Abstract
We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.
Highlights
Bone marrow contains a population of undifferentiated cells known as mesenchymal stem cells (MSCs) [1,2]
The combination of Bone marrow-derived MSCs (BMSCs) with a scaffolding material, such as hydroxyapatite (HA), can promote the formation of new bone tissue in vivo after subcutaneous transplantation with freshly isolated bone marrow cells [13] or culture-expanded BMSCs (HA/ BMSCs construct) [2,14,15,16,17], Recently, we reported a new cell transplantation technique in which BMSCs were cultured in culture medium containing Dex and ascorbic acid phosphate and transplanted as cell sheets [18]
Weaker staining was seen in passage 1 (P1), P2 and passage 3 (P3) cells, indicating that osteogenic potential of seriallypassaged BMSCs is lost by P3
Summary
Bone marrow contains a population of undifferentiated cells known as mesenchymal stem cells (MSCs) [1,2]. The combination of BMSCs with a scaffolding material, such as hydroxyapatite (HA), can promote the formation of new bone tissue in vivo after subcutaneous transplantation with freshly isolated bone marrow cells [13] or culture-expanded BMSCs (HA/ BMSCs construct) [2,14,15,16,17], Recently, we reported a new cell transplantation technique in which BMSCs were cultured in culture medium containing Dex and ascorbic acid phosphate and transplanted as cell sheets [18]. These cell sheets can be combined with HA, in which they resulted in abundant bone formation compared with conventional techniques [17]
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