranes were blocked with skim milk for 2 h and incubated with primary antibodies: anti-type II collagen (1:500 dilution), anti-MMP-13 (1:500 dilution), anti-integrin b1 (1:500 dilution), anti-p38 (1:1000 dilution), anti-p-p38 (1:1,000 dilution) and anti-b-actin (1:500 dilution) at 4 C overnight. Then the membranes were subsequently washed three times with Tween 20 in PBS and incubated with peroxidase-conjugated goat antimouse IgG secondary antibody (1:5,000 dilution) at 37 C for 2 h, followed by another three washes as described above. The membranes were developed with the ECL kit.
Statistical analysis All data were expressed as the mean 6 standard deviation (SD) and analyzed using SPSS 18.0 software (IBM, Armonk, NY, USA). Differences between groups were analyzed by single-factor analysis of variance (ANOVA). Modified Mankin scores were analyzed using the Wilcoxon signed rank test. A p-value , 0.05 was considered to indicate statistical significance. RESULTS General and histopathologic observations Four weeks after ACLT, the articular surface of normal femoral condylar cartilage was smooth, had a bright luster and did not any cracks (Fig. 2a), whereas the articular surface of OA femoral condylar cartilage had become soft, lost its original luster and bore crannies (Fig. 2b). Femoral condylar cartilage from normal and OA rabbits was stained with hematoxylin and eosin and examined under a
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Fig. 3. Expression of type II collagen and MMP-13 in normal chondrocytes and chondrocytes from rabbits with OA after LIPUS stimulation. (a, b) Western blot analysis of type II collagen and MMP-13 in normal and OA chondrocytes with b-actin as a loading control. (c) Quantitative analysis of type II collagen expression in normal chondrocytes. (d) Quantitative analysis of type II collagen expression in OA chondrocytes. (e) Quantitative analysis of MMP-13 expression in normal chondrocytes. (f) Quantitative analysis of MMP-13 expression in OA chondrocytes. The ratio between the target and b-actin was used to normalize the data for comparison. n 5 4. MMP-13 5 matrix metalloproteinase 13; OA 5 osteoarthritis; LIPUS 5 low-intensity pulsed ultrasound. *p , 0.05.
microscope. The surface of articular cartilage from normal rabbits was smooth and evenly stained with the chondrocytes arranged in normal order (Fig. 2c); however, in articular cartilage from rabbits with OA, there were fissures extending deep from the surface with significant loss of staining (Fig. 2d). The chondrocytes from rabbits with OAwere also disorganized and reduced in number (Fig. 2d). Modified Mankin scores Compared with those of the normal control group, the Mankin scores of the OA rabbit model group were
overall significantly higher (p , 0.05). Mankin scores for subgroups 1–4 of the OA rabbit model group were also significantly higher (p , 0.05) than those of the normal control group (Fig. 2h). Identification of normal and OA chondrocytes The normal chondrocytes had abundant cytoplasm and a round nucleus, which grew to full confluence within 4 to 7 d (arrows in Fig. 2e). The OA chondrocytes had hypertrophic nuclei, which were mixed with some dendrite-like cells and grew to full confluence within 9
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Fig. 4. Expression of integrin b1, p38 and p-p38 in normal and osteoarthritis chondrocytes after LIPUS stimulation. (a) Western blot analysis of integrin b1, p38 and p-p38 with b-actin as a loading control. (b) Quantitative analysis of integrin b1. (c) Ratio of p-p38 to p38. The ratio between the target and b-actin was used to normalize the data for comparison. p-p38 5 phosphorylated p38; LIPUS 5 low-intensity pulsed ultrasound; NC 5 normal control group; N 1 L 5 normal 1 LIPUS group; OA 5 osteoarthritis group; O 1 L 5 osteoarthritis 1 LIPUS group. n 5 4. *p , 0.05.
