| As a kind of chronic infectious diseases, periodontitis has a high incidence in the world. Currently, studies have suggested that periodontitis is the major cause of adult tooth loosening up and missing. Therefore, it is seriously harmful to human oral health. However, in recent years, studies have shown that hypoxia can aggravate periodontal disease process through affecting the periodontal tissue defense and repair. Although researchers have made some progress, the exact mechanism of hypoxia on periodontitis is unclear.The biological basis of periodontitis is the disturbance of bone formation and bone resorption mediated by periodontal tissue. Accompanied by direct destruction and immune damage of periodontal tissue, periodontitis ultimately leads to the loss of alveolar bone. Present studies suggest that in addition to the direct damage of pathogens, it can also induce the cells in periodontal tissues to secrete chemokines, adhesion molecules, and inflammatory cytokines. And the high levels of inflammatory mediators can mediate the immune cells adhering and gathering to the sites of inflammation. Then the precursor cells of OC, such as the PBMCs, can be induced to differentiate to OC, which can result in the destruction, degradation, and absorption of periodontal tissue.As an important component of the periodontal tissues, periodontal ligament mainly consist of hPDLF, which are not only involved in the rebuilding and regeneration of periodontal tissue, but also has a certain immunomodulatory capabilit y through the interacting with immune cells and endothelial cells. So the biological activity of hPDLF is closely related to the development of periodontitis. However, the specific role of hPDLF in periodontitis, as well as whether hypoxia can promote the periodontal disease process through affecting the biological properties hPDLF is still very unclear. In the present study, we first observed the immune response on h PDLF stimulated by different bacterial LPS; Next, we observed the different state of ET under normoxic and hypoxic conditions; Then, h PDLF and PBMCs were co-cultured under normoxic and hypoxic conditions and the secretion of inflammatory cytokines will be observed; Finally, we observed whether hypoxia can affect the osteogenic differentiation of hPDLF, and osteoclastic differentiation of PBMCs. Also the regulatory mechanisms of cell differentiation were discussed.The study is divided into three parts: First, we build experimental platform, including the separation, cultivation and identification of h PDLF and PBMCs in vitro. Second, we constructed an ET model of hPDLF under normoxic and hypoxic conditions in vitro. The secretion of cytokines and expression of TLRs will also be tested. Third, we will co-culture h PDLF with PBMCs, and observe whether hypoxic can affect the secretion of inflammatory cytokines. Meanwhile, the differentiation ability of h PDLF and PBMCs will be observed and the molecular mechanism of differentiation will be further discussed. Through our research, we hope the understanding of the role of hypoxia on periodontitis pathological lesions will be promoted.1. Separation, culture, and identification of h PDLF and PBMCs1) Separation, culture, and identification of hPDLF: Pieces of PDL were taken only from the middle of the tooth root to exclude the intermixture of gingiva and dental p ulp, then cultured by supplementing with 10% FBS and antibiotics after digested in 0.1% I collagenase. Immunohistochemical identification results: vimentin-positive and cytokeratin-negative suggested hPDLF derived from the mesenchymal, not epithelial origi n. Combining the location, morphological features, and tissue origin, we can prove that the cells we isolated and cultured are the hPDLF.2) Separation, culture, and identification of PBMCs: The method of Ficoll-Hypaque density gradient centrifugation was adopted to separate the blood cells. After low-speed centrifugation, cells were divided into four layers. The cells in the second layer are cyclic milky mononuclear cells, namely PBMCs. The isolated purity was stained by Giemsa.2. Under hypoxic conditions, endotoxin tolerance of h PDLF was reduced.1) Constructing an ET model of hPDLF under normoxic conditions in vitro: h PDLF were challenged by 1000ng/m L Pg LPS or Ec LPS followed by the same LPS stimulation from 0ng/m L to 1000ng/m L. Inflammatory cytokines concentrations were detected in cell culture supernatant. First, two kinds of initial bacterial LPS stimulation both can induce the production of six kinds of inflammatory cytokines, in which the levels of IL-8 and IL-6 have increased significantly. Second, after the two types of bacteria LPS challenge, the concentrations of IL-8 and IL-6 have decreased significantly, which suggested that both LPS can induce hPDLF to generate ET. Meanwhile, mRNA and protein levels of TLR2 and TLR4 of hPDLF were detected after LPS stimulation and challenge. And we found that TLR2 and TLR4 expressions were down-regulated in h PDLF afte inducing ET, suggesting that two kinds