| In the recent years, the research on the adult stem cells has been found to be veryimportant in the area of tissue regeneration and wound repairing. As a kind of adult stemcells in the pulp tissue, the hDPSCs have also been one of hot topic in the area of oralbiology. HDPSCs are characterized by highly proliferation and could differentiate intoodontoblast/osteoblast, adipocyte, and neurocyte in vitro. At the same time, numerousstudies also found that the hDPSCs could form the dentin/pulp-like complex by ectopictransplantation in immunocompromised mice. Therefore, it provides a preciousopportunity for establishing the bioengineered tooth and the research of pulp repair.Bacterial endotoxin LPS is the major pathogenic factor of G-bacteria. Studies show that LPS could regulate the mesenchymal stem cell differentiation by binding to itsspecific receptor TLR4. When the pulp was injured by caries, many growth factors in thedemineralized dentin matrix were released, such as TGFβl. And they could promote thehDPSCs to differentiate into odontoblast-like cell (OBLC). While the bacteria and its toxicproducts also could enter into the pulp tissue and stitimulate the hDPSCs. In this way,whether bacteria and its toxic products, such as LPS, would effect the proliferation anddirectional differentiation of hDPSCs or not, it remains unclear so far. Thus, we aimed toexplore the role of LPS in the proliferation and directional differentiation of the humandental pulp stem cell lines.We also preliminary analyzed the role of MAPK signalingpathways in LPS regulated the differentiation of hDPSCs. We have got the results asfollows:1. Isolation，culture and identification of hDPSCsThe tissue block enzymolytic method was used to isolate and culture primary humandental pulp cells. The limiting dilution was used to obtain the relative homogeneoushDPSCs. Then, we indentified the biological characteristics of hDPSCs. Flow cytometrywas used to analyse the surface markers of hDPSCs. At the same time, we also checkedthe multi-lineage differentiation capacity of hDPSCs. The results were performed inaccordance with the accepted biological characteristics of hDPSCs. It confirmed that wehad successfully established the human dental pulp stem cell lines.2. The effect of LPS on the hDPSCs proliferationIn order to study the effect of LPS on hDPSCs proliferation, hDPSCs were culturedin the two dimensions or there dimensions and stimulated by LPS. MTT assay showed thatthe role of LPS in the proliferation of hDPSCs was not very apparent in the short time（1-5d）.On the7thday, compared with the control, the LPS stimulation groups wereperformed the inhibitory effect. Then,1ug/ml LPS was used to stimulate the hDPSCswhich were seed on the HA/TCP scaffolds. Electron microscopy showed that the cells in7d groups were apparent increased comparing with3d groups. But there was no significantdifference between control and LPS stimulation group of3d and7d. On the14thday, thecells in LPS stimulation group were decreased comparing with the control group. All the results indicated LPS might not influence the proliferation of hDPSCs in the short time,but play an inhibitory role in a longer time.3. The role of LPS in directional differentiation of hDPSCsFirstly, we detected the expression of TLR4and mineralization related genes duringthe process of hDPSCs differentiation by real time PCR. We found that TLR4wasexpressed in hDPSCs and its expression reaches the peak on the7thday of hDPSCsdifferentiation. It indicated that LPS receptor TLR4might play an important role in theearly stage of hDPSCs differentiation. The expression of mineralization related genesDSPP, DMP1, ALP, and OPN were increased in different levels during the process ofhDPSCs differentiation. Then, we tested the expression of mineralization related genesand formation of the mineral nodules of hDPSCs stimulated by LPS. We found LPS couldpromote the expression of DSPP, DMP1, ALP, OPN and formation of mineral nodules. Atthe same time, we also found that LPS could promote the hDPSCs which werecompounded with HA/TCP scaffolds to secrete extracellular matrix and form collagen-likestructures. All the results proved that LPS could promote the hDPSCs to differentiate intoOBLC. These results were familiar to the process of pulp repair in vivo. It provided areliable research model for exploring the mechanism of pulp repair in vitro.4. The role of MAPK signaling pathways in LPS regulated the differentiation ofhDPSCsIn order to explore the mechanism of pulp repair, we also preliminary analyzed therole of MAPK signaling pathways in LPS regulated the differentiation of hDPSCs. Wefound that the roles of LPS in the expression of mineralization related genes and formationof the mineral nodules were significantly inhibted by blocking the ERK1/2and p38MAPK signaling pathways. However, blocking the JNK signaling pathways, the role ofLPS was not significantly influenced. In addition, it was also found that ERK and p38kinase were activated in hDPSCs in response to LPS. It meat ERK1/2and p38MAPKsignaling pathways were involved in the LPS regulated the differentiation of hDPSCs andexerted a favorable role, but JNK may not. It laid the groundwork for the research of pulprepair. All in all, LPS have an effect on the hDPSCs proliferation and directionaldifferentiation. The further study also found ERK1/2and p38MAPK signaling pathwayswere involved in the LPS regulated the differentiation of hDPSCs and might exert afavorable role. It lays the groundwork for the research of pulp repair and may provide agood idea for clinical vital pulp conservation treatment. |
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