M2 Macrophage-Conditioned Medium Inhibits Intervertebral Disc Degeneration in a Tumor Necrosis Factor-α-rich Environment


 BackgroundInflammation is the primary pathological phenomenon associated with disc degeneration; accordingly, the inflammatory cytokine tumor necrosis factor (TNF-α) plays a crucial role in disc degeneration. M1 macrophages produce proinflammatory cytokines that facilitate the progression of intervertebral disc degeneration (IDD).However, the anti-inflammatory and regenerative effects of M2 macrophages on nucleus pulposus cells (NPCs) in IDD progression remain unknown. Here, we aimed to determine the role of M2 macrophages in IDD progression. MethodsM2 conditioned medium (M2CM) was harvested and purified from THP-1 cells; it was then used for culturing human NPCs and a mouse intervertebral disc (IVD) organ culture model. NPCs and IVD organ models were divided into the following three groups: group 1 was treated with 10% fetal bovine serum to actas the control, group 2 was treated with 10 ng/ml TNF-α, and group 3 was treated with 10 ng/ml TNF-α and M2CMto act as the co-culture group. After 3 to 14 days, cell proliferation (CCK-8 assay and western blotting for proliferation markers), extracellular matrix synthesis (quantitative polymerase chain reaction, western blotting, and immunofluorescence), apoptosis (TUNEL staining and western blotting), and NPC senescence (senescence-associated beta-galactosidase staining and western blotting) were assessed.ResultsCD206 and interleukin (IL)-10 levels were increased after 48 h of induction for M2 macrophages (both p<0.01). Cell proliferation was decreased in TNF-α-treated NPCs and was inhibited by M2CM co-culture. Moreover, TNF-α treatment enhanced the apoptosis, senescence, and expression of inflammatory factor-related genes, including IL-6, MMP-13, ADAMTS-4, and ADAMTS-5, whereas M2CM co-culture significantly reversed these effects. M2CM promoted aggrecan and collagen II synthesis but reduced collagen Iα1 levels in TNF-α treatment groups. Using our established three-dimensional murine IVD organ culture model, M2CM suppressed the inhibitory effect of TNF-α of the TNF-α-rich environment. Conclusions﻿Collectively, these results indicate that M2CM promotes cell proliferation and extracellular matrix synthesis and inhibits inflammation, apoptosis, and NPC senescence. This study therefore highlights the therapeutic potential of M2CM for IDD.


Introduction
Low back pain is a global issue that affects nearly 80% of individuals at some time point during their lifetime 1 . Intervertebral disc degeneration (IDD) is considered the pathological basis of low back pain, which reduces the quality of life and generates a massive economic burden 2 . Surgical treatment is a widely accepted, ultimate solution for IDD patients with end-stage degeneration, but is associated with the risk of surgical complications, function loss, and poor clinical effects 3,4 . For most patients with earlystage IDD, current therapies are aimed at mitigating symptoms rather than fundamentally treating the pathological condition 5 . Therefore, biological therapy to restore disc morphology and function to inhibit IDD progression is highly signi cant. The pathophysiology of IDD is characterized by a reduction in the levels of extracellular matrix (ECM) components, including aggrecan and collagen II, and increase in activities of catabolic enzymes, including matrix metalloproteinase-13 (MMP-13) and a disintegrin metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS-4 and ADAMTS-5) 6 . Therefore, maintenance of the anabolism and catabolism balance in the disc may prevent or even reverse IDD.
Although the intervertebral disc (IVD) is widely accepted as an immune-privilege organ, immune homeostasis plays an important role in IVD degeneration and regeneration [7][8][9] . Various types of immune cells such as T and B cells, mast cells, and macrophages have been implicated in IDD [10][11][12] . Recently, macrophages have garnered attention in this regard with more recognition of the roles of different macrophage phenotypes, including pro-in ammatory M1, remodeling M2c, and anti-in ammatory M2a phenotypes, which have been reported in IDD 10,13 . Moreover, studies have reported cellular interactions between M0/M1 macrophages and nucleus pulposus cells (NPCs) under pathological and physiological culture conditions 14 . Furthermore, studies in animal models, including rat, mouse, and dog models, have revealed an increased number of in ltrating macrophages with a high level of in ammatory factors in IDD [15][16][17] . Moreover, a cadaver study demonstrated differences in the distribution of macrophages in different parts of the IVD, with M2 macrophages accumulating more around the granulation tissue, suggesting their remodeling potential in IDD 13 .
Macrophages display plasticity predominantly through the pro-in ammatory state of M1 polarization in the early stage of tissue injury and the anti-in ammation and healing states of M2 polarization in the later stages; the transition state has been described in various tissue injuries 18 . Although studies have increasingly focused on the roles of M0/M1 macrophages and disc cells in IDD, the role of M2 cells remains unclear. M2 macrophages help to maintain tissue homeostasis in the heart 19 , lung 20 , muscle 21 , and spinal cord 22 . We have found that M2 macrophages were also present at high levels in surgical samples of IDD patients, with a signi cantly increased trend in P rrmann grade and age (data not shown). Conditioned medium (CM) contains a mixture of different factors secreted by cells, including growth factors, cytokines, enzymes, nucleic acids, and bioactive lipids, representing a new class of therapeutics with wide applications in disease treatment and injury 23 . M2 macrophage-CM (M2CM) has been reported to inhibit in ammation and apoptosis in chondrocytes, generating a pro-chondrogenic environment by producing immunoregulatory factors [24][25][26] .
However, it remains unclear whether M2CM has anti-in ammatory and remodeling potential to alleviate IDD progression. IDD is primarily associated with in ammation; thus, the pro-in ammatorycytokine tumor necrosis factor-alpha (TNF-α) has been widely used to mimic the degeneration conditions in IDD [27][28][29] . Accordingly, we hypothesized that M2 macrophages attenuate the harmful effects of TNF-α, including the effects on the proliferation, senescence, apoptosis, and in ammation of NPCs. To test this possibility and provide a foundation for the potential therapeutic applications of M2CM in IDD, the aim of this study was to investigate the anti-in ammatory, anti-apoptotic, and anti-senescence effects of M2CM in TNF-αtreated NPCs.

