Effect of Synovial Cell Fractionation on Tenascin-C Expression in Chondrocytes Under Coculture

Abstract

Objective

This study investigated the effect of synovial cell fractionation on tenascin-C (TNC) expression in chondrocytes by coculturing human chondrocytes with synovial cells derived from osteoarthritis (OA) patients.

Design

Human cartilage and synovium were isolated from patients undergoing total knee arthroplasty. Synovial cells were classified into CD68 positive- and negative groups using western blotting. Cocultures were performed for 7 days using Cell Culture Inserts. The expression of TNC, syndecan-4 (SDC4), and anabolic and catabolic factors was measured by real-time polymerase chain reaction. TNC levels in the medium were compared using enzyme-linked immunosorbent assay. Flow cytometry examined M1 and M2 macrophage proportions in synovial cells immediately after isolation, after 7 days of monoculture, and after coculture.

Results

In the CD68 positive group, TNC and matrix metalloproteinase (MMP)-3 were significantly upregulated in cocultured chondrocytes, and SDC4 was significantly upregulated in cocultured synovial cells. TNC concentration in the medium was significantly higher in CD68 positive cocultures. M1 proportions were significantly higher in synovial cells immediately after isolation and in cocultured synovial cells than in those cultured alone.

Conclusions

Synovial cell fractionation differentially affects TNC and SDC4 expression. Macrophage-like synovial cells (MLS) increase TNC expression in chondrocytes and may contribute to OA pathology.

Keywords

osteoarthritis chondrocytes macrophage-like synovial cells coculture tenascin-C syndecan-4

Introduction

Osteoarthritis (OA) is a progressive joint disease characterized by the degeneration of articular cartilage, affecting more than 500 million people worldwide.¹ OA is associated with functional limitations and knee joint pain, which reduce patients’ quality of life.^2,3 Although the number of patients with OA is expected to continue increasing,⁴ its pathogenesis remains incompletely understood.⁵ OA is a disease of the whole joint, involving structural changes in the articular cartilage, subchondral bone, ligaments, articular capsule, infrapatellar fat pad, synovium, and periarticular muscles.⁴ The interaction between these tissues is thought to play a crucial role in OA development and progression.⁵ The relation between cartilage and synovium is particularly important, and synovitis is considered a precursor of OA.⁶ One study demonstrated a positive correlation between synovitis severity and the progression of cartilage lesions over time.⁷ In addition, it has been suggested that the initial molecular and morphological changes in OA begin in the synovial tissue, followed by alterations in articular cartilage and subchondral bone.⁸ Crosstalk between cartilage and synovium plays a major role in cartilage degradation in OA.^9,10 Therefore, elucidating the mechanisms of this crosstalk is essential for understanding OA pathogenesis and development.

Synovial cells consist mainly of fibroblast-like synovial cells (FLS) and macrophage-like synovial cells (MLS). In joint disease, homeostasis is disrupted, leading to joint destruction.¹¹ Analyses of synovial tissue in OA have reported that synovitis severity correlates with an increased proportion of MLS.¹² A transcriptomics-based study by Wang et al.¹³ constructed a crosstalk spectrum based on predicted ligand-receptor interactions between cartilage and synovium, identifying cartilage-derived tenascin-C (TNC) and synovial syndecan-4 (SDC4) as a ligand-receptor pair. However, the influence of synovial cell fractionation on these interactions remains unclear. MLS can be further classified into M1-like macrophages, which primarily contribute to tissue damage, and M2-like macrophages, which are mainly involved in tissue repair.^14
-17 An analysis of knee joint fluid from patients with OA demonstrated a significant increase in the M1 ratio. In addition, a positive correlation was observed between the M1/M2 ratio and Kellgren–Lawrence (K–L) grade, indicating that an imbalance between M1 and M2 macrophages is associated with OA progression.¹⁸ However, there have been no reports on M1/M2 ratios in synovial cells cocultured with chondrocytes. Given that synovial cells are a heterogeneous population and their proportions vary among patients, we hypothesized that synovial cell fractionation differentially affects interactions with chondrocytes. This study aimed to investigate the influence of synovial cell fractionation on TNC–SDC4 interactions by coculturing human chondrocytes with synovial cells derived from patients with OA and comparing outcomes based on synovial cell fractionation. In addition, the effect of synovial cell fractions on chondrocyte proliferation was assessed, along with the proportions of M1 and M2 macrophages in cocultured synovial cells.

