| Background and objectiveIn recent years, the mesenchymal stem cells with the self-renewal and multi-differentiation capacity in tissue regeneration project has received wide attention.And in the field of dentistry, the potential sources of some mesenchymal stem cells have been confirmed as the candidate of the oral tissue engineering, such as bone marrow mesenchymal stem cells(BMMSCs), adipose tissue, umbilical cord blood and oral tissues,etc. In2000, Gronthos etc.had first isolated the first dental stem cells from human dental pulp tissues,which were named as dental pulp stem cells(DPSCs).It created a new era for the study of dental stem cells. Subsequently, scholars have isolated other dental mesenchymal stem cells,including BMSCs,human deciduous teeth stem cells (SHED), periodontal ligament stem cells (PDLSC), epithelial root sheath cells and dental sac stem cells (DFPCs)and so on.Mesenchymal stem cells in the oral tissues with multi-differentiation capacity can differentiate to osteogenic,adipogenic, nerve, cartilage cells and myogenic,etc in the specific culture conditions.And osteogenic differentiation is one of the most obvious feature in the applications of oral tissue regeneration. Some studies have shown that DPSCs could differentiate into bone-like tissue in vitro and form ectopic pulp dentin-like composite organization while transplanted into the subcutaneous of immunodeficient mice. DPSCs is a candidate source of stem cells of the oral tissue regeneration project.However, the mesenchymal stem cells derived from the tooth tissue have the same characteristics, but there are also significant heterogeneity. In recent years, some case reports showed that the apex of young permanent teeth suffering periapical or apical abscess continuoued to develop and form after conservative root canal therapy. Traditional knowledge is difficult to explain this phenomenon.。But the new group of mesenchymal stem cells which were discovered recently that they are exist in the apical papilla of young permanent teeth may be able to explain the problem.These cells were called Stem cells from apical papilla(SCAP). When root occurs to form, the neck ring at the glaze and the outer enamel epithelium proliferate to the apical.And mesenchymal stem cells of dental papilla differentiate into odontoblasts, which form primary dentin.Then the dental papilla was surrounded by the primary dentin and developed into pulp. There is a region which is rich in cells between the pulp and apical papilla tissue. And the characteristics of the stem cells between in the pulp and apical papilla tissue are slightly different.So far many researchers have been confirmed that DPSCs with Self-renewal capacity,colony formation capacity and multi-differentiation capacity can differentiate to osteogenic,adipogenic, nerve, cartilage cells and myogenic,etc in the specific culture conditions.But the study of in vitro biological characteristics of SCAP is in the initial stage.Therefore, the purpose of this study is to compare the different biological characteristics in vitro and in vitro osteoblast/odontogenic differentiation potential of DPSCs and SCAP which were derived from two closely histological but different organizations of young permanent both to confirm that SCAP are capable of multiple lineage differentiation and showed a significantly higher proliferation rate and mineralization potential, suggesting a more excellent stem cell source. At the same time, on the basis of the first part of the experiment, we compare the effect of Mineral trioxide aggregate(MTA) on proliferation and differentiation of SCAP and DPSCs to provide new ideas and theoretical guidance for the clinical treatment of periapical lesion and the continued development of the teeth and tooth regeneration.And it provides a theoretical basis for the new conserve treatment of young permanent teeth and the tooth regeneration research.Part I Isolation and identification of stem cells from human stem cells from apical papilla and dental pulp stem cellsMethods:We removed the orthodontic teeth or third molars without apical formation.And we washed the teeth repeatedly by using the phosphate buffered saline(PBS).Then the SCAP and DPSCs were isolated from the apical papilla and pulp tissue and cultured in the medium。