Effects of low-intensity pulsed ultrasound and hyperbaric oxygen on human osteoarthritic chondrocytes
- Li-Jen Yuan†1,
- Chi-Chien Niu†1,
- Song-Shu Lin1, 2,
- Chuen-Yung Yang1,
- Yi-Sheng Chan1,
- Wen-Jer Chen1 and
- Steve WN Ueng1Email author
© Yuan et al.; licensee BioMed Central Ltd. 2014
Received: 4 November 2013
Accepted: 17 January 2014
Published: 5 February 2014
Although the individual effects of hyperbaric oxygen (HBO) and low-intensity pulsed ultrasound (LIPUS) on osteoarthritic (OA) chondrocytes have been reported, the effects of HBO combined with LIPUS treatment are unknown.
OA chondrocytes were obtained from patients undergoing knee replacement surgery. RNA was isolated for real-time polymerase chain reaction (PCR) analysis of inducible nitric oxide synthase (iNOS), type-II collagen, and aggrecan gene expression. The protein levels of MMP-3 and TIMP-1 were quantified by enzyme-linked immunosorbent assay (ELISA) after LIPUS or HBO treatment. The data are given as mean ± standard deviation (SD) of the results from three independent experiments. A p value less than 0.05 was defined as statistically significant.
Our data suggested that ultrasound and HBO treatment increased cell bioactivity of OA chondrocytes. Real-time PCR analysis showed that HBO treatment increased the mRNA of type-II collagen, aggrecan, and TIMP-1 but suppressed the iNOS expression of OA chondrocytes. LIPUS treatment increased the type-II collagen and iNOS expression of OA chondrocytes. ELISA data showed that HBO or LIPUS treatment increased TIMP-1 production of OA chondrocyte. MMP-3 production was suppressed by HBO treatment. HBO combined with LIPUS treatments resulted in additive effect in TIMP-1 production and compensatory effect in iNOS expression.
HBO combined with LIPUS treatment-induced increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio may provide an additive therapeutic approach to slow the course of OA degeneration.
KeywordsLow-intensity pulsed ultrasound Hyperbaric oxygen Osteoarthritis TIMP-1 MMP-3 Aggrecan Type-II collagen
Mechanical stimulus is thought to be one of the important factors regulating chondrocyte metabolism . Excessive mechanical stimulus has been reported to destroy articular cartilage directly and also induce other destructive factors . Conversely, insufficient mechanical stimulus, such as that due to joint immobilization, has also been associated with cartilage destruction . On the other hand, moderate (physiological) mechanical stimulus has been confirmed not only to promote articular cartilage anabolism  but also to inhibit catabolism [5, 6].
Low-intensity pulsed ultrasound (LIPUS) is a representative therapy in the orthopedic field and is clinically used to treat fractures with nonunion and to promote bone union . Application of high-intensity continuous ultrasound (1–300 W/cm2) generates considerable heat in living tissues. In contrast, LIPUS (<100 mW/cm2) has much lower intensities with nonthermogenic and nondestructive actions. Mechanical strains received in the skeleton result in the promotion of bone formation, possibly by inducing chondrocyte proliferation [8, 9]. In this context, LIPUS has been shown to enhance the endochondral ossification in the healing process of fractured bones [10, 11].
Ultrasound treatment has been tried as an approach to encourage cartilage repair . Previous in vitro work has shown that the expression levels of integrins a5 and b1, as well as chondrocytic markers, Sox5, Sox9, collagen II, and aggrecan, were increased in chondrocytes exposed to a continuous ultrasound signal at 5.0 MHz (0.14 mW/cm2) . Previous in vivo study in a New Zealand rabbit that modeled full-thickness osteochondral defects has demonstrated that exposure to LIPUS significantly improves the morphologic features and histologic characteristics of repaired cartilage . Another in vivo experimental rat osteoarthritis model also demonstrated efficacy in cartilage restoration . Exposure to LIPUS could significantly affect chondrocyte proliferation, phenotype expression, and matrix production; however, inconsistent effects were also observed.
