The Effects And Possible Mechanism Of TLR4Ligation To The Osteogenesis Potential Of PDLSCs And BMMSCs

[Background]Periodontitis is characterized by the loss of periodontal attachment and alveolar boneresorption. It has a high morbidity and is the main course of teeth loss for adults. Neitherthe non-surgical nor surgical treatment for periodontitis can make a functional andpredictable regeneration for the already lossed periodontal tissure. This may be because ofthe complicated pathogenesis, persistently existence of bacterial and the personallyspecialised immune reaction.For the rapid development of Tissue engineering medicine, people attempt to restorethe periodontal defective by tissue engineering. Seed cells are important for tissueengineering. Periodontal ligament stem cells (PDLSCs) are promised choice forperiodontal therapy. They come from periodontal ligament and can formcementum-periodontal ligament like tissue after in vivo transplantation, which isimportant for a functional periodontal regeneration.Oral cavity is an environment that consist many kinds of bacterial. For the patients of periodontitis, there are usually many reasons for bacteria plaque to form easily. Theexistence of the bacterial and their toxic products may affect the outcomes of cell therapy.Organisms recognize bacterial or other microorganisms and their products byToll-like receptors (TLRs). TLRs is a kind of conserved receptor family.10kinds of TLRssubtypes (TLR1-TLR10) have been found in human by now. Different subtypes recognizedifferent ligands containing different architecture. Periodontitis is caused by G(-) bacterial,and the main pathogenic components of G(-) bacterial is lipopolysaccharide (LPS), alsocalled endotoxin and is recognized by TLR4.Mesenchymal stem cells (MSCs) express TLRs and TLRs ligantion can affect thefunction of MSCs. Different kinds of MSCs express different kinds of TLRs and aredifferently affected by their ligation. PDLSCs is a kind of MSCs, there are still no reportsabout their TLRs expression and how TLRs ligantion influence their functions. By the way,the reports about bone marrow MSCs （BMMSCs）and TLRs are also sometimesinconsistent or even conflicting.[Aims]To compare the TLRs expression on PDLSCs and BMMSCs. Pay attention to theTLR4expression and the effects of it’s ligation to the biocharacteristics of PDLSCs andBMMSCs, especially for the osteogenesis potential. Explore the cell signalings that causethis effect for better use these two kinds of cells clinically and provid clue for the researchabout the pathogenesis of periodontitis.[Methods]1) Isolate PDLSCs and BMMSCs and identify their phenotypes. Test the expression ofTLR1-TLR10at mRNA level by real-time PCR. Test the TLR4expression at protein levelby real-time PCR. Stimulate PDLSCs and BMMSCs by LPS and detect the activation ofNF-κB pathway.2) Stimulate PDLSCs and BMMSCs by LPS and test the proliferation by cck-8andmigaration by scratch test. After osteogenesis and adipogenesis induction, test the relatedgene for bone (ALP, Runx2and SP7) and adipocytes(LPL and PPARγ) by real-time PCR.Detect the ALP activity by ALP staining. Use Alizarin red and Oil red O staining and quantification to assess the final osteogenesis and adipogenesis potential of PDLSCs andBMMSCs. Co-culture PDLSCs and BMMSCs with peripheral blood mononuclear cells(PBMNCs), and use proliferation test and apoptosis test to detect the changes ofimmunomodulatory properties.3) Use anti-TLR4antibody to block TLR4under the stimulation of LPS, then we test theosteogenesis potential of PDLSCs and BMMSCs.4) Use PDTC to block NF-κB pathway under the stimulation of LPS, then test theosteogenesis potential of PDLSCs and BMMSCs.5) Test the change of Wnt/β-catenin pathway under LPS stimulation. Then useDKK-1(（Dickkopf1）) to block Wnt/β-catenin pathway under the stimulation of LP andtest the osteogenesis potential of PDLSCs and BMMSCs.6) Use LPS locally injection to build the periodontitis model in SD rats, and use TLRneutralizing antibody or PDTC to to block the process of periodontitis. The bone loss wasassessed by Micro-CT. Use ALP and Trap staining to assess the number of osteoblasts andosteoclasts.