The growth situation and morphological characteristics of the primary cells of SCAP and DPSCs were observed under an inverted microscope.Both second generation cells were seeded on coverslips, and were detected the expression of STRO-1by using immunofluorescence technology. SCAP and DPSCs were digested by trypsin to form the cell suspension which were seeded to6-well plates. After7d,the cells were stained with crystal violet dye to observe cell clone formation.The third generation cells of SCAP and DPSCs were measured their OD values for10d by MTT assay.And the cell growth curve was drawn with time on the horizontal axis, the daily average OD value of the longitudinal axis.After induction, SCAP and DPSCs were stained by Safranin O when lipid droplet formation was observed.After the mineralized induction,SCAP and DPSCs were stained by alizarin red to detect their mineralized nodule formation capacity.After the mineralized induction,the expression of alkaline phosphatase (ALP), the bone sialophosphoprotein (BSP), osteocalcin (OC), dentin sialoprotein (DSP) of SCAP and DPSCs were detected by ImmunofluorescenceAfter the mineralized induction for0d,7d,14d,21d.The mRNA expression of the ALP, DSP, BSP, OC gene of SCAP and DPSCs were detected by Immunofluorescence to compare the capacity of osteoblast/cementoblast in vitro.Results:After culture for7d, adherent cells growed radially around the organization block. The most of two group cells presented fusiform shape as fibroblast-like. And two types of stem cells are basically the same size.The climbing films of SCAP and DPSCs were detected by anti-STRO-1immunofluorescence.The cell nuclei of two types of stem cells presented blue fluorescence.And the cytoplasm presented green fluorescent. STRO-1expressed positive.After culture for7d, crystal violet staining showed that cell colony formation were observed obviously in the6-well plates.And the colony formation rate of SCAP is higher than that of DPSCs.The growth curve of SCAP ang DPSCs was like "S".And the1-2d was incubation phase.At the logarithmic growth phase(3-8d),cells accelerated to grow.After8d,cells slowed down at the plateau phase. But the growth speed of SCAP is faster than that of DPSCs corresponding to the characteristics of slow cycle of the cells.After induction for3weeks, SCAP and DPSCs were stained by Safranin O.The results were positive,and the lipid droplet was observed.After the mineralized induction for4weeks, the jacinth mineralized nodules of two stem cells were observed by by alizarin red staining.After the mineralized induction,the expression of ALP, BSP, OC and DSPof SCAP and DPSCs were detected by Immunofluorescence.And all the results were positive. The fluorescence intensity of the SCAP group is higher than that of DPSCs.The PCR showed that with increasing induction time, the espression of the gene levels of BSP, DSP and OC were up-regulated.But that of ALP decreased with increasing induction time. And the expression of SCAP group was higher than DPSCs group.Conclusions: In this study, the SCAP and DPSCs were isolated from the apical papilla and pulp tissue and cultured in the medium successfully. Then we had compared the different biological characteristics in vitro and in vitro osteoblast/odontogenic differentiation potential of DPSCs and SCAP. It has confirmed that SCAP are capable of multiple lineage differentiation and showed a significantly higher proliferation rate and mineralization potential, suggesting a more excellent stem cell source.Part II The effects of MTA on the proliferation and differentiation of human stem cells from apical papilla and dental pulp stem cellsMethods:The methods for preparation of MTA at a concentration of20mg/ml in this study was according to Hakki.The third generation cells of SCAP and DPSCs were measured their OD values for1d,3d,5d and7d by MTT assay.The conventional medium was replaced to the medium containing0.2mg/ml MTA.After12hours starvation, the experimental group of the third generation cells of SCAP and DPSCs was added the culture medium containing0.2mg/ml MTA,but the control group not.Then the cells were collected after12h,24h,36h and48h. The mRNA expression of ALP, BSP, OC and DSP after the effect of MTA were detected by Quantitative PCR.Results:Compared with the control group, cell proliferation activity increased with time, and the increasetion of the increased proliferative activity of SCAP group is higher than the DPSCs group (P<0.05).The PCR showed that with increasing induction time, the espression of the gene levels of DSP and OC were up-regulated.But that of ALP and BSP was increased first and then decreased.And the expression of SCAP group was higher than DPSCs group.Conclusions:In this study,the concentration of calcium which was released from MTA was controlled by0.2mg/ml MTA leaching solution.Then the effects of MTA on the proliferation and differentiation of human stem cells from apical papilla and dental pulp stem cells were studied and observed,which was confirmed the biological characteristics of SCAP and DPSCs.These provided a basis to find a better source of stem cells in the tooth tissue engineering applications.And these also provided a theoretical basis for the MTA in clinical applied to deep caries and apexification. It was more important that it laid the foundation for the study of the mechanism of revascularization of the immature permanent teeth.In conclusion,in this study, we have confirmed that SCAP and DPSCs are the mesenchymal source of stem cells.Compared with DPSCs, SCAP is a more excellent stem cell source. And so far,SCAP maybe the most outstanding early odontogenic source of stem cells and has broad application prospects in the clinical application of revascularization. |
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