Photobiomodulation on human annulus fibrosus cells ... - Springer Link

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Mar 17, 2016 - Hyo Geun Nam1 & Jin Su Jeong1 & Joo Han Kim2 & Kwang Ho Lee3 & Hyuk Choi1. Received: 13 December 2015 /Accepted: 8 March 2016 ...

Lasers Med Sci (2016) 31:767–777 DOI 10.1007/s10103-016-1923-x


Photobiomodulation on human annulus fibrosus cells during the intervertebral disk degeneration: extracellular matrix-modifying enzymes Min Ho Hwang 1 & Kyoung Soo Kim 1 & Chang Min Yoo 1 & Jae Hee Shin 1 & Hyo Geun Nam 1 & Jin Su Jeong 1 & Joo Han Kim 2 & Kwang Ho Lee 3 & Hyuk Choi 1

Received: 13 December 2015 / Accepted: 8 March 2016 / Published online: 17 March 2016 # Springer-Verlag London 2016

Abstract Destruction of extracellular matrix (ECM) leads to degeneration of the intervertebral disk (IVD), which is a major contributor to many spine disorders. IVD degeneration is induced by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), which are secreted by immune cells, including macrophages and neutrophils. The cytokines modulate ECM-modifying enzymes such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human annulus fibrosus (AF) cells. The resulting imbalance in catabolic and anabolic enzymes can cause generalized back, neck, and low back pain (LBP). Photobiomodulation (PBM) is known to regulate inflammatory responses and wound healing. The aim of this study was to mimic the degenerative IVD microenvironment, and to investigate the effect of a variety of PBM conditions (wavelength: 635, 525, and 470 nm; energy density: 16, 32, and 64 J/cm 2 ) on the production of ECMmodifying-enzymes by AF cells under degenerative conditions induced by macrophage-conditioned medium (MCM), which contains pro-inflammatory cytokines such as TNF-α and IL-β secreted by macrophage during the development of intervertebral disk inflammation. We showed that the MCM-

* Hyuk Choi [email protected]


Department of Medical Sciences, Graduate School of Medicine, Korea University, 80, Guro-dong, Guro-gu, Seoul 152-703, South Korea


Department of Neurosurgery, Guro Hospital, College of Medicine, Korea University, Seoul, South Korea


Department of Advanced Materials Science and Engineering, College of Engineering, Kangwon National University, Chuncheon, South Korea

stimulated AF cells express imbalanced ratios of TIMPs (TIMP-1 and TIMP-2) and MMPs (MMP-1 and MMP-3). PBM selectively modulated the production of ECMmodifying enzymes in AF cells. These results suggest that PBM can be a therapeutic tool for degenerative IVD disorders. Keywords Photobiomodulation . Human annulus fibrosus . Intervertebral disk degeneration . Inflammation . Extracellular matrix

Introduction Degeneration of the intervertebral disk (IVD) is a major contributor to chronic low back pain (LBP). LBP is a very common condition, with an estimated 84 % of the adult population suffering from LBP during their lives [1]. In general, LBP is thought to be mediated by the compression of nerve root(s). However, LBP and sciatica have been reported even in the absence of nerve root compression. These observations have led to a reconsideration of the pathogenesis of LBP. Some studies have suggested the following possible mechanism of the pain pathway [2, 4]. The IVD is composed of two distinct regions: the inner nucleus pulposus (NP) and outer annulus fibrosus (AF). In the healthy state without sciatica, the spinal nerves are located in the outer third of the AF of the intervertebral disk. However, in the presence of sciatica conditions, the nerves are innervated deeper into the NP; this is considered to be induced by the abnormal production of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) [3, 4]. Abnormal biomechanical loading applied to AF tissue during the degenerative IVD process leads to structural deficits, such as injury and tear of the tissue. In this process, immune cells such as macrophages, T cells, and neutrophils are activated and infiltrate