Methods

Patient Selection

This study was approved by the Institutional Review Board (reference number: H2024-096). All procedures were performed in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all patients. Chondrocyte and synovial cell samples were collected during total knee arthroplasty (TKA) from patients with OA. Chondrocytes and synovial cells were isolated from patients undergoing knee replacement for advanced OA under sterile conditions. Cartilage specimens and synovial tissues were obtained from the femoral condyles and suprapatellar pouch of 33 patients who underwent TKA for OA treatment. All patients were preoperative TKA candidates with radiographically confirmed advanced knee OA (K–L grade 3 or 4). Although the cohort was clinically homogeneous in terms of surgical indication, it was heterogeneous with respect to radiographic disease severity, including both K–L grade 3 and grade 4 knees. Considering the effect of specimen volume, 11 specimens were used for real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), 12 for MTS assay, and 10 for flow cytometry. Patient characteristics are shown in Table 1 .

Table 1.

Patient Characteristics.

	Overall(n = 33)	Samples Undergoing PCR and ELISA(n = 11)	Samples Undergoing MTS assay(n = 12)	Samples Undergoing Flow Cytometry(n = 10)
Sex, Male: Female	10: 23	3: 8	5: 7	2: 8
Right-to-left	13: 20	4: 7	5: 7	4: 6
Mean age, years	73.4 ± 7.3	74.3 ± 4.8	72.6 ± 10.9	72.7 ± 5.6
Mean weight, kg	64.0 ± 12.7	62.5 ± 11.2	67.7 ± 15.6	64.0 ± 11.2
Mean height, cm	156.1 ± 8.1	157.2 ± 7.8	155.5 ± 7.6	157.1 ± 10.0
Mean BMI, kg/m²	26.2 ± 4.3	25.2 ± 3.6	27.9 ± 5.6	25.8 ± 3.4
K–L grade, 3:4	10: 23	2: 9	4: 8	4: 6

cm = centimeters; kg = kilograms; BMI = body mass index; K–L = Kellgren–Lawrence grade; PCR = polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay.

Isolation and Culture

The collected cartilage specimens were minced, and the cartilage fragments were incubated in 0.8% Pronase solution (Calbiochem, Darmstadt, Germany) dissolved in Dulbecco’s modified Eagle’s medium/Ham F-12 (DMEM/F-12) (Gibco, Grand Island, NY, USA) for 30 min at 37°C with continuous agitation in an atmosphere of 5% CO₂. After washing with DMEM/F-12, the cartilage pieces were incubated with 0.4% collagenase (Roche Diagnostics, Penzberg, Germany) in DMEM/F-12 for 90 min at 37°C with orbital mixing. The cell suspension was filtered using a 70-μm pore size nylon filter (BD Biosciences, Bedford, MA, USA) to remove tissue debris. The filtrate was centrifuged for 5 min at 1200 rpm. The cells were washed 3 times with DMEM/F-12 containing 10% fetal bovine serum (FBS). Synovial tissues were incubated with 0.2% collagenase (Roche Diagnostics, Penzberg, Germany) in DMEM/F-12 for 120 min at 37°C with orbital mixing. The cell suspension was filtered using a 70-μm pore size nylon filter (BD Biosciences, Bedford, MA, USA) to remove tissue debris. The filtrate was centrifuged for 5 min at 1200 rpm. The cells were washed 3 times with DMEM/F-12 containing 10% FBS. Chondrocytes and synovial cells were cultured in DMEM/F-12 supplemented with 10% FBS, 0.25 μg/mL amphotericin B solution (Sigma Chemical Co., St. Louis, MO, USA), 110 μg/mL kanamycin (Gibco), penicillin-streptomycin (100 IU/mL penicillin, 100 μg/mL streptomycin) (Gibco), and 25 μg/mL ascorbic acid (Sigma). For coculture experiments, 1 × 10⁵ cells were seeded into each well of 6-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) using ThinCert® Cell Culture Inserts (0.4 μm, 6-well, Greiner Bio-One, Frickenhausen, Germany). These inserts with a pore size of 0.4 μm allow diffusion of soluble factors¹⁹ ( Fig. 1 ). Chondrocytes and synovial cells were used as controls. Cells were grown at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Cocultures were maintained for 7 days, after which cells and medium were collected.

