Isolation of periodontal ligament stem cells from extracted premolars. Simplified method

Abstract

Periodontal ligament is a fibrous connective tissue of ectomesenchymal origin which contains a multipotent postnatal stem cells population with clonogenic capacity and high proliferation index. Multipotent postnatal stem cells population culture has been achieved through several extraction methods, nevertheless some technical problems have been encountered. For this reason, in the present study, 6 different methods were compared so as to improve isolation of primary periodontal ligament cells. Multipotent postnatal stem cells population were isolated from 36 healthy premolars, which were then cultured in complete Dulbecco m odified Eagle medium (BioWest) (added with L-glutamine, 10% fetal bovine serum and 1% antibiotic), in standard culture conditions. Assays for cell proliferation were conducted with 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium technique as well as cell migration through crop injury test, after which osteogenic differentiation was assessed. Cultures were performed in triplicate with each of the published techniques; thus we were able to obtain a minimum population of cells susceptible to contamination. The «simplified method» was conducted through explant enzymatic digestion and culture: with obtained multipotent postnatal stem cells population, a 14 day proliferation was observed in standard conditions of ascendant culture; in migration tests, multipotent postnatal stem cells population totally covered injured surfaces after 72 hours. CD90 positive expression was observed in over 80% of all cells; CD73 positive expression was found in 70% of cells, CD105 in 60% and vimentin in 90% of the population, as well as negative marking to CD11b and CD45. Positive osteogenic differentiation was observed after 14 days, through positive expression to RUNX2 in at least 15% of cells, and to osteocalcin in 90% as well as positive test to alizarin red. Based on obtained results we were able to affirm that the simplified method allows to isolate a population of mesenchymal cells, which might be obtained from extracted healthy premolars.