TPPU Promotes Dental Pulp-like Tissue Regeneration In Full-length Human Root Canals Via Targeted Blocking Of Soluble Epoxide Hydrolase

Objective:The dental pulp tissue maintains tooth vitality by feeling,protective functions,defence,supplying nutrients,repair and regeneration.At present,endodontic treatment is the most classic method used for treating irreversible damage of pulp,which,however,leads to loss of pulp function and repaired tooth prone to fracture due to lack of nutrition.Rencently,tissue-engineering-based strategy for regeneration has become a promising approach for endodontic treatment to preserve tooth function,and a lot of progress has been made in pulp regeneration.Among them,the revascularization of tissue engineered pulp is vital to pulp regeneration.Many studies focus on revascularization of regenerated pulp using biomaterials and cytokines.However,restoring the pulp function in full-length root canal remains a major clinical challenge.Epoxyeicosatrienoic acids(EETs)are produced by cytochrome P450.Recent studies have shown that EETs can improve tissue microcirculation and play an important role in promoting tissue regeneration.Here,we assessed the effects of soluble epoxide hydrolase inhibitor(sEHi)1-trifluoromethoxyphenyl-3-(1propionylpiperidin-4-yl)urea(TPPU)on dental pulp tissue regeneration in fulllength human root canals by increasing the levels of endogenous EET via inhibiting the degradation of EETs to promotemicrocirculation in root canals.Materials and Methods:Human dental pulp stem cells(DPSCs)culture:DPSCs were isolated from dental pulp obtained from impacted wisdom teeth or premolar for clinical reasonand were cultured.DPSCs were used at passages 3-5 in subsequent experiment.Full-length root canal preparation:the extracted mandibular first premolars were obtained.The root of each tooth was cut to a standard length of 12 mm.The apex and coronal openings of the root canal were processed to 1.0 mm and 3 mm,respectively.The effects of TPPU on the vasculogenesis and migration of human vascular endothelial cell line(HUVECs)were investigated by tube formation and wound scratch assay.The co-culture system of HUVECs and DPSCs was established to further study the the effects of TPPU on vasculogenesis and migration of endothelial cell.The effects of TPPU on differentiation of DPSCs were investigated by alizarin red and alkaline phosphatase staining.The expression of DSPP,DMP-1,ALP,RUNX-2,HIF-1α,TGF-β and VEGF in co-culture system were detected by Q-PCR.In vivo experiments,DPSCs packaged with Matrigel matrix were injected into the standard full-length root canal,then,the roots were transplanted subcutaneously in immunocompromised nude mice.In experimental group,DPSCs were pretreated with TPPU for three days,TPPU was administered in experimental group from 7 days before operation to 2W post operation,while TPPU was not used in control group.After 8 weeks,the root segments were isolated,fixed with 4%paraformaldehyde and decalcified with 10%EDTA for tissue staining.The length of regenerated tissue,odontoblast differentiation and angiogenesis were evaluated.Results:Tube formation experiments showed that the number of tubes formed from TPPUtreated HUVECs was significantly higher than that of the control group(P<0.05).The cell scratch assay showed that the TPPU-treated HUVECs exhibited significantly smaller area of gap closure at 12h and 24h than that of the control group(P<0.05).The co-culture system showed that the tube formation ability of HUVECs in TPPUtreated group was also higher than that in control group.The expression of ALP in TPPU-treated group was higher than that in control group after osteogenic induction for 7 days.Much more calcium deposits were revealed by Alizarin Red S stain in TPPU-treated group.The increased expression level of DSPP,DMP-1,ALP,RUNX2,HIF-1α and VEGF in TPPU group were identified with Q-PCR.In vivo experiments:the implanted root segments were collected 8 weeks after operation,and decalcification for histochemical staining.The results revealed that more pulp tissues were formed in the TPPU-treated group including much moredentin sialophosphoprotein(DSPP)and dentin matrix acidic phosphoprotein 1(DMP-1)positive cells compared with the control group.Conclusions:TPPU can promote the tube formation and migration of vascular endothelial cells;in co-culture system,TPPU can promote the differentiation of dental pulp stem cells into odontoblast and osteoblast.In vivo experiments show that TPPU can promote pulp tissue regeneration and promote the formation of pulp tissue.TPPU promotes fulllength pulp regeneration by enhancing angiogenesis and pulp tissue formation through the HIF-1α pathway via crosstalk between endothelial cells and DPSCs.TPPU is a potential therapeutic target for pulp tissue engineering research and clinical transformation.