The Related Study Of Reparative Dentin Formation Mechanism

ObjectivesAccording to the preliminary findings and literature review, we drew the followinghypothesis:1.The CXCR4positive dental pulp cells (CXCR4+DPCs) may bestem/precursor cells in the formation of reparative dentin;2.Hypoxia-induciblefactor-1α (HIF-1α) may be an important initiation factor for repair reaction afterinjury of dental pulp;3. Deferoxamine (DFO) may promote repair ability of dentalpulp via HIF-1α. To verify the above hypotheses, we isolated CXCR4+DPCs byimmunomagnetic cell sorting and the identification was completed at same time. Thecolony-forming and multipotent differentiation capacity of CXCR4+DPCs were alsoassessed to verify whether CXCR4+DPCs possessed stem cells properties; on theother hand, we intended to use the DFO to treat DPCs and observed the changes ofthe repair ability. Furthermore, we explored the molecular mechanism of reparativedentin formation regulated by DFO via HIF-1α with RNA interference (RNAi)techniques.Materials and methods1. CXCR4+DPCs were isolated by immunomagnetic cell sorting. The identificationwas completed at same time. The colony-forming and multipotent differentiationcapacity of CXCR4+DPCs were also assessed.2. At different time points, DPCs were treated by different concentrations of DFO invitro. The best concentration or time of DFO was decided from the effects on activity,proliferation and migration.3. As adenovirus a vector, we constructed HIF-1α shRNA, transfected DPCs andverified it. 4. The effects of DFO on the odontogenic differentiation of DPCs before/after HIF-1αgene silencing were observed in vitro.5. Pretreatment of DPCs before/after HIF-1α gene silencing with DFO, the formationof dentin-like tissue was observed.Results1. DPCs were identified successfully. DPCs possessed more clonogenic cells, and ahigher capacity of multi-directional differentiation than nonsorted DPCs.2. The activity, proliferation and migration of DPCs were improved after treatment48h with10μM DFO. Furthermore, the expression of HIF-1α was enhanced also.However, the viability or chemotaxis of DPCs was not changed.3. The efficiency of HIF-1α interference adenovirus (pAd/pENTR/shRNA-/GFP-HIF-1α) at the mRNA level was73.4%. At the protein level, it blocked the effects ofDFO on promotion the expression of HIF-1α in DPCs.4. The odontogenic differentiation of DPCs was promoted by treatment by10μMDFO for48h. Several genes associated with odontogenic differentiation wereupregulate also. However, if HIF-1α gene was interfered, the effects of DFO wereeliminated.5. The results of experiments in vivo show if DPCs were pretreated by10μM DFO for48h, they could produce more dentin-like tissue in vivo. However, if HIF-1α gene wasinterfered, DPCs hardly produced dentin-like tissue.Conclusions1. In this study, CXCR4+DPCs were isolated successfully. Furthermore, their stemcells properties were confirmed.2. The optimal concentration of DPCs is10μM and the optimal time is48h.3. HIF-1α gene interference adenovirus (pAd/pENTR/shRNA-/GFP-HIF-1α) wasconstructed successfully.4. DFO promoted odontoblast differentiation of DPCs in vitro and in vivo. When HIF-1α gene was silent, the effects of DFO would be eliminated.