Tumor necrosis factor-α inhibition restores matrix formation by human adipose-derived stem cells in the late stage of chondrogenic differentiation

Figure 1 Human adipose-derived stem cells flow cytometry analysis results and differentiation ability identification. A: Schematic diagram of TNF-α interferes with Human adipose-derived stem cells (hADSCs) chondrogenic differentiation; B: Flow cytometer identification of surface molecules of hADSCs: CD44, CD29, CD45, CD34, and CD105; C: Oil red O staining and Western blot detection of marker proteins performed after lipogenic differentiation induction of hADSCs; D: Alizarin red, alkaline phosphatase staining, and Western blot detection of osteogenic differentiation marker proteins performed after osteogenic differentiation of hADSCs. MMP-3: Matrix metalloproteinase 3; NF-κB: Nuclear factor kappa-B; ECM: Extracellular matrix; GM: Human adipose-derived stem cells grown in growth medium alone.

Figure 2 Etanercept cytotoxicity analysis and human adipose-derived stem cells chondrogenic differentiation ability identification. A: The growth curves of human adipose-derived stem cells (hADSCs) treated with chondrogenic medium containing etanercept were measured using the CCK-8 method; B: Scratch test performed to determine the migration of hADSCs treated with chondrogenic medium containing etanercept; C and D: Toluidine blue staining and Western blot detection of chondrogenic differentiation marker proteins. ^aP < 0.05. GM: Human adipose-derived stem cells (hADSCs) grown in growth medium alone; CHE: hADSCs treated with chondrogenic differentiation medium and 1 μg/mL etanercept.

Figure 3 Expression levels of tumor necrosis factor-alpha and matrix metalloproteinase 3 were detected during chondrogenic differentiation of human adipose-derived stem cells. A: Western blot detection of MMP-3; B: Elisa detection of the concentrations of tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase 3 (MMP-3) in the cell supernatant; C: Immunofluorescent staining of TNF-α and MMP-3; D: Toluidine blue staining and the Western blot detection of TNF-α and MMP-3 of human adipose-derived stem cells treated with chondrogenic medium containing interleukin-1β. ^aP < 0.05; ^bP < 0.01. TNF-α: Tumor necrosis factor-alpha; MMP-3: Matrix metalloproteinase 3; IL-1β: Interleukin-1β; GM: Human adipose-derived stem cells (hADSCs) grown in growth medium alone; CH: hADSCs treated with chondrogenic differentiation medium.

Figure 4 Detection of human adipose-derived stem cells chondrogenic differentiation marker proteins and tumor necrosis factor-alpha and matrix metalloproteinase 3 performed after treatment with chondrogenic differentiation medium containing etanercept. A: Western blot detection of Col-2 and Aggrecan of adipose-derived stem cells treated with chondrogenic differentiation medium containing etanercept; B: Toluidine blue staining; C: Elisa detection of the tumor necrosis factor-alpha and matrix metalloproteinase 3 (MMP-3) concentration in cell supernatant; D: Western blot detection of MMP-3 and NF-κB pathway. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. MMP-3: Matrix metalloproteinase 3; NF-κB: Nuclear factor kappa-B; GM: Human adipose-derived stem cells (hADSCs) grown in growth medium alone; CH: hADSCs treated with chondrogenic differentiation medium; CHE: hADSCs treated with chondrogenic differentiation medium and 1 μg/mL etanercept.

Figure 5 Detection of human adipose-derived stem cells chondrogenic differentiation marker proteins and tumor necrosis factor-alpha and matrix metalloproteinase 3 performed after treatment with chondrogenic differentiation medium containing Infliximab. A: Toluidine blue staining; B: Western blot detection of Col-2, Aggrecan and Sox-9 of adipose-derived stem cells treated with chondrogenic differentiation medium containing Infliximab; C: Western blot detection of matrix metalloproteinase 3 and tumor necrosis factor-alpha pathway; D: Western blot detection of nuclear factor kappa-B pathway. ^aP < 0.05. TNF-α: Tumor necrosis factor-alpha; MMP-3: Matrix metalloproteinase 3; IL-1β: Interleukin-1β; GM: Human adipose-derived stem cells (hADSCs) grown in growth medium alone; CH: hADSCs treated with chondrogenic differentiation medium; CHE: hADSCs treated with chondrogenic differentiation medium and 1 μg/mL etanercept; CH+Inf: hADSCs treated with chondrogenic differentiation medium and 10 μg/mL infliximab.