Study On Performance Optimization Of Three Dimensional Nano-zirconia Scaffold Combined With LMP-1 Overexpressed HDPSCs

Dental caries,trauma,periodontitis,etc.can lead to tooth defects or loss.The self-repairing ability of teeth is very limited.At present,many methods have been applying for repairing tooth defects,but various deficiencies still exist.This is a hot topic in regenerative medicine research that tissue engineering techniques are used the the multiple directional differentiation potential of stem cells and combine scaffolds to repair defective tissues and organs.In the previous experimental study,our research team successfully fabricated a three-dimensional nano-zirconia scaffold with high mechanical strength and suitable porosity,which could be used as a scaffold material for tissue defect repair.However,in the early stage of scaffold fabrication,the forming rate during the first sintering process needs to be improved during the first sintering process.Although zirconia has good biocompatibility and mechanical properties,it has poor affinity with cells and tissues.RGD peptides are short peptides consisting of 3 amino acids,which can promote cell adhesion,proliferation and differentiation.Human dental pulp stem cells(hDPSCs)are a type of mesenchymal stem cells located in the dental pulp tissue.They have multiple directional differentiation ability under a certain induction environment,and have the advantages of extensive source and convenient access.The odontogenic differentiation of hDPSCs is one of the key steps in tooth growth and endodontic tissue repair.The process is regulated by multiple growth factors and signaling pathways.The specific mechanism is not yet fully clear.LIM mineralization protein-1(LMP-1)is an intracellular non-secretory protein with osteogenic inducing activity that plays a positive regulatory role in the mineralization process.A large number of studies have shown that LMP-1 has different degrees of expression in pulp tissue,odontoblasts,and reparative dentin of growth and stimulated teeth.However,there is no relevant report on its role and mechanism in the odontogenic differentiation of hDPSCs.The purpose of this study was to enhance the carrier performance of the scaffold by improving the sintering process and modifying the surface of the scaffold;To investigate the role of LMP-1 in the odontogenic differentiation of hDPSCs and to provide new ideas and methods for the odontogenic differentiation of hDPSCs;To observe the effect of zirconia scaffold with compound RGD peptide on the adhesion,proliferation and differentiation of LMP-1 overexpressed hDPSCs and explore the feasibility of the restoration of tooth defects and application to tissue engineering.Experimental methods and results: 1.Performance optimization of three-dimensional nano-zirconia scaffoldIn this study,by prolonging the cooling time from 1360°C to 1060°C during the sintering of the scaffold,it was found that the sintering forming rate of the scaffold was obviously increased with the cooling time prolonging,and SEM showed that the number and length of the cracks on the beam of the scaffold were obviously reduced with the prolongation of the cooling time.Through the Micro-CT scan reconstruction of the scaffold,the average trabecular number and the average trabecular thickness of each group were analyzed,the compressive strength of the scaffold was also tested.It was found that the mean trabecular number,thickness and compression strength of the scaffold were significantly higher than those of the other groups when the cooling time was over 8h.FTIR analysis found that RGD peptide can be well anchored to the surface of the scaffold.CCK-8 detection found that RGD modification could promote the initial adhesion of the cells on the scaffold.2.Isolation,purification,culture and identification of hDPSCsIn this experiment,the primary cells were isolated and cultured from pulp tissue by means of tissue block combined with enzyme digestion method,and then purified and passaged for culture by limited dilution method.The clone formation ability was used to detect cells,which had strong colone unit formation capacity.The cell surface markers were detected by flow cytometry,and it was found that the cells expressed high levels of mesenchymal stem cell surface markers and negatively expressed hematopoietic cell surface markers.The staining was positive after inducing differentiation in the direction of osteogenic/odontogenic and adipogenic.It was proved that hDPSCs was successfully isolated and cultured.3.Effect of LMP-1 on proliferation and odontogenic differentiation of hDPSCsIn this experiment,the genetic engineering technology was used to construct the lentivirus and retroviral virus,which could effectively infect hDPSCs,stabilize the knockdown and overexpress LMP-1 in hDPSCs.The proliferation ability of the cells was detected by CCK-8,and it was found that the silence and overexpression of LMP-1 gene had no significant effect on the proliferation of hDPSCs.When the expression of LMP-1 in hDPSCs was silenced,the ability of mineralization nodule formation was significantly weakened.The mRNA and protein expressions of ALP,DSPP and DMP-1 were also reduced to varying degrees by qRT-PCR and Western Blot assays.After overexpression of LMP-1,the formation of mineralized nodules was enhanced,and the mRNA and protein expressions of ALP,DSPP,and DMP-1 were also significantly increased.4.In vitro study on the attachment of LMP-1 overexpressed hDPSCs by three dimensional nano-zirconia scaffoldIn this study,scaffolds optimized for previous performance were used as carriers,and hDPSCs overexpressing LMP-1 were cultured to observe that cells could adhere and proliferate well on the scaffold;.The CCK-8 experiment observed 100mg/L RGD peptide modification could promote the adhesion and proliferation of hDPSCs on the scaffold.Through ALP activity detection,it was observed that the nano-zirconia scaffold with compound RGD peptide had a synergistic effect on the osteogenic/odontogenic differentiation of LMP-1 overexpressed hDPSCs.In summary,by improving the sintering process of the scaffold,prolonging the cooling time,the initial forming rate of the scaffold sintering could be significantly improved,and the compressive strength of the scaffold could be enhanced.The use of RGD peptide modified scaffold increased the hydrophilicity of the scaffold and improved the initial cell adhesion of the scaffold.hDPSCs were.successfully isolated and identified Silenced or overexpressed the expression of LMP-1 in hDPSCs had no significant effect on cell proliferation.LMP-1 silencing could inhibit the odontogenic differentiation of hDPSCs,while overexpression of LMP-1 can promote the odontogenic differentiation of hDPSCs.100 mg/L RGD peptide-modified scaffold could promote adhesion,proliferation and differentiation of LMP-1 overexpressed hDPSCs,providing effective basis for further in vivo experiments.