| Tissue engineering is an emerging discipline in recent years,which curing the diseases,promoting the treatment effects,and improving the quality of life by regenerating new tissues or organs with normal structures and functions.The treatment of periodontal tissues diseases is one of the key issues in dental clinic and research.Although the periodontal initial therapy and operations obtained good effects,the treatment of periodontitis related bone loss is still a difficult problem.Recently,the new prospect on bio-regeneration of periodontal tissue is brought about by the progress of tissue engineering technology and periodontal ligament stem cells.Except their well studied biological properties and regenerative functions,periodontal ligament stem cells have been found to possess profound immunomodulatory capabilities.PDLSCs could not initiate allogeneic T cells proliferative responses,inhibited the mitogen-induced T cells proliferation in a dose dependent manner,and successfully restore the periodontitis related bone loss without immunological rejections of cellular immunity.In addition,periodontal ligament stem cells are capable of inhibiting the proliferation,differentiation,and chemotaxis of B lymphocytes.Immunity consists of innate immunity and adaptive immunity,and adaptive immunity is composed of cellular immunity(T lymphocytes)and humoral immunity(B lymphocytes).Although inhibiting the cellular immunity and humoral immunity,the effects of periodontal ligament stem cells on innate immunity are largely unknown.Neutrophils are important parts of innate immunity,and it is inevitable to contact neutrophils for periodontal ligament stem cells after transplantation in vivo.However,the mutual effects between periodontal ligament stem cells and neutrophils are not clear.The aim of this study is to address the interplay of periodontal ligament stem cells and neutrophils and the underlying mechanisms,thus providing experimental data for enlarging the seed cell of periodontal tissue regeneration,improving the development of periodontal tissue biological regeneration,and safe application of periodontal ligament stem cells.Part 1 Isolation,culture and identification of periodontal ligament stem cellsAim:To acquire the in vitro biological characteristics of human periodontal ligament stem cells.Methods:Normal premolars and impacted third molars of healthy individuals were extracted,and periodontal ligament stem cells were separated by enzyme digestion method and cultured as previously reported.The expression profiles of mesenchymal stem cells related markers,and the exploration of osteogenic,adipogenic,chondrogenic differentiation of periodontal ligament stem cells were performed to identify the biological characteristics of human periodontal ligament stem cells.Results:The flow cytometry assays showed that human PDLSCs expressed STRO-1 CD90 and CD 146,three mesenchymal stem cells related markers,and the positive rates are 26.2%,96.3%,and 88.6%,respectively.However,PDLSCs were negative for CD 14,CD34 and CD45,three hematopoietic lineage molecules.PDLSCs could differentiate along bone,adipose and cartilage pathways under the corresponding inductive medium.Von Kossa staining of PDLSCs in osteogenic medium for 4 weeks showed black nodular structures of calcium deposition.PDLSCs formed Oil Red O-positive lipid clusters following 4 weeks of adipogenic induction,and bead-like and ring-like lipid droplets were formed.Three weeks post chondrogenic induction,safranin O experiments showed positive staining in extracellular matrix with brown nucleus and red paticles in the cytoplasm.Immunocytochemical staining of type ? collagen displayed extensive brown dyeing.Conclusion:PDLSCs can be obtained by enzyme digestion method.PDLSCs expressed STRO-1,CD90 and CD 146,three mesenchymal stem cells related markers.However,PDLSCs were negative for CD 14,CD34 and CD45.PDLSCs could differentiate along bone,adipose and cartilage pathways.Part 2 The effect of periodontal ligament stem cells on the physiological functions of neutrophilsAim:To investigate the effect of periodontal ligament stem cells on the viability,apoptosis,expression of adhension molecules,phagocytosis function,and chemotaxis function of neutrophils.Methods:Normal premolars and impacted third molars of healthy individuals were extracted,and periodontal ligament stem cells were separated by enzyme digestion method and cultured as previously