Figure 1.

Isolation of human chondrocytes and synovial cells. Cartilage specimens and synovial tissues were incubated with collagenase P, and 1 × 10⁵ chondrocytes and synovial cells were cultured. Cocultures of chondrocytes and synovial cells were performed, with 1 × 10⁵ cells seeded into each well of 6-well plates and Transwell inserts.

Grouping by Western Blotting

After isolation of synovial cells, cells were lysed with radioimmunoprecipitation assay buffer supplemented with a cOmplete™ protease inhibitor cocktail (Roche). Proteins were separated by SDS-PAGE, and samples were adjusted to the same protein concentration before loading. Proteins were transferred to a polyvinylidene difluoride membrane and blotted. Antibodies were obtained from the following sources and used at manufacturer-recommended dilutions: monoclonal mouse anti-CD68 antibodies (1:1000, catalog M0876; Dako, Glostrup, Denmark). β-Actin was used as a loading control. Densitometric analysis was performed using ImageJ software (version 1.53m; National Institutes of Health, Bethesda, MD, USA). Patient samples used for PCR, ELISA, and MTS assay were divided into 2 groups:²⁰ CD68/β-actin > 0 as the CD68 positive group and CD68/β-actin = 0 as the CD68 negative group ( Fig. 2 ). No significant demographic differences were observed between the CD68 positive group (n = 12) and CD68 negative group (n = 11).

Figure 2.

Grouping by western blotting of CD68. (A) After synovial cell isolation, proteins were separated and blotted using anti-CD68 antibodies. β-actin was used as a loading control. (B) Densitometric analysis was performed using ImageJ software, and patients were divided into positive- and negative groups. (C) Western blot images of the samples used for polymerase chain reaction and enzyme-linked immunosorbent assay.

RNA Extraction and Complementary DNA Synthesis

After collection of cultured chondrocytes and synovial cells, total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Complementary DNA (cDNA) synthesis was performed using random primers and a first-strand cDNA synthesis kit (Roche) with 1 μg total RNA as a template. The thermal cycling conditions were as follows: 25°C for 10 min, 42°C for 60 min, 99°C for 5 min, and 4°C for 5 min.

Real-Time PCR

The expression levels of TNC, SDC4, fibronectin, anabolic factors (transforming growth factor [TGF]-β, tissue inhibitor of metalloproteinases [TIMP] 3, basic fibroblast growth factor [bFGF]), and catabolic factors (a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS] 5, matrix metalloproteinase [MMP]-3) were analyzed. In addition, interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) expression levels were evaluated in synovial cells from both the monoculture and coculture groups. TaqMan gene expression assay primer-probe pairs were obtained for the detection of TNC (assay no. Hs01115665-m1), SDC4 (assay no. Hs00161617-m1), fibronectin (assay no. Hs01549976-m1), TGF-β (assay no. Hs00998133-m1), TIMP 3 (assay no. Hs00165949-m1), bFGF (assay no. Hs00256645-m1), ADAMTS 5 (assay no. Hs00199841-m1), MMP-3 (assay no. Hs00968305-m1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (assay no. Hs99999905-m1), IL-1β (assay no. Hs01555410-m1), and TNFα (assay no. Hs01113624-g1). Quantitative analysis of cDNA was performed using an ABI Prism 7000 Sequence Detector System (Applied Biosystems, Foster City, CA, USA) and FastGene (NIPPON Genetics, Tokyo, Japan). The thermal cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase expression and compared between the CD68 positive (n = 6) and CD68 negative groups (n = 5). The expression levels of IL-1β and TNFα were compared between synovial cells cultured alone and synovial cells cocultured with chondrocytes (n = 11).

