Examination of Implanted Dental Pulp-derived Cell Sheets Cultured on Amnion

Objectives: Amnion 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 amnion as a scaffold, and applied this sheet clinically. Satisfactory results were obtained without defects such as rejection. The amnion has been demonstrated to be a suitable scaffold for cell culture, and has provided novel, useful and effective therapies in regenerative medicine. In this study, we prepared a dental pulp-derived cell sheet cultured on amnion and induced the bone differentiation of this cell sheet, subcutaneously transplanted it into the lumbar region of nude mice, and immunohistologically investigated it.
Methods: Dental pulp-derived cells were seeded on amnion and cultured for about 4 weeks in control or osteoinductive medium. The skin of the lumbar region was incised and dissected in 7-week-old male BALB/C nude mice, and the cell sheet cultured for about 4 weeks was placed in the wound and sutured. At about 4 weeks after transplantation, the mice were anesthetized with pentobarbital and examined by imaging using a soft X-ray device (Softex Co., Kanagawa, Japan). The transplants were collected from mice euthanized with an overdose of pentobarbital anesthesia, and immunohistologically investigated.
Results: Demonstrated that stem cells on a dental pulp-derived cell sheet cultured on amnion exhibited bone differentiation ability. The bone differentiation-inducing dental pulp-derived cell sheet cultured on amnion was subcutaneously transplanted into the lower back of an immunodeficient mouse, and subsequently showed a radiopaque image by soft X-ray irradiation and intense staining with Alizarin Red S and von Kossa. Immunostaining revealed that dental pulp-derived cells cultured on amnion were positive for osteocalcin.
Conclusions: Thus, calcified hard tissue was maintained even after transplantation, and that the sheet is applicable for periodontal tissue regeneration.