Ethics statement
The study was performed according to the amended declaration of Helsinki; human NP tissues were isolated from eight patients and the detailed information regarding the patients is presented in Table 1. In addition, for IVD culture models, 2-month-old male C57 mouse were used in this study. The experiment was performed according to the amended declaration of Helsinki and was approved by the Committee of Gaozhou People's Hospital (No. 2018-012). Informed consent was obtained from each patient.

Human NPisolationand human NPC culture
NPCs were isolated and harvested as previously reported 30 . Brie y, NP samples were collected and immediately transported to a cell culture room under sterile conditions. After washing with phosphatebuffered saline (PBS) three times, the NP tissues were cut into small pieces (<1 mm 3 ) and digested with 0.2% collagenase II (Sigma, USA) for 3-4 h at 37°C in a humidi ed incubator. The suspended cells were then passed through a 200-μm mesh lter and centrifuged at 150 × g for 5 min; this was followed by two washes with PBS. Finally, the cells were cultured in culture medium, consisting of F12 DMEM (HyClone), 10% fetal calf serum (Gibco), and 1% penicillin/streptomycin (Gibco) in 25-cm 2 cellculture asks at a density of 1 × 10 5 cells/mL in a humidi ed incubator at 37°C under 5% CO 2 . After 3 days, the suspended cells and medium were removed, and the adherent cells were cultured and the medium was replaced every 2-3 days. As the cells reached 70%-80% con uency, the primary cells were harvested and passaged. Passage 1 (P1) NPCs were harvested with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA; Sigma) for 1 min and subcultured at a ratio of 1:3. After the cells were gradually passaged, P3 cells were harvested or cryopreserved for further experiments.

Isolation and cultureofIVD organ culture models
Eight-week old male C57 mice were obtained from experimental animal center of Southern Medical University. The mice were euthanized, and then the tails were cut from the base segment of the tail. After soaking in Iodophor for 5 min, the tail skin was removed and tail spines were harvested under sterile condition. The Co7/8 andCo8/9IVD were harvested under sterile conditions, and the dissected IVDs were rinsed in sterile PBS and immediately placed in culture medium as mouse IVD organmodels.Every ve IVDs were cultured in 25-cm 2 cell culture bottles with 15mL culture medium containingF12 DMEM (HyClone), 10% fetal calf serum (Gibco), and 1% penicillin/streptomycin (Gibco). Samples were cultured in a humidi ed incubator at 37°C under 5% CO 2 , and the culture medium was replaced every 3 days.