ELISA

The collected medium was centrifuged at 15,000 × g for 15 min, and the supernatants were stored at –80°C until analyzed. Levels of large-subunit TNC containing the FNIII C domain were determined using an ELISA kit (IBL, Gunma, Japan) with 2 monoclonal antibodies, 4F10TT and 19C4MS. Samples were incubated in 96-well ELISA plates coated with 19C4MS for 1 h at 37°C. After washing, horseradish peroxidase-conjugated anti-TNC Fab’ fragments (4F10TT Fab’) were added. After incubation for 30 min at 4°C, the absorbance at 450 nm was determined using an ELISA plate reader. The results were calculated from the mean absorbance of duplicate wells. TNC purified from the conditioned medium of human glioma cells was used to prepare a standard curve. The TNC levels were compared between the CD68 positive (n = 6) and CD68 negative groups (n = 5).

Proliferation Assay

To evaluate the effects of synovial cell fractionation on chondrocyte proliferation, cell count estimation was performed using a proliferation assay. Cell proliferation was determined using CellTiter 96 (Promega KK, Tokyo, Japan) according to the manufacturer’s instructions. Chondrocytes were plated at a density of 1.0 × 10⁵ cells/well in 6-well plates, and synovial cells were plated at the same density in ThinCert® Cell Culture Inserts (0.4 μm, 6-well, Greiner Bio-One, Frickenhausen, Germany). Cocultures were performed in DMEM/F-12 containing 10% FBS for 4 to 7 days before the proliferation assay. Chondrocyte monocultures received the same volume of culture solution as the coculture samples. CellTiter 96 AQ One Solution Reagent was added to each well at 200 μL per well, followed by incubation for 2 h. The optical density (OD) was measured directly at 490 nm using a microplate reader. OD values were standardized between groups and compared between the CD68 positive (n = 6) and CD68 negative groups (n = 6).

Flow Cytometry

To identify the proportions of M1-like and M2-like macrophages in synovial cells, multicolor flow cytometry was used. Analyses were performed immediately after isolation, after 7 days of monoculture, and after 7 days of coculture with chondrocytes. Briefly, synovial cells were washed twice with Brilliant Stain Buffer, blocked with Fc-blocking reagent (Human BD Fc Block™), and then stained (30 min at 4°C) with the following monoclonal antibodies: anti-human CD45 (APC-Cy™7, BD), anti-human CD14 (PE-Cy™7, BD), anti-human CD80 (FITC, BD), and anti-human CD163 (APC, BD). The cells were washed again and resuspended in 100 μL of Brilliant Stain Buffer. Immediately before flow cytometry, the cells were stained with 7-aminoactinomycin D (BD) at a final concentration of 0.5 μg/mL. Cells were analyzed using a BD FACSCanto II flow cytometer. Cell debris and dead cells were excluded based on 7-aminoactinomycin D staining and forward-scatter profiles, and cells were gated based on forward- and side-scatter profiles. M1-like macrophages were defined as CD14⁺CD80⁺, and M2-like macrophages were defined as CD14⁺CD163⁺.²¹ Data were analyzed using BD FACSDiva Software ver. 6.1.3 (BD Biosciences, Franklin Lakes, NJ, USA).

Statistical Analysis

Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (R Foundation for Statistical Computing, Vienna, Austria).²² Statistical differences were assessed using Fisher’s exact test, the Mann–Whitney U test, and Kruskal–Wallis analysis. A P-value of less than 0.05 was considered significant.

Results

Gene Expression in the CD68-Positive Group

In the CD68 positive group, TNC and MMP-3 in cocultured chondrocytes were significantly upregulated compared with observations in chondrocytes alone (TNC: P = 0.026, MMP-3: P = 0.009). Additionally, SDC4 in cocultured synovial cells was significantly upregulated compared with that in synovial cells alone (P = 0.041). Other anabolic and catabolic factors tended to be upregulated in cocultured chondrocytes and synovial cells compared with those in their respective monocultures ( Fig. 3 ).

Figure 3.

