http://informahealthcare.com/cts ISSN: 0300-8207 (print), 1607-8438 (electronic) Connect Tissue Res, 2015; 56(3): 219–227 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03008207.2015.1016609

Low-intensity pulsed ultrasound stimulates matrix metabolism of human annulus fibrosus cells mediated by transforming growth factor b1 and extracellular signal-regulated kinase pathway Mei-Hsiu Chen1, Jui-Sheng Sun2, Shao-Yu Liao3, Po-An Tai4,5, Tin-Chou Li4,5, and Ming-Hong Chen4,5,6

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

1

Department of Internal Medicine, Far Eastern Memorial Hospital, Taipei, Taiwan, 2Department of Orthopedic, National Taiwan University Hospital Hsin-Chu Branch, Hsin-Chu, Taiwan, 3Department of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan, 4Division of Neurosurgery, Department of Surgery, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City, Taiwan, 5Department of Surgery, School of Medicine, Tzu Chi University, Hualian, Taiwan, and 6Department of Biomedical Engineering, Ming Chuang University, Taoyuan, Taiwan Abstract

Keywords

Purpose: There are limited strategies to restore the damaged annulus fibrosus (AF) of the intervertebral disc. Low-intensity pulsed ultrasound (LIPUS) has positive effects on the proliferation of several types of cells and the repair of damage tissue in vivo. However, scientific evidence of therapeutic effects of LIPUS on AF cells remains limited. The purpose of this study is to evaluate the feasibility of applying LIPUS to the repair of the AF. Materials and methods: We used an in vitro model of human AF cells subjected to LIPUS stimulation to examine its effects on cell proliferation and matrix metabolism. Cell viability, synthesis of collagen and glycosaminoglycan (GAG), expression of matrix metalloproteinases (MMPs) and transforming growth factor b1 and pathways involving mitogen-activated protein kinases (MAPKs) were investigated. Results: LIPUS significantly enhanced proliferation of AF cells after 5 days of treatment. LIPUS with an intensity of 0.5 W/cm2 increased the collagen and GAG synthesis and decreased the expressions of MMP-1 and -3 of human AF cells. Real-time polymerase chain reactions and western blotting analysis revealed that LIPUS could increase transforming growth factor b1 (TGF-b1) and activate extracellular signal-regulated kinase (ERK) pathway. In addition, TGF-b receptor kinase inhibitor could suppress the ultrasound-induced alterations in cell viability and matrix metabolism. Conclusions: The findings suggested that LIPUS could be useful as a physical stimulation of cell metabolism for the repair of the AF.

Annulus fibrosus, collagen, glycosaminoglycan, low-intensity pulsed ultrasound, matrix metalloproteinase, TGF-b1

Introduction Back pain is a major public health problem in modern societies. The prevalence rates of back pain ranged from 12% to 35% (1). Among the etiology of low back pain, degeneration of the intervertebral disc is a very common cause. The intervertebral discs consist of a thick outer ring of fibrous cartilage known as the annulus fibrosus (AF), which surrounds a more gelatinous core termed the nucleus pulposus. The AF is made up of 15–25 concentric layers, or lamellae, with the collagen fibers lying parallel within each layer. During the degeneration of the intervertebral discs, the number of distinct layers of the AF decreases gradually and

Correspondence: Dr. Ming-Hong Chen, Division of Neurosurgery, Department of Surgery, Taipei Tzu Chi Hospital, No. 289, Jianguo Rd., Xindian Dist., New Taipei City 23142, Taiwan. Tel: +886 2 6628 9779. E-mail: [email protected]

History Received 15 November 2014 Revised 17 January 2015 Accepted 3 February 2015 Published online 6 March 2015

the fiber bundles within layers become more irregularly distributed with increased inter-bundle space (2). The loss of distinct layers of the annulus causes its inability for a sustained response to loading and leads to the annular tears, fissures and subsequent disc protrusions/herniations (3). Early symptoms of herniated discs may begin with localized pain in the back. Pain is often temporary and the symptoms resolve with massage, analgesics or antiinflammatory medications. Although best evidence suggests there is little to support its effectiveness for improving pain or functional status, physical therapy typically has had a role in conservative management of lumbar disk herniation (4). However, with the progression of disc herniation, surgery is considered for severe or persistent pain, or neurological deficits. Discectomy via microsurgical techniques or minimal invasive procedures is an effective treatment for neurological decompression in patients suffering from a herniated nucleus pulposus. However, treatment modalities mentioned above are not directed to restore the damaged/degenerated intervertebral

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

220

M.-H. Chen et al.

