Fig 1.
PDLSCs culture & identification.
(A)single PDLSCs, (B) PDLSCs polyclonal formation, (C) a clone of PDLSCs stained by the cyanine blue, (D) PDLSCs lipid droplet formed after lipid induction, oil red O staining positive, (E) PDLSCs after osteo-induction, mineralized nodules were formed, and alizarin red staining was positive. (F) RT-PCR after mineralized induction, PDLSCs expressed osteogenic genes(COL1, OCN, RUNX-2, BSP); (G and H) Flow cytometry detection of the mesenchymal stem cell marker shows PDLSCs are positive for STRO-1 and CD146, and (I and J) negative for CD34 and CD45.
Fig 2.
(A) immunofluorscence staining of RIP1 and RIP3. (B) CO-IP, a representative immunoprecipitation (IP) and western blot (WB) results for RIP1 and RIP3 interaction. IgG as a negative control. RIP1 and β-actin input as loading control. (C) TEM, a representative normal cell (the cell had a long fusiform, cell membrane structure was complete, the nucleus was round, nucleolus was obvious and center), b representative necroptosis cell (cell membrane ruptured, organelle disappeared, chromatin consolidated and agglutinated, presenting necrotic ultrastructural characteristics), c representative necrosis cell after Nec-1 inhibited (the cell membrane was intact, and the chromatin structure returned to normal). (D) Quantification of CO-IP showing RIP1 and RIP3 interaction is significantly decreased after Nec-1 pretreatment. *p<0.05 versus LPS. (E) Annexin V, PI/FITC FCM. Quantitative analysis of apoptosis. a control group, b Pg-LPS group, c Pg-LPS+Nec-1 group.
Fig 3.
(A) Representative images of alkaline phosphatase (ALP) staining in PDLSCs treated by osteogenic induction medium for 7 days. (B) Cultured PDLSCs form calcified nodules that stained positively for Alizarin Red S staining after 4 weeks of osteogenic induction. (C) Quantitative analysis of ALP activity in PDLSCs. (D) Quantitative comparison of mineralized nodule formation. (E) The relative mRNA levels of osteogenic related gene. *: p < 0.05.
Fig 4.
Histomorphometric analysis of the newly formed cementum-like mineralized deposits tissues.
Representative hematoxylin and eosin (H&E) Staining (A) and Masson Staining (B) indicated the formation of cementum-like tissue and periodontal ligament fibers at 8 weeks after surgery. (C) The corresponding quantitative analysis of histomorphometry observation. C: cementum-like tissue; S: scaffold; PDL: periodontal ligament. *: p<0.05.