Gene expression in the CD68 positive group. (A) Chondrocyte mRNA expression. (B) mRNA expression in synovial cells. Columns and bars indicate means and standard deviations. Coculture upregulated TNC and MMP-3 expression in chondrocytes and SDC4 expression in synovial cells. AC = chondrocytes; AC_S = chondrocytes cocultured with synovial cells; S = synovial cells; S_AC = synovial cells cocultured with chondrocytes; MMP = matrix metalloproteinase; TNC = tenascin-C; SDC4 = syndecan-4; FN = fibronectin; TIMP3 = tissue inhibitor of metalloproteinase 3; TGF = transforming growth factor; FGF = fibroblast growth factor; ADAMTS5 = a disintegrin and metalloproteinase with thrombospondin motifs 5. *P < 0.05.

Gene Expression in the CD68-Negative Group

In the CD68 negative group, TNC and SDC4 in cocultured chondrocytes and synovial cells tended to be downregulated, with no significant differences. Other anabolic and catabolic factors showed a decreasing trend in cocultured chondrocytes compared with observations in chondrocytes alone. By contrast, cocultured synovial cells showed an increasing trend compared with observations in synovial cells alone ( Fig. 4 ).

Figure 4.

Gene expression in the CD68 negative group. (A) Chondrocyte mRNA expression. (B) mRNA expression in synovial cells. Columns and bars indicate means and standard deviations. Coculture did not significantly alter the mRNA expression of each factor in chondrocytes or synovial cells. AC = chondrocytes; AC_S = chondrocytes cocultured with synovial cells; S = synovial cells; S_AC = synovial cells cocultured with chondrocytes; MMP = matrix metalloproteinase; TNC = tenascin-C; SDC4 = syndecan-4; FN = fibronectin; TIMP3 = tissue inhibitor of metalloproteinase 3; TGF = transforming growth factor; FGF = fibroblast growth factor; ADAMTS5 = a disintegrin and metalloproteinase with thrombospondin motifs 5.

Gene Expression of Inflammatory Cytokines in Synovial Cells

Compared with synovial cells cultured alone, the expression of TNFα was significantly upregulated in synovial cells from the coculture with chondrocytes (P = 0.047). There was also a tendency toward increased IL-1β expression in the coculture with chondrocytes ( Fig. 5 ).

Figure 5.

Gene expression of inflammatory cytokines in synovial cells. Coculture upregulated TNFα expression in synovial cells. S=, synovial cells; S_AC = synovial cells cocultured with chondrocytes; IL-1β = Interleukin-1 beta; TNFα = tumor necrosis factor alpha. *P < 0.05.

ELISA

The concentration of TNC in the medium was significantly higher in the CD68 positive coculture than in the CD68 negative coculture (P = 0.004) ( Fig. 6 ).

Figure 6.

TNC levels in the medium. (A) TNC concentration in the coculture medium was significantly higher in the CD68 positive group than in the CD68 negative group. (B) Sub-analysis using the same samples as in panel (a), comparing only chondrocyte monoculture and synovial cell monoculture. Coculture groups (AC_S) and CD68-based subdivisions were not included in this analysis. TNC concentration in the medium was significantly higher in the chondrocyte monoculture than in the synovial cell monoculture. Columns and bars indicate means and standard deviations. AC = chondrocytes monoculture; AC_S = chondrocytes cocultured with synovial cells; S = synovial cells monoculture; TNC = tenascin-C. *P < 0.05.

Proliferation Assay

The OD of chondrocytes was significantly increased on day 7 in chondrocytes cocultured with CD68 positive synovial cells compared with that in chondrocytes cocultured with CD68 negative synovial cells (P = 0.03). No significant difference was observed between cocultured chondrocytes and chondrocytes alone ( Fig. 7 ).

Figure 7.

Proliferation assay of chondrocytes. Coculture with CD68 positive synovial cells promoted chondrocyte proliferation on day 7. AC = chondrocytes; AC_S = chondrocytes cocultured with synovial cells. Columns and bars indicate means and standard deviations. *P < 0.05.

Flow Cytometry

The proportions of M1-like macrophages in synovial cells immediately after isolation and in cocultured synovial cells were significantly higher than in synovial cells cultured alone (P = 0.002 and P = 0.001, respectively). Additionally, there was no significant difference in the proportion of M1-like macrophages between synovial cells immediately after isolation and those after coculture. The proportion of M2-like macrophages did not differ significantly among the 3 groups ( Fig. 8 ).