disc and even inevitably aggravate existing damage of the AF (5,6). Therefore, a successful surgical decompression is often followed by periods of persisting low back pain, severely affecting the quality of life (7,8). Besides, the intrinsic healing capacity of the AF to cope with damage or degenerative changes has been studied in several animal studies. The studies, thus, far concluded that the AF has only a very limited regenerative capacity (9,10). As a result, it is not surprising that the high recurrence rates after discectomy affect up to 15% of the patients (7,8). From a biomechanical point of view, capability to regenerate/repair the AF is the key issue for the successful treatment of degenerative disc diseases (11). Besides, for patients with disc degenerative disease but not requiring a surgical removal/replacement of the nucleus pulposus, restoration of the damaged AF is promising to prevent further progression of disc herniation. Thus, strategies for treating disc herniation are increasingly focusing on the regeneration or repair of the AF (12). For decades, ultrasound has been a widely used and wellaccepted physical therapy modality for the management of musculoskeletal injuries or for promoting soft tissue repair (13). Among various types of therapeutic ultrasound, lowintensity pulsed ultrasound (LIPUS), having removed the thermal component found at higher intensities, has been reported to activate several types of cells, especially in relation to tissue proliferation (14). LIPUS is effective for promoting bone union (15). Researches have also shown encouraging results with LIPUS able to promote healing in various soft tissues, such as ligaments (16), tendons (17) and cartilage (18). Nevertheless, scientific evidence of therapeutic effects of LIPUS on AF cells remains limited. Thus, there is still interest to evaluate the feasibility of enhancing regeneration/repair of the AF by LIPUS stimulation. Besides, effects of LIPUS on the metabolism of extracellular matrix (ECM) and biochemical mechanisms on repair/regeneration of the AF remain to be learned. The ECM of the AF is composed of an interlocking mesh of collagen and glycosaminoglycans (GAGs). The proportions of type I and II collagens vary gradually and inversely from the nucleus pulposus to the AF, with type I collagen mainly in the latter (19). Degeneration of the intervertebral disc results from excessive activity of tissue proteinases, which degrade collagens of the ECM (20–22). Similarly, annular repair can be a complex and highly-regulated process that is initiated, sustained and eventually terminated by a large number and variety of molecules, such as growth factors, cytokines and matrix metalloproteinases (MMPs). MMPs, a group of tightly regulated zinc-dependent enzymes, play a crucial role in the normal development, repair and remodeling of connective tissues. MMP-1 (collagenase-1), -8 (collagenase-2) and -13 (collagenase-3) capable of cleaving the intact type I collagen molecule in the extracellular environment comprise the collagenase subfamily in humans. MMP-1 has been important in the degeneration of the intervertebral disc and is capable of degrading native fibrillar collagens in the ECM (22). MMP13, significantly increased both at the mRNA and protein levels in torn rotator cuff tendons, is the only interstitial collagenase that has been suggested to degrade collagens in connective tissues in rats. The failure to properly regulate these enzymes may lead to improper or excess matrix

Connect Tissue Res, 2015; 56(3): 219–227

degeneration and plays a role in the development of pathological tendon conditions (23). In this study, we aimed to elucidate whether LIPUS stimulation can modulate the production of collagen and GAG and the expression of MMPs in human AF cells. This can contribute to understand if LIPUS stimulation can promote the repair of the AF by regulating the metabolism of ECM. The effects of ultrasound on enhancing tendon healing are well recognized in the previous studies. The mechanisms by which collagen synthesis both in protein and mRNA level are likely mediated by endogenous release of nitric oxide and upregulation of transforming growth factor b1 (TGF-b1) (24). Further more, the interactions between mitogen-activated protein kinases (MAPKs) and the expression of MMPs following TGF-b1 treatment have been shown in several types of cells. The activation of MAPK and its subsequent phosphorylation of various transcription factors have also been associated with the regulation of remarkably diverse biological processes, such as cell proliferation, cell shape changes and immune responses. Thus, apart from evaluating the MMPs and metabolism of the ECM, this study also investigated the influences of LIPUS stimulation on TGF-b1 and MAPK signaling pathway in cultured human AF cells.

Materials and methods Primary culture of human AF cells Surgical specimens of the AF were collected from six patients (all male, mean age was 34.6, range from 23 to 52 years, receiving microsurgical discectomy) with full ethical permission (Table 1). Written informed consents for participation in this study were obtained from all participants. Techniques for the AF cell culture used in this study followed previous reports (25,26). Each tissue was cut into small pieces (approximately 1.0 mm3). Then, the AF tissues were washed twice with phosphate buffered saline (PBS), and digested with 0.4% pronase and 0.025% collagenase type II overnight. The isolated cells were seeded in 10 cm dishes. Seven milliliters of Dulbecco’s Modified Eagle Medium and Ham’s F-12 (DMEM/F12) with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml) were added to each plate. The cells grew rapidly and the confluence culture was subcultured by trypsin digestion. The cultures were incubated under 37  C, 95% humidity and 5% CO2 environment. Then, the AF cells were harvested and placed into 6-well culture plates at a density of 2  105 cell/plate with 1 ml DMEM/F12 in each well. All the procedures for collecting specimens received approval of the Ethic Committee of Cathay General Hospital (approval number: CT099028). Table 1. Clinical features of each case in this study. Patient 1 2 3 4 5 6

Age

Diagnosis

Disc level

23 27 52 31 33 42

HIVD HIVD HIVD HIVD HIVD HIVD

L4/5 L5/S1 L5/S1 L5/S1 L4/5 L4/5

HIVD: herniated intervertebral disc.

Surgical procedures Microsurgical Microsurgical Microsurgical Microsurgical Microsurgical Microsurgical

discectomy discectomy discectomy discectomy discectomy discectomy

Low-intensity pulsed ultrasound

DOI: 10.3109/03008207.2015.1016609

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

Low-intensity pulsed ultrasound (LIPUS) treatment The AF cells derived from human were cultured at a density of 2  105 cells/plate in 60 mm dishes for 24 h and subjected to LIPUS treatment. Therasound 3.5 ultrasound apparatus (Rich-Mar Corporation, Inola, OK), at a frequency of 1.0 MHz, was used to stimulate the AF cells. The prepared culture dishes filled with a total volume of 10 ml of culture medium were securely placed and floated over the water surface of a thermostatically controlled water bath (37  C). The transducer head just touched the surface of the medium. The distance between the transducer head (working surface area: 2 cm2) and the AF cells was approximately 10 mm. The pulsed ultrasound stimulation (frequency of 1 MHz, duty cycle 20%.) was applied to the AF cells at the intensity of 0.1, 0.3, 0.5 and 0.7 W/cm2 (spatial averaged, temporal averaged [SATA] intensity) for different experimental groups. The AF cells were stimulated for 5 min each day for 5 days (starting from the next day after seeding of cells). Control samples were prepared in the same manner without ultrasound treatment. To create a relative thermostatic and sterile condition, the whole experimental system was placed in 37  C water bath in laminar flow chamber under ultrasound stimulation. MTT (tetrazolium) assay for measuring the AF cell proliferation The AF cells were cultured in 6-well plates with 2  105 cells/ plate and 1 ml culture medium. At predetermined time point, MTT assay was performed. The mitochondria activity of the AF cells after LIPUS treatment was determined by colorimetric assay, which detected the conversion of 3 -(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, Sigma catalog no. M2128, Sigma, St. Louis, MO) to formazan, then read by a Spectra MAX340 ELISA reader (Molecular Device, Sunnyvale, CA) at 540 nm. The results were recorded as optical density. Sircolä collagen assay for total collagen in culture medium The SircolÔ collagen assay (Biocolor, Newtownabbey, Northern Ireland, UK) is a method to measure the collagens released into the medium from the cultured cell. After the AF cells were treated by LIPUS for 5 days, 200 ml sample of from each group was removed to a 1.5-ml centrifuge tube and 1.0 ml SircolÔ dye reagent was added into each tube. The mixed contents were kept at room temperature for 30 min. The tubes were then drained off and discarded. The remaining pellets were mixed with 1.0 ml alkali reagent to form solutions. Two hundred microliters aliquots of the alkali dye solutions from the assay tubes were transferred to a 96-well plate and the plate was read with spectrophotometer at 555 nm.