Harvest of human M2CM and treatment of samples
The THP-1cells were seeded at a density of 2×10 6 in175-cm 2 cell culture bottle for 24h, and then treated with PMA (100 ng/mL; RD, Littleton, CO) for 24h. Thereafter, they were changed into serum-free media and treated with or without IL-4 (100 ng/mL; RD) for another 24h. The supernatant medium was replaced with serum-free medium and cultured for an additional 24h. Finally, the corresponding supernatant conditioned medium was obtained and centrifuged for 15 min at 4°C at 600× g to remove cellular debris and repeated at 1500× g. The harvested supernatant conditioned medium was de ned as M2 macrophage-conditioned medium (M2CM) in the study ( Figure 1A). After culturing NPCs or IVD organ models in medium for 24 h, they were divided into three groups as follows: group 1 was treated with 10% FBS culture medium as the control, group 2 was treated with 10 ng/mL TNF-α, and group 3 was treated with 10 ng/mL TNF-α and 30% M2CM as the co-culture group. The entire procedure is shown in Figure   1A.

Cell proliferation assay
Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Japan) was used as previously described to measure cell proliferation 28 . Brie y, NPCs were seeded in 96-well plates (2× 10 3 cells/well) and different groups were incubated for 24, 48, and 96h. After removing the culture medium and M2CM, 10 μL of CCK-8 solution was added to 100 μL of fresh medium and the mixture was incubated at 37°C for 1 h. Finally, the samples were added to 96-well plates for nal measurements. The absorbance of the solution was measured at 450 nm using a microplate absorbance reader (Bio-Rad, USA). A blank 96-well plate was used for zero setting. All experiments were performed four times for each sample.
2.6 Cell apoptotic ratedetermination using the TUNEL assay Cell apoptosis was con rmed using the TUNEL assay (Promega, Madison, WI) according to the manufacturer's instructions and previous reports 28 . Brie y, the cells were washed with PBS three times and xed in 4% paraformaldehyde for 30 min. Thereafter, the cells were washed with PBS three times and incubated with TdT-mediated dUTP for 1 h in the dark. The cells with the entire nuclear area labeled redwere de ned as apoptotic cells. The cell nuclei were stained with DAPI solution (1:1000; Invitrogen) for 2 min at room temperature in the dark. The cells were counted and averaged for three different sets of experiments. The positivity of cell apoptosis was analyzed using Image-Pro Plus software (Version 5.1; Media Cybernetics, Inc., USA).All experiments were performed four times for each sample. Quanti cation was performed by counting the number of SA-β-gal-positive cells and thetotal number of cells from three randomly selected areas for each sample. All analyses were carried out in triplicate.

Immuno uorescence microscopy
NPCs were plated in at-bottomed 24-well plates (1 × 10 4 /well) and xed with 4% paraformaldehyde for 30 min, permeabilized with 0.2% triton X-100 in PBS (PBS-T) for 10 min, blocked with PBS containing 5% FBS, and incubated with antibodies against collagen II (1:100; Abcam, UK) at 4°C overnight. As a negative control, the cells were incubated with antibody diluents without antibodies under similar conditions. After washing, the cells were incubated with anti-rabbit secondary antibody (Jackson, USA) at a dilution of 1:100 for 1 h at room temperature. Following this, the cells were washed three times and the cell nuclei were stained with DAPI solution (1:1000; Invitrogen) for 2 min at room temperature. The samples were examined and photographed using a uorescence microscope (FV-1000; Olympus). For quantitative examination, the immunostaining results for the cells were analyzed using Image-Pro Plus software (Version 5.1; Media Cybernetics, Inc.).

Real-time PCR analysis
After incubation under different conditions for 2days, the total RNA was extracted from NPCs or IVDs, using TRIzol (Invitrogen) according to the manufacturer's instructions. The RNA concentration was determined by spectrophotometry and RNA was reverse-transcribed using the PrimeScript™ RT Master Mix (TaKaRa, China). qPCR was performed in triplicate in 96-well plates, using the SYBR Premix Ex Taq Kit; the nal volume of the reaction mixture was 20μL. All primers were obtained from Sangon (Shanghai, China) and are listed in Supplementary Table 1. qPCR was performed using the One Step SYBR* PrimeScript RT-PCR Kit (TaKaRa, China). GAPDHwas used for normalization. The cycle threshold values were obtained and data were normalized to GAPDH expression using the 2 -△△Ct method.

