The Role Of Wnt5a In The Osteo/Dentogenic Differentiation Of Hdpscs In Hypoxia

Periapicalperiodontitis in young permanent teeth mainly develops from pulp inflammation or pulp necrosis,and pulp infection can cause inflammation or lesions in the periapical tissue through the wide apical foramen.As the pulp and periapical tissues of young permanent teeth are loose and enriched with blood,once inflammation occurs,the infection is easy to spread,and the inflammation is easy to control and repair if treated in time.Apexification and Apical barriers are used to induce or artificially form an apical barrier for the purpose of apical closure.Pulp regeneration therapy regenerates functional pulp tissue by inducing the differentiation of stem cells introduced endogenously or exogenously into the root canal to promote the continued development of dentin,the pulp-dentin complex,and the root of the tooth.Human dental pulp stem cells(h DPSCs)are a type of MSC-derived cells that are considered to be an ideal seed cell for pulp regeneration therapy due to their strong proliferative capacity and easy accessibility.Currently,the microenvironment for in vitro culture of DPSC is a 21% oxygen concentration microenvironment,i.e.normoxia.However,the microenvironment of DPSC in vivo is hypoxia.Oxygen concentration directly affects biological processes such as cell proliferation,multidirectional differentiation potential,apoptosis levels,and damage repair.Wnt5 a,a signaling molecule of the non-canonical WNT signaling pathway,has regulatory roles in cell proliferation,migration,and differentiation at various stages of tooth development.Some in vitro studies have found that Wnt5 a regulates osteogenic/odontogenic differentiation of DPSCs,yet studies in hypoxic environments have not been reported;therefore,this study was conducted to investigate the regulation of osteogenic/odontogenic differentiation ability of h DPSCs by Wnt5 a in hypoxia environments in vitro.Objective: By constructing a 3% oxygen concentration hypoxic environment and overexpressing Wnt5 a,the effect of Wnt5 a on osteogenic/odontogenic differentiation of h DPSCs was explored in vitro.Methods: 1.Freshly extracted human isolated teeth were collected and h DPSCs were extracted from the dental pulp.h DPSCs were identified by flow cytometry for cell surface antigens,and their ability to induce osteogenic and lipogenic differentiation was identified using Alizarin Red S(ARS)staining and Oil Red O staining.2.3% oxygen concentration was constructed using a three-gas incubator for culturing h DPSCs,and the CCK-8 method was used to detect the effect of3% oxygen concentration on the proliferation ability of h DPSCs.Adenovirus-mediated Wnt5a(Ad-Wnt5a)transfection of h DPSCs was used to overexpress Wnt5 a.3.Three groups were set up: Normoxia group(21% O2),Hypoxia group(3% O2),and Hypoxia(3% O2)+ Wnt5 a group.Alkaline phosphatase(ALP)staining and ARS staining were performed on days 14 and 21 after osteogenic induction culture to detect alkaline phosphatase and mineralized nodule formation.Cellular RNA and protein were extracted on day 7,and RT-q PCR was performed to detect the m RNA expression of osteogenic/dentinogenic related genes Alp,Osx,Ocn,Opn,Col 1,Runx2,Dmp1,Dspp.Western Blot was performed to detect the protein expression of RUNX2,DMP1,DSPP.Results: 1.The morphology of the isolated cultured cells was shuttle-shaped with colony-like growth.The flow cytometry results showed positive expression of CD73,CD90,CD146,and negative expression of CD31,CD45,which were consistent with the characteristics of h DPSCs.After 21 days of osteogenesis induction,ARS staining showed red calcified nodule formation;after 21 days of lipogenesis induction differentiation,Oil Red O staining showed red lipid droplet formation,indicating that h DPSCs have the ability to induce differentiation into osteogenic and lipogenic cells.2.CCK-8 results showed that 3% oxygen concentration had no significant effect on the proliferation ability of h DPSCs.h DPSCs transfected with Ad-Wnt5 a and Ad-GFP showed green fluorescent expression under the microscope.RT-q PCR and Western blot results showed that HIF1-α expression was elevated in the hypoxia group and somewhat reduced in the transfected Wnt5 a group(P <0.05),The expression of Wnt5 a was reduced in the hypoxia group and significantly increased in the transfected Wnt5 a group,indicating the successful overexpression of Wnt5 a.3.ALP and ARS staining results showed that the 3% oxygen concentration microenvironment had an inhibitory effect on alkaline phosphatase and calcified nodule formation in h DPSCs,which was partially counteracted by transfection with Wnt5 a,demonstrating that Wnt5 a could protect the osteogenic differentiation activity of h DPSCs in a hypoxic environment.4.RT-q PCR results on day 7 showed that the m RNA expression of Alp,Osx,Col 1,Opn,Ocn,Runx2,Dmp1,and Dspp was lower in the hypoxic group compared to the normoxic group(P < 0.05),indicating that the expression of h DPSCs osteogenic/odontogenic genes decreased at 3% oxygen concentration;after overexpression of Wnt5 a,it was observed that the m RNA levels of Osx,Col 1,and Dspp were significantly higher in the hypoxia group compared to the normoxia group(P < 0.05).The m RNA levels of Osx,Col 1,and Dspp were significantly higher than those of the hypoxic and normoxic groups,and the expression of Alp,Opn,Ocn,Runx2,and Dmp1 were higher than those of the normoxic and hypoxic groups(P < 0.05),indicating that the overexpression of Wnt5 a up-regulated the expression of h DPSCs osteogenic/odontogenic-related genes in the 3% oxygen concentration microenvironment.Western Blot showed that all groups of h DPSCs expressed proteins RUNX2,DMP1 and DSPP,with the lowest expression in the hypoxic group and increased expression in the overexpression group of Wnt5 a,indicating that Wnt5 a protected the expression of osteogenic/odontogenic related proteins in h DPSCs under hypoxia environment.Conclusion: In in vitro experiments,3% oxygen concentration microenvironment inhibited the osteogenic/odontogenic differentiation ability of h DPSCs,and overexpression of Wnt5 a can protected the osteogenic/odontogenic differentiation ability of h DPSCs under low oxygen environment to some extent,which has potential application in dental tissue engineering.