Osteogenic ability of rat bone marrow concentrate is at least as efficacious as mesenchymal stem cells in vitro

Abstract

Cell therapy using bone marrow concentrate (BMC) or purified and expanded mesenchymal stem cells (MSCs) has been shown to have a promising osteogenic capacity. However, few studies have directly compared their relative osteogenic ability. The aim of this study was to compare the osteogenic ability of BMC isolated by density gradient centrifugation with bone marrow-derived MSCs in vitro using the cells of 3-month-old Sprague-Dawley rats. The isolated cells were seeded onto 24-well plates (1 × 10⁵ cells/well) and cultured in control growth media, osteogenic media with dexamethasone, or media without dexamethasone (which simulated the in vivo tissue environment). Alkaline phosphatase activity at week 2, osteocalcin using quantitative real-time polymerase chain reaction at week 4, and Alizarin red staining at week 4 were evaluated. In the osteogenic media with dexamethasone, BMC showed equivalent (osteocalcin) or even greater (Alizarin red staining) osteogenic ability compared to MSCs, suggesting that cross-talk among various cells in the BMC leads to greater osteogenesis. Furthermore, in the osteogenic media without dexamethasone, BMC showed equivalent (osteocalcin) or a trend for greater (Alizarin red staining) bone formation than MSCs alone. Our results suggest that BMC has at least comparable bone regeneration potential to MSCs. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2500-2506, 2019.

Keywords: bone marrow concentrate; bone regeneration; cell therapy; mesenchymal stem cell; osteogenesis.

Fig. 1

Experimental outline of the study. Rat bone marrow cells were collected from the femurs and tibias. BMC and MSCs were isolated and cultured in 3 different kinds of media. Osteogenic ability was evaluated by ALP activity assay, osteocalcin (qRT-PCR), and Alizarin red staining.

Fig. 2

The immunophenotype of the isolated rat MSCs was characterized by flow cytometry (CD90+/CD146+/CD44+/CD45−/CD31−/CD11b−).

Fig. 3

Tri-lineage differentiation was confirmed by Alizarin red staining (osteogenesis) (A, B), Oil red O staining (adipogenesis) (C, D), and toluidine blue staining (chondrogenesis) (E, F), respectively.

Fig. 4

ALP activity assay. All the groups of MSCs showed significantly higher ALP activity than the corresponding groups of BMC. *p <0.05, **p <0.01, ***p <0.005

Fig. 5

Osteocalcin (qRT-PCR). The osteogenic media with DEX group of MSCs showed significantly higher osteocalcin level than the other groups of MSCs, and also showed the comparable osteocalcin level to that of BMC. **p <0.01, ***p <0.005

Fig. 6

Alizarin red staining. The osteogenic media with DEX group of BMC showed significantly stronger staining than the other groups of BMC, and also showed significantly stronger staining than that of MSCs. The osteogenic media without DEX group of BMC showed a trend of stronger staining than that of MSCs. **p <0.01, ***p <0.005