dye for 30 min. The solutions were then centrifuged at 12 000 rpm for 10 min. The supernatants were removed and pellets were resuspended with 0.5 ml dissociation reagent. Aliquot 100 ml of sample was added into a 96-well absorbance plate and the plate was read with a spectrophotometer at 656 nm. RNA isolation and real-time polymerase chain reaction After the AF cells had been treated with LIPUS for 5 days, RNA was isolated from the AF cells by an acid quanidine method using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA) and reversely transcribed to cDNA. Real-time polymerase chain reactions (RT-PCRs) were performed using Roche Light Cycler, utilizing SYBR Green reagents (Light Cycler-DNA Master SYBR Green I kit; Roche, Basel, Switzerland) according to the manufacturer’s instructions. The amplification program consisted of initial denaturation at 95  C for 10 min, followed by 40 cycles of denaturation at 95  C for 15 s, an annealing phase at 60  C for 5 s and an elongation phase at 72  C for 15 s. Amplification of PCR products was quantified during PCR by measurement of fluorescence associated with bindings of double-stranded DNA to the SYBR Green dye incorporated in the reaction mix. Oligonucleotide sequences for the specific primers used in this study are summarized in Table 2. SB431542 (Sigma, St. Louis, MO), a TGF-b1 receptor kinase inhibitor, was used to evaluate the role of TGF-b1 in the expression of type I collagen, MMP-1 and MMP-13 under LIPUS treatment. Western blotting analysis To evaluate the protein expression of p-ERK of AF cells with/ without the influences of LIPUS, AF cells were incubated for 24 h, 48 h, and 5 days. The effects of TGF- b1 inhibitor (SB431542) and extracellular signal-regulated kinase (ERK) specific inhibitor (PD98059) on the expression of p-ERK were also evaluated with western blotting analysis after incubation of the AF cells for 48 h. PD98059 (Sigma, St. Louis, MO), the ERK specific inhibitor, was also used to evaluate the effect of suppressing ERK pathway on cell proliferation. For western blot analysis, the AF cells were washed twice with PBS and lysed in 20 ml of radioimmunoprecipitation assay (RIPA) buffer at 4  C. For protein phosphorylation analysis, a phosphatase inhibitor cocktail

Table 2. Forward and reverse primers and probes used for RT-PCR analysis. Gene

Primer/probe sequences

COL1A1

50 -CAGACCAACAACCCAAACTCAAT-30 50 -TGCACTTTTGGTTTTTGGTCAC-30 50 -TGACAAAACCAAAGACATCACACAC-30 50 -CGCCAGGAATTGTTGCTATATTTC-30 50 -TGACTTTTAAAACATAGTCTATGTTCA-30 50 -TCTTGGATTGATTTGAGATAAGTCATAGC-30 50 -ACAGTTGATAGACTCCGAGAAATGC-30 50 -ACCATTTGAGTGTTCGAGGGA-30 50 -TTCATTGACCTCAACTACAT-30 50 -GAGGGGCCATCCACAGTCTT-30

TGF-b1 MMP-1

Sulfated glycosaminoglycans (sGAG) assay The amount of sulfated glycosaminoglycan (sGAG) in the culture supernatant was determined by the dimethylmethylene blue (DMMB) assay (Blyscan GAG assay, Biocolor, Newtownabbey, Northern Ireland, UK). After treatment by LIPUS for 5 days, 50 ml medium was mixed with 1 ml Blyscan

221

MMP-13 GAPDH

COL1A1, collagen, type I, alpha 1; MMP, matrix metalloproteinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

222

M.-H. Chen et al.

Connect Tissue Res, 2015; 56(3): 219–227

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

(Sigma, St. Louis, MO) was added. The lysates were centrifuged at 12 000 rpm for 20 min at 4  C. The protein samples were separated by SDS-PAGE on 10% gel and then transferred to nitrocellulose membranes followed by blocking with 5% bovine serum albumin for 1 h at room temperature. The membranes were then incubated overnight at 4  C with antibodies specific to the p-ERK (extracellular signalregulated kinases) (1:1000) (Cell Signaling Technology, Inc., Danvers, MA). After four washes, the membrane was incubated with anti-rabbit IgG conjugated with horseradish peroxidase for 1 h at room temperature. Detection was performed with luminal chemiluminescent systems. Quantitative data were obtained using a computing densitometer and Multi-Gauge software version 3.0 (Fuji Photo Film Co., Ltd., Tokyo, Japan). Statistical analysis All the experimental data were expressed as mean ± standard deviation of six repeats of experiments. Statistical significance of differences was evaluated using Student’s t-test. Non-parametric one sample Wilcoxon test was used for the analysis of RT-PCR results. The level of statistical significance was set at p50.05.

