| Periodontal disease which generates damage to the periodontal and oral health is one of the most common oral diseases. Periodontitis, with the destruction of periodontal supporting tissues including the periodontal ligament, the alveolar bone and the cementum, is a kind of infectious disease of periodontal tissues caused by the plaque biofilm and could finally lead to the formation of periodontal pocket and inflammation, the progressive loss of adhesion, the alveolar bone absorption and the tooth loss. Even though it also has close relationship with the whole body health, there is no efficient therapy to cure periodontal disease at present.Periodontal ligament stem cells(PDLSCs) are a kind of mesenchymal stem cells existing in periodontal tissues, which have been confirmed as important seed cells for periodontal regeneration. As is known to all, cellular and extracellular signals in stromal microenvironment together regulate stem cell self-renewal and multi-directional differentiation. Inflammation not only changes the balance of stromal microenvironment of stem cells, but also affects the regulatory function of endogenous signals. The regeneration ability of PDLSCs in inflammatory microenvironment is weaker than that of normal cells. So how to restore the declined osteogenic capacity of PDLSCs caused by inflammatory microenvironment becomes a new way of periodontal regeneration.Previous studies have found that epigenetic mechanism is closely related with the changes of the microenvironment, and histone deacetylation plays an important role in the process of periodontal disease’s occurrence and development. The dynamic balance of histone deacetylation depends on histone deacetylases(HDACs) and histone acetyltransferases(HATs). Histone deacetylase inhibitors(HDACIs/HDIs) have been found to promote osteoblast differentiation and inhibit osteoclast formation and the production of inflammatory mediators, producing positive regulatory effect on bone regeneration.So in this study, firstly we isolated and purified human PDLSCs from healthy and periodontitis periodontal ligament tissues respectively, and compared the expression of HDACs in the two kinds of PDLSCs. Then we researched on the principle of how HDACIs(NaB and TSA) affect the osteogenic capacity of PDLSCs in inflammatory microenvironment by suppressing HDACs functions. Finally we constructed the periodontitis model in SD rats and tested the influence of NaB and TSA on the alveolar bone loss in periodontitis by local injection. This study provided a new way of periodontal regeneration treatments based on stem cell therapy and explored new potential drugs for the clinical treatment for periodontitis.Part I To isolate and identify the two kinds of PDLSCs from healthy and periodontitis periodontal tissues and compare their biological characteristicsObjective To isolate and identify H-PDLSCs and P-PDLSCs in vitro and compare their biological characteristics.Methods(1) H-PDLSCs and P-PDLSCs were achieved from the normal and periodontitis periodontal tissues by limiting dilution technique.(2) The two types of PDLSCs phenotype identification were analyzed by flow cytometry analysis.(3) The proliferation potential was determined by MTT and colony-forming assays.(4) ALP, alizarin red and oil red O staining were used to detect and compare the multi-differentiation potential of H-PDLSCs and P-PDLSCs.Results(1) We successfully achieved H-PDLSCs and P-PDLSCs. There was cell-cloning formation when cultured by limited dilution method.(2) H-PDLSCs and P-PDLSCs both were positive to MSC surface markers STRO-1, CD105, CD90, CD146 and negative to hematopoietic markers CD34, CD45.(3) MTT and colony-forming assays found that the proliferation potential and colony-forming rate of P-PDLSCs were higher than H-PDLSCs.(4) Both H-PDLSCs and P-PDLSCs had multi-differentiation potential. The osteogenic capacity of P-PDLSCs was lower when compared with H-PDLSCs, while the adipogenic ability of the two types of PDLSCs had no obvious difference.Conclusions(1) H-PDLSCs and P-PDLSCs were mesenchymal stem cells and had self-renewal and multi-differentiation potential characters.(2) Inflammatory microenvironment could influence the biological characteristics of stem cells and the osteogenic capacity of P-PDLSCs was significantly decreased.Part II Influence of NaB and TSA on the osteogenic differentiation capacity of P-PDLSCsObjective To compare the expression of HDAC1-11 between PDLSCs in normal and inflammatory microenvironment, screen appropriate concentrations of the two HDACIs(NaB and TSA)and investigate the influence on the osteogenic capacity of H-PDLSCs and P-PDLSCs.Methods(1) The expression of HDAC1-11 was detected by RT-PCR technique and the greatest specific changed HDAC was screened. Then western blot analysis was used to verify the result and the chosen HDAC-X was used for subsequent experiments.(2) The appropriate drug concentrations of NaB and TSA applied in our study were determined by western blot analysis.(3) The influence of NaB and TSA on proliferation potential of PDLSCs was tested by MTT assays and the changes of cell morphology were observed with an inverted microscope.(4) ALP staining, alizarin red staining and western blot were used to investigate the osteogenic capacity changes of PDLSCs in every group.(5) The application of NaB and TSA in P-PDLSCs cell sheets was detected by immunohistochemical staining.(6) P-PDLSCs were treated with NaB and TSA separately, and then western blot was used to test the expression of HDAC9 and acetylated histones.Results(1) RT-PCR results showed that the expression of HDAC1-11 in inflammatory PDLSCs was generally higher than that in H-PDLSCs, among which HDAC9 expression increased stably in all experiments, and western blot analysis showed P-PDLSCs had a higher expression of HDAC9 protein.(2) We selected NaB100μM and TSA100 n M as the drug concentrations in our study by western blot analysis.(3) MTT results showed that NaB and TSA had no influence on PDLSCs proliferation potential. The cell morphology was normal and cell growth conditions were good, and there were no dead cells when observed with an inverted microscope.(4) ALP staining, alizarin red staining and western blot analysis results showed that NaB and TSA could enhance the mineralization ability and promote the expression of osteogenesis related proteins in H-PDLSCs and P-PDLSCs.(5) NaB and TSA significantly enhanced the secretion of osteogenesis related indicator Runx2 in extracellular matrix, according to the results of immunohistochemical staining.(6) The expression of H3K9, H3K18 and H4K16(especially H4K16) was elevated by NaB. The expression of H3K18 was enhanced by TSA.Conclusions(1) PDLSCs in inflammatory microenvironment generally had a higher expression of HDACs and the histone deacetylation condition was out of balance.(2) NaB and TSA could significantly enhance the osteogenic capacity of H-PDLSCs and P-PDLSCs.(3) As the broad-spectrum HDACIs, NaB and TSA could target the acetylated sites of H4K16, H3K18 and so on.Part III Study of NaB and TSA on ectopic osteogenesis of P-PDLSCs in nude mice and in situ periodontal regeneration of SD ratsObjective To investigate the influence of NaB and TSA on ectopic osteogenesis of P-PDLSCs and in situ periodontal regeneration.Methods(1) First we cultured PDLSCs and fabricated the cell sheets. The cell sheets co-cultured with HA were transplanted subcutaneously into immunocompromised mice for 8 weeks. The tissue regeneration ability was assessed by H&E staining.(2) Local injection with LPS was used to construct periodontitis model in SD rats. We observed the changes of the alveolar bone after injection with NaB and TSA by micro-CT.Results(1) NaB and TSA can significantly promote the new bone formation of P-PDLSCs.(2) Micro-CT results showed that both NaB and TSA could obviously inhibit the alveolar bone loss caused by periodontitis.Conclusions NaB and TSA could significantly improve the osteogenic ability of P-PDLSCs and obviously inhibit the alveolar bone loss caused by periodontitis. |
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