Statistical analysis
The results are shown as mean ± standard deviation(SD).Student's t-test or analysis of variance (ANOVA)was used to determine the statistical difference between groups. Mann-Whitney non-parametric test was performed to compare mean positivity between groups.All statistical analyses were carried out using SPSS software (V11.0; SPSS, Inc., Chicago, IL). Differences were considered statistically signi cant at P<0.05.All quantitative results were calculated from a minimum of three biological replicates.

Identi cation of polarized M2 macrophages
THP-1 cells were cultured in a cell culture bottle in suspension medium and treated with PMA for 24 h; the cells gradually adhered to the bottom of the culture bottle as M0 macrophages ( Figure 1B). Thereafter, the cells differentiated into M2 macrophages in the presence of IL-4 for another 24h in serum-free medium, exhibiting a homogeneous elongated spindle-like morphologywith slender tentacles ( Figure  1B).The expression of M2 macrophage markers CD206 and IL-10 was signi cantly upregulated in M2induced macrophages compared with that in M0 macrophages (both p<0.01, Figure 1C and D).

M2CM promoted the proliferation of TNF-α-treated NPCs
The CCK-8 assay was performed to evaluate the proliferative potential of NPCs. The optical density (OD) of TNF-α-treated NPCs was signi cantly lower than that of the control group at 24, 48, and 96h (all p <0.05; Figure 2A). Interestingly, in the TNF-α-treated groups, co-culture with M2CM resulted in a higher OD than that observed in cells treated with only TNF-α (all p <0.05; Fig. 2A). Additionally, a signi cantly lower number of NPCs was detected in the co-culture group than in the TNF-α-treated groups at 24, 48, and 96 h (p< 0.05, p< 0.05, and p< 0.01, respectively; Figure 2B). In contrast, in TNF-α-treated NPCs, a higher cell number was observed in the M2CM co-culture group, with a signi cant difference at all time points (all p < 0.05; Figure 2B). Furthermore, western blotting for the cell proliferation markers PCNA and cyclinD1 in the control group and TNF-α-treated NPCs with or without M2CM co-culture showed signi cant downregulation in the expression of both markers in TNF-α-treated NPCs (p <0.01 and p <0.05) compared with that in the control group ( Figure 2C); however, co-culture of TNF-α-treated NPCs with M2CM upregulated the expression of PCNA and cyclinD1 (both p <0.05; Figure 2D and E).

M2CM protected TNF-α-treated NPCs from apoptosis
The effect of M2CM on TNF-α-induced apoptosis of NPCs was assessed using the TUNEL assay. Upon treatment with TNF-α for 3days, the apoptotic rate gradually increased in NPCs compared with that in the control group (p <0.001), whereas co-culture with M2CM signi cantly mitigated this effect (p <0.01; Figure  3A, B). As shown in Figure 3C-E, western blotting revealed that the expression of the pro-apoptotic protein Bax was upregulated, whereas the expression of the anti-apoptotic protein Bcl-2 was downregulated in TNF-α-treated NPCs (p <0.01 andp <0.05, respectively); however, these effects were reversed upon coculturing with M2CM (both p <0.05).

M2CM upregulated ECM components in TNF-α-treated NPCs
To evaluate the effect of M2CM on ECM biosynthesis, immuno uorescence staining of collagen II in NPCs was performed ( Figure 4A). After treatment with TNF-α for 3days, the OD value signi cantly decreased (p <0.01; Figure 4B); however, this effect was considerably inhibited in the co-culture group (p <0.05; Figure 4B). Western blotting was performed to analyze the changes in aggrecan and collagen I expression ( Figure 4C). Aggrecan expression was also downregulated in the TNF-α-treated group (p <0.05; Figure 4D) but was upregulated in the co-culture group (p <0.05; Figure 4D), whereas collagen I expression displayed the reverse pattern (both p <0.05; Figure 4E). Finally, we investigated ECM-related gene expression by evaluating the expression of genes encoding collagen type Iα1, collagen type IIα1, and aggrecan. Although the expression of ECM-related genes (aggrecan and collagen IIα1) was downregulated in TNF-α-treated NPCs (p <0.001 and p <0.01, respectively), M2CM signi cantly eliminated this inhibitory effect (both p <0.05; Figure 4F, G). Conversely, the expression of collagen type Iα1 was further upregulated in the TNF-α-treated group (p <0.05; Figure 4H); co-culturing with M2CM signi cantly inhibited this increase (p <0.05; Figure 4H). Together, these results indicated that M2CM potentially promotes matrix synthesis in TNF-α-treated NPCs.

