Application of stem-cell media to explant culture of human periosteum: An optimal approach for preparing osteogenic cell material

Abstract

As part of our clinical tests on bone regeneration using cultured periosteal sheets, here, we prepared cultured periosteal sheets in two types of stem-cell culture media, STK1 and STK3. Human periosteum was expanded either in 1% human serum–supplemented STK1 for 28 days, in 1% human serum–supplemented STK1 for 14 days followed by 1% human serum–supplemented STK3 for 14 days (1% human serum–supplemented STK1+3), or in 10% fetal bovine serum–supplemented Medium 199 for 28 days (control). Cultured periosteal sheet diameter and DNA content were significantly higher, and the multilayer structure was prominent in 1% human serum–supplemented STK1 and 1% human serum–supplemented STK1+3. The messenger RNA of osteoblastic markers was significantly upregulated in 1% human serum–supplemented STK1+3. Osteopontin-immunopositive staining and mineralization were evident across a wide area of the cultured periosteal sheet in 1% human serum–supplemented STK1+3. Subcutaneous implantation in nude mice following expansion in 1% human serum–supplemented STK1+3 produced the highest cultured periosteal sheet osteogenic activity. Expansion in 1% human serum–supplemented STK1+3 successfully induced cultured periosteal sheet growth while retaining osteogenic potential, and subsequent osteoblastic induction promoted the production of homogeneous cell material.