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Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

Liu Y, Zhao Y, Zhang X, Chen T, Zhao X, Ma GE, Zhou Y - PLoS ONE (2013)

Bottom Line: We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs.Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo.These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Prosthodontics, School and Hospital of Stomatology, Peking University, Beijing, China.

ABSTRACT
Human adipose-derived stromal cells (hASCs) are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI) and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS). The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI) or between purified hASCs and purified hASCs+OI (P>0.05). Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

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Osteogenic potential of human adipose-derived stromal cells (hASCs) and purified hASCs in vitro.A) hASCs and purified hASCs stained positive for alkaline phosphatase (ALP) after 7 days of osteoinduction (OI). The scale bar represents 100 µm. B) hASCs and purified hASCs were positive for Alizarin Red staining after 14 days of OI. The scale bar represents 100 µm. C) The ALP activities of hASCs, purified hASCs and hBMMSCs were significantly elevated after 7 days of OI; however, the ALP activities of both hASCs and purified hASCs were significantly lower than that of hBMMSCs (P<0.05). There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. D) The mineralization activities of hASCs, purified hASCs and hBMMSCs were significantly increased after 14 days of OI. There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. *P<0.05 compared with hASCs without OI; **P<0.05 compared with purified hASCs without OI; ***P<0.05 compared with hBMMSCs without OI; #P<0.05 compared with hBMMSCs after 7 days of OI.
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pone-0056002-g005: Osteogenic potential of human adipose-derived stromal cells (hASCs) and purified hASCs in vitro.A) hASCs and purified hASCs stained positive for alkaline phosphatase (ALP) after 7 days of osteoinduction (OI). The scale bar represents 100 µm. B) hASCs and purified hASCs were positive for Alizarin Red staining after 14 days of OI. The scale bar represents 100 µm. C) The ALP activities of hASCs, purified hASCs and hBMMSCs were significantly elevated after 7 days of OI; however, the ALP activities of both hASCs and purified hASCs were significantly lower than that of hBMMSCs (P<0.05). There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. D) The mineralization activities of hASCs, purified hASCs and hBMMSCs were significantly increased after 14 days of OI. There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. *P<0.05 compared with hASCs without OI; **P<0.05 compared with purified hASCs without OI; ***P<0.05 compared with hBMMSCs without OI; #P<0.05 compared with hBMMSCs after 7 days of OI.

Mentions: To compare the osteogenic potential of hASCs and purified hASCs in vitro, the cells were induced in osteogenic medium (OM) for 7 days or 14 days. Alkaline phosphatase (ALP) staining, Alizarin Red staining, ALP activity assay and quantitative Alizarin Red assays were then performed to assess the osteogenic potential of the cells. Positive results for ALP staining (Fig. 5A) and Alizarin Red staining (Fig. 5B) confirmed the osteogenic potential of both hASCs and purified hASCs. Human fibroblasts were used as negative controls and human bone marrow MSCs (hBMMSCs) as positive controls. ALP activity assay (Fig. 5C) showed the ALP activities of both hASCs and purified hASCs to be significantly elevated after 7 days of OI (P<0.05). However, there were no significant differences (P>0.05) between hASCs and purified hASCs or between hASCs that underwent OI (hASCs+OI) and purified hASCs that underwent OI (purified hASCs+OI). Quantitative Alizarin Red assay (Fig. 5D) showed the mineralization activity of both hASCs and purified hASCs to be significantly increased after 14 days of induction in OM (P<0.05), but there were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI (P>0.05). In addition, we found that after 7 days of OI, the ALP activities of both hASCs and purified hASCs were significant lower than that of hBMMSCs (P<0.05). After 14 days of OI, the mineralization activity of both hASCs and purified hASCs were slightly lower than that of hBMMSCs; however, the difference was not significant (P>0.05).


Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.

