Novel Molecule Nell-1 Promotes Angiogenic Differentiation Of Dental Pulp Stem Cells

ObjectivesNell-1(Nel-like 1)is a novel signaling molecule,which plays an important positive regulatory role in osteoblast differentiation,bone formation and bone regeneration.Our recent study reported that Nell-1 could enhance odontoblastic differentiation,the formation of dentin,and neural-like differentiation of human dental pulp cells.There are several studies having confirmed that coculture of HUVECs with DPSCs enhanced angiogenic differentiation of them in vitro and vivo.We performed this study to assess the effects of Nell-1 on the angiogenic differentiation of human dental pulp stem cells(DPSCs)alone or coculture with human umbilical vein endothelial cell(HUVECs),and we also want to detect whether Nell-1 play a role in pulp exposure model.Materials and methods1.Isolation,cultivation and identification of human dental pulp stem cellsDPSCs were obtained from third molars with no caries and periodontal diseases.The expression of mesenchymal stem cell markers and hematopoietic markers were tested by flow polychromatic cytometric analysis.Osteogenic differentiation assay and adipogenic differentiation assay was performed on DPSCs to test the multilineage differentiation capacity of DPSCs.2.The effect of Nell-1 on angiogenic differentiation of human dental pulp stem cells alone or coculture with human umbilical vein endothelial cell in vitroFirstly,we conducted immunofluorescence to ascertain the location of Nell-1 on DPSCs and HUVECs.In order to determine the proliferation and angiogenic differentiation ability of Nell-1 on DPSCs alone or coculture with HUVECs,we completed quantitative real-time polymerase chain reaction(qRT-PCR),enzyme-linked immunosorbent assay(ELISA),Matrigel angiogenesis assay and western blot to detect the expression levels of angiogenic markers.3.The effect of Nell-1 on angiogenic differentiation of dental tissues in vivoFirstly,we conducted double immunofluorescence to detect the distribution and relationship of Nell-1 with angiogenetic markers,including VEGF and Flk-1 in rat pulp tissues.Then,collage sponge containing or not containing Nell-1 protein was added into the pulp cavity of rat molars.And normal pulp tissue was used as positive control group.The number of blood vessels between different group were observed and compared using immunohistochemical by building rat pulp exposure model.Results:1.Human DPSCs were successfully isolated and culturedHuman dental pulp cells obtained by enzymatic digestion showed long spindle-shaped appearance and grew with adherence.Odonto/osteogenic and adipogenic lineage was successfully confirmed by positive staining with Alizarin red and oil red O staining after 20 days being induced by osteogenesis/adipogenesis-inducing medium.The results of the flow cytometric analysis confirmed that DPSCs were positive for CD90(98.3%),CD44(99%),CD 105(99.4%).In addition,almost all cells population negative for CD34(4.53%),CD45(14%).2.Nell-1 enhances angiogenic differentiation of dental pulp stem cells in vitroDPSCs and HUVECs were treated with 0,50,100 and 200ng/mL Nell-1 for 1,3,5,and 7days.We found that Nell-1 has no effect on DPSCs and HUVECs’ proliferation using CCK-8.Cell immunofluorescence was performed to locate the subcellular distribution of Nell-1 in DPSCs and HUVECs.We observed that Nell-1 is greatly expressed in the DPSCs and HUVECs’ nucleus.We proved that Nell-1 could promote the angiogenic markers expression on DPSCs alone and coculture with HUVECs by qRT-PCR and Western Blot.We also found that Nell-1 could enhance the constitution of vessel-like structures on DPSCs coculture with HUVECs.3.Nell-1 promotes angiogenic differentiation of dental pulp stem cells in vivoDouble immunofluorescence of normal pulp tissue sections was used to observe the distribution of Nell-1,VEGF and Flk-1 in dental pulp.The co-expression of Nell-1 and angiogenic markers were revealed by immunofluorescence staining method in rat pulp tissues.In rat pulpitis model,immunohistochemical staining of CD31 and CD34 demonstrated there had more vascular lumens in Nell-1 group comparing with collagen group.Conclusion:The results showed that Nell-1 promotes angiogenic differentiation of dental pulp stem cells.The findings of this experiment provide a theoretical basis for a comprehensive understanding of the role of Nell-1 in pulp regeneration.