Effects of adipose-derived mesenchymal stem cell conditioned medium on human tenocytes exposed to high glucose

Abstract

Introduction: Diabetic tendinopathy is a common invalidating and challenging disease that may be treated using stem cells. However, the effects of adipose-derived mesenchymal stem cell conditioned medium (ASC-CM) in diabetic tendinopathy have never been explored.

Objectives: The present study evaluated the effects of ASC-CM on morphology, cell viability, structure, and scratch wound closure of human tenocytes (HTNC) exposed to high glucose (HG).

Design: Experimental study.

Methods: HTNC were exposed to HG (25 mM) for 7, 14 and 21 days with or without ASC-CM for the last 24 h. CM was collected from 4 × 10⁵ ASCs, centrifuged for 10 min at 200 g and sterilized with 0.22 μm syringe filter.

Results: At 7 days, HG-HTNC had decreased cell viability [72 ± 2%, p < 0.01 versus normal glucose (NG)] compared to NG-HTNC (90 ± 5%). A further decrement was detected after 14 and 21 days (60 ± 4% and 60 ± 5%, both, p < 0.01 versus NG and p < 0.01 versus HG7). While NG-HTNC evidenced a normal fibroblast cell-like elongated morphology, HG-HTNC showed increased cell roundness. In contrast, HG-HTNC exposed to ASC-CM showed a significant increase in cell viability, an improved cell morphology and higher scratch wound closure at all HG time points. Moreover, the exposure to ASC-CM significantly increased thrombospondin 1 and transforming growth factor beta 1 (TGF-β1) content in HG-HTNC. The TGF-β1 elevation was paralleled by higher Collagen I and Vascular Endothelial Growth Factor in HG-HTNC.

Conclusion: ASC-CM may restore the natural morphology, cell viability and structure of HTNC, promoting their scratch wound closure through TGF-β1 increase.

Keywords: adipose-derived mesenchymal stem cells; conditioned medium; diabetes; tendinopathy; thrombospondin-1; transforming growth factor beta 1.

Figure 1.

(a) Representative images of optical microscopy showing ASCs isolated from µFAT (Day 0), then after 4, 7 and 10 days (Day 4–7–10); scale bar: 20 µm; magnification 10×, (b) ASCs cell viability, expressed as OD values at 570 nm ± SD. ** p < 0.01 versus Day 4 and (c) representative immunofluorescence images of CD90/CD73/CD105 expression shown in red, with the nucleus stained in blue by DAPI (4′,6-diamidino-2-phenylindole). Scale bar: 10 µm; magnification 20×. The relative quantization was expressed by the percentage of positive cells (red) on total cells counted (blue) ± SD. Results are expressed as mean ± SD of nine observations. ASC, adipose-derived mesenchymal stem cell; µFAT, microfragmented adipose tissue; OD, optical density.

Figure 2.

(a) Representative optical microscope images of HTNC morphology in NG or HG (6, 13 and 20 days) followed by 24 h of αMEM as control (NG, HG₇, HG₁₄, HG₂₁) or ASC-CM. Scale bar: 20 µm; magnification 10×, (b) HTNC cell viability in NG or HG (6, 13 and 20 days) followed by 24 h of αMEM as control (NG, HG₇, HG₁₄, HG₂₁) or ASC-CM. HTNC metabolic activity was measured as OD at 570 nm and calculated as (mean OD treatment/mean OD control) × 100 and (c) representative images of wound healing in HTNC at T0 (day 6, 13, 20) and after 24 h in serum free αMEM as control or serum free ASC-CM. The percentage of scratch wound closure at T24 was measured by ImageJ and normalized against the wound area at T0; scale bar: 10 µm; magnification 20×. Results are expressed as mean ± SD of nine observations. ASC-CM, adipose-derived mesenchymal stem cell conditioned medium; HG, high glucose; HTNC, human tenocytes; αMEM, α-minimum essential medium; NG, normal glucose; OD, optical density.

Figure 3.

(a) Total TGF-β1 (pg/ml ± SD), active TGF-β1 (pg/ml ± SD) and TSP-1 levels (ng/ml ± SD) in ASC-CM; **p < 0.01 versus active TGF-β1, (b) total TGF-β1 (pg/ml ± SD), (b and c) Col I (ng/ml) and (b and d) VEGF (pg/ml) levels in HTNC after exposure to NG or HG (6, 13 and 20 days) followed by 24 h of αMEM as control (NG, HG₇, HG₁₄, HG₂₁) or ASC-CM. Results are expressed as mean ± SD of nine observations. *p < 0.05 and **p < 0.01 versus NG; °°p < 0.01 versus HG at the same time point; ^^p < 0.01 versus HG₇; ^§§p < 0.01 versus HG₇ + ASC-CM, ^#p < 0.05 versus HG₁₄ + ASC-CM. ASC-CM, adipose-derived mesenchymal stem cell conditioned medium; Col I, Collagen I; HG, high glucose; NG, normal glucose; TGF-β1, transforming growth factor beta 1; TSP-1, thrombospondin 1; αMEM, α-minimum essential medium; VEGF, Vascular Endothelial Growth factor.

Figure 4.

Representative images of immunocytochemistry showing active TGF-β1 in HTNC after exposure to NG or HG (6, 13 and 20 days) followed by 24 h of αMEM as control (NG, HG₇, HG₁₄, HG₂₁) or ASC-CM, with relative quantization reported as % of positive cells (red for TGF-β1, blue for the nucleus) on total cells counted. Results are expressed as mean ± SD of nine observations. Scale bar: 10 µm; magnification 20×. **p < 0.01 versus NG; °°p < 0.01 versus HG at the same time point; ^^p < 0.01 versus HG₇; ^§§p < 0.01 versus HG₇ + ASC-CM. ASC-CM, adipose-derived mesenchymal stem cell conditioned medium; HG, high glucose; HTNC, human tenocytes; NG, normal glucose; TGF-β1, transforming growth factor beta 1; αMEM, α-minimum essential medium.

Figure 5.

(a) TSP-1 content (ng/ml ± SD) in HTNC after exposure to NG or HG (6, 13 and 20 days) followed by 24 h of αMEM as control (NG, HG₇, HG₁₄, HG₂₁) or ASC-CM; (b) Pearson correlation analysis between active TGF-β1 levels in HTNC (% of positive cells/total cells counted) and TSP-1 levels (ng/ml) in HTNC. Results are expressed as mean ± SD of nine observations. **p < 0.01 versus NG; °p < 0.05 and °°p < 0.01 versus HG at the same time point; ^^p < 0.01 versus HG₇; ^§§p < 0.01 versus HG₇ + ASC-CM. ASC-CM, adipose-derived mesenchymal stem cell conditioned medium; HG, high glucose; NG, normal glucose; TGF-β1, transforming growth factor beta 1; TSP-1, thrombospondin 1; αMEM, α-minimum essential medium.