The Role Of EphrinB2-EphB4 Signaling Pathway In Regeneration Of Inflammatory Bone Defect

Background and PurposesBone defects in the oral and maxillofacial region usually interfere with normal masticatory function and often have devastating esthetic,emotional and social impact on patients.Thus,physiological and functional reconstruction of the damaged tissues is highly expected.Continuously occurring on existing bone surfaces,bone remodeling starts from old bone resorption by osteoclasts,followed by new bone regeneration in the resorption lacunae by osteoblasts.It is essential to preserve a certain balance between bone resorption and bone formation to maintain the structural integrity and mechanical function of the skeleton throughout life and especially during bone repair after injury.To that end,a complex and delicate communication should be established between osteoblasts and osteoclasts.This tightly coupled communication is prerequisite to ensure that microscopic skeletal damages are repaired and the aged bone replaced to maintain a functional skeletal system.In our previous in vitro study,we found that the treatment with TNF-a at lower concentrations promotes osteogenic differentiation and enhances expression levels of osteogenic transcription factors and bone marker genes.On the contrary,higher dose of TNF-a results in activating of NF-?B signaling pathway and inhibits osteogenic differentiation.In addition,long-term treatment with TNF-a shows dose-dependent suppression of osteogenesis differentiation.The important role of signaling pathway between osteoblasts and osteoclasts in bone remodeling has been demonstrated,while its effect on inflammatory bone defect regeneration remains poorly understood.During the process of bone remodeling,macrophage-colony stimulating factor(M-CSF)and receptor activator of nuclear factor kappa-B ligand(RANKL)produced by osteoblasts stimulate their receptors on osteoclast precursors,activate downstream molecular pathways,and promote the differentiation of osteoclasts.On the other hand,tartrate-resistant acid phosphatase(TRAP)released by osteoclasts has been reported to stimulate bone formation.In 2006,Zhao and colleagues reported that an Eph-ephrin bidirectional signaling mediates interaction between osteoclast and osteoblast populations.Ephs are a large family of tyrosine kinase receptors which can be divided into EphAs and EphBs based on gene sequence and binding affinity to the ephrin ligands.Acting as contact-dependent repellent molecules in both prenatal and postnatal organisms,Ephs play various roles in boundary formation,axon guidance,angiogenesis,bone formation and bone homeostasis.Briefly in maintaining homeostasis of bone remodeling,osteoclasts express ephrinB2,a transmembrane ligand,while osteoblasts express the EphB4 receptor.The reverse signaling through ephrinB2 into osteoclast precursors suppresses osteoclast differentiation,on the contrary,the forward signaling into osteoblasts mediating by EphB4 promotes osteogenic differentiation.In addition,in vivo overexpression of EphB4 in osteoblasts increases bone mass by enhancing bone formation and decreasing bone resorption in a mouse model.Furthermore,EphB4 was reported to be important in osteogenic differentiation and migration of human bone marrow-derived mesenchymal stem cells(hBM-MSCs),and ephrinB2-EphB4 signaling successfully promotes osteoblastic mineralization in vitro.At present,the inflammatory molecules in the inflammatory microenvironment are divided into two categories:the endogenous inflammatory mediators,including TNF alpha,IL-1,PGE2,etc;and exogenous inflammatory mediators,including bacterial lipopolysaccharide,mechanical tension or shear force,etc.TNF alpha is now recognized as one of the most important mediators in inflammatory microenvironment of all the endogenous inflammatory mediators.The action principle of TNF alpha is the activation of target cells in NF-kB and MAPKS pathway,such as p38,ERK and JNK signaling pathway regulating cell proliferation,differentiation and nflammation.RNA interference(RNAi)is the sequence-specific post-transcriptional gene silencing in biology,which is considered to be a very effective research tool in gene engineering fields.Employing this technique,we can stably and high-effectively block the expression of target gene to silence this gene.Double strand RNA(dsRNA)and short hairpin RNA(shRNA)have already been widely and successfully utilized in study of gene function and disease therapy research.Lentiviral vector is one of viral vectors which are commonly used to mediate RNAi.It can efficiently infect both the cells with split or non-split growth,and does not induce the immune response from the host.It is a powerful tool for gene silencing with a longer expression duration.As discussed above,the