Osteogenic ability study on co-culture of adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells

Abstract

Objective To assess the osteogenic ability after co-culture BMSC and ADSC in vivo and in vitro. Methods ADSC and BMSC were obtained by adherent screening method and enzymatic digestion method. Flow cytometry was used to confirm the phenotypes of ADSC and BMSC. Oil red O was used to induce MSC to fat. Alkaline phosphatase (ALP) and alizarin red staining were used in osteogenic group. This sample was divided into four groups, no-induced stem cells group; BMSC osteogenic induction group; ADSC osteogenic induction group; co-culture of BMSC and ADSC osteogenic induction group. ALP activities and Calcium absorbance were determined during different periods of osteogenic introduction. OCN and Runx2 expression level were tested via RT-PCR and western blot methods after osteogenic induction for 2 weeks. Furthermore, cells in each group were seeded on HA/CS/PLLA composite scaffolds, and the scaffolds with cells were planted into bone defects in rat models. The rats were sacrificed by overdose anesthesia at 8 weeks after surgery and the scaffolds were removed for further analysis. Results Oil red O staining demonstrated red after adipogenic induction. Alkaline phosphatase and Alizarin red staining showed flaky red under condition of osteogenic induction. There had no statistical change among each group after osteogenic induction for 3 days, and ALP activity significantly increased after osteogenic induction for 5 days. Meanwhile, the ALP activity in co-culture of BMSC and ADSC group was markedly higher than the other three groups. However, there had no significant change in A value of calcium absorbance among each group after osteogenic induction for 7 days, while it increased at 14th day and ALP activity in co-culture of BMSC and ADSC group was significantly higher than the other three groups. After osteogenic induction for 2 weeks, the mRNA expression of OCN and Runx2 in co-culture of BMSC and ADSC group was 78.24±8.11 and 1 180.13±121.16 respectively, and the protein expression of OCN and Runx2 was 6.54±0.59 and 4.43±0.51. These mRNA and protein expression level in co-culture of BMSC and ADSC group enhanced significant compared with the other 3 groups. Histological assay demonstrated that the new bone tissues formed in co-culture of BMSC and ADSC group were 497.75±7.44 μm2, which was larger than that in the other 3 groups at 8 weeks after implantation. Conclusion Co-culture BMSC and ADSC may up-regulated the osteogenic ability in vivo and in vitro. Key words: Mesenchymal stem cells; Bone marrow; Adipose tissue; Coculture techniques; Tissue engineering