Previous report suggested that VEGF induced by HBO is through c-Jun/AP-1 activation and through simultaneous activation of ERK and JNK pathways in umbilical vein endothelial cells . HBO-suppressed ERK1/2 and p38 MAPK mediate nitric oxide-induced apoptosis on human degenerated intervertebral disc cells . In OA chondrocytes, the MAP kinases, AP-1, and NF-κB transcription factors have been shown to play a predominant role in the expression of metalloproteinases (MMPs) and inflammatory genes and protein . Our previous study demonstrated that attenuation of apoptosis and enhancement of proteoglycan synthesis in rabbit cartilage defects by HBO treatment are related to the suppression of IL-1β and nitric oxide (NO) production . HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70 .
Although the individual effect of HBO or LIPUS on the chondrocytes have been reported, the effect of HBO combined with LIPUS treatment is still controversial. We harvested the articular cartilage from patients who receive total knee arthroplasty (TKA). We investigate whether the beneficial effect on OA will be synergistic up-regulation (such as aggrecan, type-II collagen, and TIMP-1 expression) and the subversive effect will be complementary compensation (such as iNOs expression) after HBO combined with LIPUS treatment.
The experimental protocol was approved by the Human Subjects Institutional Review Board of the Chang Gung Memorial Hospital.
Cell isolation and cell culture
Articular cartilage specimens (tibial plateaus and femoral condyles) were obtained from 20 Ahlbäck grade IV or Kellgren and Lawrence grade IV OA patients who receive TKA surgery. The specimen was obtained under aseptic conditions, and the cartilage was dissected on ice. The chondrocytes were released from the articular cartilage by sequential digestion with 1 mg/ml collagenase (Sigma, St. Louis, MO, USA) in Dulbecco's minimal essential medium (DMEM/F-12) (Gibco, Grand Island, NY, USA) containing 5% fetal bovine serum (FBS) and incubated at 37°C until the fragments were digested. The isolated chondrocytes were centrifuged (1,000 rpm for 5 min), washed with PBS, and seeded in T-75 tissue culture flasks (Falcon, BD Biosciences, Drive Franklin Lakes, NJ, USA) in 15 ml of medium (DMEM/F-12) supplemented with 20% (v/v) FBS and antibiotics (mixture of 100 U/ml of penicillin and 100 μg/ml of streptomycin; Gibco). The cultures were incubated in a humidified atmosphere of 5% CO2/95% air until cell confluence.
Cell exposure to intermittent HBO
About 3 × 104 cells are platted on the 35-mm cell culture dish (Falcon) with medium (DMEM/F-12) containing 10% FBS and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Control cells were maintained in 5% CO2/95% air (non-HBO) through the experimental protocol. All hyperoxic cells were exposed to 100% O2 for 25 min then to air for 5 min at 2.5 atmospheres absolute (ATA) in a hyperbaric chamber (Sigma II, Perry Baromedical Corporation, Riviera Beach, FL, USA) with a total treatment of 90 min per 48 h.
Cell exposure to LIPUS treatment
About 3 × 104 cells are platted on the 35-mm cell culture dish with medium (DMEM/F-12) containing 10% FBS and incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. A UV-sterilized transducer (Exogene 3000; Smith & Nephew Inc., Memphis, TN, USA) that generated 1.5-MHz US in a pulsed-wave mode (200-μs pulse burst width with repetitive frequency of 1 kHz at an intensity of 30 mW/cm2) was immersed vertically into each culture well and placed to just contact the surface of the medium. The distance between the transducer and the cells was approximately 5–6 mm. The exposure time was 20 min per 48 h.
Cell exposure to LIPUS combined with HBO
The cells were treated with LIPUS first and then with HBO as previously described.
RNA extraction and real-time PCR analysis
At 24 h after each treatment, cellular RNA was isolated using an RNeasy mini kit (Qiagen, Valencia, CA, USA) and reverse-transcribed into cDNA with the ImProm-II reverse transcription system (Promega, Madison, WI, USA). For real-time PCR detection of iNOs, aggrecan, and type-II collagen RNA transcripts, cDNA was analyzed on an ABI PRISM 7900 sequence detection system using the TaqMan PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The cycle threshold (Ct) values were obtained, and the data was normalized to GAPDH expression using the ΔΔCt method to calculate relative mRNA levels of each target gene.
