A Study Of RhBMP4 Enhances Tendon And Ligament(T/L)-lineage Differentiation Of HPDLSC With Multi-differentiation Capacity

OBJECTIVE Stem cell-guarded periodontal regeneration is a novel and more effective therapy strategy for periodontal diseases-induced tissue destruction. Periodontal ligament stem cells(PDLSCs) are an ideal stem cell source for periodontal regeneration. Bone morphogenetic protein 4(BMP4) has been known to have potential in maintaining the stemness and multi-potency of PDLSCs. However, the effect of BMP4 on the tendons/ligaments(T/L)-lineage differentiation ability remains unknown.MATERIALS AND METHODS 1. human PDLSCs(h PDLSCs) were obtained from periodontal ligament tissues by an explant culture. The stemness potential of h PDLSCs were identified by PCR analysis for CD-146, c-Myc, Nanog and Oct-4A and by FCM for CD146 and STRO-1. The osteogenic differentiation capability was valued by osteogenic induction. Cells were analyzed by reverse transcription PCR to detect ligament / tendon markers SCX, Six1, Six2 and TNC. 2. To elucidate the effect of an in vitro culture condition on the multilineage differentiation capability of h PDLSCs, we explored the expression of Runx2, Tenascin C(TNC), Collagen 1α1(COL1α1) and Collagen 1α2(COL1α2) in h PDLSCs with or without the addition of recombinant human bone morphogenetic protein-4(rh BMP4) or BMP4 overexpression or recombinant human noggin(rh Noggin) by reverse transcription PCR, realtime PCR and western blot.3. To investigate the mechanism of BMP4 promotes h PDLSCs into ligament / tendon differentiation. Realtime PCR and westrn blot were used to test the m RNA and protein expression levels of tendon-specific transcription factor Scleraxis(SCX), and immunofluorescence was used to analysis expression location. Search relationship between BMP4 and SCX from STRING database. VISTA software was used to analyze whether there Smad binding site SCX promoter.RESULTS BMP4 can transiently enhance the T/L-lineage differentiation of h PDLSCs, which maintain the capacity of multi-lineage differentiation, through nuclear accumulation of scleraxis(SCX, a tendon-specific transcription factor).