| ObjectivesDental pulp tissue engineering consists of three elements:cells,signaling molecules,and biological scaffolds.Our previous studies have found that neural EGFL like 1(NELL-1)has the ability of anti-inflammatory,and can induce human dental pulp stem cells to differentiate into odontoblast-like cells,contribute to neurogenesis and angiogenesis.Therefore,it can be selected as a signaling molecule for dental pulp tissue engineering.Poly(lactic-co-glycolic acid)(PLGA)can be made into nanoparticles(NPs)for loading drugs,controlling the release,and maintaining biological activity.Fibrin hydrogel is a suitable injectable scaffold material for pulp tissue engineering.The tooth has a special anatomical structure,and new tissue must grow into the tooth gradually from the apical.Therefore,this study attempted to construct a scaffold progressively degraded from the apical to the coronal side by adjusting the composition of fibrin gel,combined with dental pulp stem cells and PLGA particles coated with NELL-1,to verify the potential of NELL-1 application in the field of dental pulp regeneration.Methods1.Dental pulp stem cells were isolated and cultured.The stem cell characteristics of the cells were detected by detecting the ability of cell clone formation,osteogenic and adipogenic differentiation,and the expression of cell surface markers.2.PLGA particles coated with NELL-1 were prepared using the double emulsion solvent evaporation method.The surface morphology and structure of the particles were observed under the electron microscope.The particle size,polydispersity index(PDI),zeta potential,recovery rate,encapsulation rate,drug loading rate,and release rate of the particles were detected.3.Hydrogels with appropriate coagulation time were prepared.And we detected the survival and proliferation of cells in different types of hydrogels and the degradation of different types of hydrogels.And finally,the appropriate fibrinogen concentration was selected for subsequent experiments.4.The animal model of semi-orthotopic pulp regeneration was constructed.The samples were taken after 2 weeks,and the pulp regeneration was observed by hematoxylin-eosin(HE)staining.Results1.Dental pulp stem cells were successfully isolated and their stem cell properties were demonstrated.2.PLGA particles were successfully prepared and observed to be spherical with smooth surfaces under the electron microscope,and most of the particles were damaged after 30 days of degradation.The average particle size was(462.0±2.83)nm,the polydispersity index was(0.218±0.0090),and the zeta potential was(-22.77±1.39)mV.The recovery rate was(73.6±0.59)%,the encapsulation rate was(53.81 ± 1.10)%,and the drug loading rate of protein was(12.18±0.230)%.Less than 10%of the protein was released on the first day,the burst release was not obvious,and less than 15%of the protein was released on the 15th day.The results of the CCK8 assay showed that PLGA particles at concentrations below 10 mg/mL were not toxic to cells.3.Fibrin hydrogels were successfully prepared and thrombin of 5 mg/mL was found to mediate gel formation in 2-3 min,which is a suitable operation time.The survival rate of cells in hydrogels formed with different concentrations of fibrinogen was higher.The lower the concentration of fibrinogen was,the faster the proliferation rate of cells in hydrogels formed with fibrinogen was,the faster the gel degradation was.4.The results by in vivo experiments have demonstrated that NELL-1 can induce regenerated tissue formation,and fibrin hydrogels that degrade gradually can induce further tissue growth.ConclusionsThis study confirmed the role of NELL-1 protein in promoting dental pulp regeneration and laid a foundation for the clinical application of NELL-1 protein.At the same time,the promoting effect of progressive degradation scaffolds on tissue induction was also observed.However,further attempts and studies are needed for the construction and application of scaffolds that degrade gradually with tissue growth. |
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