Results

Figure 1. Effects of LIPUS of different intensities on the AF cells. The results showed that the viability of AF cells increased with 0.5 W/cm2 LIPUS 3 days and 5 days after pulsed ultrasound stimulation, while at an intensity of 0.7 W/cm2, pulsed ultrasound stimulation resulted in a decrease of cell viability at 3 day culture. After 5 days of culture, the viability of AF cells increased significantly under the stimulation of pulsed ultrasound at the intensities of 0.1, 0.3, 0.5 and 0.7 W/cm2 (n ¼ 6; *p50.05).

LIPUS-induced proliferation of AF cells In the pilot study, the effects of pulsed-ultrasound of different intensities (0.1, 0.3, 0.5 and 0.7 W/cm2) on the AF cells were evaluated at day 3 and day 5. The results showed that viability of the AF cells increased with 0.5 W/cm2 LIPUS 3 days after pulsed ultrasound stimulation, while at an intensity of 0.7 W/cm2, pulsed ultrasound stimulation resulted in a decrease of cell viability at 3-day culture. After 5 days of culture, viability of the AF cells increased significantly under the stimulation of pulsed ultrasound at the intensities of 0.1, 0.3, 0.5 and 0.7 W/cm2 (Figure 1). With the stimulation of 0.5 W/cm2 LIPUS, the AF cells showed the significant increase in cell viability at day 5. LIPUS of 0.5 W/cm2 was therefore used for the following experiments. LIPUS stimulated collagen and GAG synthesis of AF cells After 5 days of LIPUS treatment, medium was collected and the collagen content was evaluated by SircolÔ collagen assay kit. The results showed that LIPUS with an intensity of 0.5 W/cm2 increased the collagen synthesis of the AF cells (69.01 ± 0.55 mg/ml versus 53.93 ± 0.15 mg/ml) (Figure 2A). In addition, sGAG production of the AF cells was also evaluated. After 0.5 W/cm2 LIPUS for 5 days, sGAG content in the medium (25.18 ± 1.08 mg/ml) was higher than the control group (19.86 ± 1.23 mg/ml) (Figure 2B). The results suggested that LIPUS stimulated the AF cells in the production of ECM including collagen and GAG. LIPUS stimulated the expression of type I collagen and suppressed the expression of MMP-1 and MMP-13 at transcriptional level Intervertebral disc is a highly specialized cartilaginous tissue, containing mainly two types of collagens (I and II).

The proportions of I and II varied gradually and inversely across the intervertebral disc, with mainly type I in the AF (19). Along various matrix metabolic enzymes, MMP-1 and MMP-13 are main enzymes that cleave intact interstitial collagen molecules. For further investigating the effects of LIPUS on matrix metabolism, the mRNA expression of type I collagen, MMP-1 and MMP-13 were analyzed by RT-PCR (Figure 2C). When stimulated with 0.5 W/cm2 LIPUS, the mRNA expression of type I collagen in the LIPUS treatment group (189.7 ± 27.2% of control group) was significantly stronger than in the control group on the fifth day. On the other hand, mRNA expressions of MMP-1 and MMP-13 significantly decreased in the LIPUS treatment group (31.1 ± 5.3% and 62.9 ± 5.3% of control group for MMP-1 and MMP-13, respectively). LIPUS-induced early expression of TGF-b1 The AF cells were treated with 0.5 W/cm2 LIPUS for 5 min and the mRNA expression of TGF-b1 was evaluated with RTPCR at 6, 12 and 24 h. The results showed that LIPUS significantly increased the expression of TGF-b1 6 h after stimulation. The expressions of TGF-b1 were 139.9 ± 4.8%, 432.4 ± 61.3% and 816.5 ± 162% of control groups for 6, 12 and 24 h, respectively (Figure 2D). LIPUS-induced early expression of TGF-b1 and the expression kept increasing for 24 h. TGF-b1 receptor kinases inhibitor regulated the mRNA expression of type I collagen, MMP-1 and MMP-13 of AF cells Previous researches showed that TGF-b1 stimulated the synthesis of type I and II collagens (27). In addition, TGF-b1 was found to be associated with MMP-13 production

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

DOI: 10.3109/03008207.2015.1016609

Low-intensity pulsed ultrasound

223

Figure 2. Effects of 0.5 W/cm2 LIPUS on the production of collagen and sGAG and the mRNA expressions of type I collagen, MMP-1, MMP-13 and TGF-b1. (A) and (B) The results showed that LIPUS with an intensity of 0.5 W/cm2 increased the collagen synthesis (69.01 ± 0.55 mg/ml versus 53.93 ± 0.15 mg/ml) (A) and sulfated glycosaminoglycan (sGAG) production (25.18 ± 1.08 mg/ml versus 19.86 ± 1.23 mg/ml) of the AF cells (n ¼ 6, *p50.05). (C) When stimulated with 0.5 W/cm2 LIPUS, the mRNA expression of type I collagen increased and the expressions of MMP-1 and MMP13 decreased. The results showed significant difference by non-parametric one sample Wilcoxon test. (D) LIPUS significantly increased the expression of TGF-b1 6 h after stimulation. The expression kept increasing for 24 h.

in many cell types. Therefore, we investigated the effects of TGF-b1 inhibitors on the expression of type I collagen, MMP1 and MMP-13. As shown in Figure 3(A), pretreatment with SB431542 (a TGF-b1 receptor kinase inhibitor, 10 mM) significantly down-regulated LIPUS-induced mRNA expression of type I collagen after 5 days (from 173.6 ± 11.1% to 103.4 ± 12.4%). Interestingly, using the TGF-b1 receptor kinase inhibitor (SB431542) also reversed the inhibited expressions of MMP-1 and MMP-13 (from 37.8 ± 13.4% to 107.5 ± 16.3% and from 33.2 ± 5.1% to 102.1 ± 11.9%, respectively). LIPUS-induced proliferation of AF cells was reversed by TGF-b1 receptor kinase inhibitor Besides the role in matrix metabolism, TGF-b1 plays an important role in mediating cell proliferation during tissue repair (28). In this study, we also examined if TGF-b1 mediated the proliferation of AF cells. As the results of MTT

