Photobiomodulation of extracellular matrix enzymes in human nucleus pulposus cells as a potential treatment for intervertebral disk degeneration

Intervertebral disc (IVD) degeneration is associated with imbalances between catabolic and anabolic responses, regulated by extracellular matrix (ECM)-modifying enzymes such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs). Potential contributing factors, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, derived from infiltrated, activated macrophages within IVD tissues, can trigger abnormal production of ECM-modifying enzymes and progression of IVD degeneration. Novel therapies for regulating ECM-modifying enzymes can prevent or ameliorate IVD degeneration. Photobiomodulation (PBM), known to regulate wound repair, exhibits regenerative potential by modulating biological molecules. This study examined the effects of PBM, administered at various wavelengths (630, 525, and 465 nm) and energy densities (16, 32, and 64 J/cm2), on the production of ECM-modifying enzymes in replicated degenerative IVD. Our results showed that PBM selectively inhibited the production of ECM-modifying enzymes in a dose- and wavelength-dependent manner, suggesting that it could be a novel tool for treating symptomatic IVD degeneration.


Materials and Methods
Isolation and culture of human NP cells. Human NP cells were obtained from IVD tissues removed from consenting patients during surgical procedures. The tissues were obtained according to the regulations and all experimental protocols were approved by the institutional review board of Korea University Hospital (KUGH17208-001). Written informed consent was obtained from the patients. All methods were carried out in accordance with relevant guidelines and regulations. IVD tissue specimens were placed into sterilized Ham's F-12 medium (Gibco-BRL) supplemented with 5% fetal bovine serum (FBS; Gibco-BRL) and 1% penicillin/ streptomycin (P/S; Gibco-BRL). After washing the tissues, the definitive NP regions were dissected and digested for 60 min in F-12 medium containing 1% P/S, 5% FBS, and 0.2% Pronase (Calbiochem, La Jolla, CA, USA), followed by incubation for 24 h in a medium containing 0.025% collagenase. Cells were filtered using a sterile nylon mesh (70-μm pore size) to remove tissue debris and isolate human NP cells. The isolated NP cells were cultured in 75-cm 2 cell culture flasks (VWR Scientific Products, Bridgeport, NJ, USA) in a humidified atmosphere with 5% CO2 at 37 °C.

Differentiation of human monocytic leukemia THP-1 cells into activated macrophage-like cells and generation of macrophage-conditioned medium (MCM). The human monocytic leukemia
THP-1 cell line (ATCC TIB202; ATCC, Manassas, VA, USA) was seeded into 75-cm 2 cell culture flasks containing Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 160 nM phorbol myristate acetate (PMA), 1% FBS, and 1% P/S. After 48 hours, activated macrophage-like THP-1 cells continued to secrete proinflammatory cytokines, such as IL-1β and TNF-α, which can trigger degenerative conditions. The cells were washed with phosphate buffered saline (PBS; Gibco-BRL), and cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) containing 1% FBS and 1% P/S for 48 hours. To justify the effectiveness of NF-κB inhibitor, cells were pre-treated with BAY11-7082 (Sigma-Aldrich) for 1 hour, followed by treatment for 48 hours with MCM and BAY11-7082. The supernatant (containing the potential contributing factors) was stored at −80 °C until an enzyme-linked immunosorbent assay (ELISA) and further experiments were done.
Macrophage-mediated degenerative response in PBM-irradiated human NP cells that mimic degenerative IVD conditions. Human NP cells were plated at a density of 5 × 10 4 cells per well in 6-well culture plates containing DMEM/F12 supplemented with 1% FBS and 1% P/S. After 48 hours, the medium was removed and MCM was added for an additional 48 hours. A range of wavelengths (465, 525, and 630 nm) and SCieNtifiC REPORTS | (2018) 8:11654 | DOI:10.1038/s41598-018-30185-3 doses (16, 32, and 64 J/cm2) were used to apply PBM to each separate group. This irradiation parameter was determined by our previous studies 26,27 . The supernatant was then harvested, and the production profiles were analyzed using ELISA. mRNA expression levels were analyzed by qRT-PCR. All the irradiation experiments were performed on a clean surface at 37 °C in a humidified atmosphere with 5% CO2. An indium gallium aluminum phosphide (InGaAIP) light-emitting diode (LED) (630, 525, and 465 nm) (Photron Co., Ltd., Anseong-si, Gyeonggi-do, Korea) was used as light source. We have developed three distinct devices, each for a particular wavelength of LED. The PBM platform was controlled by the ATmega128 microcomputer unit (Mouser Electronics Inc., Kwun Tong, KL, Hong Kong, China) to maintain the atmospheric conditions. Figure 1 depicts the schematic diagram of experimental design for degenerative conditions and the effects of PBM (Fig. 1). The phototherapy and experimental treatment parameters are listed in Tables 1-3. Enzyme-linked immunosorbent analysis (ELISA). The concentrations of IL-1β, TNF-α, MMP-1, MMP-3, TIMP-1, TIMP-2, ADAMTS-4, and ADAMTS-5 were measured in the supernatant using commercially available ELISA kits (R&D Systems) according to the manufacturers' protocols.

