Effects of enamel matrix derivative on the proliferation and osteogenic differentiation of human gingival mesenchymal stem cells

Abstract

IntroductionGingiva-derived mesenchymal stem cells (GMSCs) have recently been harvested and applied for rebuilding lost periodontal tissue. Enamel matrix derivative (EMD) has been used for periodontal regeneration and the formation of new cementum with inserting collagen fibers; however, alveolar bone formation is minimal. Recently, EMD has been shown to enhance the proliferation and mineralization of human bone marrow mesenchymal stem cells. Because the gingival flap is the major component to cover the surgical wound, the effects of EMD on the proliferation and mineralization of GMSCs were evaluated in the present study.MethodsAfter single cell suspension, the GMSCs were isolated from the connective tissues of human gingiva. The colony forming unit assay of the isolated GMSCs was measured. The expression of stem cell markers was examined by flow cytometry. The cellular telomerase activity was identified by polymerase chain reaction (PCR). The osteogenic, adipogenic and neural differentiations of the GMSCs were further examined. The cell proliferation was determined by MTS assay, while the expression of mRNA and protein for mineralization (including core binding factor alpha, cbfα-1; alkaline phosphatase, ALP; and osteocalcin, OC; ameloblastin, AMBN) were analyzed by real time-PCR, enzyme activity and confocal laser scanning microscopy.ResultsThe cell colonies could be easily identified and the colony forming rates and the telomerase activities increased after passaging. The GMSCs expressed high levels of surface markers for CD73, CD90, and CD105, but showed low expression of STRO-1. Osteogenic, adipogenic and neural differentiations were successfully induced. The proliferation of GMSCs was increased after EMD treatment. ALP mRNA was significantly augmented by treating with EMD for 3 hours, whereas AMBN mRNA was significantly increased at 6 hours after EMD treatment. The gene expression of OC was enhanced at the dose of 100 μg/ml EMD at day 3. Increased protein expression for cbfα-1 at day 3, for ALP at day 5 and 7, and for OC at week 4 after the EMD treatments were observed.ConclusionsHuman GMSCs could be successfully isolated and identified. EMD treatments not only induced the proliferation of GMSCs but also enhanced their osteogenic differentiation after induction.

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“…Furthermore, GMSC isolated from inflamed tissues demonstrate similar multilineage differentiation potential as compared to those retrieved from healthy tissue [59] . As well, investigators have found that GMSC are non-tumorigenic and maintain normal karyotype and telomerase activity [33,60] . Multiple studies have demonstrated that GMSC tend to proliferate at a higher rate than either PDLSC or BMSC [7,33,61] .…”

“…Wu et al examined the use of enamel matrix derivative (EMD) treatment to promote GMSC proliferation and osteodifferentiation, finding a dose dependent increase in Runx2, Alp and Ocn expression with EMD treatment [60] . Conversely, GMSC cultured in adipogenic medium express PPARγ-2 [7] and form lipid globules characteristic of adipogenic differentiation [57] .…”