into the AF. This hypothesis is verified through studies on blood vessel invasion and CD68+ macrophage, neutrophil, and T cell (CD4+ and CD8+) infiltration into herniated disks [5, 6]. These invading immune cells express pro-inflammatory cytokines such as TNF-α and IL-1β. These molecules can also trigger a vicious cycle of inflammation and dysregulation of the extracellular matrix (ECM). We have previously demonstrated that IL-1β-stimulated AF cells secrete several inflammatory mediators [7]. In this study, we used inflammation in vitro model using the macrophage-conditioned medium (MCM) including a several of pro-inflammatory cytokines, which is already proven the previous studies [8–11]. The AF is composed of ECM with collagen type I and fibrocartilaginous tissue. ECM remodeling is believed to be regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [12]. The IVD in its normal state has a constant ratio of MMPs to TIMPs. However, a higher ratio is found in degenerative disks compared to controls [13]. Additionally, pro-inflammatory cytokines are generally known to play a major role in matrix degeneration by upregulating proteolytic enzymes such as MMPs [14]. Furthermore, several studies have reported that in diseased IVDs, dramatic increases in the levels of MMPs, including MMP-1, MMP-3, MMP-7, and MMP-9, promote the degenerative response [15, 16]. Based on these molecular pathogenic mechanisms, a logical approach to ameliorate or restore symptomatic disk degeneration would be to modulate the production of ECM-modifying enzymes. In the last few decades, PBM has been widely applied in studies of cell proliferation, wound healing, acute/chronic inflammation, and pain control [17–20]. PBM showed an antiinflammatory effect through regulation of cyclooxygenase-2 (COX-2)-related transcription factor expression [21]. Cellular PBM studies showed that PBM regulates reactive oxygen species (ROS) and NF-κB expression in mouse embryonic fibroblasts [22, 23]. Moreover, PBM induced a decrease in expression of IL-1β mRNA and influx of neutrophils in a lipopolysaccharide (LPS)-induced rat inflammation model [24]. PBM is also involved in changes in MMP activity and tendon healing through collagen synthesis in ECM. However, the mechanism by which PBM affects inflammation and ECM modulation is largely unknown, moreover, these studies only investigated a narrow wavelength range of 630–660 nm, and there is no applied PBM study on symptomatic IVD degeneration in the spine to date. For wider application and to verify the effect of PBM, a variety of ranges and doses must be evaluated. Our group studied a spectrum range of 470, 525, and 635 nm, and doses of 16, 32, and 64 J/cm2. We demonstrated inflammation in an in vitro model with AF cells in MCM and applied a wide spectrum and dosage of PBM to these cells to investigate the modulating effect on ECM-modifying enzymes.

Lasers Med Sci (2016) 31:767–777

Materials and methods AF cell isolation and culture Human AF cells were isolated from disk tissues removed during elective surgical procedures on 5 female patients. This study was conducted with the approval of the institutional review board of Korea University Hospital (KUGH12202-001), and their written informed consent was obtained from the subjects. The disk tissues were collected from patients with degenerative spinal disease (degenerative grade II-III). Tissue specimens were cultivated in sterilized Ham’s F-12 medium supplemented with 1 % penicillin/streptomycin (P/S; Gibco-BRL) and 5 % fetal bovine serum (FBS; Gibco-BRL). The disk tissues were washed three times with Hank’s balanced salt solution (HBSS; Gibco-BRL) containing 1 % P/S, and an incision was made in the AF part of the disk. The tissues were subjected to 60 min of digestion in F12 medium containing 1 % P/S, 5 % FBS, and 0.2 % pronase (Calbiochem, La Jolla, CA, USA), followed by a day of incubation in medium containing 0.025 % collagenase. A sterile nylon-mesh filter (pore size 70 μm) was used for tissue debris removal, and cells were isolated. The supernatant was centrifuged at 676g for 5 min and resuspended in F-12 medium containing 10 % FBS and 1 % P/S. The cells were cultured in 75cm2 cell culture flasks (VWR Scientific Products, Bridgeport, NJ, USA) at 37 °C in a humidified atmosphere with 5 % CO2. Macrophage-like THP-1 cell culture/differentiation and MCM collection The human leukemia monocyte cell line THP-1 (ATCC TIB202; ATCC, Manassas, VA, USA) have been widely used to investigate immune responses in the macrophage-like state [25], and can be differentiated with phorbol myristate acetate (PMA) treatment into macrophage-like cells, which can secrete pro-inflammatory cytokines such as TNF-α and IL-1β. The treated THP-1 cells were cultured in RPMI 1640 medium (ATCC) containing FBS, 1 % P/S, and 0.05 mM 2mercaptoethanol (2 ME; Sigma Chemical Co., St. Louis, MO, USA), with medium supplementation every other day. The cultured THP-1 cells were centrifuged at 676g for 5 min, and the supernatant was resuspended using 5 mL of RPMI 1640 medium. To induce THP-1 cell differentiation, 5 × 106 cells were seeded in 75-cm2 culture flasks with RPMI 1640 medium containing 160 nM PMA. After 72 h, PMA-treated THP-1 cells differentiated into macrophage-like THP-1 cells, with pro-inflammatory cytokine secretion and attachment to the cell culture plate. The cells were washed three times using phosphate-buffered saline (PBS), and supplemented with 25 mL of DMEM/F12 medium containing 1 % FBS and 1 % P/S. The cells were cultured for 24 or 48 h, and MCM was collected. The supernatant was stored at −80 °C for ELISA and other experiments.

Lasers Med Sci (2016) 31:767–777

MCM stimulation of AF cells in a degenerative spinal microenvironment After cell isolation from disk tissue, AF cells were cultured in F-12 medium, and the medium was aspirated and washed three times with PBS at pH 7.2. Then, AF cells were trypsinized and the supernatant was removed after centrifugation at 108g for 5 min. AF cells were resuspended by adding MCM obtained from activated macrophage-like THP-1 cells, and 2.0 × 106 AF cells/mL were seeded in 6-well culture plates and maintained at 37 °C in a humidified atmosphere with 5 % CO2. To determine conditions needed for induction of inflammation and attachment to the culture plate, the cells were cultured for 24, 48, or 72 h, and the supernatants were stored at −80 °C for ELISA and other experiments. Design and implementation of the custom-made PBM system