Quantitative real-time polymerase chain reaction (qPCR). Human NP cells were lysed with
Trizol reagent (Invitrogen), RNA was extracted, and cDNA synthesized (Life Technologies) according to the manufacturer's instructions. The quantity and quality of the RNA were determined using a Nanodrop 2000 Spectrophotometer (Thermo Scientific). qRT-PCR was performed for MMP1 and MMP3 using the SYBR Green PCR Master mix (Applied Biosystems). mRNA expression was analyzed using the 2 −∆∆Ct method, in which values  were expressed as the mean fold change normalized to that of the housekeeping gene GAPDH. Naive NP cells, and NP cells exposed to MCM, were used as controls for NP cells irradiated by PBM.
Immunofluorescence staining of nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) p65 protein. Human NP cells were plated on a glass-bottom confocal dish and exposed to MCM for 48 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature, blocked with 5% bovine serum albumin (BSA; Millipore) in PBS, and then incubated with the primary antibodies overnight at 4 °C in 5% BSA. Anti-NF-κB p65 mouse monoclonal antibody (Santa Cruz) was used to detect the NF-κB p65 protein. Goat anti-mouse Alexa 555 secondary antibodies (Invitrogen) and 5% BSA were used for the secondary incubation in PBS for 1 h at room temperature. After washing in PBS, the plate was counterstained with 4′,6-diamidino-2-phenylindol (DAPI, Invitrogen). Images were acquired using the EVOS FL Auto cell imaging system (Thermo Fisher Scientific Inc., USA).
Total collagen measurement (Sircol assay). Human NP cells were stimulated with or without MCM for 48 hours. The amount of total soluble collagen in the supernatants was quantified using the Sircol collagen assay (Biocolor, Belfast, UK). Sirius red dye (500 μL), an anionic dye that reacts specifically with the basic side-chains of collagen during assays, was added to 100 μL of the supernatant and incubated with gentle rotation for 30 min at room temperature. The sample and Sirius red dye mixture was centrifuged at 13,475 g for 10 min, and the collagen-dye complex was washed with an acid-salt wash reagent to remove the unbound dye from the surface of the complex. The collagen-dye complex was re-centrifuged at 13,475 g for 10 min, and the precipitate was collected and re-solubilized in 0.5 M sodium hydroxide. The sample was transferred into a 96-microwell plate. The total soluble collagen concentration was estimated using a spectrophotometer at 555 nm (Beckman Coulter, Fullerton, CA, USA).

Cell cytotoxicity and lactate dehydrogenase assay (LDH).
Measurements of the release of lactate dehydrogenase (LDH) were performed per manufacturer's instructions. After the cells were exposed to PBM, the exposure medium was collected for quantitating the release of lactate dehydrogenase. Viability was calculated with respect to that of the controls (human NP cells treated with MCM). If the human NP cells were damaged by PBM therapy, these cells would show a tendency toward increased LDH production.
Statistical analysis. Data were expressed as the mean ± standard deviation for four or five individual experiments. One-way analysis of variance (ANOVA) and Bonferroni's correction post hoc test were used to assess the differences in the experimental groups. The normal distribution of each subgroup was assessed by the Shapiro-Wilk test. For data not showing normal distribution, we used Kruskal-Wallis with Dunn's multiple comparison test. All statistical analyses were performed using SPSS software (version 21.3, IBM SPSS Statistics Inc., Chicago, IL, USA). A p-value < 0.05 was considered statistically significant.

