Differentiation Of Stem Cells From The Apical Papilla On PGA Scaffolds With Different Arrangement

Objective In this study,human stem cells from the apical papilla(SCAPs)were obtained,cultured and identified in vitro,and their expression of tendon ligament and osteogenic related genes were detected.The differentiation of SCAPs on different arrangement PGA scaffolds was studied by in vitro and in vivo experiments.Methods 1?Human SCAPs were isolated and cultured by enzyme digestion method,and cell morphology and growth characteristics were observed and recorded.Cell proliferation was measured.Colony formation experiment of plate clone was carried out.Cytoimmunohistochemistry was used to identify the expression of keratin and vimentin.Flow cytometry was used to identify stem cell surface marker molecules(including STRO-1,CD146,CD90,CD34,CD105 and CD45).Osteoblast and chondroblast differentiation were induced and identified in vitro.Gene expression analysis of tendon and ligament related genes and embryonic stem cells in SCAPs.2?Scanning electron microscopy was used to observe the growth of SCAPs inoculated in randomly or linearly arranged PGA scaffolds.Ordinary complete culture medium and osteogenic mineralization induction solution were used to culture SCAPs-linear PGA scaffolds and SCAPs-randomly arranged PGA scaffolds for 21 days,and the expression of the genes related to tendon ligament and osteogenic genes were detected.3?Two weeks of SCAPs-linear alignment PGA scaffolds,SCAPs-random alignment PGA scaffolds,PDLSCs-linear PGA scaffolds,PDLSCs-randomly arranged PGA scaffolds were implanted subcutaneously in the back of nude mice,samples were obtained 14 weeks later,and histological staining was performed to compare the formed tissues.Results 1?Primary cultured human SCAPs are spindle-shaped or fusiform,with similar cell size and morphology,clustered;The results of the growth curve of SCAPs showed an S-shaped shape,entered the logarithmic growth phase on the third day,and entered the plateau phase on the seventh day;SCAPs can form colony colony cell clusters with a formation rate of(8.9±0.6)%.The cells show colony growth under light microscope.Cellular immunohistochemistry showed that SCAPs positively expressed wavy protein,while negative expression of keratin;SCAPs positive expression Mesenchymal stem cell surface marker molecules STRO-1,CD146,CD90,CD34,CD105,negative expression of stem cell surface marker molecule CD45;SCAPs osteogenic induction after the formation of macroscopic nodules,alizarin red staining after the reddish brown calcium nodules,Von Kossa stained dark brown;SCAPs induced cartilage induced cell clusters can be stained blue;RT-PCR detection showed that SCAPs expressed SCX,SIX1,SIX2,Col-?,Col-?,NANOG,C-MYC,TNC,EYA2 gene.Compared with human PDLSCs,SCAPs expressed higher levels of SCX,SIX1,SIX2,NANOG,EYA2,while their Col-?,Col-?,C-MYC,TNC expression levels were similar.2?The cells were seeded on randomly or linearly arranged PGA scaffold with good adhesion and moderate extension to secrete large amounts of extracellular matrix.Randomly arranged scaffolds promote the expression of osteogenic genes,linear alignment of scaffolds promoted tendon genes expression.3?The transplanted nude mice were sampled 14 weeks later.Histological examination showed that the randomly arranged group,the linearly arranged group SCAPs and PDLSCs all formed ligament-like tissue.The ligament-like tissue formed by the random arrangement group was disordered,and the ligaments formed by the linear alignment group were formed.The tissue is approximately wavy and the direction is similar to the direction in which the stent is arranged.The tissues formed by SCAPs and PDLSCs in the stents in the randomly arranged group and the linearly arranged group showed only partial mineral deposition,but the stents in the randomly arranged group showed darker red color than those in the linearly arranged group.However,there was no typical osteoblastic morphology around the mineral nodules.Conclusion The human SCAPs obtained in vitro have strong proliferation and differentiation ability.when they were constructed differently on different arrangements of PGA scaffold,linear arrangements of which promoted their differentiation into the tendon/ligament tissues,and random ones promoted their differentiation into osteoblasts.