Figure 8.

Percentage of M1 and M2 macrophages in synovial cells. Proportions of M1 and M2 macrophages in synovial cells immediately after isolation and after 7 days of coculture with chondrocytes were comparable. The proportion of M2 macrophages did not differ significantly among the 3 groups. *P < 0.05.

Discussion

The most important finding of this study was that coculture with CD68 high-expressing synovial cells led to upregulated TNC in chondrocytes and SDC4 in synovial cells, whereas the expression of these factors did not change significantly in coculture with CD68 low-expressing synovial cells. TNC in chondrocytes and SDC4 in synovial cells have been reported to interact;¹³ however, this interaction could be differentially influenced by the fraction of synovial cells. It has been demonstrated that mechanical stress, including mechanical insults, induces increased expression of MMP-3 in chondrocytes.^23,24 In addition, the progression of osteoarthritis is driven by the combined effects of mechanical stress and biochemical changes, and the interplay between these factors modulates catabolic and inflammatory responses in cartilage.²⁵ Synovial cells are thought to secrete molecules that induce specific signaling events in chondrocytes,²⁶ and studies have reported that MLS plays an important role in crosstalk between chondrocytes and synovial cells.⁹ The present study suggests that MLS—but not FLS—plays a major role in TNC expression in OA and demonstrates that different fractions of synovial cells exert distinct effects. In addition to TNC expression, MMP-3 expression was upregulated in chondrocytes cocultured with CD68 high-expressing synovial cells. It has been reported that TNC is involved in the upregulation of MMP-3,^27,28 whereas other studies have demonstrated increased MMP-3 expression in chondrocytes cocultured with macrophages.²⁹ In studies of synovial fluid in OA, TNC and MMP-3 expression were positively correlated,^30,31 and our results are consistent with these findings. MMPs, including MMP-3, are key contributors to OA progression by promoting cartilage degradation, inhibiting matrix synthesis, and triggering inflammatory responses.³² MMP-3, specifically, plays a crucial role in OA by facilitating cartilage degradation, promoting synovitis, and perpetuating a vicious cycle with inflammatory responses.³³ This study suggests that MLS affects the interaction between TNC in chondrocytes and SDC4 in synovial cells and is involved in the pathogenesis of OA.

TNC levels in synovial fluid are increased in patients with OA, and TNC concentrations have been positively correlated with OA progression.³⁰ In the present study, TNC concentration in the coculture medium was significantly higher in the CD68 positive group than in the CD68 negative group, suggesting that MLS is primarily responsible for TNC in synovial fluid in OA. Additionally, TNC concentration in the medium was significantly higher in the chondrocyte culture group than in the synovial cell culture group. As OA progression correlates with the severity of synovitis,⁷ and TNC levels in synovial fluid increase with OA progression,³⁰ the synovium has been hypothesized to be the primary source of TNC in synovial fluid; however, our findings suggest that TNC expression per cell is higher in chondrocytes. This apparent discrepancy may be explained by differences in cellular density and surface area between cartilage and synovial tissue, and by the possibility that chondrocytes may contain a higher amount of TNC per unit surface area compared to synovial tissue. Because the present study evaluated isolated cells under standardized in vitro conditions, our results reflect relative cellular TNC expression rather than the total tissue-level contribution to synovial fluid TNC in vivo.

By contrast, the proliferative activity of chondrocytes was significantly higher in the CD68 positive group than in the CD68 negative group in the MTS assay. A previous study on chondrocyte proliferation in cocultures of chondrocytes and synovial cells using the MTT assay found that chondrocyte proliferation was significantly greater in coculture than in monoculture.³⁴ TNC has been reported to promote chondrocyte proliferation in osteoarthritic cartilage.³⁵

In the present study, the synovial cell fraction also differentially affected chondrocyte proliferation, suggesting that MLS may influence chondrocyte proliferation in addition to TNC-mediated inflammation. Selective depletion of macrophages from synovial cells has been reported to induce infiltration of other inflammatory cells and exacerbate synovitis,³⁶ suggesting that macrophages are essential for joint homeostasis and play a critical role in chondrocyte proliferation.

