The Study Of Nanofibrous Spongy Microspheres In Dentin Pulp-like Tissue Regeneration

Caries and dental pulp diseases are common oral problems which are high morbidity and serious damages. Current treatment is to remove the leisure, sterilize and repair the defect with synthesis materials such as silver amalgam, compose resin or gutta-percha etc. However, these material cannot repair the dental tissue with total biological function even they recovered part of the morphology of the teeth. These treatments can cause tooth discoloration, increased brittleness, and ultimately tooth loss which will effect masticatory and digestive function even psychology development and social activity in the long term. Tissue engineering technology provides a new approach to achieve pulp-dentin regeneration. Based on the basic principles of biology and engineering, dental tissue engineering also harnesses cells, scaffolds, and morphogens, alone or in combinations. The scaffold is vital for tissue engineering. It is challenging to design a scaffold that not only delivering cells into the irregular-shaped tooth canal, but also directing cells differentiation and angiogenesis in this encaged small space with minimal collateral blood circulation from the apical foramen. To achieve this goal, a novel injectable cell carrier nanofibrous spongy microspheres(NF-SMS) was synthesized from star-shaped poly(L-lactic acid)-block-poly(L-lysine) copolymers. In this study, we seeded human dental pulp stem cells(h DPSCs) on NF-SMS to detect the effect of NF-SMS on ondontogenic differentiation of h DPSCs. Secondly, we tried to combine NF-PLLA, NF-SMS and h DPSCs to form experimental pulp-dentin complex. Thirdly, we set up a hypoxia instrument for h DPSCs 3D culture and detected the hypoxia effect on h DPSCs on NF-SMS. All the results we got were shown that NF-SMS was a good cell carrier and enhanced h DPSCs proliferation and ondontogenic differentiation which is a desirable scaffold for dental pulp-dentin tissue engineering. Hypoxia precondition can be a good method to support the h DPSCs on NF-SMS to form pulp-like tissue.Part 1. NF-SMS enhanced h DPSCs proliferation and odontogenic differentiation To detect the odontogenic differentiation ability of h DPSCs on NF-SMS, the cell growth curve was made, and ALP activity, calcium quantity and odontogenic differentiation related genes expression levels were detected. The results showed that h DPSCs on NF-SMS were better odontogenic differentiated than those on NF-MS and S-MS. The DSPP positive staining was shown in subcutaneously injection of h DPSCs/NF-SMS complexes. It confirmed that NF-SMS enhanced h DPSCs odontogenic differentiation and would be a commendable scaffold in pulp-dentin complex regeneration.Part2. The study of experimental pulp-dentin complex regeneration by h DPSCs seeded NF-SMS and NF-PLLA scaffoldsTo make an experimental pulp-dentin complex, we combined NF-PLLA and NF-SMS together. h DPSCs seeded NF-PLLA and BMP-7 released NF-PLLA scaffold were tried to form dentin part, the subcutaneous implantation results showed the dentin like tissue comfirmed by the DSPP immunohistological staining. NF-SMS and h DPSC complex were subcutaneously injected into nude mice generated neo tissue which were more blood vessles than the counterparts. It hints the NF-SMS is the potential scaffold for the pulp regeneration. When the microspheres and h DPSCs complex were putting into the NF-PLLA dish, the DSPP stainning did not show the positive results in the interface between the scaffold and the microspheres area. We did not get the pulp-dentin complex though this method. The better microenvironment still needs to be explored.Part3. Pulp-like tissue regeneration by Hypoxia primed h DPSCs on NF-SMSThe instruments for 3D cell culture in hypoxia situation were set. The effect of the self-made instrument can be compared with the hypoxia chamber purchased in the market by comparing cell growth curves. To choose the hypoxia sensitive microspheres and hypoxia pre-condition, the VEGF gene expression of h DPSCs on different microspheres were detected and HIF-1α and VEGF immunofluenrence staining were taken. The models of subcutaneous injection, roots subcutaneous implantation and pulp in situ renegeration in nude rats were used to comfirm the possibility of h DPSCs on NF-SMS after hypoxia primed regenerate pulp-like tissues. The results displayed that h DPSCs on NF-SMS were more sensitive compare with those on NF-MS or S-MS. h DPSCs on NF-SMS expressed HIF-1α and VEGF in hypoxia culture condition and kept for several days. The results of subcutaneous injection showed that there were much more blood vessels in hypoxia group than that in normoxia group. The roots subcutaneous implantation model showed that pulp-like tissue regenerated in the roots, more blood vessels in hypoxia group than that in normoxia group and there were DSPP possitive staining in the interface between dentin and pulp-like tissue. The pulp regeneration in situ model displayed that NF-SMS could go into root canals and degradation after neo tissue regeneration in nude rat molar. The neo tissue of hypoxia groups were more blood vessels and expressed DSPP in the pulp-dentin interface. Taken together, hypoxia preprimed h DPSC/NF-SMS complexes were high potential in the application of pulp tissue regeneration.