Knockdown of decorin in human bone marrow mesenchymal stem cells suppresses proteoglycan layer formation and establishes a pro-inflammatory environment on titanium oxide surfaces

Abstract

Osseointegration is essential for successful implant treatment. However, the underlying molecular mechanisms remain unclear. In this study, we focused on decorin (DCN), which was hypothesized to be present in the proteoglycan (PG) layer at the interface between bone and the titanium oxide (TiOx) surface. We utilized DCN RNA interference in human bone marrow mesenchymal stem cells (hBMSCs) to investigate its effects on PG layer formation, proliferation, initial adhesion, cell extension, osteogenic capacity, fibrotic markers, and immunotolerance to TiOx in vitro. After 14 days of cultivation, we observed no PG layer was detected, and the osteogenic capacity was suppressed in DCN-depleted hBMSCs. Furthermore, the conditioned medium upregulated the expression of M1 macrophage markers in human macrophages. These results suggest that endogenous DCN plays a crucial role in PG layer formation and that the PG layer alters inflammation around Ti materials.

Fig. 1

Characterization of N-BMSCs and sh-BMSCs. A mRNA and B protein expression analysis of DCN. C Representative flow cytometry analysis of cell surface markers. D Alizarin red S staining, E Oil Red O staining, and F Alcian blue staining after osteogenic induction, adipogenic induction, and chondrogenic induction culture. **p < 0.01

Fig. 2

A Transmission electron micrograph of the cell–TiOx interface. The ruthenium-positive areas were detected at 14 days of N-BMSC cultivation (arrowheads). B Proliferation of N-BMSCs and sh-BMSCs on TiOx on days 1, 2, 3, 7, and 14. C Initial cell adhesion of N-BMSCs and sh-BMSCs to TiOx at 1, 2, and 3 h. This figure is representative of three independent experiments. **p < 0.01

Fig. 3

Immunofluorescence staining analysis of N-BMSCs and sh-BMSCs after 24 h of cultivation on TiOx. A Expression of vinculin. Green: F-actin, Red: vinculin, Blue: DAPI. B Area, C major, D minor, E aspect ratio, F Feret, and G minimum Feret were measured to evaluate cell adhesion and extension. **p < 0.01

Fig. 4

Osteogenic capacity of N-BMSCs and sh-BMSCs. Gene expression of A DCN, B Col1a1, C Runx2, D ALPL, and E OCN after 7 and 14 days of cultivation on TiOx. Mineralized nodule formation by alizarin red S staining (F) and quantitative analysis (G). This figure is representative of three independent experiments. **p < 0.01

Fig. 5

Gene expression of A Acta2, B FN1, and C TGF-β1 after 7 and 14 days of cultivation on TiOx. Western blotting analysis of extracellular matrix after 14 days of cultivation on TiOx (D) and semi-quantification (E–H). This figure is representative of two independent experiments. *p < 0.05, **p < 0.01

Fig. 6

Gene expression related to immunotolerance in N-BMSCs and sh-BMSCs after 14 days of TiOx (A–C) cultivation and in CM-treated hMps derived from N-BMSCs and sh-BMSCs (D–J). Protein expression levels of IL-6 and IL-10 in the supernatant of BMSCs cultured on TiOx (K, L) and after stimulating hMps (M, N). **p < 0.01

Fig. 7

Decellularization assay analysis of N-BMSCs and sh-BMSCs. Gene expression of A Col1a1, B Runx2, C ALPL, D OCN, E Acta2, F FN1, and G TGF-β1 after 14 days of cultivation on decellularized TiOx. SEM-EDX analysis of decellularized Ti surfaces (H, I). This figure is representative of two independent experiments. *p < 0.05, **p < 0.01