Activated macrophage-like cells induce degeneration in human NP cells by modulating ECMmodifying enzymes and preferentially distributing the NF-κB p65 protein.
To determine whether macrophage-like THP-1 cells secrete proinflammatory cytokines that initiate degeneration of human NP cells, we analyzed the production of IL-1β and TNF-α in MCM using ELISA. Additionally, to determine the effects of NF-κB inhibition on protein and gene expression of ECM-modifying enzymes and total collagen, we treated human NP cells using BAY11-7082, which reduces NF-κB activation by inhibiting the IκBα phosphorylation.

Group Description
(1) Control Naive human NP cells (2) Macrophage-conditioned medium (MCM) Potential contributing factors derived from activated macrophage-like THP-1 cells (3) Degenerative conditions Human NP cells exposed to MCM (4) Degenerative conditions + phototherapy Human NP cells exposed to MCM with PBM  Table 2. Treatment parameters.
MCM showed a significantly higher expression of IL-1β and TNF-α than that in naive NP cells ( Fig. 2A,B). To investigate the expression of ECM-modifying enzymes in human NP cells exposed to MCM (NPM), the gene and protein expression of MMP-1, MMP-3, TIMP-1, and TIMP-2 were measured in NPM by qRT-PCR and ELISA. The secretion of collagen, which is upregulated in the early stages of IVD degeneration in human NP cells, was identified by the Sircol assay. The production of MMP-1, MMP-3, TIMP-1, and TIMP-2 in NPM was markedly increased compared with that in naive NP cells (Fig. 2D,E,H,I). NPM also exhibited upregulated genetic expression of MMP1 and MMP3 (Fig. 2F,G). Similarly, NPM showed a marked increase in total collagen secretion (Fig. 2C). BAY11-7082 treatment on NPM was able to attenuate the protein production and gene expression of all target factors used in this study compared with NPM ( Fig. 2C-I). Additionally, our fluorescence images revealed that NF-κB p65 protein is preferentially distributed in the nucleus under the presence of MCM rather than in the cytoplasm, where it is associated with the catabolic response by acting as a transcription factor, whereas in the absence of MCM, it was present in the cytoplasm (Fig. 3A). Quantitatively, the p65 activity calculated from the average intensity value in inflamed NP cells was shown to have an increasing trend by potential contributing factors derived from macrophages and most of the detected activity was located in the nucleus at 45 and 60 min compared with naïve NP cells (Fig. 3B).
These results indicate that potential contributing factors, derived from activated macrophages, induce degenerative conditions in human NP cells via an increased production of ECM-modifying enzymes, secretion of collagen, and gene expression of catabolic enzymes such as MMP1 and MMP3. First, we measured the gene and protein expression of MMP-1, known as collagenase-1 in IVD tissues, by qRT-PCR and ELISA. Our mRNA results show that all doses of PBM at 630 nm more significantly suppressed the mRNA expression of MMP1 than that of NPM without PBM (Fig. 4D). These effects result in an inhibited protein production of MMP-1 on NPM, except for that observed at 32 J/cm 2 (Fig. 4A). PBM at 525 nm with 16 and 32 J/ cm 2 had inhibitory effects in production of MMP-1 (Fig. 4B), but all of doses did not significantly bring about a change in mRNA levels (Fig. 4E). At a wavelength of 465 nm, NP cells were regulated by PBM at the mRNA level at all of the doses (Fig. 4F). However, production of the MMP-1 protein did not change significantly during irradiation with PBM at 465 nm (Fig. 4C). Additionally, there was no difference in the production of TIMP-2 as the endogenous inhibitor of MMP-1 (Fig. 4G-I).
These results demonstrated that PBM at 630 nm with 16 and 64 J/cm 2 had an inhibitory effect on degenerative NP cells through regulation of both mRNA and protein, and human NP cells were regulated during the production of MMP-1 protein at 525 nm with 16 and 32 J/cm 2 .