M2CM decreasedsenescence and in ammatory factor levels in TNF-α-treated NPCs
Cell senescence was analyzed via SA-β-gal staining and determination of senescence-associated gene expression. The number of SA-β-gal-positive NPCs increased upon TNF-α treatment compared with that in the control group after 7 days (p <0.01; Figure 5A and B). However, when co-culturing TNF-α-treated cells with M2CM, the number of SA-β-gal-positive cells signi cantly decreased (p <0.05; Figure 5A and B). With respect to cell senescence-related genes, TNF-α signi cantly upregulated the expression of p16, p21, and p53 compared with that in the control group (p <0.01,p <0.01,and p <0.05, respectively; Figure 5C-E);however, M2CM downregulated the expression of these markers after TNF-α treatment (all p <0.05; Figure 5C-E). The effect of TNF-α on the secretion of in ammatory factors (IL-6) and extracellular proteases (MMP-13, ADAMTS-4, and ADAMTS-5) by NPCs was assessed after treatment for 48h. TNF-α signi cantly upregulated the expression of all four in ammatory genes compared with that in the control group (p <0.05,p <0.001,p <0.01, and p <0.001, respectively; Figure 5F-I);however, M2CM signi cantly downregulated these markers after TNF-α treatment (all p <0.05; Figure 5F-I).

M2CM decreased IVD degeneration in TNF-α-treated IVD organ cultures
To evaluate the effect of M2CM on ECM biosynthesis, HE staining of mouse IVD organ models was performed. After 2 weeks of culture, the nucleus pulposus (NP)tissue in the control group displayed a spherical morphology and accounted for 50% of the disc area, and NPCs displayed a stellar or spherical morphology. In contrast, in the TNF-α-treated group, the NP constituted 25%-50% of the disc area, and large, spherical NPCs were separated by dense areas of a proteoglycan matrix ( Figure 6A). However, these degeneration effectswere reversed in both NP and NPCs ( Figure 6A).We further analyzed the annulus brosus (AF) and annulus brosus cells (AFCs) and found thatthe ruptured or serpentine bers constituted<25% of the AF. Furthermore, broblasts, comprising>75%-90% of cells, were detected in the control group, whereas the ruptured or serpentine bers constituted >50% of the AF. Moreover, chondrocytes, comprising >75% of the cells, were detected in the TNF-α-treated group ( Figure 6A). Notably, with co-culture in M2CM, the degeneration of both AF and AFCs was inhibited ( Figure 6A). Finally, the border between the NP and AF resulted from minimal, moderate, and severe interruption in the control, co-culture, and TNF-α-treated groups, respectively ( Figure 6A). The modi ed histological grading system of IVD was used for further quantitative analysis, and the results demonstrated that the TNF-αtreated group displayed signi cant degeneration-related morphologicalchanges with higher scores than the control group (p <0.05, Figure 6B), whereas the co-culture group displayed a signi cant decreasewithlower scores (p <0.05, Figure 6B). Furthermore, we investigated ECM synthesis by evaluating the expression of genes encoding collagen type IIα1 and aggrecan. Although the expression of ECM proteins (aggrecan and collagen IIα1) was decreased in the TNF-α-treated mouse IVD model (both p <0.01, Figure 6C and D), M2CM signi cantly abolished this inhibitory effect (both p <0.05, Figure 6C and D).