of LPS both can induce h PDLF to generate ET through downregulating the TLR2 and TLR4.2) Under hypoxic conditions, changes of ET in h PDLF have been observed: Based on the ET platform of h PDLF under normoxic conditions, inflammatory cytokines produced by hPDLF were detected after inducing ET by two kinds of LPS under hypoxic condition. It was found that two kinds of the initial LPS stimulation can increase the concentration of IL-6 and IL-8. However, hPDLF were not generated ET after LPS challenge. Meanwhile, compared with LPS initial stimulation, two kinds of TLR expression levels did not change after the re-stimulation. These results suggested that hypoxia can inhibit ET of hPDLF. And, high levels of inflammatory cytokines will be sustained when hPDLF were continuously stimulated by exogenous pathogens under hypoxic conditions.3. Under hypoxic and co-cultured conditions, the secretions of inflammatory cytokines and the abilities of differentiation of PBMCs and h PDLF have been detected.1) The concentrations of inflammatory cytokines have been detected in cocultured cells under normoxic and hypoxic conditions: hPDLF and PBMCs co-cultured under normoxic and hypoxic conditions for 24 h, supernatants were collected to detect inflammatory cytokines. It was found that other five kinds of inflammatory cytokines concentrations were significantly increased when h PDLF cocultured with PBMCs, except IL-12p70. There is no difference of IL-8, IL-6, and IL-12p70 expression between the normoxic and hypoxic conditions. The expression of TNF-α in hypoxic condition is higher than normoxia. However, the expression of IL-1β and IL-10 in normoxic condition is higher than hypoxia. These results indicate that oxygen concentrations have the capacity to regulate the kinds and amounts of inflammatory cytokines produced by cocultured cells. Compared with PBMCs cultured alone under normoxic and hypoxic culture conditions, either indirect or direct co-culture can reduce the levels of TNF-α, especially under hypoxic conditions. Changes of other cytokines were not statistically significant. It still needs further confirmation to see whether hypoxia can increase other inflammatory mediators to enhanced immune injury.2) Under co-culture conditions, the abilities of h PDLF differentiated into OB and PBMCs differentiated into OC have been detected. ALP and TRAP staining were respectively used to observe the differentiation of the cells. It was found that the hypoxia and co-cultured conditions weakened the differentiation ability of h PDLF to OB, but promoted PBMCs to differentiate into OC. PBMCs cultured alone did not form a single typical OC, indicating that OC formation requires the interactions between the two cells. When periodontitis occurs, hypoxia can inhibit hPDLF differenting into OB and promote PBMCs differentiating into OC, thereby enhancing the pathological lesions of periodontal tissues.3) The mechanisms of hypoxia affecting the hPDLF and PBMCs differentiation: We first examined the expressions of transcription factors HIF-1α and NF-κB in h PDLF and found that the expression of HIF-1α protein was increased when the h PDLF was upon hypoxia for 3h. The role of HIF-1α in regulation of both cells differentiation was further investigated by suppressing or enhancing the expression of HIF-1α. It was found that the inhibition of HIF-α suppressed the differentiation of h PDLF into OB and enhanced the differentiation of PBMCs into OC. However, the overexpression of HIF-α promoted h PDLF differentiating into OB. These results demonstrated that HIF-1α plays the protective role under hypoxic condition and it was not in a HIF-1α-dependent way that hypoxia promote osteoclastic differentiation during the interactions between h PDLF and PBMCs. It needs to be further discussion that the mechanisms of hypoxia aggravate periodontal tissue damage by promoting osteoclastic differentiation.In this research, we have successfully isolated and identified h PDLF and PBMCs. We found that LPS stimulation and challenge can regulate the expression of TLR2 and TLR4 and induce the generation of ET in hPDLF. However, hypoxia can inhibit the generation of ET. This suggests that exogenous pathogenic stimuli may act directly on the weakened or missing ET in hPDLF, thereby enhancing the secretions of inflammatory cytokines and increasing the periodontal tissue damage. Then, the interactions between the hPDLF and PBMCs under hypoxic condition were discussed. We found that hypoxia weakened the differentiations of h PDLF into OB and enhanced the differentiations of PBMCs into OC. Further studies have showed that the expressions of HIF-1α were upregulated in hPDLF upon hypoxic condition, but not involved in the regulation. In our research, we firstly confirmed that LPS can induce h PDLF to generate ET and to some extent revealed the mechanisms of hypoxia enhancing the injury of periodontitis. Our research provides a new theoretic foundation for the intervention of periodontal diseases. |
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