A significantly increased M1 macrophage ratio has been observed in synovial fluid from patients with OA, indicating that an imbalance between M1 and M2 macrophages contributes to OA pathogenesis.¹⁸ In the present study, synovial cells immediately after isolation and after 7 days of coculture were predominantly M1 macrophages. Furthermore, in the cocultured synovial cells, which are considered to be M1-dominant, the gene expression level of TNFα was significantly higher than in synovial cells cultured alone. This finding is consistent with previous studies showing that M1 macrophages predominantly secrete TNFα.^37,38 The M1 and M2 proportions in synovial cells after coculture were similar to those immediately after isolation, suggesting that coculture in this study replicated in vivo conditions. However, in synovial cells cultured alone for 7 days, the M1 population decreased, resulting in an M1/M2 ratio closer to that of M2 macrophages. Therefore, the M1 and M2 ratios in MLS suggest potential interactions with chondrocytes. Inflammation has been implicated in OA progression,³⁹ yet the detailed mechanisms remain unclear. This study suggests that cartilage–synovium interactions do not uniformly induce TNC-mediated inflammation and may have variable effects across patients. Previous studies have proposed TNC as a potential biomarker for OA and have demonstrated a significant correlation between TNC concentration and K–L grade.³⁰ In our study, we observed that TNC levels may be associated with highly inflammatory OA phenotypes, which may provide additional insight into the potential role of TNC in OA pathophysiology. From a clinical perspective, elevated TNC levels may reflect not only the degree of cartilage degeneration but also the inflammatory status of the synovium, particularly M1 macrophage polarization. Early detection of increased TNC may therefore help identify a highly inflammatory OA phenotype and enable risk stratification or earlier intervention aimed at maintaining synovial homeostasis, including modulation of the M1/M2 macrophage balance, which may contribute to suppressing OA progression. However, the prognostic significance of distinguishing highly inflammatory from non-inflammatory OA phenotypes remains unclear, and it is currently unknown whether early identification of elevated TNC can modify disease course or long-term outcomes. Therefore, further studies are needed to confirm these findings. This study has several limitations. First, the sample size was small. Second, this study evaluated isolated chondrocytes and synovial cells under standardized in vitro conditions and did not account for differences in tissue volume, cell number, or surface area between cartilage and synovium. Therefore, our findings reflect relative cellular TNC expression and may not directly represent the total contribution of each tissue to TNC levels in synovial fluid in vivo. Third, only chondrocytes and synovial cells derived from patients with OA were analyzed, preventing examination of normal cartilage or synovium. Fourth, the study focused solely on cartilage and synovium, without considering interactions with other intra-articular tissues. To further address these limitations, future studies should evaluate our findings under conditions that more faithfully recapitulate the joint niche by employing organoid models of cartilage, synovium, and other intra-articular tissues to study interactions with other joint tissues. Such organoid platforms have been reported to simulate physiological and pathological crosstalk among multiple joint tissues,^40,41 and therefore represent effective approaches to complement the limitations of the present study. Finally, owing to the use of Transwell inserts for coculture, there is a risk that certain factors may not pass through if cells reach confluence in the inserts.⁴² However, in a preliminary experiment, when 1 × 10⁵ synovial cells were seeded in 12-well plates, confluence was observed after 7–9 days, suggesting that this effect was minimal.

Conclusion

Synovial cell fractionation differentially influences the interaction between TNC and SDC4, increases TNC in chondrocytes, and may be involved in OA pathology and chondrocyte proliferation. Additionally, MLS play a key role in TNC regulation, providing important insights into the pathophysiology of OA.

Footnotes

Acknowledgements

We would like to thank Katsura Chiba (Mie University) for excellent technical support.

ORCID iD

Masahiro Hasegawa

Ethical Considerations

This study was approved by the Institutional Review Board of the Mie University Graduate School of Medicine (reference number: H2024-096).

Consent to Participate

Informed consent of all patients was obtained.

Author Contributions

Writing—original draft preparation, G.K.; writing—review and editing, M.H. and K.I.-Y.; analysis and investigation, T.I.; project administration, M.H. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Data can be obtained by contacting the corresponding author upon request.

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