Enhanced derivation of osteogenic cells from murine embryonic stem cells after treatment with ionic dissolution products of 58S bioactive sol-gel glass

Abstract

Embryonic stem (ES) cells represent a potentially useful cell source for tissue regeneration. Previously, using factors known to enhance differentiation and mineralization of primary osteoblasts, we were able to generate cell populations enriched with osteoblasts from a murine ES cell source. Dexamethasone was a potent inducer of osteoblast differentiation and the timing of stimulation markedly increased the proportion of osteoblast lineage cells. This study examined whether inorganic stimuli derived from bioactive glasses could affect the differentiation of osteoblasts in an ES-cell based system. Previous work has demonstrated the ability of soluble ions released from bioactive glasses undergoing dissolution in vitro to stimulate gene expression characteristic of a mature phenotype in primary osteoblasts. We report here on the potential of soluble extracts prepared from 58S sol-gel bioactive glass to further enhance lineage-specific differentiation in murine ES cells. Differentiation of ES cells into osteogenic cells was characterized by the formation of multilayered, mineralized nodules. These nodules contained cells expressing the transcription factor runx2/cbfa-1, and deposition of osteocalcin in the extracellular matrix was detected by immunostaining. When differentiating cells were placed in an osteoblast maintenance medium supplemented with soluble extracts prepared from bioactive glass powders, we observed increased formation of mineralized nodules (98 +/- 6%, mean +/- SEM) and alkaline phosphatase activity (56 +/- 14%, mean +/- SEM) in a pattern characteristic of osteoblast differentiation. This effect of the glass extracts exhibited dose dependency, with alkaline phosphatase activity and nodule formation increasing with extract concentrations. Compared with medium supplemented with dexamethasone, which had previously been used to enhance osteoblast lineage derivation, the glass extracts were as effective at inducing formation of mineralized nodules by murine ES cells. When glass extracts were used in combination with dexamethasone, a further increase in the number of nodules was observed (110 +/- 16%; cf. 83 +/- 7% for dexamethasone alone). This study demonstrates the capacity of an entirely inorganic material to stimulate differentiation of ES cells toward a lineage with therapeutic potential in tissue-engineering applications.