Discussion
M2 macrophages can downregulate pro-in ammatory cytokines and promote tissue remodeling by producing anti-in ammatory cytokines, including transforming growth factor beta (TGF-β), vascular endothelial growth factor, IL-10, and insulin-like growth factor 18, 33,34 . Furthermore, M2CM has the potential to regenerate numerous tissues; it exerts different effects such as promoting retinal neovascularization, odontogenic differentiation, and epithelial-mesenchymal transition. Importantly, in cartilage cells, which are close to the intervertebral disc cells, macrophages acting as immune cells are of importance in the symptomology and structural progression of osteoarthritis 33,35,36 . However, whether it exerts similar effects in NPCs during IDD progression has remained unknown. To our knowledge, no study has focused on the protective effect of M2CM in IDD to date. Hence, consistent with other studies, we mimicked the in ammatory environment using TNF-α to promote apoptosis, cell senescence, and ECM catabolism in NPCs. Thereafter, we compared these effects in cells with or without TNF-α treatment cultured in M2CM. Together, our results show that the protective effects of M2CM in IDD were mediated through the promotion of cell proliferation; synthesis of ECM; and inhibition of in ammation, apoptosis, and NPC senescence, thereby alleviating IDD. Our results provide fundamental evidence regarding the clinical application of M2CM therapies.
Macrophage reprogramming, characterized by the transformation of M1 to M2 macrophages, is reportedly an effective treatment alternative for osteoarthritis 18, 36 . In cartilage degeneration, the pro-in ammatory M1 macrophages contribute to osteoarthritis, whereas M2 macrophages can reverse this effect or favor chondrogenesis. In IDD, a mixed population of M1 and M2 macrophages was observed in both patients and mouse models, suggesting that both cell types contribute to IDD pathophysiology 13,37 .
Recent studies have reported interactions between macrophages and IVD cells 14,38 . Although M2 macrophages are known as wound-healing and remodeling macrophages, and provide a protective environment in injured tissues, their potential anti-in ammatory effects and ability to mediate remodeling in IDD have remained unknown. In this study, we successfully induced the differentiation of THP-1 cells to M2 macrophages, as previously described 14,32 . The change in cell morphology and signi cant upregulation of the M2 surface markers CD206 and IL-10 indicated that polarization was successful. In the conditioning medium, we used 30% M2CM in the total culture medium in accordance with a previously reported similar co-culture system 14,36 . Thus, we successfully induced the generation of M2 macrophages and harvested M2CM by supplementing IL-4 to THP-1 cells.
Disc degeneration results in a concomitant reduction in the number of NPCs, which are responsible for the production of NP matrix components. Therefore, regenerative therapies depending on the stimulation of cell proliferation are promising and feasible. NPC proliferation decreased in the TNF-α-treated group; however, proliferation was restored upon M2CM co-culture. Furthermore, the increase in cell number was con rmed to be due to an increase in cell proliferation rather than the inhibition of cell death. These data are critical for the development of potential treatments aimed at inhibiting the reduction in cell number observed during IDD 39 . This pro-proliferative effect can be potentially attributed to several cytokines, including TGF-β1, ARG-1, and chemokine ligand 18 (CCL-18), which reportedly promote cell proliferation 24,40,41 . However, further studies are still needed to determine the detailed mechanism underlying the observed effects.
Another important nding of this study is that M2CM decreased the incidence of NPC apoptosis. Excessive disc cell apoptosis, leading to decreased cell density and ECM catabolism, exerts a detrimental effect in IDD 42,43 . Here, the TUNEL assay showed that the increased apoptotic rate of NPCs treated with TNF-α was signi cantly reversed upon co-culturing with M2CM. Furthermore, the expression of the proapoptotic protein Bax and the anti-apoptotic protein Bcl-2 was signi cantly upregulated and downregulated, respectively, in TNF-α-treated NPCs, and these changes were signi cantly inhibited upon co-culturing with M2CM. Notably, aberrant apoptosis of NPCs is considered a major cellular phenomenon associated with IDD 44 . Together, these results indicate that the apoptotic rate of TNF-α-treated NPCs was reduced upon co-culturing with M2CM.
The balance between ECM anabolism and catabolism by disc cells is disrupted by pro-in ammatory cytokines during IDD 45 . Hence, inhibition of these in ammatory cytokine-mediated pathological processes might promote NPC synthesis and result in increased ECM deposition. In this study, we used exogenous TNF-α stimulation to mimic the degeneration environment, and we found that M2CM increases ECM synthesis by promoting both gene and protein expression of aggrecan and collagen IIα1.
However, the downregulation of collagen Iα1 expression suggests that the inhibitory effect of M2CM during brosis further facilitates IVD regeneration. Together, these results indicate that M2CM promotes ECM synthesis in TNF-α-treated NPCs. Several cytokines such as TGF-β1, ARG-1, and CCL-18 have been reported in M2CM with pro-chondrogenic effects 18 . Hence, we speculate that these factors may be key regulators in the M2CM responsible for protecting TNF-α-treated NPCs in our study. Nonetheless, further studies are required to isolate and identify these factors for clinical application.
NPCs maintain normal NP tissue homeostasis 46 . However, the gradual increase in NPC senescence during IDD has a detrimental effect by decreasing the number of functional cells; thus, the inhibition of NPC senescence is considered an important strategy for IDD treatment. SA-β-gal is a senescent cell biomarker 47 . In our study, a signi cantly lower percentage of SA-β-gal-positive cells were observed in the co-culture group than in the TNF-α group, indicating a protective role of M2CM on NPC senescence. Our results suggest that M2CM can attenuate premature senescence of NPCs in an in ammatory microenvironment. The telomere-based p53-p21-pRb pathway and the stress-based p16-pRb pathway are the predominant pathways in IDD 44 . Our results suggest that both pathways mediate the effect of TNF-α on NPCs in vitro, and that the extrinsic factor p16 plays a particularly crucial role, as indicated by its marked upregulation. Moreover, co-culture with M2CM led to the downregulation of pro-in ammatory cytokines (IL-6) and extracellular proteases (MMP-13, ADAMTS-4, and ADAMTS-5), which are widely accepted as risk factors during disc degeneration. Together, these ndings further strengthen our conclusion.
Finally, three-dimensional organ culture models for IVD have been widely used to investigate the effects of speci c treatment strategies in a controlled environment 48 . Coccygeal animal disc models are most commonly used owing to their availability and similarity to human IVDs, and the mouse model is highly similar to the human lumbar disc with respect tothe disc height, anteroposterior width, NP, disc torsion mechanics, axial compression mechanics, and glycosaminoglycan content 49 . Herein, the mouse IVD organ culture model was used to assess the biological effect of M2CM in TNF-α-added medium. A modi ed histological scoring system was used to analyze the protective effect of M2CM 50 . We found that treatment with only TNF-α signi cantly increased the score in the IVD organ culture model, whereas co-culturing with M2CM inhibited this effect. Furthermore, our results indicate that degenerative culture conditions considerably attenuated the downregulation of collagen IIa and ACAN expression, thus demonstrating the regeneration potential of M2CM in the three-dimensional model.
Although the present results highlight the potential of M2CM in disc repair therapy, this study also has some limitations. First, the degenerated environment in IDD is multifactorial and di cult to mimic completely with only exogenous TNF-α supplementation. Second, the microenvironment regulated by proand anti-in ammatory macrophages is usually in a dynamic balance, and M1/M2 is associated with the two major and opposing activities of macrophages 51 . Hence, the predominant role of M2 cells likely involvesinhibiting or maintaining a balance with M1 macrophages. Therefore, it is di cult to analyze this potential, which may limit the applicability of our results to some extent. Furthermore, although the present conclusion is based on evidence obtained from invitro and IVD culture models, the therapeutic effects on disc degeneration in vivo still need to be assessed in an animal model. Furthermore, the precise cytokines and mechanism of action of M2CM remain unclear and warrant further investigation; our future work will focus on elucidating the underlying mechanisms. Finally, the sources of NPCs from disc cells of different species may interfere with M2CMto some extent, as the primary NPCs originate from different degenerating IVD samples.

