Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

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.

Calcined Hydroxyapatite with Collagen I Foam Promotes Human MSC Osteogenic Differentiation

“…Even though HAP has a low solubility rate [50], and thus does not release high amounts of stimulation ions, the HAP construct was shown to have good biocompatibility and osteoinductive properties both in vitro and in vivo [59,60]. These results are in good accordance with our obtained data.…”

Calcium Concentration in Culture Medium as a Nondestructive and Rapid Marker of Osteogenesis

“…Therefore, the development of a reliable, rapid, easy, and nondestructive method is imperative. During cultivation of the construct, the calcium deposited in the matrix is supplied from the culture medium and regulated by the osteogenic activity of the osteoblasts 31–34 . Thus, we hypothesized that decreased calcium in the medium would reflect the amount of calcium deposited.…”

“…We used serially passaged cells for the construction of β-TCP/BM-MSCs in the present study, since osteogenesis decreases with cell passaging 34 . To verify whether calcium concentration can be a nondestructive marker of osteogenesis, we needed a simple experimental model in which BM-MSC osteogenesis decreases obviously.…”

Polycaprolactone-Based Scaffolds Facilitates Osteogenic Differentiation of Human Adipose-Derived Stem Cells in a Co-Culture System

“…For the co-cultured groups, ASC/HOB in the ratio of 2:1 showed the most positive enhancement in terms of cell growth and differentiation into osteogenic-like cells with its polygonal morphology as observed through microscopic visualization. This indicates that paracrine activity of co-cultured ASC/HOB in the correct ratio increase the ASC ability to differentiate into osteogenic cells and this may be enhanced by the use of PCL-HA composites, particularly HA [ 15 ] which has been shown to support bone formation as compared to the use of PCL alone [ 16 ] as HA has been reported to induce greater surface roughness, which increase surface area resulting in more cells attachment particularly suitable for differentiation along osteogenic lineage. In addition, surface roughness has been shown to influence the regulation of osteogenic differentiation [ 17 ].…”