Effects of PBM on gene and protein expression of MMP-3 and its endogenous inhibitor TIMP-1.
We used qRT-PCR and ELISA to examine the regulatory effects of PBM on protein production and genetic expression of MMP-3 and its endogenous inhibitor TIMP-1 in NPM.
Our results show that PBM selectively modulated the mRNA expression of MMP3 at all of the tested wavelengths in dose-dependent manner. However, the change in protein production of MMP-3 was not observed at all the tested wavelengths ( Fig. 5A-C). At the wavelengths of 525 and 465 nm, MMP3 mRNA was significantly down-regulated by PBM at the doses of 16, 32, and 64 J/cm 2 , respectively (Fig. 5E,F); however, the differences in protein production of MMP-3 and TIMP-1 were not significantly different (Fig. 5H,I). Although PBM, at the dose of 64 J/cm 2 and at 630 nm, induces an upregulation in the mRNA expression of MMP3 (Fig. 5D), its protein production was not changed. Interestingly, the production of its endogenous inhibitor TIMP-1 was significantly upregulated by PBM at 630 nm with 32 J/cm 2 (Fig. 5G).
These results indicate that PBM can regulate the genetic expression of MMP3 in dose-and wavelength-dependent manner, but cannot inhibit the levels of the MMP-3 protein. However, PBM at 32 J/cm 2 and 630 nm may exert positive effects via elevating the secretion of the TIMP-1 protein; this is associated with decreased MMP-3 activity in degenerative conditions.

Cytotoxicity assessment using LDH secretion in human NP cells irradiated with PBM. Measurement
of lactate dehydrogenase (LDH) release is a common method used in cytotoxicity assays. Because PBM can damage cells, we tested PBM at the dose of 64 J/cm 2 at all the assessed wavelengths. In this study, 64 J/cm 2 was the  . The lactate dehydrogenase (LDH) assay used to assess LDH released by human NP cells treated with PBM. Human NP cells exposed to MCM were irradiated using PBM at 64 J/cm 2 , which is the maximum dose used in this study. The data show that none of the wavelengths, used in this study, were cytotoxic to human NP cells. Values are mean ± SE of three or four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with control, and line indicates comparison with each group. maximum dose, and it enhanced LDH release from human NP cells exposed to MCM. As shown in Fig. 6, PBM at 525 nm did not significantly up-regulate LDH release from human NP cells. Additionally, irradiation at 630 and 465 nm showed positive effects on the viability of human NP cells (Fig. 6). Together, Fig. 7 depicts a schematic summary of the IVD degeneration in vitro model used in this study and effects of PBM on human NP cells (Fig. 7).