assay shown in Figure 3(B), pretreatment of the AF cells with SB431542 (a TGF-b1 receptor kinase inhibitor) significantly reversed the LIPUS-induced proliferation of AF cells. The optical density at 540 nm for MTT assay was 0.37 ± 0.02, 0.52 ± 0.01, 0.33 ± 0.02 and 0.36 ± 0.03 for control, LIPUS, SB431542 and LIPUS + SB431542 groups. LIPUS-induced proliferation of AF cells involved the activation of ERK pathway MAPKs constitute key steps as final effectors of signal transduction pathways directly to activate transcription factors in the cytoplasm and nucleus. Subsets of MAPKs, such as ERK, MAPK-p38 and JNK can be activated by mechanical stress (29). The effects of LIPUS on AF cells were evaluated by western blot at 24 h, 48 h and 5 days. As shown in Figure 4(A), exposure of AF cells to LIPUS increased ERK phosphorylation, but not JNK and p38 (data not shown), at 24 h and these effects sustained for 48 h following ultrasound

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

224

M.-H. Chen et al.

Connect Tissue Res, 2015; 56(3): 219–227

Figure 3. TGF-b1 receptor kinases inhibitor (SB431542) regulated the mRNA expression of type I collagen, MMP-1, MMP-13 and cell viability of the AF cells. (A) Pretreatment with SB431542 (10 mM) for the AF cells down-regulated LIPUS-induced mRNA expression of type I collagen after 5 days. SB431542 also reversed the inhibited expressions of MMP-1 and MMP-13. The differences between LIPUS and LIPUS + SB431542 groups were statistically significant (p50.05) in the mRNA expression of type I collagen, MMP-1 and MMP-13 (173.6 ± 11.1% versus 103.4 ± 12.4%, 37.8 ± 13.4% versus 107.5 ± 16.3%, 33.2 ± 5.1% versus 102.1 ± 11.9% for COL1A1, MMP-1 and MMP-13 expression without and with SB431542, respectively). (B) Similarly, pretreatment of the AF cells with SB431542 reversed the LIPUS-induced proliferation of the AF cells (n ¼ 6, *p50.05).

stimulation. However, the effects subsided after 5 days. Using PD98059 (an ERK inhibitor), suppression of ERK pathways could be confirmed even under the simulation of LIPUS (Figure 4C). Nonetheless, the AF cells pretreated with TGF-b1 receptor kinase inhibitors (SB431542) only slightly inhibited the phosphorylation of ERK (Figure 4B). However, with the pretreatment of ERK inhibitors, LIPUS-induced proliferation of AF cells decreased significantly on the fifth day (Figure 4D).

Discussion Ultrasound is widely used in clinical medicine for diagnosis, physical therapy and tissue destruction. For enhancing soft tissue healing, LIPUS has been reported to improve the resolution of chronic inflammatory process, circulation in chronically ischemic muscle, the recovery of conduction block of the median nerve, angiogenesis of the skin and healing of ligaments and tendons (14,16,30–32). The SATA intensity unit, which determines the strength of an ultrasound beam, is the spatial average intensity averaged over both the on-time and off-time of the pulsed ultrasound. This is a measurement for LIPUS to determine the energy delivered to the tissue and is expressed in units of watts per square centimetre (W/cm2). In this study, LIPUS stimulation at the range of 0.1–0.7 W/cm2 was used. Several previous researches reported LIPUS with an intensity of 30 mW/cm2, the usual

setting used commercially for fracture repair, improved proliferation of various cells. Yet, a previous report suggested that low intensity ultrasound accelerated proliferation and maturation of osteoblast-like cells at an intensity range between 0.05 and 0.3 W/cm2 (33). Dalla-Bona et al. also reported LIPUS with 150 mW/cm2 was more effective in increasing cell number and collagen synthesis than LIPUS with 30 mW/cm2 (34). In this study, our results demonstrated that LIPUS stimulated proliferation of the AF cells as the intensity increased from 0.1 to 0.5 W/cm2 (Figure 1). At the intensity of 0.7 W/cm2, LIPUS might induce the detachment of AF cells from the culture plates and decrease the cell viability. It is reasonable that different frequencies, intensities and durations can result in various biologic responses. Before one can establish that LIPUS treatment has consistent therapeutic efficacy on the repair of AF, additional protocols need to be tested both in vitro and in vivo to define the optimal conditions. However, LIPUS with an intensity of 0.5 W/cm2 was used for our later studies to understand the mechanisms underlying the AF repair. Intervertebral disc degeneration is characterized by changes in the biochemical composition and mechanical integrity of the intervertebral disc. In the degenerated intervertebral disc, the content of GAG decreases and that of denatured collagen increases. Because of the biochemical content change, subsequent dehydration leads to tearing of the AF. Therefore, it is fundamental to restore the production

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

DOI: 10.3109/03008207.2015.1016609

Figure 4. LIPUS-induced proliferation of the AF cells involved in the activation of ERK pathway. (A) Exposure of AF cells to LIPUS increased ERK phosphorylation at 24 h and the effect sustained for 48 h following ultrasound stimulation. (B) After incubation of the AF cells for 48 h, activation of ERK pathway by LIPUS was suppressed with the treatment of TGF-b1 receptor kinases inhibitor (SB431542). (C) Using PD98059 (an ERK inhibitor), suppression of ERK pathways could be confirmed even under the simulation of LIPUS. (D) Cell viability evaluated by MTT assay showed LIPUS-induced proliferation of AF cells was suppressed by blocking ERK signaling pathway by PD98059 (n ¼ 6, *p50.05).

of GAG and metabolic balance of collagen for AF repair. It has been reported that collagen production can increase when rat tenocytes, human fibroblasts, osteoblasts or monocytes are exposed to LIPUS (27,35,36). Other studies reported the positive effects of mechanical stimulation on ECM metabolism of the intervertebral disc (37,38). Miyamoto et al. also reported that collagen synthesis by bovine AF cells was increased by the application of LIPUS (39). Our results confirmed that 0.5 W/cm2 LIPUS promoted collagen production of human AF cells by 28% (Figure 2A). In addition, RTPCR results further demonstrated that expression of type I collagen, the predominant type of collagen in the AF, could be enhanced by LIPUS stimulation (Figure 2C). Interestingly, the expressions of MMP-1 and MMP-13 decreased under the treatment of LIPUS (Figure 2C). Although MMPs have been implicated in the excessive breakdown of the ECM during disc degeneration, they are important regulators of ECM network remodeling. Among various MMPs, MMP-1 and MMP-13 can cleave intact interstitial collagen molecules. MMP-1 has been important in the degeneration of the intervertebral disc and is capable of degrading native fibrillar collagens in the ECM (40). Also, stress deprivation in vitro

Low-intensity pulsed ultrasound

225

results in an immediate and significant increase in MMP-13 mRNA expression and protein synthesis. This increase in MMP-13 production also results in a significant decrease in the material properties. An increase in these enzymes capable of degrading type I collagen can weaken the structure of the annulus (21). Even though, we are not sure if decreased expression of MMPs may be beneficial for annular repair. As the disorganized and denatured collagen increases during the degeneration/injuries of the annulus, expression of MMPs should play a crucial role in remodeling the ECM of the annulus. However, with increased collagen synthesis and decreased expression of MMP-1 and MMP-13 under LIPUS stimulation, the equilibrium of collagen metabolism should proceed toward the anabolic direction. Another important component of ECM is GAG. GAGs have an important role in retaining water content within ECM and affecting the degeneration processes of the intervertebral disc. Previous studies showed mechanical stimulation on cartilages had a tendency to promote the release of newly synthesized proteoglycans into the culture medium (41). Iwashina et al. reported that LIPUS stimulation significantly up-regulated the [35S]-sulfate incorporation and proteoglycan synthesis in rabbit AF cells (25). Our results also showed LIPUS of 0.5 W/cm2 intensity enhanced the production of sulfated GAG up to 26.6% in the human AF cells (Figure 2B), which was compatible with the previous studies. Besides MMP, several factors including cytokines, TGF-b1, nitric oxide regulate the controlled metabolism of ECM, which is essential in tissue repair and remodeling. TGF-b1 has many biological functions, such as mediating fibroblast proliferation, differentiation, migration, adhesion and ECM production in disease and normal states (42). Previous studies have showed that TGF-b1 promotes proliferation and matrix synthesis of cells from the intervertebral disc (43). Tsai et al. reported that ultrasound treatment upregulated TGF-b gene expression and stimulated type I and type III collagens synthesis from cultured tenocytes (27). A study by Hiyama et al. suggested that nucleus pulposus cells also showed an increase of TGF-b1 production and increased expression of TGF-b1 type I receptor when stimulated with LIPUS for 4 days (44). In the present study, LIPUS stimulation induced an early expression of TGF-b1 of the human AF cells. The mRNA expression of TGF-b1 increased with time from 6 to 24 h (Figure 2D). For tenocytes, previous studies showed that ultrasound stimulated proliferation and regulated matrix metabolism through the cross-talk between TGF-b and ultrasound-induced MAPKs signaling pathways (45). For cells isolated from the nucleus pulposus, LIPUS and TGF-b1 showed synergistic effects on the synthesis of proteoglycans (44). However, our results showed the effects of LIPUS on the human AF cells in both increasing proliferation and altering the expressions of type I collagen, MMP-1 and MMP-13 could be reversed by pretreatment with a TGF-b1 receptor kinase inhibitor (Figure 3A and B). This suggested LIPUS-induced proliferation of AF cells and alterations in matrix metabolism were dependent on the expression of TGF-b1. TGF-b1 is known to function as modulators of ECM proteins and to cause induction of both collagen gene activation and protein formation via Smad proteins (46).

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

226

M.-H. Chen et al.

The crosstalk mechanisms between Smad and MAPK pathways in expression of MMP following TGF-b1 treatment have been shown in different types of cells. MAPK pathways are the key steps as final effectors of signal transduction directly to activate transcription factors in the cytoplasm and nucleus. It has been reported in different types of cells that subsets of MAPKs, such as ERK, MAPK-p38 and JNK can be activated by mechanical stress. Smad 2, p38 MAPK and ERK are required for TGF-b1-induced MMP-13 expression in osteoblasts (47). In addition, ERK-mediated pathways are mostly involved in proliferation, differentiation and anti-apoptosis. Our results also showed that 0.5 W/cm2 LIPUS treatment could stimulate ERK pathway rather than MAPK-p38 or JNK at 24 and 48 h (Figure 4A). Using SB431542, LIPUS-induced activation of ERK pathway can be suppressed (Figure 4B). When the AF cells were pretreated with PD98059 (an ERK inhibitor, 10 mM), LIPUS-induced proliferation of the AF cells was significantly inhibited (Figure 4D). The results suggested that LIPUS stimulation could induce the expression of TGF-b1 and modulate proliferation and matrix metabolism via the cross-talk between TGF-b1 and ERK pathways. In summary, this present study suggested that LIPUS stimulated proliferation and matrix metabolism of the AF cells. The mechanisms by which cell proliferation and matrix metabolism were stimulated are likely mediated by upregulation of TGF-b1 and activation of ERK pathway. Thus, LIPUS seemed to hold considerable promise for simulating the repair/regeneration of the AF cells. However, multiple cell types can be present in the AF cells (48) and the severity and duration of disc degeneration can affect the heterogeneity of different cell types in the AF cells. These factors can further have influence on the effects of LIPUS on the AF cells. In addition, results obtained in this study were based on an in vitro model that should not be extrapolated directly into an in vivo condition. Besides, in order to apply LIPUS percutaneously for patients suffering from disc diseases, a device may need to be developed and further in vivo studies are mandatory.