to 12 d (arrows in Fig. 2f). Immunohistochemical staining of type II collagen in normal chondrocytes (Fig. 2e) was much deeper than that in OA chondrocytes (Fig. 2f). The mean density of type II collagen was significantly higher in normal chondrocytes than in OA chondrocytes (p , 0.05) (Fig. 2g). Expression of type II collagen and MMP-13 in normal and OA chondrocytes after 20 min of LIPUS stimulation at four different intensities To determine an appropriate LIPUS intensity for normal and OA rabbit chondrocytes, we examined the expression of type II collagen and MMP-13 in chondrocytes after LIPUS stimulation at four different intensities (20, 30, 40 and 50 mW/cm2). Western blot analysis revealed that in normal and OA rabbit chondrocytes, type II collagen expression was significantly increased (p , 0.05) and MMP-13 expression was significantly decreased (p , 0.05) after LIPUS stimulation at all four intensities. In normal chondrocytes, type II collagen expression was significantly increased (p , 0.05) in the 40 mW/cm2 intensity group compared with all other groups, whereas MMP-13 expression was significantly decreased (p , 0.05) in the 40 mW/cm2 intensity group compared with the
20 mW/cm2 intensity (p , 0.05), 50 mW/cm2 intensity (p , 0.05) and control (p , 0.05) groups. However, no significant difference (p . 0.05) was observed compared with the 30 mW/cm2 intensity group. In OA chondrocytes, type II collagen expression was significantly increased (p , 0.05) in the 30 mW/cm2 intensity group compared with the 40 mW/cm2 intensity group (p , 0.05), 50 mW/cm2 intensity group (p , 0.05) and control group (p , 0.05). However, no significant difference (p . 0.05) was observed compared with the 20 mW/cm2 intensity group. MMP-13 expression was also significantly decreased (p , 0.05) in the 30 mW/cm2 intensity group compared with all other groups (Fig. 3). Integrin b1 expression and p38 phosphorylation in normal and OA rabbit chondrocytes after 20 min of LIPUS stimulation We examined integrin b1 expression and p38 phosphorylation in normal and OA rabbit chondrocytes after LIPUS stimulation. LIPUS at 40 and 30 mW/cm2 was used to stimulate normal and OA rabbit chondrocytes, respectively. Integrin b1 expression significantly increased after LIPUS stimulation (p , 0.05) in both normal and OA chondrocytes, whereas expression in
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Fig. 5. Expression of integrin b1 and p-p38 in normal chondrocytes and OA chondrocytes after treatment with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. (a–d) Western blot analyses of integrin b1 and p38 with b-actin as a loading control in normal and OA chondrocytes. (e) Quantitative analysis of integrin b1 after treatment with 10, 30 or 50 mM GRGDSP in normal and OA chondrocytes. (f) Quantitative analysis of p-p38 after treatment with 1, 5 or 10 mM SB203580 in normal and OA chondrocytes. The ratio between the target and b-actin was used to normalize the data for comparison. n 5 3. OA 5 osteoarthritis. *p , 0.05.
OA chondrocytes was significantly higher than in normal chondrocytes (p , 0.05). The level of p38 phosphorylation increased significantly after LIPUS stimulation in normal chondrocytes (p , 0.05), but significantly decreased after LIPUS stimulation in OA chondrocytes (p , 0.05). The level of p38 phosphorylation was significantly higher in OA chondrocytes than normal chondrocytes (p , 0.05) (Fig. 4). Expression of integrin b1 and p-p38 in normal and OA rabbit chondrocytes after treatment with integrin and p38 inhibitors The expression of integrin b1 and p-p38 in normal and OA chondrocytes was examined after treatment with inhibitors. Integrin b1 expression in normal and OA rabbit chondrocytes significantly decreased (p , 0.05) after treatment with 10, 30 or 50 mM GRGDSP, with the lowest expression in chondrocytes at 50 mM GRGDSP (p , 0.05). Expression of p-p38
in normal and OA rabbit chondrocytes significantly decreased (p , 0.05) after treatment with 1, 5 or 10 mM SB203580, with the lowest expression at 10 mM (p , 0.05) (Fig. 5). Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes after integrin inhibitor treatment Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes was examined after treatment with the integrin inhibitor GRGDSP at 50 mM. After GRGDSP treatment, type II collagen expression significantly increased (p , 0.05), and MMP-13 expression significantly decreased (p , 0.05) in normal and OA chondrocytes. The phosphorylation level of p38 significantly increased (p , 0.05) in normal chondrocytes, but significantly decreased (p , 0.05) in OA chondrocytes. Expression of type II collagen expression significantly increased (p , 0.05) and that
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Fig. 6. Expression of type II collagen, MMP-13, p38 and p-p38 in NC with or without LIPUS stimulation after treatment with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. (a) Western blot analysis of type II collagen, MMP-13, p38 and p-p38 with b-actin as a loading control. (b) Quantitative analysis of type II collagen. (c) Quantitative analysis of MMP-13. (d) Ratio of p-p38 to p38. The ratio between the target and b-actin was used to normalize the data for comparison. MMP-13 5 matrix metalloproteinase 13; NC 5 normal chondrocytes; p-p38 5 phosphorylated p38; LIPUS 5 low-intensity pulsed ultrasound. n 5 4. *p , 0.05.
