Cementum-periodontal ligament complex regeneration using the cell sheet technique

Background and Objective: In the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament-cementum complex. Material and methods: Human periodontal ligament cells were isolated and then cultured in dishes coated with a temperature-responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed. Results: After 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral-like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum-like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum-like tissue. Conclusion: This study suggests that the multilayered temperature-responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament-cementum complex.