Conditioned Medium From Stem Cells of Human Exfoliated Deciduous Teeth Alleviates Mouse Osteoarthritis by Inducing sFRP1-Expressing M2 Macrophages

Abstract

Intravenous administration of conditioned medium from stem cells of human exfoliated deciduous teeth (SHED-CM) regenerates mechanically injured osteochondral tissues in mouse temporomandibular joint osteoarthritis (TMJOA). However, the underlying therapeutic mechanisms remain unclear. Here, we showed that SHED-CM alleviated injured TMJ by inducing anti-inflammatory M2 macrophages in the synovium. Depletion of M2 by Mannosylated Clodrosome abolished the osteochondral repair activities of SHED-CM. Administration of CM from M2-induced by SHED-CM (M2-CM) effectively ameliorated mouse TMJOA by inhibiting chondrocyte inflammation and matrix degradation while enhancing chondrocyte proliferation and matrix formation. Notably, in vitro, M2-CM directly suppressed the catabolic activities while enhancing the anabolic activities of interleukin-1β-stimulated mouse primary chondrocytes. M2-CM also inhibited receptor activator of nuclear factor NF-κB ligand-induced osteoclastogenesis in RAW264.7 cells. Secretome analysis of M2-CM and M0-CM revealed that 5 proteins related to anti-inflammation and/or osteochondrogenesis were enriched in M2-CM. Of these proteins, the Wnt signal antagonist, secreted frizzled-related protein 1 (sFRP1), was the most abundant and played an essential role in the shift to anabolic chondrocytes, suggesting that M2 ameliorated TMJOA partly through sFRP1. This study suggests that secretome from SHED exerted remarkable osteochondral regeneration activities in TMJOA through the induction of sFRP1-expressing tissue-repair M2 macrophages.

Keywords: Wnt antagonist; dental pulp stem cell; mesenchymal stem cell; osteoarthritis; osteochondral regeneration; secretome of M2 macrophage; temporomandibular joint.

Graphical Abstract

Figure 1.

M2 macrophages induced by SHED-CM are required to alleviate mouse TMJOA. (A) Mice were subjected to forced mouth opening (FMO) to keep the mandible in the maximal opened position. (B) Experimental design. Mechanical stress was applied to the TMJs by FMO for 3 hours/day. White, black, red arrows and a black triangle represent FMO, CM treatment, m-Clo administration, and animal sacrifice, respectively. (C, D) Representative images of immunofluorescence staining and quantitative analysis of CD206⁺/DAPI⁺ cell ratio in the TMJ synovium (n = 6-8/group), scale bar = 50 µm. (E) Representative computed tomography images of osteochondral interfaces and sagittal views of the mandible condyles on day 12. (F) Quantification of BV/TV and Tb.Th in the posterior subchondral bone (n = 4-5/group). (G) HE and TB staining of the condyles, scale bar = 200/100 µm in low/high magnification. (H, I) Ratio of TB-positive area (H) and modified Mankin score (I) in the posterior cartilage (n = 4-5/group). Data are presented as the mean ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: P, posterior; A, anterior; BV/TV, bone volume/total volume; Tb.Th, trabecular thickness; HE, hematoxylin and eosin; TB, toluidine blue.

Figure 2.

SHED-CM induces M2 polarization of BMMs in vitro. (A) Representative flow cytometry plots of F4/80 and CD206 expressions in M0, M2 (IL-4), or M2 (SHED-CM). (B) qRT-PCR analysis of the relative mRNA expressions in M0, M2 (IL-4), M2 (SHED-CM), or M1 (n = 3/group). (C) Schematic diagram of M0/M1 mixture preparation procedures. (D, E) Representative flow cytometry plots and quantitative analysis of iNOS and Arg1 expressions in M0, M0/M1 mixture, and M1 treated with DMEM or SHED-CM (n = 3/group). Data are presented as the mean ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001, n.s., not significant. Abbreviations: IL-4, interleukin 4; iNOS, inducible nitric oxide synthase; Arg1, arginase1; IFNγ, interferon-γ; LPS, lipopolysaccharide.