Conclusions The findings of the current study provide evidence that LIPUS can effectively stimulate matrix metabolism of the AF cells through the up-regulation of TGF-b1 and ERK pathways. Although, further studies will undoubtedly necessary in optimizing the delivery of LIPUS in clinical applications, this study provides the theoretical basis for this potential therapeutic modality in the repair of the AF cells.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported in part by a grant from Cathay General Hospital Research Aid and a grant from National Taiwan University Hospital Hsin-Chu Branch.

References 1. Maniadakis N, Gray A. The economic burden of back pain in the UK. Pain 2000;84:95–103.

Connect Tissue Res, 2015; 56(3): 219–227

2. Postacchini F, Bellocci M, Massobrio M. Morphologic changes in annulus fibrosus during aging. An ultrastructural study in rats. Spine (Phila Pa 1976) 1984;9:596–603. 3. Edwards W, Ordway N, Zheng Y, McCullen G, Han Z, Yuan H. Peak stresses observed in the posterior lateral anulus. Spine (Phila Pa 1976) 2001;26:1753–9. 4. Luijsterburg P, Verhagen A, Ostelo R, van Os T, Peul W, Koes B. Effectiveness of conservative treatments for the lumbosacral radicular syndrome: a systematic review. Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc 2007;16:881–99. 5. Carragee E, Han M, Suen P, Kim D. Clinical outcomes after lumbar discectomy for sciatica: the effects of fragment type and anular competence. J Bone Joint Surg Am 2003;85-A:102–8. 6. Hegewald A, Ringe J, Sittinger M, Thome C. Regenerative treatment strategies in spinal surgery. Front Biosci J Virtual Libr 2008;13:1507–25. 7. Atlas S, Keller R, Wu Y, Deyo R, Singer D. Long-term outcomes of surgical and nonsurgical management of sciatica secondary to a lumbar disc herniation: 10 year results from the maine lumbar spine study. Spine (Phila Pa 1976) 2005;30:927–35. 8. Atlas S, Keller R, Wu Y, Deyo R, Singer D. Long-term outcomes of surgical and nonsurgical management of lumbar spinal stenosis: 8 to 10 year results from the maine lumbar spine study. Spine (Phila Pa 1976) 2005;30:936–43. 9. Ahlgren B, Lui W, Herkowitz H, Panjabi M, Guiboux J. Effect of anular repair on the healing strength of the intervertebral disc: a sheep model. Spine (Phila Pa 1976) 2000;25:2165–70. 10. Rousseau M, Ulrich J, Bass E, Rodriguez A, Liu J, Lotz J. Stab incision for inducing intervertebral disc degeneration in the rat. Spine (Phila Pa 1976) 2007;32:17–24. 11. Wilke H-J, Heuer F, Neidlinger-Wilke C, Claes L. Is a collagen scaffold for a tissue engineered nucleus replacement capable of restoring disc height and stability in an animal model? Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc 2006;15:S433–8. 12. Bron JL, Helder MN, Meisel H-J, van Royen BJ, Smit TH. Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges. Eur Spine J Off Publ Eur Spine Soc Eur Spinal Deform Soc Eur Sect Cerv Spine Res Soc 2009;18: 301–13. 13. Speed CA. Therapeutic ultrasound in soft tissue lesions. Rheumatol Oxf Engl 2001;40:1331–6. 14. Young S, Dyson M. Effect of therapeutic ultrasound on the healing of full-thickness excised skin lesions. Ultrasonics 1990;28: 175–80. 15. Hadjiargyrou M, McLeod K, Ryaby J, Rubin C. Enhancement of fracture healing by low intensity ultrasound. Clin Orthop Relat Res 1998;(355 Suppl):S216–29. 16. Sparrow K, Finucane S, Owen J, Wayne J. The effects of lowintensity ultrasound on medial collateral ligament healing in the rabbit model. Am J Sports Med 2005;33:1048–56. 17. Maffulli N, Longo U. Conservative management for tendinopathy: is there enough scientific evidence? Rheumatol Oxf Engl 2008;47: 390–1. 18. Cook S, Salkeld S, Patron L, Doughty E, Jones D. The effect of low-intensity pulsed ultrasound on autologous osteochondral plugs in a canine model. Am J Sports Med 2008;36:1733–41. 19. Eyre DR, Muir H. Types I and II collagens in intervertebral disc. Interchanging radial distributions in annulus fibrosus. Biochem J 1976;157:267–70. 20. Anderson DG, Izzo MW, Hall DJ, Vaccaro AR, Hilibrand A, Arnold W, Tuan RS, Albert TJ. Comparative gene expression profiling of normal and degenerative discs: analysis of a rabbit annular laceration model. Spine 2002;27:1291–6. 21. Goupille P, Jayson MI, Valat JP, Freemont AJ. Matrix metalloproteinases: the clue to intervertebral disc degeneration? Spine 1998; 23:1612–26. 22. Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM. Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc. Spine 2000;25:3005–13. 23. Sharma P, Maffulli N. Tendon injury and tendinopathy: healing and repair. J Bone Joint Surg Am 2005;87:187–202. 24. Chao Y-H, Tsuang Y-H, Sun J-S, Chen L-T, Chiang Y-F, Wang C-C, Chen M-H. Effects of shock waves on tenocyte proliferation and

DOI: 10.3109/03008207.2015.1016609

25.

26.

27.

Connect Tissue Res Downloaded from informahealthcare.com by Nyu Medical Center on 05/29/15 For personal use only.

28.

29. 30. 31.

32. 33.

34. 35.

36.

extracellular matrix metabolism. Ultrasound Med Biol 2008;34: 841–52. Iwashina T, Mochida J, Miyazaki T, Watanabe T, Iwabuchi S, Ando K, Hotta T, Sakai D. Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate. Biomaterials 2006;27: 354–61. Sato M, Asazuma T, Ishihara M, Ishihara M, Kikuchi T, Kikuchi M, Fujikawa K. An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method. Spine 2003;28: 548–53. Tsai W-C, Pang J-HS, Hsu C-C, Chu N-K, Lin M-S, Hu C-F. Ultrasound stimulation of types I and III collagen expression of tendon cell and upregulation of transforming growth factor beta. J Orthop Res Off Publ Orthop Res Soc 2006;24:1310–16. Chen Y-J, Wang C-J, Yang KD, Kuo Y-R, Huang H-C, Huang Y-T, Sun YC, Wang FS. Extracorporeal shock waves promote healing of collagenase-induced Achilles tendinitis and increase TGF-beta1 and IGF-I expression. J Orthop Res Off Publ Orthop Res Soc 2004; 22:854–61. Lai J, Pittelkow MR. Physiological effects of ultrasound mist on fibroblasts. Int J Dermatol 2007;46:587–93. Hogan RD, Burke KM, Franklin TD. The effect of ultrasound on microvascular hemodynamics in skeletal muscle: effects during ischemia. Microvasc Res 1982;23:370–9. Lu H, Qin L, Cheung W, Lee K, Wong W, Leung K. Low-intensity pulsed ultrasound accelerated bone–tendon junction healing through regulation of vascular endothelial growth factor expression and cartilage formation. Ultrasound Med Biol 2008;34:1248–60. Young S, Dyson M. The effect of therapeutic ultrasound on angiogenesis. Ultrasound Med Biol 1990;16:261–9. Hsu S-K, Huang W-T, Liu B-S, Li S-M, Chen H-T, Chang C-J. Effects of near-field ultrasound stimulation on new bone formation and osseointegration of dental titanium implants in vitro and in vivo. Ultrasound Med Biol 2011;37:403–16. Dalla-Bona DA, Tanaka E, Inubushi T, Oka H, Ohta A, Okada H, Miyauchi M, Takata T, Tanne K. Cementoblast response to lowand high-intensity ultrasound. Arch Oral Biol 2008;53:318–23. Doan N, Reher P, Meghji S, Harris M. In vitro effects of therapeutic ultrasound on cell proliferation, protein synthesis, and cytokine production by human fibroblasts, osteoblasts, and monocytes. J Oral Maxillofac Surg Off J Am Assoc Oral Maxillofac Surg 1999; 57:409–19; discussion 420. Reher P, Doan N, Bradnock B, Meghji S, Harris M. Therapeutic ultrasound for osteoradionecrosis: an in vitro comparison between 1 MHz and 45 kHz machines. Eur J Cancer Oxf Engl 1990 1998;34: 1962–8.

Low-intensity pulsed ultrasound

227

37. Handa T, Ishihara H, Ohshima H, Osada R, Tsuji H, Obata K. Effects of hydrostatic pressure on matrix synthesis and matrix metalloproteinase production in the human lumbar intervertebral disc. Spine 1997;22:1085–91. 38. Matsumoto T, Kawakami M, Kuribayashi K, Takenaka T, Tamaki T. Cyclic mechanical stretch stress increases the growth rate and collagen synthesis of nucleus pulposus cells in vitro. Spine 1999;24:315–19. 39. Miyamoto K, An HS, Sah RL, Akeda K, Okuma M, Otten L, Thonar EJ, Masuda K. Exposure to pulsed low intensity ultrasound stimulates extracellular matrix metabolism of bovine intervertebral disc cells cultured in alginate beads. Spine (Phila Pa 1976) 2005;30: 2398–405. 40. Takahashi M, Haro H, Wakabayashi Y, Kawa-uchi T, Komori H, Shinomiya K. The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene. J Bone Joint Surg Br 2001;83: 491–5. 41. Steinmeyer J, Knue S. The proteoglycan metabolism of mature bovine articular cartilage explants superimposed to continuously applied cyclic mechanical loading. Biochem Biophys Res Commun 1997;240:216–21. 42. Hsu C, Chang J. Clinical implications of growth factors in flexor tendon wound healing. J Hand Surg 2004;29:551–63. 43. Walsh AJL, Bradford DS, Lotz JC. In vivo growth factor treatment of degenerated intervertebral discs. Spine 2004;29:156–63. 44. Hiyama A, Mochida J, Iwashina T, Omi H, Watanabe T, Serigano K, Iwabuchi S, Sakai D. Synergistic effect of lowintensity pulsed ultrasound on growth factor stimulation of nucleus pulposus cells. J Orthop Res Off Publ Orthop Res Soc 2007;25: 1574–81. 45. Chao Y-H, Tsuang Y-H, Sun J-S, Cheng C-K, Chen M-H. The cross-talk between transforming growth factor-beta1 and ultrasound stimulation during mechanotransduction of rat tenocytes. Connect Tissue Res 2011;52:313–21. 46. Javelaud D, Mauviel A. Crosstalk mechanisms between the mitogen-activated protein kinase pathways and Smad signaling downstream of TGF-beta: implications for carcinogenesis. Oncogene 2005;24:5742–50. 47. Selvamurugan N, Kwok S, Alliston T, Reiss M, Partridge NC. Transforming growth factor-beta 1 regulation of collagenase-3 expression in osteoblastic cells by cross-talk between the Smad and MAPK signaling pathways and their components, Smad2 and Runx2. J Biol Chem 2004;279:19327–34. 48. Feng G, Yang X, Shang H, Marks IW, Shen FH, Katz A, Arlet V, Laurencin CT, Li X. Multipotential differentiation of human anulus fibrosus cells: an in vitro study. J Bone Joint Surg Am 2010;92: 675–85.

Low-intensity pulsed ultrasound stimulates matrix metabolism of human annulus fibrosus cells mediated by transforming growth factor β1 and extracellular signal-regulated kinase pathway.

There are limited strategies to restore the damaged annulus fibrosus (AF) of the intervertebral disc. Low-intensity pulsed ultrasound (LIPUS) has posi...
584KB Sizes 5 Downloads 5 Views