Examination of Bone Differentiation for Human Dental Pulp-Derived Cells Cultured On Amniotic Membrane
Amniotic membrane as a cell culture scaffold is a useful membrane in regenerative medicine. We established a method to create a cultured oral mucosal epithelial sheet using the amniotic membrane as a scaffold, and applied this sheet clinically. Satisfactory results were obtained without defects such as rejection. The amniotic membrane has been demonstrated to be a suitable scaffold for cell culture, and has provided novel, useful and effective therapies in regenerative medicine. Additionally, this cell culture system was applied to the culture of periodontal membrane-derived cells. Periodontal ligament-derived cells proliferated on the amniotic membrane, and showed desmosomes and intercellular junctions, such as tight junctions, and thus successfully created a cultured periodontal ligament sheet (ref. 1, 2).
Dental pulp-derived cells can be readily collected from wisdom teeth discarded after extraction as medical waste; their proliferation ability is high compared with that of periodontal ligament cells and the risk of bacterial infection is low.
In this study, we prepared a dental pulp-derived cell sheet cultured on an amniotic membrane, and investigated it histologically and immunohistochemically.
Materials and methods
Extracted wisdom teeth were cut across the cement-enamel border. Only the pulp tissue was aseptically collected. The pulp tissue was minced, followed by primary culture in 10% FBS DMEM. After three or four passages, the dental pulp-derived cells were seeded onto an amniotic membrane and cultured for about 2 weeks at 10% FBS DMEM, followed by histological examination using hematoxylin-eosin (H-E) and immunohistochemistry. The dental pulp-derived cells were seeded onto an amniotic membrane and cultured for about 4 weeks in osteoinductive medium. After osteogenic induction, alizarin red S staining was conducted. This study and the use of dental pulp tissue and amniotic membranes were approved by the Medical Ethics Committee of Kyoto Prefectural University of Medicine (RBMR-C-1207).
Results
A single-layered structure of dental pulp cells cultured on amniotic membrane was observed by H-E staining. Localized vimentin-, Ki-67-, Zo-1-, desmoplakin-, CD29-, CD44-, CD105-, CD146-, and STRO-1-positive cells were observed by immunostaining. Dental pulp cells cultured on amniotic membrane were strongly stained with alizarin red S when cultured in osteoinductive medium.
Conclusion
Amniotic membrane-cultured dental pulp-derived cells were positive for Ki-67 (cell proliferation marker) and vimentin (mesenchymal cell marker), suggesting that dental pulp-derived cells proliferate on the amniotic membrane and maintain the characteristics of dental pulp. Additionally, dental pulp-derived cells cultured on the amniotic membrane expressed Zo-1 (tight junction structural protein) and desmoplakin (found on desmosomes), suggesting the presence of tight intercellular adhesion. Furthermore, they expressed CD29, CD44, CD105, CD146, and STRO-1 (mesenchymal stem cell markers), suggesting the presence of mesenchymal stem cells in the dental pulp-derived cells on the amniotic membrane. Finally, from the result of alizarin red S, bone differentiation of the dental pulp-derived cells was carried out, and the possibility of bone regeneration was suggested. In summary, the amniotic membrane formed a scaffold appropriate for culturing dental pulp cells, showing that the preparation of cultured cell sheets is possible. Dental pulp cells proliferated on the amniotic membrane and may have retained the property of the dental pulp. The possibility of applying the cultured cell sheet containing mesenchymal stem cells for wound healing after surgery and periodontal tissue regeneration was suggested.
References
1. Yamamoto T, Amemiya T, et al. Usefulness for a cultured human oral epithelial cell sheet on human amniotic membrane following removal of minor salivary gland tumor surgery. J Oral Tissue Engin; 5: 54-58, 2007.
2. Adachi K, Amemiya T, et al. Human periodontal ligament cell sheets cultured on amniotic membrane substrate. Oral Dis, in press.