Extracellular Vesicles Secreted by Osteogenic-Differentiated Mesenchymal Stem Cells Promote Bone Formation In Rat Calvarial Defect

Jun 29, 2022, 4:10 PM
10m
Room: S2

Room: S2

Speaker

Prof. Mustafa, Kamal (University of Bergen)

Description

Background:
Mesenchymal stem cell (MSCs) -based therapy has a promising potential in bone tissue regeneration. Although, growing evidence has suggested that paracrine mechanisms may be involved in the underlying mechanism of MSC transplantation, and extracellular vesicles (EVs) are an important component of this paracrine role. However, information on the influence of different microenvironmental stimuli of MSCs culture conditions on the osteogenic effects of EVs is scarce. The main purpose of this study was to determine whether EVs derived from MSCs under normoxic (Normo-EVs), Hypoxic (Hypo-EVs) and chemically osteogenic induced MSCs (Osteo-EVs) show greater effects on osteogenic differentiation potential in vitro and on the bone formation of calvarial defects in vivo, and whether findings are associated with various proteins profile.
Methods:
Undifferentiated MSCs were incubated under normoxic and hypoxic culture conditions, and 7-days of chemically osteogenic induced MSCs were incubated under normoxic conditions, for 72 h. Conditioned media were collected and concentrated onto 100 kDa centrifugal filters (UF), followed by separation of EVs using size-exclusion chromatography method (SEC). The Normo-EVs, Hypo-EVs, and Osteo-EVs recovery were characterized by size distribution using DLS, morphology using TEM, and flow cytometry analysis of tetraspanin CD63 and CD81. The proteomic composition of different groups of EVs was characterized by LC-MS/MS. We evaluated the in vitro effect of EVs groups (10 μg/ml) on the proliferation, scratch assay, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) by Alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, and qRT-PCR for osteogenic related genes, respectively. Furthermore, Osteo-EVs (50 μg/ml) were combined with collagen membrane scaffolds (MEM) to repair critical-sized calvarial bone defects in rats, and the efficacy was assessed using in vivo and ex vivo µCT, and histological examination. The in vitro release of Osteo-EVs from MEM scaffolds and their internalization by cultured MSCs were also examined.
Results:
Using UF-SEC, we could isolate and characterize EVs from all groups. We found that all EVs groups could profoundly enhance the proliferation, and migration of cultured hMSCs. However, Osteo-EVs were shown greater effects on the in vitro osteogenic differentiation of hMSCs as detected by higher mRNA expression levels of late markers of osteogenesis-related genes, BSP and OC, and calcium deposit using ARS. In addition, Osteo-EVs/MEM combination scaffolds could enhance greater bone formation after 4 weeks as compared to native MEM loaded with serum-free media. In vitro assay showed that the Osteo-EVs could release from the MEM scaffold and could be internalized by cultured hMSCs.
Conclusions:
We suggest that EVs derived from chemically osteogenic induced MSCs can significantly enhance both the osteogenic differentiation activity of cultured hMSCs and the osteoinductivity of MEM scaffolds. These results indicate that Osteo-MSC secreted nanocarriers-EVs combined with MEM scaffolds can be used for repairing bone defects.

31451708199

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