MMP-3 ELISA assay
The cells were plated at 3 × 104 cells per 35-mm tissue culture dish (Falcon) in 2.5 ml of medium (DMEM/F-12) containing 5% FBS. The level of TIMP-1 in the conditioned media after each treatment was determined using a commercial immunoassay kit (Quantikine Human TIMP-1, R&D System, Minneapolis, MN, USA). At intervals of 48, 96, and 144 h, 200 μl of conditioned media was accumulated and tested according to the manufacturer's instructions. The measurements were performed in triplicate.
TIMP-1 ELISA assay
The cells were plated at 3 × 104 cells per 35-mm tissue culture dish in 2.5 ml of medium (DMEM/F-12) containing 5% FBS. The level of TIMP-1 in the conditioned media after each treatment was determined using a commercial immunoassay kit (Quantikine Human TIMP-1, R&D System). At intervals of 48, 96, and 144 h, 200 μl of conditioned media was accumulated and tested according to the manufacturer's instructions. The measurements were performed in triplicate.
Effect of HBO and LIPUS on MMP-3 production
Effect of HBO and LIPUS on TIMP-1 production
Effect of HBO and LIPUS on gene expression of iNOs, aggrecan, and type-II collagen
The secretion of proteolytic enzymes by the cartilage has been confirmed to contribute to the loss of extracellular matrix in OA. MMPs are capable of degrading the macromolecules of connective tissue matrices and have been considered the major proteases responsible for the pathologic destruction of tissue . Moreover, an imbalance between MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is responsible for the pathogenic sequence of cartilage degradation .
To elucidate whether mechanical stimulation by LIPUS combined with HBO treatment is chondrocyte-protective, we studied the effect of LIPUS combined with HBO treatment at several intensities on the protein expression of MMPs and TIMPs. LIPUS may potentially protect articular cartilage by inhibiting MMP-13 and MMP-1 mRNA expression in an intensity-dependent manner . TIMP-1 mRNA expression was inhibited significantly by LIPUS stimulation of the articular cartilage explants at 67 mW/cm2 but up-regulated by the stimulation of the cultured chondrocytes at 30 mW/cm2. In the present study, the MMP-3 production was significantly down-regulated by the HBO treatment but not by the LIPUS stimulation (Figure 1). The TIMP-1 production in OA chondrocytes was significantly up-regulated by the HBO or LIPUS treatment (Figure 2). We further showed that HBO combined with LIPUS treatment resulted in an additive effect in the TIMP-1 production (Figure 2). HBO combined with LIPUS treatment-induced increase of the anabolic factor (TIMP-1)/catabolic factor (MMP-3) ratio may provide a therapeutic approach to slow the course of OA chondrocyte degeneration.
Nitric oxide (NO) is a highly reactive nitrogen radical implicated in multiple biological processes, including regulation of vascular tone, platelet and leukocyte adhesion, neurotransmission, mediation of excessive vasodilatation, and cytotoxic actions of macrophages against microbes and tumor cells . Ultrasound stimulates NF-κB activation and iNOS expression in cultured preosteoblasts . Exposure to LIPUS increases NO and prostaglandin release, which are required for mechanically induced bone formation . However, apoptosis of chondrocytes can be induced by NO [10, 16, 27]. HBO treatment prevents NO-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70 . In the present study, HBO suppressed while LIPUS increased the gene expressions of iNOS in OA chondrocytes (Figure 3). HBO combined with LIPUS treatment resulted in compensatory effect in iNOS expression in OA chondrocytes (Figure 3), thus may prevent NO-induced apoptosis.
