Takeshi Amemiya

Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders

Background/aims: To determine outcomes of transplants of cultivated autologous oral epithelial cells in patients with severe ocular surface disorders. Methods: The eyes (n = 6) of four patients with Stevens-Johnson syndrome (three eyes) or chemical burns (three eyes) were studied. Autologous oral epithelial cells, grown for 2–3 weeks on a denuded amniotic membrane carrier in the presence of 3T3 fibroblasts, were air lifted. The resultant sheet was transplanted onto the damaged eye, and acceptance of the sheet by the corneal surface was confirmed 48 hours after surgery. The success of ocular surface reconstruction, graft survival, changes in visual acuity, and postoperative complications were assessed and the quality of the cultivated oral epithelial sheet was evaluated histologically. Results: At 48 hours after transplant, the entire corneal surface of all six eyes was free of epithelial defects indicating complete survival of the transplanted oral epithelium. Visual acuity was improved in all eyes. During follow up (mean 13.8 (SD 2.9) months), the corneal surface remained stable, although all eyes manifested mild peripheral neovascularisation. Conclusions: Autologous oral epithelial cells grown on denuded amniotic membrane can be transplanted to treat severe ocular surface disorders.

Hydrostatic Pressure Induces Cytokine Production in Human Periodontal Ligament Cells

Abstract Periodontal tissue has a unique structure in that the human periodontal ligament (hPDL) lies between the hard tissues of cementum and alveolar bone. Although the role of cytokines in hPDL function is not clearly understood, we investigated the effect of mechanical stress as hydrostatic pressure (HP) on cytokine expression in hPDL cells. The hPDL cells were obtained from a healthy maxillary third molar. After the 3rd to 4th passage, the cells were exposed to HP ranging from 1 MPa to 6 MPa as previously described. The expression of cytokine mRNA was determined by RT-PCR and cytokines in the culture supernatants were assessed by enzyme-linked immunosorbent assay (ELISA). The exposure to 6 MPa of HP caused no morphological changes of hPDL cells, and did not affect cellular viability. No expression of IL-1β, IL-6, IL-8, TNF-α, RANK, RANKL or OPG mRNA was observed in the control cells under atmospheric pressure, whereas in hPDL cells treated with HP, enhancement of IL-6, IL-8, RANKL and OPG mRNA expression was observed between 10 and 60 minutes after the exposure to HP. After the exposure to HP, the production of IL-6 and TNF-α were induced significantly in hPDL cells, but IL-1β and IL-8 were not produced. These results suggest that hPDL cells may play a role in the production of cytokines in response to mechanical stress in vivo .

Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects.

Evaluation of a dental pulp-derived cell sheet cultured on amniotic membrane substrate

OBJECTIVE Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. METHODS Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. RESULTS DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. CONCLUSIONS DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.