distributing the LED diode dosage evenly on the cells, the apparatus was designed for application of an 8° LED lens (Edison Opto Corp., Ltd., Chung-Ho, Taipei 235, Taiwan). The distance between the LED array and the 6-well microplates was 1.8 cm, and the diameter of the LED diode beam was designed for 3.38 cm, so that it could fully cover the 6-well plate surface area. In PBM application, various ranges of wavelengths and doses were applied separately to each group. All irradiation experiments were performed on a clean surface at 37 °C in a humidified atmosphere with 5 % CO2. During irradiation, a black cover was placed over the culture plate to block light infiltration, and LED diode arrays were controlled by ATmega128 (Mouser Electronics Inc., Kwun Tong, KL, Hong Kong, China) to maintain atmospheric conditions. Figure 1 depicts the in vitro PBM culture system. The PBM irradiation and experimental treatment parameters are listed in Tables 1, 2, and 3. Biochemical analysis, enzyme-linked immunosorbent assay (ELISA)

An indium gallium aluminum phosphide (InGaAIP) LED (635, 525, and 470 nm) (Photron Co., Ltd., Anseong-si, Gyeonggi-do, Korea) was used as a light source. The LED controller unit array (Top of Machine Company Co., Ltd., Gunpo-si, Gyeonggi-do, Korea) was designed to fit on a 6-well cell culture plate. For

Concentrations of TNF-α, IL-1β, MMP-1, MMP-3, TIMP-1, and TIMP-2 in the supernatant were measured using commercially available ELISA kits (R&D Systems) according to the manufacturer’s protocols. Primary capture antibody was

Fig. 1 Schematic diagram of the experimental design on PBM culture system. The in vitro PBM culture system composed of LED diode, 8° LED lens, LED module fitting cap, and 6-well plate. The spot area

(9 cm2) was calculated by a radius of 6-well cell culture plate. And we controlled the size of LED spot wider by using 8 degree concave lens, for applying evenly the dosage of LED light on the cells


Lasers Med Sci (2016) 31:767–777

Table 1 Device information Manufacturer Model Identifier

LED diode (PHOTRON CO., LTD, Anseong-City, Gyeonggi-do, Korea) 635 nm Red LED, 525 nm Green LED, 470 nm Blue LED

Emitter Type Spatial Distribution of Emitters

InGaAIP LED Four emitters spaced apart in square pattern

Beam Delivery System

Free air

added in 96-well plate. Diluted standards and samples to desired concentrations in blocking solution add 100 μl per well to the ELISA plate. The plate was incubated at room temperature for 2–4 h. And then the plate was washed three times using washing buffer. Secondary detection antibody was added in the primary coated 96-well plate and incubated at room temperature for 1 h. Streptavidin-HRP and substrate solution was added in the plate at room temperature for 20 min. To determine the concentration of each protein level in the supernatant, we used microplate reader to measure the absorbance of the samples at 450 and 570 nm wavelengths.

treatment group), AFMR, AFMG, and AFMB (with MCM + PBM group). Each of the experimental group has five samples, and an identical experiment procedure was applied for three to four times. Hence, we used total over 100 samples used. Differences between numerical variables among the groups were analyzed using one-way ANOVA followed by a post hoc Scheffe’s method. All statistical analyses were performed using the SPSS software (version 21.3, IBM SPSS Statistics Inc., Chicago, IL, USA). A p value < 0.05 was considered statistically significant.

Cell cytotoxicity test

Results Cell cytotoxicity was measured using Cytotoxicity Detection Kitplus LDH as described by the manufacturer. LDH activity released from the cytosol of damaged cells was measured to ensure that the PBM did not influence the observed results. To determine the percentage cytotoxicity, we calculated the average absorbance values of the triplicate samples and controls. And then samples were added in 96-well plate. After LDH reaction mixture was added in the plate, it was incubated at room temperature for 25 min. To determine the concentration of LDH level in the samples, we used microplate reader to measure the absorbance of the samples at 490 or 492 nm wavelength. Statistical analysis The experimental group consists of the naïve AF cells group (without MCM treatment group), AFM group (with MCM Table 2

Quantification of pro-inflammatory cytokines in MCM and time course of ECM-modifying enzyme production by MCM-stimulated AF cells To determine whether MCM obtained from macrophage-like cells can induce a degenerative microenvironment, TNF-α and IL-1β, which are major initiating factors during IVD degeneration, were quantified. THP-1 cells are pre-monocytes and have the potential to differentiate into macrophage-like cells. THP-1 cells are in suspension and do not adhere to the cell culture plate. Through PMA treatment, THP-1 cells differentiate into macrophage-like cells and become adherent. At the terminal differentiation stage after 72 h of PMA treatment, MCM shows significantly high levels of TNF-α (0.50 ± 0.02 ng/mL, p < 0.001) and IL-1β (0.11 ± 0.008 ng/mL, Table 3

Irradiation parameters

Treatment parameters

Parameter [unit]


Parameter [unit]


Wavelength [nm] Spectral bandwidth [nm] Operating mode Optical frequency [GHz] Luminous flux [lm] Average radiant power [mw] Aperture diameter [cm] Beam divergence [deg] Beam profile Beam shape

635, 525, 470

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