Yoshinori Shinohara

Effective Bone Engineering with Periosteum-derived Cells

Bone augmentation via tissue engineering has generated significant interest. We hypothesized that periosteum-derived cells could be used in place of bone marrow stromal cells (which are widely used) in bone engineering, but the differences in osteogenic potential between these 2 cell types are unclear. Here, we compared the osteogenic potential of these cells, and investigated the optimal osteoinductive conditions for periosteum-derived cells. Both cell types were induced, via bFGF and BMP-2, to differentiate into osteoblasts. Periosteal cells proliferated faster than marrow stromal cells, and osteogenic markers indicated that bone marrow stromal cells were more osteogenic than periosteal cells. However, pre-treatment with bFGF made periosteal cells more sensitive to BMP-2 and more osteogenic. Transplants of periosteal cells treated with BMP-2 after pre-treatment with bFGF formed more new bone than did marrow stromal cells. Analysis of these data suggests that combined treatment with bFGF and BMP-2 can make periosteum a highly useful source of bone regeneration.

Subcultured odontogenic epithelial cells in combination with dental mesenchymal cells produce enamel-dentin-like complex structures.

We showed in a previous study that odontogenic epithelial cells can be selectively cultured from the enamel organ in serum-free medium and expanded using feeder layers of 3T3-J2 cells. The subcultured odontogenic epithelial cells retain the capacity for ameloblast-related gene expression, as shown by semiquantitative RT-PCR. The purpose of the present study was to evaluate the potential of subcultured odontogenic epithelial cells to form tooth structures in cell–polymer constructs maintained in vivo. Enamel organs from 6-month-old porcine third molars were dissociated into single odontogenic epithelial cells and subcultured on feeder layers of 3T3-J2 cells. Amelogenin expression was detected in the subcultured odontogenic epithelial cells by immunostaining and Western blotting. The subcultured odontogenic epithelial cells were seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells, and transplanted into athymic rats. After 4 weeks, enamel–dentin-like complex structures were present in the implanted constructs. These results show that our culture system produced differentiating ameloblast-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.

Mixing conditions for cell scaffolds affect the bone formation induced by bone engineering with human bone marrow stromal cells, β‐tricalcium phosphate granules, and rhBMP‐2

Bone regenerative medicine via tissue engineering is expected to be an alternative treatment for conventional autogenous bone graft, as it is less invasive. One of the best triads for bone engineering is bone marrow stromal cells, calcium phosphate ceramics, and bone morphogenetic protein (BMP). However, the optimal mixing conditions for BMP-induced osteoblasts and ceramic granules remain unclear. Therefore, we investigated the effect of the mixing conditions for cell scaffolds on the bone-forming potential. The cells were mixed with beta-tricalcium phosphate (beta-TCP) granules followed by osteoblast induction with recombinant human BMP-2 (rhBMP-2) (first mixture), or were first induced with rhBMP-2 on plastic dishes and then mixed with the beta-TCP granules (last mixture) just prior to the operation. Both the first and last mixtures were transplanted into nude mice subcutaneously, with the amount of bone formation analyzed histomorphometrically. In addition, cell numbers and alkaline phosphatase (ALP) activity before transplantation was determined in both the mixtures. In vitro analyses revealed that cell numbers were greater in the last mixture, whereas ALP activity was greater in the first mixture. In vivo analyses revealed that the first mixture was much more osteogenic than the last mixture with respect to new bone formation and osteocalcin synthesis. These data suggest that cell-scaffold mixing conditions have a significant influence on the bone-forming capacity via bone engineering and that first mixture might be the optimal condition for rhBMP-2-induction of human osteoblasts.

Enamel Tissue Engineering Using Subcultured Enamel Organ Epithelial Cells in Combination with Dental Pulp Cells

We describe a strategy for the in vitro engineering of enamel tissue using a novel technique for culturing enamel organ epithelial (EOE) cells isolated from the enamel organ using 3T3-J2 cells as a feeder layer. These subcultured EOE cells retain the capacity to produce enamel structures over a period of extended culture. In brief, enamel organs from 6-month-old porcine third molars were dissociated into single cells and subcultured on 3T3-J2 feeder cell layers. These subcultured EOE cells were then seeded onto a collagen sponge in combination with primary dental pulp cells isolated at an early stage of crown formation, and these constructs were transplanted into athymic rats. After 4 weeks, complex enamel-dentin structures were detected in the implants. These results show that our culture technique maintained ameloblast lineage cells that were able to produce enamel in vivo. This novel subculture technique provides an important tool for tooth tissue engineering.

Effect of Vitronectin Bound to Insulin-Like Growth Factor-I and Insulin-Like Growth Factor Binding Protein-3 on Porcine Enamel Organ-Derived Epithelial Cells

The aim of this paper was to determine whether the interaction between IGF, IGFBP, and VN modulates the functions of porcine EOE cells. Enamel organs from 6-month-old porcine third molars were dissociated into single epithelial cells and subcultured on culture dishes pretreated with VN, IGF-I, and IGFBP-3 (IGF-IGFBP-VN complex). The subcultured EOE cells retained their capacity for ameloblast-related gene expression, as shown by semiquantitative reverse transcription-polymerase chain reaction. Amelogenin expression was detected in the subcultured EOE cells by immunostaining. The subcultured EOE cells were then seeded onto collagen sponge scaffolds in combination with fresh dental mesenchymal cells and transplanted into athymic rats. After 4 weeks, enamel-dentin-like complex structures were present in the implanted constructs. These results show that EOE cells cultured on IGF-IGFBP-VN complex differentiated into ameloblasts-like cells that were able to secrete amelogenin proteins and form enamel-like tissues in vivo. Functional assays demonstrated that the IGF/IGFBP/VN complex significantly enhanced porcine EOE cell proliferation and tissue forming capacity for enamel. This is the first study to demonstrate a functional role of the IGF-IGFBP-VN complex in EOE cells. This application of the subculturing technique provides a foundation for further tooth-tissue engineering and for improving our understanding of ameloblast biology.

Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections.