To develop a differentiation screening method using 3D culture with ES cells, EBs were cultured for 21 days, in a culture media of collagen gel and chemicals affecting differentiation. The chemicals consisted of cytokines, EGF, FGF, IL-6, IL-12, TNF-α and mLIF, vitamins such as L-ascorbic acid, cholecalciferol and folic acid, and BMP, Emdogain^®, four adrenocortical hormones such as Dexamethasone, Betamethasone, Betamethasone Valerate and Betamethasone Dipropionate. The results demonstrated that the myocardial pulsation rate decreased with IL-6, TNF-α, L-ascorbic acid or cholecalciferol, but there was no significant difference observed with other chemicals. Regarding the amount of osteocalcin, FGF induced 360% of that in the control, BMP induced 310% in the control and Cholecalciferol induced 180% that of that in the control. For other combinations, there were no significant differences observed. XPS also showed a tendency toward similar results. The results of this study have shown that the effects of chemical substances on ES cell differentiation can be determined with the 3D culture method by collagen.

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Bisphosphonates(BPs), synthetic analogs of pyrophosphate, are potent inhibitors of bone resorption and are being successfully used with increasing frequency in the treatment of osteoporosis. We investigated whether BPs (etidronate, alendronate and risedronate) affect the proliferation and the mRNA expression of type I collagen, osteocalcin and alkalinephosphatase on human osteoblast-like Saos-2 cells. When alendronate and risedronate were co-cultured with Saos-2 cells, proliferations were deceased compared with control and etidoronate. The mRNA expression was analyzed using a reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNA, type I of collagen, osteocalcin and alkalinephosphatase were decreased by treatment with alendronate and risedronate but not etidronate. These results indicate that N-BPs may influence the activity of osteoblasts not but non-N- BPs.

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