reported.Neutrophils were isolated and cultured as document statement.Under cell-cell contact culture or Transwell culture conditions,different ratios of periodontal ligament stem cells and neutrophils were co-cultured together.Then neutrophils were got and examined their viability,apoptosis,expression of adhension molecules,phagocytosis functions,and chemotaxis functions.Enzyme-linked immunosorbent assays were used to detect the concentration of IL-6 in the supernatant of co-culture of periodontal ligament stem cells and equal amount neutrophils post 24 hours.The apoptosis of neutrophils was investigated after culture with different concentrations of IL-6.Anti-IL-6 antibody was added into the co-culture of periodontal ligament stem cells and neutrophils,then the apoptosis of neutrophils was examined.Results:Periodontal ligament stem cells could promote the viability of resting neutrophils and IL-8-activated neutrophils.However,periodontal ligament stem cells-mediated viability promotion of neutrophils did not rise with the amount increase of periodontal ligament stem cells.There is no significant difference between periodontal ligament stem cells-mediated viability promotion of resting neutrophils and IL-8-activated neutrophils.Periodontal ligament stem cells could decrease the apoptosis of resting neutrophils and IL-8-activated neutrophils,and there is no significant difference between periodontal ligament stem cells-mediated apoptosis decreasement of resting neutrophils and IL-8-activated neutrophils.Periodontal ligament stem cells could decrease the apoptosis of neutrophils under cell-cell contact culture or Transwell culture conditions.The concentration of IL-6 in supernatant of periodontal ligament stem cells alone is low,whereas is elevated significantly post 24 hours co-culture of periodontal ligament stem cells and neutrophils.In addition,IL-6 could inhibit the apoptosis of neutrophils.Anti-IL-6 antibody is able to counteract the periodontal ligament stem cells-mediated apoptosis decreasement of neutrophils.Periodontal ligament stem cells did not influence the expression of adhension molecules,phagocytosis function,and chemotaxis function of neutrophils.Conclusion:Periodontal ligament stem cells could promote the viability and decrease the apoptosis of resting neutrophils and IL-8-activated neutrophils,which was achieved by the secretion of IL-6.The expression profiles of adhension molecules,phagocytosis function,and chemotaxis function of neutrophils were not affected by periodontal ligament stem cells.Part 3 Effect of neutrophils on biological characteristics of periodontal ligament stem cellsAim:To study the effect of neutrophils on biological characteristics of periodontal ligament stem cells.Methods:Normal premolars and impacted third molars of healthy individuals were extracted,and periodontal ligament stem cells were separated by enzyme digestion method and cultured as previously reported.Periodontal ligament stem cells of 80%confluence were digested and plated with the density of 5.0 ×104 at 37 ? and 5%CO2 for 2 hours to make the cells adhenrence.Periodontal ligament stem cells were co-cultured with equal amount of neutrophils for 24 hours.Then periodontal ligament stem cells were obtained and examined the following items:colony-forming efficiency,cell proliferation rate,multi-potential differentiation,expression of mesenchymal stem cell markers,ultra-structure,and karyotype analysis.Results:The colony-forming efficiency of periodontal ligament stem cells alone and co-cultured periodontal ligament stem cells were 28/5.0 ×103 and 19/5.0×103 respectively,and the Brdu positive rate were 71%±11.2%and 47%±9.1%respectively,there was significant difference between the two groups.Both periodontal ligament stem cells alone and co-cultured periodontal ligament stem cells could differentiate along bone and adipose pathways,and were positive for STRO-1,CD 190,and CD 146,and the expression rates were 29.6%,98.8%,and 89.5%,respectively.Moreover,two groups had the same manifestation of ultra-structure and G-band karyotype.Conclusion:Neutrophils could decrease the colony-forming efficiency and cell proliferation rates of periodontal ligament stem cells markedly,but did not affect the multi-potential differentiation,expression of mesenchymal stem cell markers,ultra-structure,and karyotype analysis of periodontal ligament stem cells. |
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