HBO treatment increased PG synthesis in vivo[19, 20]. However, the effect of LIPUS to stimulate chondrocyte matrix synthesis is still controversial. Several in vitro studies have been undertaken to characterize the effects of LIPUS on chondrocytes in both monolayer and 3D model systems. These studies report the up-regulation of aggrecan and collagen II genes [28–30] and GAG synthesis . However, conflicting reports suggest that LIPUS induces, at best, a transient effect on chondrocyte culture systems in terms of GAG and collagen II production  and aggrecan gene expression . In the present study, similar results suggested that the aggrecan and type-II collagen mRNA expression in the OA chondrocytes were significantly up-regulated by HBO treatment. However, there was no additive effect in aggrecan and type-II collagen mRNA expression by HBO combined with LIPUS treatment (Figures 4 and 5).
In this paper, the author combined a chemical factor (hyperbaric oxygen) and a mechanical factor (LIPUS) treatment. The weighting of these two factors are equal in the combined treatment, and the induced increase of the (TIMP-1)/catabolic factor (MMP-3) ratio may provide an additive therapeutic approach to slow the course of OA degeneration. Although the effects of the combined factors are better than those of a single factor, the optimal combination ratio of these two factors needs further investigation.
HBO combined with LIPUS treatment resulted in an additive effect in the TIMP-1 production and a compensatory effect in iNOS expression. Therefore, we will apply similar techniques of HBO combined with LIPUS therapy in future studies of cartilage injury models. The advantage of HBO combined with LIPUS treatment is that it is a useful tool for clinics and a more applicable clinical therapy.
This research was supported in part by grants from the National Science Council and Chang Gung Memorial Hospital (CMRPG381441), Taiwan, Republic of China.
- Sun HB: Mechanical loading, cartilage degradation, and arthritis. Ann N Y Acad Sci. 2010, 121: 37-50.View ArticleGoogle Scholar
- Fujisawa T, Hattori T, Takahashi K, Kuboki T, Yamashita A, Takigawa M: Cyclic mechanical stress induces extracellular matrix degradation in cultured chondrocytes via gene expression of matrix metalloproteinases and interleukin-1. J Biochem. 1999, 125: 966-975. 10.1093/oxfordjournals.jbchem.a022376.View ArticlePubMedGoogle Scholar
- Leong DJ, Gu XI, Li YH, Lee JY, Laudier DM, Majeska RJ, Schaffler MB, Cardoso L, Sun HB: Matrix metalloproteinase-3 in articular cartilage is up-regulated by joint immobilization and suppressed by passive joint motion. Matrix Biol. 2010, 29: 420-426. 10.1016/j.matbio.2010.02.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Smith RL, Lindsey DP, Dhulipala L, Harris AHS, Goodman SB, Maloney WJ: Effects of intermittent hydrostatic pressure and BMP-2 on osteoarthritic human chondrocyte metabolism in vitro. J Orthop Res. 2011, 29: 361-368. 10.1002/jor.21250.View ArticlePubMedGoogle Scholar
- Torzilli PA, Bhargava M, Park S, Chen CTC: Mechanical load inhibits IL-1 induced matrix degradation in articular cartilage. Osteoarthritis Cartilage. 2010, 18: 97-105. 10.1016/j.joca.2009.07.012.PubMed CentralView ArticlePubMedGoogle Scholar