Discussion
We show that potential contributing factors, derived from activated macrophages, induced an upregulation of ECM-modifying enzymes, production of collagen, and preferential activation of the NF-κB p65 protein in human NP cells in vitro, mimicking conditions of degenerative IVD. We confirmed that PBM selectively ameliorated these degenerative conditions via modulation of genes and proteins, associated with production of ECM-modifying enzymes, in a dose-and wavelength-dependent manner.
The onset of IVD degeneration is characterized by angiogenesis of vascular structures into otherwise avascular IVD tissues. Using this route, circulating immune cells, including monocytes, neutrophils, and macrophages, infiltrate these IVD tissues. This hypothesis is supported by the results of studies showing infiltration of CD68 + macrophages, neutrophils, and T cells (CD4 + and CD8 + ), along with invading blood vessels, in herniated discs 16,39 . Proinflammatory cytokines, such as IL-1β and TNF-α, secreted by these infiltrating immune cells, play a major role during progression of the disease. Various studies showed that upregulated expression of proinflammatory cytokines, including IL-1β, TNF-α, IL-6, and IL-17, is observed in degenerative tissues of IVD 16,40-42 . Our results similarly show that potential contributing factors, secreted by activated macrophage THP-1 cells, include proinflammatory cytokines such as IL-1β and TNF-α. IL-1β and TNF-α possess numerous functions, such as stimulating the secretion of inflammatory mediators and inducing the expression of adhesion molecules on endothelial cells; these are responsible for angiogenesis, nerve ingrowth, and chemo-attraction of neutrophils 3 . When these cytokines bind to their receptors including IL-1 receptor type 1 (IL-1R1) and TNF receptor superfamily member 1 A (TNFR1), the resulting intracellular complex leads to activation of the IκB kinase (IKK). IKK phosphorylates the inhibitory IκBα protein, resulting in the nuclear translocation of NF-κB subunits, such as transcription factors p65 and p50, which control the expression of numerous inflammatory and catabolic genes [43][44][45] . A study reported that inactivation of the NF-κB pathway including inhibition of IKK and stabilization of IκB by prolactin treatment significantly alleviated the progression of IVD degeneration through increasing the collagen components. Our previous studies and other reports demonstrated that stimulation with TNF-α and IL-1β induce the upregulation of various catabolic enzymes, including MMP-1, -2, -3, -9, -13, -14, and a disintegrin and metalloproteinase with thrombospondin motifs (ADMATS)-4 and -5, in IVD cells 18,26,46,47 . These enzymes promote the degradation of ECM components, such as collagen and aggrecan, during IVD degeneration. Clinically, in a healthy state, angiogenesis of vascular structures in the IVD is blocked by high levels of sulfate bonding generated by ECM components and aggrecan 8 . However, in herniated and degenerative IVD, there is a marked increase in the expression of MMPs and ADAMTS. These can induce continuous structural breakdown of ECM components 48,49 . Our results indicate that human NP cells exposed to MCM also showed a dramatic increase in the protein and gene expression of MMP-1 and MMP-3. Furthermore, our immunofluorescence images show that under the influence of potential contributing factors derived from macrophages, the NF-κB p65 protein translocated into the nucleus rather than into the cytoplasm of human NP cells. These results show that potential contributing factors derived from macrophages can induce degenerative conditions in human NP cells via upregulation of ECM-modifying enzymes. Some studies reported that blocking the IL-1β-mediated expression of gene encoding MMPs in IVD cells can restore expression of aggrecan and prevent proteoglycan depletion 50,51 . Hence, regulation of these enzymes may be a biological therapeutic target for the treatment of IVD.
Numerous studies have investigated the effects of PBM in ameliorating or treating various diseases. PBM can lead to reduction of inflammation, cell proliferation, increased synthesis of ATP, and regulation of ECM components [25][26][27]35,[52][53][54] . Low-level light irradiation suppresses the inflammatory response in human adipose-derived stem cells by modulating the activity of cyclic AMP and NF-κB 55 . Another study showed that treatment with a low-level laser improved tendon healing by regulating MMP activity and collagen synthesis 38,52 . Various studies have shown that PBM alters the gene and protein expression of MMPs and TIMPs. This modulates collagen production via regulation of the intracellular downstream signaling of c-Jun N-terminal kinase-mitogen-activated protein kinase (JNK-MAPK) and extracellular signal-regulated protein kinase (ERK) pathways 53,56,57 .
For these photo-biological effects, a large proportion of photons must be absorbed on electronic absorption band, which is enabled by specific chromophores. Absorption of a particular chromophore depends significantly on the frequency range. The primary chromophore for red light (600-700 nm) is generally cytochrome c oxidase, which is a multi-subunit membrane-associated enzyme that is the end point of mitochondrial respiratory chain 58 . Increasing evidence suggests that flavins, flavor-proteins, or cryptochromes, in case of ROS generation, are photo-acceptors at shorter (visible) wavelengths 59 , such as green and blue light, although the extent of expression of cryptochromes in mammalian cells is yet to be clarified. Additionally, light of this wavelength range is known to be primarily absorbed by opsins (OPNs). The main signaling pathways induced by OPNs are strongly linked to the family of transient receptor potential (TRP) cation channels such as TRPV1 (capsaicin receptor and the vanilloid receptor 1) 60 . TRPV1 has been shown to be activated by various wavelengths of light including green, red, and near-infrared 61,62 . Interestingly, this cation channel is thought to be a major factor in painful IVD. At present, the leading hypothesis to explain symptomatic IVD degeneration is related to the fact that the TRP cation channel in the dorsal root ganglion (DRG) is expressed by neurogenic factors, such as β-nerve growth factor (β-NGF) and brain-derived neurotrophic factor (BDNF) within degenerative IVD tissues 3 .
As discussed above, there is an "optical window" within tissues ensuring effective infiltration of light, and specific photo-acceptor molecules or chromophores for each wavelength may be considered for applying the effects of PBM. In summary, we hypothesized that PBM may control the gene and protein expression of MMPs or TIMPs in human NP cells. Thus, we investigated the effects of PBM, at various wavelengths (630, 525, and 465 nm) and doses (16,32, and 64 J/cm 2 ), on the regulation of ECM-modifying enzymes in MCM-stimulated human NP cells. Our data show that PBM at 630 and 525 nm successfully and dose-dependently suppressed the expression of MMP-1 without altering TIMP-2 production. Consistent with our results, other study observed that photo irradiation at 660 nm showed upregulation of collagen and down regulation MMP-1 in vitro 63 .
PBM at 525 and 465 nm diminished gene expression of MMP3 in a dose-dependent manner. These results were considered that PBM, applied over a longer period, may alter its protein production. There is possible mechanism for these effects on MMP-3. Recent study showed that phototherapy had the greatest impact on raising mitochondrial membrane potential which will lead to a drop in the levels of reactive oxygen species (ROS) generated in the mitochondria of oxidatively stressed cells 64 . Furthermore, other study showed that inhibition of ROS by N-acetyl-cysteine or diphenylene iodonium significantly suppressed the expression of MMP-3 in lipopolysaccharide (LPS)-stimulated microglia 65 . Thus, we considered that inhibitory effects of PBM on MMP-3 might be modulated by this possible mechanism. However, further studies are needed to elucidate these mechanisms.
PBM at the doses of 32 J/cm 2 at 630 nm revealed that its inhibitory effects occur via the upregulation of TIMP-1. TIMP-1can attach to alternate or active MMP sites, thereby inhibiting MMPs. Consistent with our result for TIMP-1, recent study showed that phototherapy at 660 nm induced significant increased release of TIMP-1 proteins in stressed fibroblast cells 66 . Later on, an increase in the amount of TIMPs might protect the newly synthesized collagen from proteolytic degradation by MMPs. Our results show that PBM exerts different regulatory effects; these depend not only on the properties of PBM, but also on the target protein. Similar to that, the biphasic dose response or Arndt-Schulz curve in PBM has been shown in various in vitro studies and animal models. This phenomenon suggested that insufficient power density that fails to reach the threshold for regulation of gene or protein will have no effect on pathology. In addition, excessive power density may have inhibitory effects or negate the beneficial response induced at optimal power density. Various studies have shown that low-and medium-dose of PBM promoted cell growth, whereas high intensity negated the beneficial effects of PBM in various types of cells 67 . In this study, doses of 16 and 32 J/cm 2 at 525 nm achieved a significant effect on MMP-1 production and MMP3 gene expression; this effect was lost when 64 J/cm 2 was delivered. Additionally, a dose of 16 J/ cm 2 at 465 nm reduced the MMP1 gene expression levels, whereas higher doses with same frequency promoted it. Doses of 32 J/cm 2 at 630 and 465 nm were optimal for the modulation of TIMP-1 and MMP-3 production, respectively, although other doses, examined in this study, negated these effects.
Taken together, understanding the mechanisms of additional photo-acceptors and identification of effective doses (considering the biphasic dose-response for target proteins and genes) would be necessary for clinical application. In addition, the parameters used in this study may not be practically applicable in clinics yet. Since light needs to be delivered to the target tissues or cells with sufficient energy, exploring the optimal dose may be required for clinical application. Thus, fusion of PBM irradiation with light delivery system (for example, photosensitizer and/or light guidance system) may be suggested as a strategy for clinical practice.

Conclusions
In this study, we show that PBM inhibits the macrophage-mediated production of ECM-modifying enzymes in human NP cells in a dose-and wavelength-dependent manner. We conclude that PBM may be a novel tool for the treatment of symptomatic disc degeneration.