Figure 3.

M2-CM restores injured subchondral bone and cartilage matrix in TMJOA. (A) Representative computed tomography images of the mandible condyles. (B) Quantification of BV/TV and Tb.Th in the posterior subchondral bone (n = 9-10/group). (C) TRAP and TB staining of TMJs, scale bar = 100 µm in TRAP, 200/100 µm in low/high magnification of TB. (D, E) Quantification of TRAP-positive cells and TB-positive areas (n = 8-10/group). Data represent mean ± SD. *P < .05, **P < .01, ***P < .001. Abbreviations: P, posterior; A, anterior; TB, toluidine blue; TRAP, tartrate-resistant acid phosphatase; BV/TV, bone volume/total volume; Tb.Th, trabecular thickness.

Figure 4.

M2-CM suppresses catabolic and promotes anabolic activities in TMJOA. Immunohistochemical analysis of iNOS, IL-1β, and MMP13 was performed according to the schedule in (A) and that of SOX9 and PCNA was performed according to the schedule in (B). White arrows, black arrows, and black triangles represent FMO, CM treatment, and animal sacrifice, respectively. (C) Representative images (high magnification is shown in the box at the bottom right) and (D) quantitative analysis of the number of iNOS-, IL-1β-, and MMP13-positive chondrocytes on day 4 (n = 4-6/group) and SOX9- and PCNA-positive chondrocytes on day 12 (n = 4-5/group) in the TMJ condylar cartilage, scale bar = 50 µm. The number of cells was counted at ×200 magnification and depicted as the percentage of positively stained cells. Data are presented as the mean ± SD. *P < .05, **P < .01, ***P < .001. Abbreviations: iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; MMP13, matrix metalloproteinase 13; SOX9, SRY-box transcription factor 9; PCNA, proliferating cell nuclear antigen.

Figure 5.

M2-CM directly suppresses catabolism but accelerates anabolic reactions in IL-1β-stimulated mouse primary chondrocytes. (A) Representative immunofluorescence images and (B) the ratio of the number of iNOS-, MMP13-, Col II-, and ACAN-positive chondrocytes in DAPI-positive cells (n = 3/group), scale bar = 50 µm. (C) qRT-PCR analysis of the expression levels of the indicated mRNA (n = 3/group). The results are presented relative to their levels in cells treated with IL-1β in DMEM. Data are presented as mean ± SD. *P < .05, **P < .01, ***P < .001, ****P < .0001. Abbreviations: iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; MMP13, matrix metalloproteinase 13; Col II, collagen II; ACAN, aggrecan; DAPI, 4ʹ,6-diamidino-2-phenylindole.

Figure 6.

Secretome analysis of M2-CM and M0-CM. (A) In M2-CM, a total of 2824 proteins were identified, 41 of which were highly expressed by more than 3-fold compared to those in M0-CM. (B) Scatter plot of the differential protein profiles in M2-CM and M0-CM based on normalized abundances of proteins. Five therapeutic factors are indicated as red squares. (C) Five proteins in M2-CM were identified as potential therapeutic factors for TMJOA.

Figure 7.

Inhibition of sFRP1 abrogates the treatment effects of M2-CM on inflamed chondrocytes and TMJOA cartilage. (A) Representative immunofluorescent images and (B) the ratio of the number of iNOS-, MMP13-, and Col II-positive chondrocytes in DAPI-positive cells (n = 3/group), scale bar = 50 µm. (C) Experimental design of WAY administration in vivo. White, black, red arrows and a black triangle represent FMO, M2-CM treatment, WAY administration, and animal sacrifice, respectively. (D, E) TB staining and quantification of TB-positive areas (n = 6/group), scale bar = 100 µm. Data are presented as the mean ± SD. *P < .05, n.s., not significant. Abbreviations: sFRP1, secreted frizzled-related protein 1; iNOS, inducible nitric oxide synthase; MMP13, matrix metalloproteinase 13; Col II, collagen II; DAPI, 4ʹ,6-diamidino-2-phenylindole; TB, toluidine blue.