Conclusion
TNF-α successfully induced premature senescence of NPCs and exerted detrimental effects, including the proliferation and senescence of NPCs, and the expression of matrix macromolecules in NPCs. Our study revealed that M2CM positively in uences NPCs and IVD culture models in an anti-in ammatory microenvironment by increasing cell proliferation, decreasing cellular senescenceand apoptosis, and promoting ECM accumulation. Our study provides novel insights into the therapeutic potential of M2CM in IDD. Further studies are warranted to provide adequate evidence regarding the therapeutic potential of resident IVD cells producing su cient ECM for IDD treatment.

Availability of Data and Materials
The details of information used and analyzed for the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate The study was performed according to the amended declaration of Helsinki; human NP tissues were isolated from eight patients and the detailed information regarding the patients is presented in Table 1. In addition, for IVD culture models, 2-month-old male C57 mouse were used in this study. The experiment was performed according to the amended declaration of Helsinki and was approved by the Committee of Gaozhou People's Hospital (No. 2018-012). Informed consent was obtained from each patient.     were analyzed in the three groups. All data are expressed as mean ± SD; n=3 donors, *p<0.05, **p<0.01. collagen Iα1(G), and collage IIα1(H) in NPCs were compared among different groups after 3 days of culture. All data are expressed as mean ± SD, n = 3 donors, * p< 0.05, ** p< 0.01, *** p< 0.001.