Cell characterization and LPS-induced inflammation of hPDLCs in vitro
hPDLCs grew out from the tissue explant after 7 and 10 days of culture (Figure 1, A). Spindle shapes were observed, and a number of cells were distributed in a circinate pattern with rapid proliferation (Figure 1, B). The cells were vimentin positive (Figure 1, C) and keratin negative (Figure 1, D) according to immunochemistry staining, indicating that these primary cells were of mesenchymal origin.
We then examined whether LPS induced the inflammation in hPDLCs without affecting cell viability. hPDLCs were exposed to increasing concentrations of LPS (at range = 0-500 μg/ml) for 24, 48 and 72 h and cell viability was determined. According to the results of the MTT assay, the proliferation of hPDLCs showed a significant reduction after treated with 100 or 500 μg/ml LPS for 24 h, 48 h and 72 h compared with the control group (P<0.001). While the other experimental groups, which were treated with 0.1, 1.0 or 10 μg/ml LPS, showed no significant difference in cell proliferation and viability compared with the control group (P>0.05) (Figure 1, E).
Since the high concentration (100 and 500 μg/ml) of LPS treatment would affect the bioactivity of hPDLCs which could be excluded for the modeling, we then investigated the inflammatory response of the hPDLCs under 0.1, 1.0, 10 μg/ml LPS treatment. The mRNA expressions of inflammatory cytokines were exhibited using real-time PCR. The results indicated that 10 μg/ml LPS induced the expression of pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1) and TNF-α in hPDLCs, compared with the control group (P<0.05). However, the mRNA expression of pro-inflammatory cytokines in those 0.1 and 1.0 μg/ml LPS groups showed no statistical significant up-regulation compared to the control group (P>0.05) (Figure 1, F). According to the results of this section, 10 μg/ml would be chosen as the working concentration of LPS for the inflammation induction model of hPDLCs in vitro.
The pro-inflammatory effects of different dynamic cyclic stress on LPS-induced inflammatory hPDLCs
Given that 10 μg/ml LPS would induce the inflammation model, we then investigated the inflammation status of the hPDLCs under both 10 μg/ml LPS and different dynamic cyclic stress. From the results of real-time PCR, among the different loading groups of dynamic cyclic stress, all the LPS(+) groups showed significant higher mRNA expression of the pro-inflammatory cytokines including IL-1β, IL-6, IL-8, MCP-1 and TNF-α compared to the corresponding loading groups of LPS(-) (P<0.05). LPS(+)/0-150 kPa dynamic cyclic stress loading treatment up-regulated all the pro-inflammatory cytokines (P<0.05), while LPS(+)/0-50 kPa and LPS(+)/0-90 kPa treatment showed no significant effect on the expressions of those cytokines, compared to LPS(+)/0 kPa group. Among the LPS(-) groups, the mRNA expression of IL-1β, MCP-1 and TNF-α showed an increasing tendency as the loading increased, which came to a head in the LPS(-)/0-150 kPa group. In addition, LPS(-)/0-150 kPa loading significantly up-regulated the expression of IL-6 and IL-8 compared with both the LPS(-)/0-50 kPa and LPS(-)/0-90 kPa groups (P<0.05) (Figure 2, A).
Since IL-1β and TNF-α are two of the most important inflammatory factors in the progress of periodontitis, we characterized the effects of different dynamic cyclic stress on these protein expressions in LPS-induced inflammatory hPDLCs to extend our observations at the mRNA level. Consistent with the gene expression pattern, Western blotting analysis demonstrated that after treated with LPS, the expression level of proteins IL-1β and TNF-α in hPDLCs showed obvious enhancement no matter which range of the cyclic stress was loaded. Especially compared to the LPS (-)/0-90 kPa group, the LPS(+)/0-90 kPa group exhibited significant more expression of TNF-α protein. Meanwhile, both IL-1β and TNF-α expressed higher in the 0-150 kPa group than the other loading groups, no matter the hPDLCs was treated with or without LPS. What’s more, the LPS(+)/0-150 kPa treatment induced the highest expression of IL-1β and TNF-α protein than all the other groups, which showed the exacerbation of inflammatory status after over loading on the LPS-induced hPDLCs(Figure 2, B and C).
The osteoblastic effects of different dynamic cyclic stress on LPS-induced inflammatory hPDLCs
Among the four loading groups without LPS treatment, after 5 days of 0-90 kPa dynamic cyclic stress loading, the mRNA expressions of osteoblastic cytokines alkaline phosphatase (ALP), COL-1 and osteocalcin (OCN) were up-regulated to the greatest extent, compared with the 0 and 0-50 kPa groups. Then they declined to the basal line after 5 days of 0-150 kPa dynamic cyclic stress loading. Meanwhile, the mRNA expression of RUNX-2 in LPS(-)/0-150 kPa group was also promoted as with the LPS(-)/0-90 kPa group, compared with the LPS(-)/0 kPa and LPS(-)/0-50 kPa groups. However, there was no significant difference in any expression of the osteoblastic cytokines between LPS(-)/0 kPa and LPS(-)/0-50 kPa groups. Similar to the expression pattern of ALP, COL-1, OCN and RUNX-2, osteopontin (OPN) and osterix (OSX) also showed significant higher mRNA level in the LPS(-)/0-90 kPa group than the other LPS(-) groups. What’s more, the expression of these two osteogenic related genes decreased distinctly in the LPS(+)/0-150 kPa group among all the other groups and reached a statistical significance. But no significant difference among the other LPS(+) groups after the different loadings was found (Figure 3, A).
In accordance with the results of real-time PCR, Western blotting analysis revealed that the expression level of RUNX-2 protein reached the peak at LPS(-)/0-90 kPa group compared with the other LPS(-) loading groups. However, the RUNX-2 protein level showed no significant difference among the different loading groups in LPS-induced hPDLCs. Moreover, the expression of COL-1 protein had no obvious change among the different dynamic cyclic stress groups no matter treated with LPS or not after 5 days (Figure 3, B and C).
The osteoclastic effects of different dynamic cyclic stress on LPS-induced inflammatory hPDLCs
Having observed the changes in osteoblastic cytokines after the treatment of different dynamic cyclic stress and LPS, we then investigated the expression changes of the pro-osteoclastic cytokines. RT-PCR results suggested that the mRNA levels of pro-osteoclastic cytokines, including RANKL, macrophage colony-stimulating factor (M-CSF), CTSK, PTHLH in LPS-induced hPDLCs were up-regulated compared to the corresponding loading groups without LPS respectively, and expressed the statistical difference at the 0-150 kPa group. Among four LPS(-) groups, it could be seen that the mRNA level of pro-osteoclastic cytokines went up following the loads increased. Similar to the LPS(-) groups, the expression of these osteoclastic markers was significantly promoted in the LPS(+)/0-90kPa groups, and reached the highest level in the LPS(+)/0-150 kPa group after 5 days(Figure 4, A).
The result of Western blotting showed that LPS(+)/0-90 kPa and LPS(+)/0-150 kPa dynamic cyclic stress treatment could up-regulate the expression level of RANKL protein, compared with other LPS(+) groups. In addition, only 0-150 kPa dynamic cyclic stress treatment without LPS could also promote the expression of RANKL protein to an extreme high level than the other LPS (-) groups. There was no obvious effect of osteoclasts after the cyclic stress under 90 kPa regardless the LPS treatment or not. Nevertheless, the synergistic effect of a smaller cyclic stress and LPS treatment on promoting the osteoclasis in hPDLCs was nearly equal to the much more dynamic cyclic stress treatment without LPS (Figure 4, B and C).