The Study On The Effect Of Mammalian Target Of Rapamycin On Differentiation Of Lipopolysaccharide-regulated Human Dental Pulp Stem Cells

Dental pulpitis is one of prevalent and frequent diseases in human oral. At present wehave not found therapies that could completely recover the function of tooth. Applicationof tissue engineering clinically brings new hope for recovery of tooth vitality and function.When successfully isolated and cultured dental pulp stem cell (DPSCs) withself-renewable capacity and multi-directional differentiated potential, people foundDPSCs could form dental pulp-dentin complex, thus DPSCs became one of important seedcells for tissue engineering. In the process of making bioengineered teeth, themicro-environment, interaction between cells, intracellular or extracellular signalingmolecules and other factors as well as seed cells and scaffold materials are importantfactors in cell growth and differentiation, which further affect tissue engineering. Under the inflammatory microenvironment of pulp tissue, pro-inflammatorycytokines and bacterial component could influence the biological function of DPSCs.Toll-like Receptor4, as the special receptor of lipopolysaccharide (LPS), plays vital rolesin initiation of innate immunity and inflammatory response. With the development ofpulpitis, various toxic products including LPS produced by gram-negative bacteria wereincreased. However, at present there is little study about the effect and mechanism of LPSand its receptor TLR4on DPSCs.The mammalian target of rapamycin (mTOR) signaling could regulate cell autophagy,growth, immune, inflammation, apoptosis, protein synthesis and energy metabolism bycross-talking with other signaling ways which is crucial for growth factors and energystate, thus mTOR is the hot signaling way in recent years. The studies about the effect ofmTOR on DPSCs differentiation have been focused by researchers, but it is not clearabout the biological mechanism of mTOR on stem cells stimulated with bacterial factorssuch as DPSCs stimulated with LPS. As we all known that human pulpal tissueinflammation is the main pulp disease, and bacteria is one of important pathogenic factors,thus it is of great significance to expound the effect of bacterial component ondifferentiation of DPSCs and related intracellular molecular mechanism. In present study,we aimed to study the effect of LPS-activated TLR4signaling on differentiation of DPSCsand the mechanism of mTOR in this process based on DPSCs separation, cultivation andidentification in vitro by stimulation of LPS and mTOR special inhibitor rapamycin,which is important to understanding the role of mTOR on LPS-regulated differentiationand elucidation of the repair mechanism of injured pulp. Meanwhile, the study providesnew ideas for tissue engineering based on DPSCs and and new methods for future vitalpulp therapy.Main results as followed:1. The expression and change on LPS receptor TLR4and mTOR in differentiation ofDPSCsTo acquire highly purified DPSCs, human dental pulp cells were primary cultured with tissue modified tissue explant technique, and then cells were collected and preparedfor limiting dilution procedures to obtain single cell clones. Our result showed that themorphology of DPSCs were typical and homogeneous, and flow cytometry analysisdemonstrated a typical pattern for mesenchymal stem cells (MSCs) by the positiveexpression rate of Stro-1and other marker genes; the effects on mineralization andadipogenic differentiation of identified DPSCs were observed by related culture mediumfor14days. The results showed that mineralization nodules or lipid droplet were formedby Alizarin red staining analysis and Oil O staining, which suggested that DPSCspossessed the potential of osteogenesis and adipogenesis. The result of this part indicatedthat our identified DPSCs have self-renewable capacity, multi-directional differentiatedpotential and the characterization of MSCs, which further demonstrated that application ofDPSCs have broad prospects in tooth tissue engineering and provided solid basis forfurther study based on DPSCs.To explore the expression of LPS receptor TLR4and mTOR in the process of DPSCsdifferentiation, we cultured DPSCs identified above after24h of basal medium culture andRNA extraction, and founded that the expression of TLR4and mTOR could be detectedby reverse transcriptase polymerase chain reaction (RT-PCR). In addition, the DPSCswere cultured by mineralization induced medium for7days,10days and14daysrespectively. The Real-time PCR results showed that expression of TLR4and mTORgenes were gradually upregulated in the mineralization of DPSCs during2weeks, whichindicated that TLR4and mTOR were probably participated in the differentiation ofDPSCs.2. The effect of LPS on the expression of TLR4and mTOR during DPSCsdifferentiationTo further confirm whether TLR4and mTOR signal pathways were involved inLPS-regulated DPSCs, we incubated DPSCs at different induction conditions for7days,10days and14days. The groups are as followed: control group was cultured withmineralized medium, and LPS-stimulated group was culturing in the mineralized medium containing1μg/mL LPS. Real-time PCR results showed that expression levels of TLR4mRNA and mTOR mRNA were increased significantly compared to control group, at thesame time, LPS-induced TLR4mRNA and mTOR mRNA were upregulated in atime-dependent manner. The date indicated that TLR4and mTOR signalings may beinvolved in LPS-regulated differentiation of DPSCs.3. The effect of mTOR on differentiation of LPS-stimulated DPSCsTo better understanding the effect of LPS on differentiation of DPSCs and themolecular mechanism of mTOR involved in, we cultured DPSCs in mineralized mediumcontaining various stimuli for7days,10days, and14days respectively and divided intothree groups as followed：control group was cultured with mineralized medium; LPSgroup was culturing in mineralized medium containing with1μg/mL LPS; andRapa+LPS group was incubated in mineralized medium containing with1μg/mL LPSand1μM Rapa. Result of ALP staining and Alizarin red staining demonstrated that ALPactivity and mineralized nodules in LPS group were much stronger than in the controlgroup, however, rapamycin could attenuate LPS-induced effect. It was also observed thatexpression of odontoblastic genes DSPP, BSP and OCN were downregulated obviouslyin Rapa+LPS-treated group compared with LPS-stimulated group by Real-time PCR.And expression of DSPP in LPS-stimulated group was distinctly higher than the controlgroup; LPS-stimulated group expressed higher BSP gene, which is significant differencecompared with control group at days10and14; however, OCN gene in LPS group wassignificantly lower compared to control group at14days while showing opposite resultat7days.Meanwhile, DPSCs were cultured in mineralized medium containing various stimulias above for21days. The SEM micrographs and Quantitative analysis of alizarin redrevealed an appreciably larger amount of small globular mineral deposits and calciumcontent in LPS-treated group compared with control group, which could be decreased inRapa+LPS-stimulated group. All above results demonstrated that an effectiveconcentration of LPS induced differentiation of DPSCs, while rapamycin distinctly abrogated LPS-induced effect, which indirectly confirmed that mTOR may be involved inLPS-induced differentiation of DPSCs.In conclusion, our study demonstrated that LPS-activated TLR4signaling induceddifferentiation of DPSCs, and mTOR may play important roles in LPS-regulateddifferentiation, which is essential to elucidation of the repair mechanism of injured pulp.Meanwhile, the study provides new ideas for tissue engineering based on DPSCs and newmethods for future vital pulp therapy.