gms | German Medical Science

Objectives: Inducing lineage specific differentiation of host cells is acknowledged to be a crucial step for accelerating bone repair. In this regard, the extracellular vesicles (EVs), an emerging cell-to-cell communicator, have aroused increasing attention as they could transfer a complex cargo of biologically active molecules to target cells and thereby promoting osteogenic differentiation. This study aims to explore the therapeutical potential of EVs derived from osteoblasts that were stimulated by dexamethasone (OB-EVsDex) for 14 days in the regulation of osteo-differentiation and proliferative activities of porcine osteoblasts, ultimately improving osteogenesis and bone formation.

Methods: The OB-EVsDex were characterized and identified via Nano Tracking Analysis (NTA) and trans electron microscopy (TEM) analysis. The OB-EVsDex were taken up by osteoblasts confirmed by up-take-assays. The cell attachment and proliferation on the bone scaffold after stimulation with OB-EVsDex were evaluated via Calcein AM staining, DAPI staining, and CCK-8 assays. Cytoskeletal staining and scanning electron microscope (SEM) observation were further performed. The key osteogenesis-related gene expression levels of type 1A collagen (Col 1A), ALP, BMP-2, osteocalcin (OCN), osteopontin (OPN), osteonectin (ON), runt-related transcription factor 2 (RUNX2) were examined in OB-EVsDex-treated osteoblasts through quantitative real-time PCR (qRT-PCR); meanwhile multiple mineralization markers were evidenced.

The experiments were carried out in the following experimental groups:

1.

Scaffold + osteoblasts (negative control)

2.

Scaffold + BMP-2 + osteoblasts (positive control)

3.

Scaffold + OB-EVsDex + osteoblasts

4.

Scaffold + OB-EVsDex + BMP-2 + osteoblasts

Results and conclusion: Cellular uptake assays indicated that fluorescently labeled OB-EVsDex were engulfed into the cytoplasm of OBs. Besides, in vitro proliferation assays verified that the cell number in the OB-EVsDex+BMP-2 group is significantly higher than that of only OB-EVsDex or BMP-2. Moreover, the osteogenesis-related gene expression levels of Col 1A, ALP, BMP-2, OCN, OPN, ON were significantly upregulated and elevated by induction of combination of OB-EVsDex and BMP-2 than solely BMP-2 or OB-EVsDex treatment. Mineralization experiments revealed that the combination of OB-EVsDex and BMP-2 markedly resulted in a substantial increase in the formed mineralization markers (calcium deposition) than that of OB-EVsDex or BMP-2 treatment alone.

Dexamethasone-stimulated OB-EVsDex positively upregulated crucial osteogenic genes and osteoblastic differentiation but also noticeably promoted in vitro proliferation. This role exhibited by OB-EVsDex was comparable to BMP-2 treatment alone. More importantly, the combined application of OB-EVsDex and BMP-2 maximumly hasten osteogenic differentiation and biomineralization, resulting in more effective bone formation.