The efficacy of vitamin C in the formation of periodontal ligament stem cell sheet: an in-vitro study

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Periodontitis is a complex genetic disease mostly ends by tooth loss; consequently, the regeneration of a whole tooth structure may be required. The conventional tissue engineering technique is based on scaffold strategy, which may trigger inflammation. Cell sheet-based bioengineering ignores the use of scaffold to overcome its drawbacks.


The aim of the present study was to investigate the efficacy of vitamin C in forming periodontal ligament stem cell (PLSC) sheet.

Materials and methods

Periodontal ligament tissue was extracted from human-impacted wisdom teeth of healthy individuals aged 18–25 years. Following the culturing procedure, PLSCs were tested for colony-forming unit fibroblast assay, immunocytofluorescence detection of STRO-1 marker and multilineage differentiation. For PLSCs sheet formation, vitamin C (20 µg/ml) was added to the culture medium (experimental group). In another group (the control group) PLSCs were cultured without the addition of vitamin C (control group). Both groups were tested for alkaline phosphatase (ALP) enzyme activity and alizarin red stain.


PLSCs revealed their capability to form clonogenic cell clusters. They were also positive for STRO-1 marker. In addition, positive reaction for ALP activity, alizarin red and oil red O stains indicated their multilineage differentiation potentiality. Addition of vitamin C resulted in the formation of PLSC sheets after 12 days of culture. The cells at the edge of the dish were wrapped, indicating the formation of PLSC sheet. PLSC sheet displayed an increase in ALP activity and alizarin red stain positivity, whereas the control group did not reveal either of the two stains.


The study indicated that periodontal ligament cells have ultimate stem cell properties, and confirmed the efficacy of vitamin C to form human PLSC sheet. Thus, periodontal ligament cells will become the most beneficial source for periodontal regeneration.

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