Characterization And Effect In Periodontal Regeneration Of The Alveolar Socket-derived Human Periodontal Ligament Stem Cells

Periodontal regeneration, which needs regeneration of both hard and soft tissues, has been a challenge for decades. It is a hot spot that the applications of tissue engineering in the field of periodontal regeneration in recent years. The source of seeding cells is a key element on the tissue engineering. It has been proved that there are stem cells residing in the periodontal ligament on the root surface (r-PDLSCs). They reported that r-PDLSCs had the potential to form cementum/ PDL-like tissue in vivo and was useful for the periodontal regeneration. Periodontal ligament cells are heterogeneous cell populations. We speculate that stem cells in periodontal ligament have different characteristics according to the position. In this study, we isolated stem cells from human periodontal ligament on the alveolar bone surface(a-PDLSCs)successfully and identified the stem-cell characteristics and the effect in periodontal regeneration. Compared with r-PDLSCs and bone marrow mesenchymal stem cells (BMSCs), we evaluated the differences between the two kinds of periodontal ligament stem cells. Key findings are as follows:Part I: Isolation, culture and biological characterization of periodontal ligament stem cells derived from alveolar bone surface.In order to prove the distribution of periodontal ligament after extraction, we simulated the normal extraction procedure at SD rat molar. H&E staining showed that there was a part of periodontal ligament embedding on the alveolar bone surface after extraction. We labeled SD rats in situ with BrdU and evaluated the BrdU long-term adsorption capacity of periodontal ligament stem cells by immunofluorescence assay. The results showed the asymmetry distribution of the stem cells in periodontal ligament. The BrdU-positive cells in PDL on the site of alveolar bone were more than that on the site of root. Through enzymatic digestion and tissue-culture, we isolated periodontal ligament stem cells from periodontal ligament on the alveolar bone surface. The results of colony-forming, MTT and flow cytometry analysis showed that the proliferation ability of a-PDLSCs was stronger than BMSCs, similar with r-PDLSCs. The results of flow cytometry for epitope profile showed that a-PDLSCs expressed mesenchymal stem cell markers positively and expressed hematopoietic markers negatively. Immunofluorescence staining showed that r-PDLSCs and a-PDLSCs both expressed mineralization markers OCN and COL?. Alkaline phosphatase activity assay exhibited that a-PDLSCs expressed higher than r-PDLSCs. Quantitative analysis on the RNA levels for the differentiation markers showed that a-PDLSCs expressed on higher levels for the mineralization markers than r-PDLSCs; and two kinds of periodontal ligament stem cells expressed higher for the tendon- specific transcription factor than BMSCs.These results suggest that there are stem cells residing in the periodontal ligament derived from the alveolar bone surface. Because of the high proliferation capability and stronger mineralization potential, a-PDLSCs can be used as seed cells in the study of periodontal regeneration.Part?: Differentiation potential and effect in the periodontal regerneration of a-PDLSCsIn this study, on the condition of osteogenic induction, adipogenic induction and non-induction, the results of the differentiation potential for r-PDLSCs, a-PDLSCs, BMSCs showed that both of a-PDLSCs and r-PDLSCs can form mineralized nodules and lipid in vitro. The quantitative assay showed that a-PDLSCs had stronger potential in osteogenic / adipogenic differentiation. Cells mixed with CBB and coverd with Matrigel were implanted subcutaneously in SCID mice. The histological results after 6 weeks showed that r-PDLSCs and a-PDLSCs implants both generated periodontal ligament-like tissues and mineralized tissues, and the shape and area of mineralized tissues were differences. The mineralized tissues generated by a-PDLSCs were significantly larger than r-PDLSCs; and the shape was similar to that regenerated by BMSCs. In order to evaluate the ability on mineralization and repairing for the bone defect, the critical-size defects in calvarial bone were created in NOD=SCID mice. R-PDLSCs and a-PDLSCs were implanted into the defects. Micro-CT results show that, r-PDLSCs and a-PDLSCs groups both formed new bone in the defect area. The quantitative analysis showed the area of new bone formation generated by a-PDLSCs was larger than r-PDLSCs. These results suggested a stronger bone defect repair capacity of a-PDLSCs.The results on differentiation and regeneration capacity suggested that a-PDLSCs, which had greater differentiation and bone regeneration and repair capabilities, would be widely used in the field of periodontal tissue engineering.