of MMP-13 significantly decreased (p , 0.05) after LIPUS stimulation in normal and OA chondrocytes. When GRGDSP was used, there was no significant change in the expression of type II collagen and MMP-13 in normal and OA chondrocytes (p . 0.05) after LIPUS stimulation. The phosphorylation level of p38 did not significantly change (p . 0.05) in normal chondrocytes and OA chondrocytes after LIPUS stimulation (Figs. 6 and 7). Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes after p38 MAPK inhibitor treatment Expression of type II collagen, MMP-13, p38 and p-p38 in normal and OA rabbit chondrocytes was examined after treatment with the p38 MAPK inhibitor. Type II collagen expression significantly increased in normal and OA chondrocytes (p , 0.05). Type II collagen expression did not change significantly in normal chondrocytes (p . 0.05), but significantly decreased in OA chondrocytes (p , 0.05) after LIPUS stimulation following SB203580 treatment. MMP-13 expression significantly decreased (p , 0.05) in normal and OA chondrocytes after SB203580 treatment. On exposure to LIPUS, MMP-13 expression significantly decreased in OA chondrocytes (p , 0.05), but LIPUS
had no significant effect on normal chondrocytes treated with SB203580 (p . 0.05). The phosphorylation level of p38 significantly decreased (p , 0.05) in normal and OA chondrocytes treated with SB203580. LIPUS stimulation significantly decreased the phosphorylation level of p38 in OA chondrocytes (p , 0.05), but had no significant effect in normal chondrocytes (p . 0.05) after LIPUS stimulation following SB203580 treatment (Figs. 6 and 7). DISCUSSION In this study, we sought to determine whether LIPUS affects ECM production through an integrin–p38 MAPK signaling pathway in normal and OA chondrocytes. We found that LIPUS significantly increases ECM production and decreases MMP-13 expression through an integrin–p38 MAPK signaling pathway. We found that after LIPUS stimulation, type II collagen expression significantly increased (p , 0.05), but MMP-13 expression significantly decreased (p , 0.05). We suggested that the increased levels of MMP-13 degraded type II collagen in the ECM of chondrocytes to cause OA and that LIPUS prevented the type II collagen from being degraded by MMP-13 in both normal and OA chondrocytes.
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Fig. 7. Expression of type II collagen, MMP-13, p38 and p-p38 in OA chondrocytes with or without LIPUS stimulation after treatment with the integrin inhibitor GRGDSP or the p38 inhibitor SB203580. (a) Western blot analysis of type II collagen, MMP-13, p38 and p-p38 with b-actin as a loading control. (b) Quantitative analysis of type II collagen. (c) Quantitative analysis of MMP-13. (d) Ratio of p-p38 to p38. The ratio between the target and b-actin was used to normalize the data for comparison. MMP-13 5 matrix metalloproteinase 13; p-p38 5 phosphorylated p38; OA 5 osteoarthritis; LIPUS 5 low-intensity pulsed ultrasound. n 5 4. *p , 0.05.