- Trindade MCD, Shida J, Ikenoue T, Lee MS, Lin EY, Yaszay B, Yerby S, Goodman SB, Schurman DJ, Smith RL: Intermittent hydrostatic pressure inhibits matrix metalloproteinase and pro-inflammatory mediator release from human osteoarthritic chondrocytes in vitro. Osteoarthritis Cartilage. 2004, 12: 729-735. 10.1016/j.joca.2004.05.008.View ArticlePubMedGoogle Scholar
- Duarte LR: The stimulation of bone growth by ultrasound. Acta Orthop Traumatol Surg. 1983, 101: 153-159. 10.1007/BF00436764.View ArticleGoogle Scholar
- Ikeda K, Takayama T, Suzuki N, Shimada K, Otsuka K, Ito K: Effects of low-intensity pulsed ultrasound on the differentiation of C2C12 cells. Life Sci. 2006, 79: 1936-1943. 10.1016/j.lfs.2006.06.029.View ArticlePubMedGoogle Scholar
- Wiltink A, Nijweide PJ, Oosterbaan WA, Hekkenberg RT, Helders PJ: Effect of therapeutic ultrasound on endochondral ossification. Ultrasound Med Biol. 1995, 21: 121-127. 10.1016/0301-5629(94)00092-1.View ArticlePubMedGoogle Scholar
- Nolte PA, Klein-Nulend J, Albers GH, Marti RK, Semeins CM, Goei SW, Burger EH: Low-intensity ultrasound stimulates endochondral ossification in vitro. J Orthop Res. 2001, 19: 301-307. 10.1016/S0736-0266(00)00027-9.View ArticlePubMedGoogle Scholar
- Warden SJ, Bennell KL, McMeeken JM, Wark JD: Acceleration of fresh fracture repair using the sonic accelerated fracture healing system (SAFHS): a review. Calcif Tissue Int. 2000, 66: 157-163. 10.1007/s002230010031.View ArticlePubMedGoogle Scholar
- Tien YC, Lin SD, Chen CH, Lu CC, Su SJ, Chih TT: Effects of pulsed low-intensity ultrasound of human child chondrocytes. Ultrasound Med Biol. 2008, 34: 1174-1181. 10.1016/j.ultrasmedbio.2007.12.019.View ArticlePubMedGoogle Scholar
- Hasanova GI, Noriega SE, Mamedov TG, Thakurta SG, Turner JA, Subramanian A: The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds. J Tissue Eng Regen Med. 2011, 5: 15-822.View ArticleGoogle Scholar
- Cook SD, Salkeld SL, Popich-Patron LS, Ryaby JP, Jones DG, Barrack RL: Improved cartilage repair after treatment with low-intensity pulsed ultrasound. Clin Orthop. 2001, 391 (Suppl): S231-S243.View ArticlePubMedGoogle Scholar
- Huang MH, Yang RC, Ding HJ, Chai CY: Ultrasound effect on level of stress proteins and arthritic histology in experimental arthritis. Arch Phys Med Rehabil. 1999, 80: 551-556. 10.1016/S0003-9993(99)90198-3.View ArticlePubMedGoogle Scholar
- Samali A, Orrenius S: Heat shock proteins: regulators of stress response and apoptosis. Cell Stress Chaperones. 1998, 3: 228-236. 10.1379/1466-1268(1998)003<0228:HSPROS>2.3.CO;2.PubMed CentralView ArticlePubMedGoogle Scholar
- Niu CC, Yuan LJ, Chen LH, Lin SS, Tsai TT, Liao JC, Lai PL, Chen WJ: Beneficial effects of hyperbaric oxygen on human degenerated intervertebral disk cells via suppression of IL-1β and p38 MAPK signal. J Orthop Res. 2011, 29: 14-19. 10.1002/jor.21195.View ArticlePubMedGoogle Scholar
- Medzhitov R: Toll-like receptors and innate immunity. Nat Rev Immunol. 2001, 1: 135-145. 10.1038/35100529.View ArticlePubMedGoogle Scholar
- Yuan LJ, Ueng SWN, Lin SS, Yeh WL, Yang CY, Lin PY: Attenuation of apoptosis and enhancement of proteoglycan synthesis in rabbit cartilage defects by hyperbaric oxygen treatment are related to the suppression of nitric oxide production. J Orthop Res. 2004, 22: 1126-1134. 10.1016/j.orthres.2004.01.006.View ArticlePubMedGoogle Scholar
- Ueng SWN, Yuan LJ, Lin SS, Niu CC, Chan YS, Wang IC, Yang CY, Chen WJ: Hyperbaric oxygen treatment prevents nitric oxide-induced apoptosis in articular cartilage injury via enhancement of the expression of heat shock protein 70. J Orthop Res. 2013, 31: 376-384. 10.1002/jor.22235.View ArticlePubMedGoogle Scholar
