Chondrogenic differentiation capacity of mechanically stimulated MSCs is increased via downregulation of CD73

INTRODUCTION: The bone regeneration process is characterized by hematoma formation, inflammation, angiogenesis, chondrogenesis, osteogenesis and bone remodeling. Mesenchymal stem cells (MSCs) are crucial during the early phase of bone healing, possibly acting via migration, proliferation and differentiation into functional cells of the chondrogenic or osteogenic lineage. In addition to biological factors, mechanical boundary conditions determine the path of bone regeneration. During the early phase of bone healing moderate compression (15-30%) stimulates healing in vivo. In vitro, cyclic-compressive loading of MSCs affected the expression of molecules involved in angiogenesis and matrix remodelling, both key processes of bone healing. Investigations regarding cell autonomous alterations in MSCs due to loading are still missing. Therefore, we aimed to analyse MSC stem cell characteristics after dynamic compression. Mechanical loading led to a decreased CD73 expression and increased chondrogenic differentiation. The mechanisms behind CD73 associated changes due to mechanical loading however, remained unknown. METHODS: Bone marrow-MSCs from 10-12 weeks old male Lewis rats were embedded in a fibrin/trabecular bone construct in passage 3 and placed in a bioreactor, where cyclic-compressive stimulation of the cells was applied (20% compression, 1Hz, 72h). Afterward cells or RNA were isolated by enzymatic digestion of the construct or by using Trizol®, respectively. Cell surface marker expression was analyzed by flow cytometry. Gene expression of cell surface and differentiation markers and adenosine receptors was investigated by quantitative RT-PCR and normalized to β-actin, Eef1a1 and GAPDH and statistically analyzed using REST© 2008. Differentiation assays were performed as described elsewhere with and without adenosine or, adenosine 5’-(α,βmethylene)diphosphate (APCP; CD73 inhibitor). Differentiated cells were detected by measuring alkaline phosphatase (ALP) activity and by staining with alizarin red (AR) (osteogenic), sudan IV (adipogenic) and alcian blue (chondrogenic). Proliferation was validated by means of MTS. Results from at least three independent experiments were analyzed for statistical significance using a paired two-sided Student's t test (p < 0.05). RESULTS: Cyclic-compressive loading led to down regulation of CD73 on protein (CD73 MFI normalized to isotype control: loaded = 2.35, unloaded = 5.04; p = 0.017; Fig. 1A) and mRNA level compared to the controls (CD73 relative expression: E = 0.496; p = 0.011). Among the chondrogenic (Sox9, collagen II, aggrecan, fibromodulin), osteogenic (Runx2, ALP, osteopontin, bone sialoprotein, collagen I, osteocalcin) and adipogenic (Ppar-γ, lipoprotein lipase, fatty acid-binding protein 4, fatty acid synthase) differentiation markers, fibromodulin was significantly downregulated in loaded compared to unloaded MSCs (Fmod relative expression: E = 0.377; p = 0.028). After differentiation in appropriate media, however, chondrogenic differentiation ability was increased after mechanical loading. (area alcian blue: loaded = 52.9 %, unloaded = 34.2 %, p = 0.082, n=3). Osteogenic and adipogenic differentiation were unaffected by mechanical stimulation. Next, the putative involvement of CD73 in chondrogenic differentiation was investigated. Supplementation of chondrogenic media with adenosine 5’-(α,β-methylene)diphosphate (APCP; CD73 inhibitor) stimulated chondrogenic differentiation of MSCs, which could be reversed by adenosine (area alcian blue: loaded = 52.9 %, loaded+APCP = 69.5 %, loaded+adenosine = 45.3 % ploaded vs. loaded+APCP = 0.044, n=3; Fig.2). Cell viability was unaffected by APCP and adenosine. MSCs expressed the four known adenosine receptors (A1R, A2aR, A2bR, A3R) on mRNA level. After mechanical stimulation of MSCs, the A2A receptor was significantly down regulated (A2aR relative expression: E = 0.366; p = 0.013; Fig.1B).