We further observed that inhibition of integrin by GRGDSP suppressed MMP-13 expression, which may prevent the degradation of type II collagen and decrease the activity of p38 in both normal and OA chondrocytes. Therefore, we speculate that integrin may have a negative effect on chondrocytes without LIPUS stimulation. We also found that integrin b1 expression in both normal and OA chondrocytes significantly increased (p , 0.05) after LIPUS stimulation. In fact, integrin plays an important role in mechanical signal transduction in chondrocytes by participating in the proliferation, differentiation and migration of chondrocytes (Huang et al. 2004; Humphries 2000; Loeser et al. 2000; Luo et al. 2013; Stamenovic and Wang 2000). Fibronectin fragment (Fn-f), on one hand, stimulates MMP production to cause chondrocyte degeneration; on the other hand, integrin binds to Fn-f (Guo et al. 2009; Yasuda 2010). Therefore, we deduced that the increased integrin expression caused by LIPUS in both normal and OA chondrocytes may be ascribed to the binding to Fn-f to eliminate the negative effect of Fn-f on chondrocyte degradation. In this case, integrins might have a beneficial effect on chondrocytes. To sum up, integrins played different roles under different situations. We found that after LIPUS stimulation, phosphorylation levels of p38 increased significantly (p , 0.05) in normal chondrocytes, but decreased significantly
(p , 0.05) in OA chondrocytes. This indicates that when integrin was activated by LIPUS, the downstream p38 MAPK of the integrin–p38 MAPK signaling pathway was activated in normal chondrocytes, but inhibited in OA chondrocytes. In fact, p38 activity is much higher in OA chondrocytes, and inhibition of p38 can curb apoptosis and hypertrophic terminal differentiation of cultured OA chondrocytes (Reilly et al. 2005; Takebe et al. 2011), whereas p38 can be an effector kinase of mechanotransduction in various cell types and is essential to normal articular cartilage (Sondergaard et al. 2010). On the other hand, in adult articular tissues, p38 is known to play an important role in the inflammatory process (Joos et al. 2009), and some evidence suggests that p38 is a major signaling molecule in the induction of MMP-13 and the activity of p38 can have negative effects on cartilage matrix maintenance (Julovi et al. 2011; Mengshol et al. 2000; Pei et al. 2006). In addition, moderate mechanical stress such as knee loading reduces MMP-13 activity through the p38 MAPK signaling pathway in normal articular cartilage (Hamamura et al. 2013). Therefore, we speculated that LIPUS inhibited phosphorylation of p38 in OA chondrocytes, but facilitated phosphorylation of p38 in normal chondrocytes to promote adaptation to a moderate mechanical stress. We found that after application of the integrin inhibitor GRGDSP, levels of type II collagen, MMP-13 and p38 phosphorylation did not significantly change
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in both normal and OA chondrocytes after LIPUS stimulation, suggesting again that p38 MAPK was downstream of integrin and that inhibition of integrin expression disrupted the regulation of p38 and ECM production by LIPUS. We also found that p38 inhibition affected both normal and OA chondrocytes and the effects of LIPUS on normal chondrocytes. Expression of type II collagen significantly increased and expression of MMP-13 significantly decreased in normal and OA chondrocytes treated with the p38 inhibitor SB203580, indicating that p38 may be a negative factor in both normal and OA chondrocytes without LIPUS stimulation. Our results also indicated that expression of type II collagen and MMP-13 did not significantly change in normal chondrocytes after LIPUS stimulation following SB203580 treatment, but in OA chondrocytes, LIPUS stimulation significantly increased type II collagen expression and decreased MMP-13 expression. Thus, we deduced that p38 was mainly a protective factor in normal chondrocytes, but mainly an inflammatory and injurious factor in OA chondrocytes under LIPUS stimulation. We found that inhibition of p38 disrupted the protective effects of LIPUS on ECM production in normal chondrocytes. Because LIPUS had the same inhibitory effect on p38 as SB203580 in OA chondrocytes, the inhibition of p38 by SB203580 had no impact on the effects of LIPUS on ECM production in OA chondrocytes. These results indicate that p38 of normal and OA chondrocytes has different roles under different conditions. For the protective effect of LIPUS, p38 has a more important role in normal chondrocytes than in OA chondrocytes under LIPUS simulation. Of note, the protective effect of LIPUS on chondrocytes is exerted not only through the p38 MAPK pathway, but also through other pathways such as PI3K/Akt pathway, as described in our previous study (Cheng et al. 2014). Therefore, we speculated that the p38 MAPK pathway and PI3K/Akt pathway might have a synergistic effect on ECM production of chondrocytes under LIPUS stimulation, which should be investigated in a further study. CONCLUSIONS Low-intensity pulsed ultrasound was found to have effects on the expression of type II collagen and MMP13 through the integrin–p38 MAPK signaling pathway. Therefore, we deduced that the integrin–p38 MAPK signaling pathway plays an important role in LIPUSmediated ECM production in chondrocytes. Our findings further revealed the mechanism underlying the effects of LIPUS on chondrocytes and provide some basis for the development of OA treatments using LIPUS.
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Acknowledgments—We thank Medjaden Bioscience Limited for editing and proofreading this article. We also thank the National Natural Science Foundation of China (Grant 81272151) for financial support.
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