- Blain EJ: Mechanical regulation of matrix metalloproteinases. Front Biosci. 2007, 12: 507-527. 10.2741/2078.View ArticlePubMedGoogle Scholar
- Kafienah W, Al-Fayez F, Hollander AP, Barker MD: Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach. Arthritis Rheum. 2003, 48: 709-718. 10.1002/art.10842.View ArticlePubMedGoogle Scholar
- Ito A, Aoyama T, Yamaguchi S, Zhang X, Akiyama H, Kuroki H: Low-intensity pulsed ultrasound inhibits messenger RNA expression of matrix metalloproteinase-13 induced by interleukin-1β in chondrocytes in an intensity-dependent manner. Ultrasound Med Biol. 2012, 38: 1726-1733. 10.1016/j.ultrasmedbio.2012.06.005.View ArticlePubMedGoogle Scholar
- Nathan C, Xie QW: Nitric oxide synthases roles, tolls, and controls. Cell. 1994, 78: 915-918. 10.1016/0092-8674(94)90266-6.View ArticlePubMedGoogle Scholar
- Hou CH, Lin J, Huang SC, Hou SM, Tang CH: Ultrasound stimulates NF-κb activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts. J Cell Physiol. 2009, 220: 196-203. 10.1002/jcp.21751.View ArticlePubMedGoogle Scholar
- Tang CH, Yang RS, Huang TH, Lu DY, Chuang WJ, Huang TF, Fu WM: Ultrasound stimulates cyclooxygenase-2 expression and increases bone formation through integrin, focal adhesion kinase, phosphatidylinositol 3-kinase, and Akt pathway in osteoblasts. Mol Pharmacol. 2006, 69: 2047-2057. 10.1124/mol.105.022160.View ArticlePubMedGoogle Scholar
- Yuan LJ, Niu CC, Lin SS, Chan YS, Yang CY, Chen WJ, Ueng SWN: Additive effects of hyperbaric oxygen and platelet-derived growth factor-BB in chondrocytes transplantation via up-regulation expression of platelet-derived growth factor-β receptor. J Orthop Res. 2009, 27: 1439-1446. 10.1002/jor.20889.View ArticlePubMedGoogle Scholar
- Zhang ZJ, Huckle J, Francomano CA, Spencer RGS: The effects of pulsed low-intensity ultrasound (PLIUS) on chondrocyte viability, proliferation, gene expression and matrix production. Ultrasound Med Biol. 2003, 29: 1645-1651. 10.1016/j.ultrasmedbio.2003.08.011.View ArticlePubMedGoogle Scholar
- Mukai S, Ito H, Nakagawa Y, Akiyama H, Miyamoto M, Nakamura T: Transforming growth factor-beta(1) mediates the effects of low-intensity pulsed ultrasound in chondrocytes. Ultrasound Med Biol. 2005, 31: 1713-1721. 10.1016/j.ultrasmedbio.2005.07.012.View ArticlePubMedGoogle Scholar
- Zhang ZJ, Huckle J, Francomano CA, Spencer RGS: The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: an explant study. Ultrasound Med Biol. 2002, 28: 1547-1553. 10.1016/S0301-5629(02)00659-2.View ArticlePubMedGoogle Scholar
- Kopakkala-Tani M, Leskinen JJ, Karjalainen HM, Karjalainen T, Hynynen K, Toyras J, Jurvelin JS, Lammi MJ: Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes. Biorheology. 2006, 43: 271-282.PubMedGoogle Scholar
- Korstjens CM, van der Rijt RHH, Albers GHR, Semeins CM, Klein-Nulend J: Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro. Med Biol Eng Comput. 2008, 46: 1263-1270. 10.1007/s11517-008-0409-9.View ArticlePubMedGoogle Scholar
- Zhang ZJ, Huckle J, Francomano CA, Spencer RGS: The effects of pulsed low-intensity ultrasound on chondrocyte viability, proliferation, gene expression and matrix production. Ultrasound Med Biol. 2003, 29: 1645-1651. 10.1016/j.ultrasmedbio.2003.08.011. 1222View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.