We aim to examine the influence of platelet rich plasma (PRP) and spatial cues in cartilage/bone matrix forming proteins, and to evaluate the mitotic and chemotactic potential of PRP on human mesenchymal stem cells (hMSCs). Directed cell migration towards PRP gradients was assessed in chemotactic chambers, and recorded by time-lapse microscopy. hMSCs cultured in three-dimensional (3D) scaffolds were visualized by scanning electron microscopy; Hoechst dye was used to confirm cell confluence in 3D-constructs and monolayers before experimental treatment. MSCs were treated with 10% PRP lysate or 10% PRP lysate supplemented with TGF-b-based differentiation medium. The expression of cartilage (COL2A1, Sox9, ACAN, COMP), and bone (COL1A1, VEGF, COL10A1, Runx2) fundamental genes was assessed by real time PCR in monolayers and 3D-constructs. PRP had mitotic (p \ 0.001), and chemotactic effect on hMSCs, Ralyleigh test p = 1.02E-10. Two and three-week exposure of MSCs to PRP secretome in 3Dconstructs or monolayers decreased Sox9 expression (p \ 0.001 and p = 0.050) and COL2A1, (p = 0.011 and p = 0.019). MSCs in monolayers exposed to PRP showed increased ACAN (p = 0.050) and COMP (p \ 0.001). Adding TGF-b-based differentiation medium to PRP increased COMP, and COL2A1 expression at gene and protein level, but merely in 3D-constructs, p \ 0.001. TGF-b addition to monolayers reduced Sox9 (p \ 0.001), aggrecan (p = 0.004), and VEGF (p = 0.004). Cells exposed to PRP showed no changes in hypertrophy associated genes in either monolayers or 3Dconstructs. Our study suggests hMSCs have high-degree of plasticity having the potential to change their matrix-forming phenotype when exposed to PRP and according to spatial configuration. Keywords Joint repair Á Human mesenchymal stem cells (hMSCs) Á Platelet rich plasma (PRP) Á 3D-cultures Abbreviations 2D 2-dimensional 3D 3-dimensional ACAN Aggrecan BM-MSC Bone marrow-derived stem cells COL10A1 Type X collagen COL1A1 Type 1 collagen COL2A1 Type 2 collagen COMP Cartilage oligomeric matrix protein ECM Extracellular matrix GAPDH Glyceraldehyde-3-phosphate deshydrogenase IGF Insulin growth factor ITS Insulin transferrin selenium OD Optical density Arantza Infante and Eva Rubio-Azpeitia contributed equally to the authorship of this manuscript.
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