The Role Of CAMP/PKA Signal Pathway In The Proliferation And Differentiation Of Stem Cells From The Apical Papilla

In recent years, tooth tissue engineering has become an important branch of the tissue engineering. It promotes the development of bioroot and the regeneration of pulp/dentin.During recent years, tooth tissue engineering has gained increasing insight and become a hotspot of the academicians. Stem cell plays a critical role in tissue engineering, and as a new dental stem cell, stem celsl derived from the apical papilla(SCAPs) were found by Sonoyama in 2006. The cell is located at the root apex of the incompletely developed tooth root. SCAPs are a population mesenchymal stem cells, which have a greater proliferation capacity, migration ability and differentiation potential when compared with PDLSC and DPSC. Besides, their immunogenic are weaker than DPSC. As a new dental progenitor cell, these characteristics of the SCAPs extend the range of its applications in tissue engineering for the future.Cyclic adenosine monophosphate/protein kinase A signal pathway has been described in many cells. The signal pathway may modulate the proliferation and differentiation of cells, such as BMMSC and DPSC. SCAPs and BMMSC are both mesenchymal stem cells. In addition, SCAPs and DPSC are dental stem cells. c AMP/PKA signal pathway can interact with other signal pathways such as NF-κB and BMP. Moreover, NF-κB and BMP can regulate the proliferation and differentiation of SCAPs. Therefore, we hypothesize that the c AMP/PKA signal pathway may play an important role in the proliferation, migration and differentiation of SCAPs. However, it is not clear whether the c AMP/PKA signal pathway may modulate proliferation, migration and differentiation of SCAPsObjective The aim of this study was to observe whether the c AMP/PKA was in SCAPs and to investigate the role of c AMP/PKA signal pathway in the proliferation, migration and differentiation of SCAPs.Methods SCAPs were cultured by enzyme digestion method. We used flow cytometry to identify the surface markers of SCAPs. After mineralization-inducing and adipogenic inducing, the multiple differentiation potential of SCAPs was identified by Alizarin red staining and Oil red O staining. PCR and QPCR were used to check whether the c AMP/PKA was in SCAPs. SCAPs were exposed to various concentrations of Forskolin or H-89 respectively. Cell proliferation ability and migration capacity were checked by MTT assay and wound healing test. After adding activator(Forskolin) and inhibitor(H-89) into the mineralization-inducing medium, we measured calcium deposition by Alizarin red staining. QPCR was performed to measure the m RNA expression of mineralization-related genes.Results1 Isolation, culture and identification of SCAPsHuman health third molars of the 16-18 year human with incompletely developed roots were collected extraction due to orthodontic/impactic reasons. The tissue was isolated bluntly. The cells were culured by enzyme digestion method. Flow cytometry showed that the cells expressed stem cell surface markers highly. However, the epidermal cell surface marker was low expressed. After mineralization-inducing and adipogenic inducing, calcium deposition and lipid droplet were found in the cells. The cells were capable of differentiating into osteo/odontogenic and adipogenic cell.2 The c AMP/PKA pathway in SCAPsPCR showed that the SCAPs expressed the c AMP receptor PKA. Activating the c AMP/PKA signaling, the PKA m RNA expression of SCAPs increased. These results indicated that the c AMP/PKA signaling was in the SCAPs.3 The influence of c AMP/PKA signal pathway on the proliferation of SCAPs2.5μmol/l Forskolin enhanced the proliferation ability of SCAPs. However, higher concentrations of Forskolin(10μmol/l, 20μmol/l) markedly inhibited proliferation of the cell. 1μmol/l H-89 inhibited the proliferation of SCAPs at the first day and the third day.In contrast, it enhanced the proliferation capacity of SCAPs at the fifth day and the seventh day. After inhibiting the signaling by H-89(5μmol/l, 10μmol/l), the SCAPs exhibited higher proliferation compared with normal untreated cells. 20μmol/l H-89 promoted proliferation of the SCAPs at the third day. However, the cells exhibited lower proliferation at the fifth day and the seventh day after inhibiting the signaling by H-89(20μmol/l).4 The influence of c AMP/PKA signal pathway on the migration of SCAPsThe SCAPs stimulated by Forskolin(10μmol/l, 20μmol/l) presented a lower migration capacity. On the contrary, the migration capacity of SCAPs was enhanced after inhibiting the signaling by H-89(5μmol/l, 10μmol/l).5 The influence of c AMP/PKA signal pathway on the differentiation of SCAPsActivating the c AMP/PKA signal pathway, the SCAPs exhibited an enhanced calcium deposition. Besides, the m RNA expression of ALP, OCN, OSX, RUNX2 was upregulated sharply in c AMP/PKA pathway-activated SCAPs. The pathway-inhibited SCAPs exhibited a decreased calcium deposition and downregulated expression of odonto/osteogenic genes, except for ALP.Conclusion The c AMP/PKA signal pathway could influence the proliferation and odonto/osteogenic differentiation of SCAPs.In summary, the study stated the effects of c AMP/PKA signal pathway on the proliferation and odonto/osteogenic differentiation of SCAPs. Moreover, it provided theoretical foundation for the application of SCAPs in tooth tissue engineering.