Liu Y, Zhao Y, Zhang X, Chen T, Zhao X, Ma GE, Zhou Y - PLoS ONE (2013)

Osteogenic potential of human adipose-derived stromal cells (hASCs) and purified hASCs in vitro.A) hASCs and purified hASCs stained positive for alkaline phosphatase (ALP) after 7 days of osteoinduction (OI). The scale bar represents 100 µm. B) hASCs and purified hASCs were positive for Alizarin Red staining after 14 days of OI. The scale bar represents 100 µm. C) The ALP activities of hASCs, purified hASCs and hBMMSCs were significantly elevated after 7 days of OI; however, the ALP activities of both hASCs and purified hASCs were significantly lower than that of hBMMSCs (P<0.05). There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. D) The mineralization activities of hASCs, purified hASCs and hBMMSCs were significantly increased after 14 days of OI. There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. *P<0.05 compared with hASCs without OI; **P<0.05 compared with purified hASCs without OI; ***P<0.05 compared with hBMMSCs without OI; #P<0.05 compared with hBMMSCs after 7 days of OI.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569421&req=5

pone-0056002-g005: Osteogenic potential of human adipose-derived stromal cells (hASCs) and purified hASCs in vitro.A) hASCs and purified hASCs stained positive for alkaline phosphatase (ALP) after 7 days of osteoinduction (OI). The scale bar represents 100 µm. B) hASCs and purified hASCs were positive for Alizarin Red staining after 14 days of OI. The scale bar represents 100 µm. C) The ALP activities of hASCs, purified hASCs and hBMMSCs were significantly elevated after 7 days of OI; however, the ALP activities of both hASCs and purified hASCs were significantly lower than that of hBMMSCs (P<0.05). There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. D) The mineralization activities of hASCs, purified hASCs and hBMMSCs were significantly increased after 14 days of OI. There were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI. *P<0.05 compared with hASCs without OI; **P<0.05 compared with purified hASCs without OI; ***P<0.05 compared with hBMMSCs without OI; #P<0.05 compared with hBMMSCs after 7 days of OI.
Mentions: To compare the osteogenic potential of hASCs and purified hASCs in vitro, the cells were induced in osteogenic medium (OM) for 7 days or 14 days. Alkaline phosphatase (ALP) staining, Alizarin Red staining, ALP activity assay and quantitative Alizarin Red assays were then performed to assess the osteogenic potential of the cells. Positive results for ALP staining (Fig. 5A) and Alizarin Red staining (Fig. 5B) confirmed the osteogenic potential of both hASCs and purified hASCs. Human fibroblasts were used as negative controls and human bone marrow MSCs (hBMMSCs) as positive controls. ALP activity assay (Fig. 5C) showed the ALP activities of both hASCs and purified hASCs to be significantly elevated after 7 days of OI (P<0.05). However, there were no significant differences (P>0.05) between hASCs and purified hASCs or between hASCs that underwent OI (hASCs+OI) and purified hASCs that underwent OI (purified hASCs+OI). Quantitative Alizarin Red assay (Fig. 5D) showed the mineralization activity of both hASCs and purified hASCs to be significantly increased after 14 days of induction in OM (P<0.05), but there were no significant differences between hASCs and purified hASCs or between hASCs+OI and purified hASCs+OI (P>0.05). In addition, we found that after 7 days of OI, the ALP activities of both hASCs and purified hASCs were significant lower than that of hBMMSCs (P<0.05). After 14 days of OI, the mineralization activity of both hASCs and purified hASCs were slightly lower than that of hBMMSCs; however, the difference was not significant (P>0.05).

Bottom Line: We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs.Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo.These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Prosthodontics, School and Hospital of Stomatology, Peking University, Beijing, China.

ABSTRACT
Human adipose-derived stromal cells (hASCs) are a promising cell source for bone tissue engineering. However, before the clinical application of hASCs for the treatment of bone defects, key questions require answers, including whether pre-osteoinduction (OI) and flow cytometric cell purification are indispensible steps for in vivo bone formation by hASCs. In this study, hASCs were purified by flow cytometric cell sorting (FCCS). The osteogenic capabilities of hASCs and purified hASCs with or without pre-osteoinduction were examined through in vitro and in vivo experiments. We found that pre-OI enhanced the in vitro osteogenic capacity of hASCs. However, 8 weeks after in vivo implantation, there were no significant differences between hASCs and hASCs that had undergone OI (hASCs+OI) or between purified hASCs and purified hASCs+OI (P>0.05). Interestingly, we also found that purified hASCs had an osteogenic potential similar to that of unpurified hASCs in vitro and in vivo. These results suggest that FCCS and in vitro pre-OI are not requirements for in vivo bone formation by hASCs.

Show MeSH
Related in: MedlinePlus