ephrinB2-EphB4 bidirectional signaling system shows a significant regulatory effect on bone formation and bone remodeling under physiological conditions,which is a potential target for the development of novel therapeutics to enhance bone regeneration.However,a high percentage of patients visiting dental clinics suffer from local chronic inflammation such as periodontitis,peri-implantitis,and periapical periodontitis and it is not clear whether EphB4 receptor and ephrinB2 ligand exerts similar effect on bone defect regeneration in an inflammatory and non-inflammatory microenvironment.In this study,in vivo expression of ephrinB2 or EphB4 in mandibular bone defects created in an inflammatory mouse model was knocked down using siRNAs specifically targeting ephrinB2 or EphB4,respectively.Bone formation and bone resorption in the bone defects were evaluated to fully elucidate the function of ephrinB2-EphB4 bidirectional signaling in an inflammatory microenvironment in order to open up a new idea for clinical research and development of new periodontal therapy,and to provide new drug targets for the therapy of periodontal disease,so it has important clinical significance and scientific research value.Materials and MethodsPart 1 Establishment of an in vivo inflammatory microenvironment via intraperitoneal injections of TNF-? into miceMethodThirty 6-8-week-old male C57BL/6 mice were randomly assigned into 3 groups to receive intraperitoneal injections of TNF-? at the doses of 0.5?g/kg,3?g/kg or 5?g/kg after bone defect model was performed,respectively.The injections were performed every other day.At 3,7,10 and 14 days after the first injection,blood samples were collected from the angular vein of the animals,and the serum concentration of TNF-?was determined using the mouse TNF-alpha Platinum ELISA kit(eBioscience,San Diego,CA,USA)according to the protocols provided by the manufacturers.Another 36 male C57BL/6 mice were subjected to bone defects and intraperitoneal injections of TNF-? at the doses of 0?g/kg,0.5?g/kg,3?g/kg or 5?g/kg at 3,7 and 14 days after surgery.The newly formed bone area was expressed as a percentage(area of newly formed bone/area of original wound)and was measured with the Image-Pro Plus 6.0 software.ResultAfter injected with TNF-a intraperitoneally,blood samples were collected from the C57BL/6 mice and subjected for the determination of serum TNF-a level.We found that animals receiving injections of TNF-a at the dose of 0.5?g/kg only showed a slight increase in the serum TNF-a level.In mice receiving injections of TNF-a at the dose of 3?g/kg,serum TNF-a level were continuously increased at 3 days,7 days and 10 days after the injection.However at 14 days after the injection,the increase in serum TNF-a level was less prominent compared with 0?g/kg group(p>0.05).In contrast,in animals receiving injections of TNF-a at the dose of 5?g/kg,serum TNF-a level was dramatically increased from 3 days after the first injection,and reached a steady plateau state thereafter.Therefore intraperitoneal injections of TNF-a at the dose of 5?g/kg were used to establish an inflammatory microenvironment in the following in vivo experiments.New bone tissue could be observed in all of the groups at 3 days after surgery,however,no statistically significant difference in the newly formed bone area was detected among these groups(p>0.05).At 7 days and 14 days after surgery,the newly formed bone area was significantly lower in the 3?g/kg group and 5?g/kg group than in the control(0?g/kg)group(p<0.05).In contrast,the newly formed bone area was the same in the 0.5?g/kg group when compared with the control group(p>0.05).Part 2 Construction and Packagement of the lentiviral vector mediating RNA interference of target gene ephrinB2 or EphB4Method1.Synthesis of small interfering RNAs(siRNAs)specifically targeting ephrinB2 or EphB4According to the mouse gene sequence in GenBank,siRNAs specifically targeting ephrinB2 or EphB4 were designed and synthesized by ThermoFisher Scientific,Inc.(Shanghai,China).A scrambled siRNA with no homology to any known mouse or human gene was also synthesized to serve as a negative control.Synthesized siRNAs were duplexed and ligated into the pcDNA6.2-GW/EmGFP-miR using the BLOCK-iT Pol ? miR RNAi Expression Vector Kit with EmGFP.To validate siRNA-mediated knockdown of ephrinB2 or EphB4 mRNA,pcDNA6.2-GW/EmGFP-miR plasmids containing the siRNA inserts were cotransfected along with pcDNA3.1(+)-ephrinB2 or pcDNA3.1(+)-EphB4 into HEK-293 human embryonic kidney cells.A quantitative real-time reverse transcription-PCR(qRT-PCR)assay was performed by ThermoFisher Scientific,Inc.(Shanghai,China)to detect the mRNA levels of mouse ephrinB2 or EphB4.2.Preparation of lentiviral particles expressing siRNAs targeting mouse ephrinB2 or EphB4After siRNA validation,selected siRNAs specifically targeting mouse ephrinB2 or EphB4 were introduced into the pDONR221 vector by in vitro recombination with the BP-clonase ? enzyme mix(ThermoFisher Scientific,Shanghai,China).The resulted BP-recombination plasmids were then transferred into the lentiviral expression vector pLenti6.3/V5-DEST by in vitro recombination with the LR-clonase ? enzyme mix(ThermoFisher Scientific,Shanghai,China).The resulted lentiviral vectors were named as pLenti6.3-efnb2siRNA and pLenti6.3-ephb4siRNA,respectively.A lentiviral vector encoding the above-mentioned scrambled siRNA,pLenti6.3-ctrl,was also created to be used as a negative control.Result1.Synthesis of small interfering RNAs(siRNAs)specifically targeting ephrinB2 or EphB4The sequences specificity were confirmed by DNA sequencing and it is indicated that lentiviral vector plasmids to ephrinB2 and EphB4 genes(including one negative control)have been successfully constructed.Among the four siRNAs targeting ephrinB2,siRNA 1#demonstrated the highest inhibition efficiency,which successfully down-regulated the mRNA level of ephrinB2 by 87%.Similarly,among siRNAs targeting EphB4,siRNA 2#showed the highest inhibition efficiency,with the mRNA level of EphB4 down-regulated by 77%.Therefore,siRNA 1#targeting ephrinB2 and siRNA 2#targeting EphB4 were chosen for the lentivirus preparation experiment and were used in the following in vivo studies.2.Preparation of lentiviral particles expressing siRNAs targeting mouse ephrinB2 or EphB4After a series of in vitro recombination assays,selected siRNAs were successfully transferred into the lentiviral expression vector pLenti6.3/V5-DEST,and were named as pLenti6.3-efnb2siRNA and pLenti6.3-ephb4siRNA,respectively.pLenti6.3-efnb2siRNA,pLenti6.3-ephb4siRNA and the negative control pLenti6.3-ctrl were then pseudotyped with the pLPl,pLP2 and pLP/VSVG envelope glycoproteins,respectively.After filtration,centrifugation and titration,the lentiviral particles were diluted to 1x108 TU/ml with opti-MEM and stored at-80? for further use.Part 3 The role of ephrinB2-EphB4 signaling pathway in regeneration of inflammatory bone defectMethodMandibular bone defects were created in male C57BL/6 mice receiving intraperitoneal injections of TNF-a at the dose of 5?g/kg every other day.The bone defects were treated with pLenti6.3-efnb2siRNA,pLenti6.3-ephb4siRNA or pLenti6.3-ctrl lentiviral particles,respectively.Mice were sacrificed at 7,14 and 21 days after surgery,and the newly formed bone was isolated.The gene expression of osteogenic differentiation markers Runx2,Osterix,ALP,OC and BSP and osteoclastogenic differentiation maker NFATcl in healing tissue of bone defect were examined by quantitative real-time polymerase chain reaction(PCR).Selected protein expressions of osteogenic differentiation markers were examined by western blot and immunohistochemistry respectively and the decalcified tissues were subjected to histological examination.ResultPCR analysis showed that mRNA levels of these above-mentioned bone-related genes were significantly lower in the pLenti6.3-ephb4siRNA group than in the pLenti6.3-ctrl group.In contrast,no significant difference in expression levels of bone-related genes was detected between the pLenti6.3-efnb2siRNA group and the pLenti6.3-ctrl group.Furthermore,NFATcl mRNA levels were significantly increased in the pLenti6.3-ephb4siRNA group when compared with those in the pLenti6.3-ctrl group at 7 and 14 days after surgery.However,no significant difference was detected in NFATcl mRNA levels between the pLenti6.3-efnb2siRNA group and the pLenti6.3-ctrl group.The protein levels of Runx2 were significantly lower in the pLenti6.3-ephb4siRNA group when compared with those in the pLenti6.3-ctrl group at 7 and 14 days after surgery.Similarly,the protein level of BSP was decreased in the pLenti6.3-ephb4siRNA group when compared with that in the pLenti6.3-ctrl group at 14 days after surgery.H&E staining,TRAP staining and bone histomorphometry showed that bones were thinner and the number of giant osteoclasts was higher in EphB4 siRNA group than in control group,whereas there was no significant difference in osteoblastic and osteoclastic differentiation between ephrinB2 siRNA mice and control mice.Conclusions:1.An in vivo inflammatory microenvironment was established successfully in mice via intraperitoneal injections of TNF-a at the dose of 5?g/kg.2.Lentiviral particles encoding siRNAs targeting mouse ephrinB2 or EphB4 named pLenti6.3-efnb2siRNA and pLenti6.3-ephb4siRNA,were successfully prepared and titrated.3.EphB4 plays an irreplaceable role in bone regeneration in an inflammatory microenvironment,whereas the functional loss of ephrinB2 can be effectively compensated,most possibly by other ephrins with similar chemical structures.