[Results]1) PDLSCs and BMMSCs positively express Stro-1, CD44, CD146, CD90, CD105,CD29and negatively express CD31, CD45. PDLSCs and BMMSCs quantitativelydifferently express TLRs subtypes, except for TLR7. For PDLSCs, the mostly expressed isTLR3, and for BMMSCs, the mostly expressed are TLR3and TLR4. They both expressTLR4at protein level, and after LPS stimulation NF-κB is activated.2) LPS can improve the proliferation of PDLSCs and BMMSCs but decrease theirmigration propertiy. LPS can also strengthen the adipogenesis potential of PDLSCs andBMMSCs. For PDLSCs, under LPS stimulation, the ALP staining showed lower ALPavtivity, the osteogenesis realated genes are decreased and Alizarin red staining revealedweakend ossification property. For BMMSCs, under LPS stimulation, the ALP stainingshowed lower ALP avtivity. The osteogenesis realated genes ALP is decreased; Runx2isunchanged; SP7is increased. Alizarin red staining revealed unchanged ossificationproperty. TLR4ligation didn’t change the immunomodulatory properties of PDLSCs and BMMSCs. But, the TLR3ligation can strengthen the immunomodulatory properties ofPDLSCs, not for BMMSCs.3) anti-TLR4antibody can partially reverse the decreased osteogenesis potential ofPDLSCs under LPS stimulation, but had no effect for the osteogenesis potential of BMMSCs.4) PDTC can partially reverse the decreased osteogenesis potential of PDLSCs underLPS stimulation, but had no effect for the osteogenesis potential of BMMSCs.5) Wnt/β-catenin pathway is activated after LPS stimulation. DKK-1can partiallyreverse the decreased osteogenesis potential of PDLSCs under LPS stimulation, but hadno effect for the osteogenesis potential of BMMSCs.6) Micro-CT shows that LPS can cause obvious periodontal bone loss, and the boneloss in LPS+anti-TLR4group and LPS+PDTC group were significantly reduced (P<0.05).ALP staining shows that the number of osteoblasts was greatly elevated in the TLR4antibody group and PDTC group. Trap staining shows that the number of osteoclastsbetween groups was nearly the same.[Conclusions]1) PDLSCs and BMMSCs express functional TLR4. PDLSCs and BMMSCsquantitatively differently express TLRs subtypes. The expression of TLR4is verified atthe protein level. After LPS stimulation, the NF-κB pathway was activated.2) TLR4ligation can change the biocharacteristics of PDLSCs and BMMSCs. TLR4ligantion can improve the proliferation but decrease the migration property of PDLSCsand BMMSCs. For PDLSCs, LPS can weaken the osteogenesis potential but strengthenthe adiopogenesis potential. For BMMSCs, LPS can strengthen the adipogenesis potentialbut had no effect to the osteogenesis potential. LPS had no effect to theimmunomodulatory properties of PDLSCs and BMMSCs. Interestingly, the TLR3ligationcan improve the immunomodulatory properties of PDSLCs, not for BMMSCs.3) Block TLR4by anti-TLR4antibody can partially reverse the osteogenesispotential of PDLSCs under LPS stimulation. We proved that though LPS functions byTLR4, but in the long process of ex vivo osteogenesis induction anti-TLR4antibody can’tget an100%blockage. 4) NF-κB pathway participates the process of weaken the osteogenesis potential ofPDLSCs. PDTC can partially reverse the decreased osteogenesis potential of PDLSCsunder LPS stimulation, but had no effect for the osteogenesis potential of BMMSCs.5) Wnt pathway participates the process of weaken the osteogenesis potential ofPDLSCs. DKK-1can partially reverse the decreased osteogenesis potential of PDLSCsunder LPS stimulation, but had no effect for the osteogenesis potential of BMMSCs.6) TLR4neutralizing antibody and NF-κB blocking can effectively reduce the boneloss caused by LPS injection in rats. The difference of bone level between groups ismainly caused by the elevated bone forming ability because of the osteoclasts numberbetween groups is nearly the same.