Adipose-derived mesenchymal stem cells treated with growth differentiation factor-5 express tendon-specific markers

Abstract

Objectives: Adipose-derived mesenchymal stem cells (ADMSCs) are a unique population of stem cells with therapeutic potential in the treatment of connective tissue injuries. Growth differentiation factor-5 (GDF)-5 is known to play a role in tendon repair and maintenance. The aim of this study was to investigate the effects of GDF-5 on proliferation and tendonogenic gene expression of rat ADMSCs.

Methods: ADMSCs were treated in culture with different concentrations of GDF-5 (0-1000 ng/mL) for 12 days. Biochemical, temporal, and concentration kinetic studies were done. Extracellular matrix (ECM) synthesis, tendonogenic differentiation, and matrix remodeling gene and protein expression were analyzed.

Results: GDF-5 led to increased ADMSC proliferation in a dose- and time-dependent manner. ADMSCs demonstrated enhanced ECM (collagen type I, decorin, and aggrecan) and tendonogenic marker (scleraxis, tenomodulin, and tenascin-C) gene expression with 100 ng/mL of GDF-5 (p < 0.05). ECM and tendon-specific markers showed time-dependent increases at various time points (p < 0.05), although decorin decreased at day 9 (p < 0.05). GDF-5 did alter expression of matrix remodeling genes, with no specific trends observed. Western blot analysis confirmed dose- and time-dependent increases in protein expression of tenomodulin, tenascin-C, Smad-8, and matrix metalloproteinase-13.

Conclusion: In vitro GDF-5 treatment can induce cellular events leading to the tendonogenic differentiation of ADMSCs. The use of combined GDF-5 and ADMSCs tissue-engineered therapies may have a role in the future of tendon repair.

FIG. 1.

The concentration-dependent (a) and time-dependent (b) effect of growth differentiation factor-5 (GDF-5) on the proliferation rate of adipose-derived mesenchymal stem cells (ADMSCs). For concentration kinetics, cells were harvested at day 4. For time kinetics, cells were treated with 100 ng/mL of GDF-5. Cell viability was measured as absorbance using MTT [3-(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide] assay. The proliferation rate of ADMSCs was significantly increased with increasing time and concentrations of GDF-5 (*p < 0.001 and ^#p < 0.01).

FIG. 2.

(a) Proteoglycan (glycosaminoglycan [GAG]/DNA) ratio and (b) hydroxyproline (OHP/DNA) content of ADMSCs treated with different concentrations of GDF-5 for 4 days. The GAG/DNA ratio was significantly increased at 10 and 100 ng/mL of GDF-5 (*p < 0.05). The OHP/DNA ratio exhibited a trend indicative of an increase in response to GDF-5 but was not statistically different at any concentration.

FIG. 3.

(a) Proteoglycan (GAG/DNA) ratio and (b) hydroxyproline (OHP/DNA) content of ADMSCs treated with or without 100 ng/mL of GDF-5 for 3, 6, 9, or 12 days. GAG/DNA was not statistically different from the control in this study. The OHP/DNA ratio was increased compared to control at day 12 (*p < 0.05).

FIG. 4.

Concentration-dependent effect of GDF-5 treatment on expression of tendonogenic markers of ADMSCs. Expression of scleraxis and tenomodulin was significantly increased at the concentration of 100 ng/mL of GDF-5. Expression of tenascin-C significantly increased at the concentration of 1000 ng/mL of GDF-5 (^#p < 0.01 and *p < 0.05).

FIG. 5.

Concentration-dependent effect of GDF-5 treatment on expression of genes for macromolecular components of extracellular matrix and cell adhesion molecules. Expression of collagen I (Col I) was increased at the concentration of 100 ng/mL of GDF-5. Expression of aggrecan was increased at 100 ng/mL of GDF-5, and decorin was increased at 1 and 10 ng/mL of GDF-5 (^#p < 0.01 and *p < 0.05).

FIG. 6.

Concentration-dependent effect of GDF-5 treatment on expression of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) genes. Expression of MMP-13 was increased at 10 and 1000 ng/mL of GDF-5. In contrast, expression of TIMP-2 was decreased at 100 and 1000 ng/mL of GDF-5 (^#p < 0.01 and *p < 0.05).

FIG. 7.

Time-dependent kinetics of GDF-5 on ADMSCs for gene expression of (a) scleraxis, (b) tenomodulin, and (c) tenascin-C. Expression of scleraxis was significantly increased at days 3 and 6. Expression of tenomodulin was increased significantly at day 6 and slightly decreased at day 12. Expression of tenascin-C was increased at days 9 and 12 (^#p < 0.01 and *p < 0.05).

FIG. 8.

Time-dependent kinetics of GDF-5 on ADMSCs for gene expression of (a) Col I, (b) Col III, (c) aggrecan, and (d) decorin. Expression of Col I was increased at day 12, while Col III increased at days 3 and 9. Expression of aggrecan significantly increased at days 6, 9, and 12. In contrast, expression of decorin decreased at day 9 (^#p < 0.01 and *p < 0.05).

FIG. 9.

Time-dependent kinetics of GDF-5 on ADMSCs for gene expression of (a) MMP-3, (b) MMP-13, and (c) TIMP-2. Expression of MMP-3 was decreased at day 3 and increased at day 6. MMP-13 was decreased at day 12, and TIMP-2 was increased at day 9 (^#p < 0.01 and *p < 0.05).

FIG. 10.

Concentration-dependent effect of GDF-5 on the protein expression for tendonogenic markers and MMP-13. Western blot revealed that expression uniformly increased when compared to controls for the proteins in question.

FIG. 11.

Time-dependent effect of GDF-5 on protein expression of tendonogenic markers and MMP-13. Tenomodulin increased at days 3, 6, and 12; Smad-8 increased at days 3, 6, and 9; tenascin-C increased at days 3, 6, and 12 but decreased at day 9; and MMP-13 increased at days 3 and 9 but decreased at days 6 and 12.