Introduction The periosteum has recently been

Introduction The periosteum has recently been recognized as a promising source of immature progenitor cells that are not yet committed to specific cell lineages and osteogenic progenitors. For osteogenic cell processing in tissue engineering, the periosteum has generally been utilized to prepare dispersed cells (Hutmacher and Sittinger, 2003). The initial advantage of this conventional method is that periosteal sheet-derived cells grow faster in dispersed, single cell cultures than in explant cultures. However, one crucial disadvantage is the frequent contamination by non-progenitor cells, such as fibroblastic cells that often inhibit the differentiation of the progenitor cells in vitro (Iwasaki et al., 1995; Solchaga et al., 1998). The second advantage of this expansion culture is that the fibroblastic contamination can be easily removed by cell fractionation based on cell-surface antigens. Compared with this popular cell-dispersion method, an alternate method that uses explant culture from harvested periosteal tissue segments, which we have followed in our clinical practice, may be perceived to increase the risk of fibroblastic contamination and lengthen the processing time. Due to the limitations of permitted human surgeries, it is difficult to harvest an intact niclosamide layer, the lower osteoblastic-cell layer of the periosteum, from human alveolar bone tissues in routine clinical practice (Kawase, 2010). Therefore, taken together with the inability to perform cell fractionations, it would appear unlikely that this methodology could successfully transfer sufficient numbers of osteoblastic cells to the culture condition. However, we believe the cellular multilayered tissue-like periosteal sheets prepared by explant culture are substantially superior to the monolayer formed from dispersed periosteal sheet-derived cells because the multilayered periosteal sheets possess a tougher mechanical property than the mono-layered periosteal cells and are more amenable to handling. These are attractive features for any grafting material. In addition, this methodology also has the advantage of minimizing manipulation so that the risk of possible cell damage or transformation is reduced. In previous basic studies (Kawase et al., 2009, 2010a), we demonstrated that when cultured periosteal sheets are subcutaneously implanted into nude mice, their original periosteum tissue segments function as nuclei of mineralization to promote the formation of bone-like tissue. Furthermore, in previous clinical studies (Yamamiya et al., 2008; Okuda et al., 2009; Nagata et al., 2012), we demonstrated the successful regeneration of alveolar bone using this periosteal-sheet preparation in periodontal regenerative therapy. Although pre-osteoblastic or more mature osteoblastic cells can be harvested for culture, these cells are generally slow to grow and difficult to expand in vitro. In contrast, we have recently found in our prepared periosteal sheets a group of highly proliferative cells that reside around capillary blood vessels contained within the region of the original periosteal segment (Kawase, 2010; Kawase et al., in press). We speculate that these cells could be immature progenitor cells that are either not yet committed to a specific lineage or constitute somewhat immature osteogenic progenitor cells when derived from bone-related tissues. Relevant to this point, we have been able to generate cells of osteoblast lineage by treating periosteal sheets with conventional commercial methods of osteogenic induction, so we know that immature osteogenic cells are present. If this is the case, then it is possible that culture media developed and formulated to maintain or expand stem cells would be useful for the expansion of these immature progenitor cells. In this manner, the combination of a specific medium and our developed culture protocol should function as a promising cell-expansion system to improve the effectiveness and clinical utility of cultured periosteal sheets when used as osteogenic grafting materials.