Roles of human periodontal ligament stem cells in osteogenesis and inflammation in periodontitis models: Effect of 1α,25-dihydroxyvitamin D3

Periodontitis is a chronic inflammatory disease caused by Porphyromonas gingivalis and other bacteria, and human periodontal ligament stem cells (hPDLSCs) are a promising candidate for the treatment of periodontal supporting tissue defects. This study aimed to investigate the effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)2VitD3] on osteogenic differentiation of hPDLSCs in an in vitro periodontitis model and whether it can improve inflammatory status. hPDLSCs were in vitro isolated and identified. After treatment with 1,25(OH)2VitD3 and ultrapure pure Porphyromonas gingivalis lipopolysaccharide (LPS-G), the viability of hPDLSCs was detected using Cell Counting Kit-8, the expressions of osteogenic markers and inflammatory genes using Western blotting and quantitative reverse transcription PCR (qRT-PCR), the levels of inflammatory factors in cells using enzyme linked immunosorbent assay (ELISA), and the fluorescence signal intensity of osteoblastic markers and inflammatory genes in cells using immunofluorescence assay. It was found that 1,25(OH)2VitD3 reversed the inhibition of hPDLSCs proliferation by LPS-G; LPS-G exhibited inhibitory effect on ALP, Runx2, and OPN expressions, and such inhibitory effect was significantly weakened when co-acting with 1,25(OH)2VitD3. Meanwhile, LPS-G upregulated the expressions of inflammatory genes IL-1β and Casp1, whereas 1,25(OH)2VitD3 antagonized such an effect and improved the inflammatory status. In conclusion, 1,25(OH)2VitD3 can reverse the inhibitory effect of LPS-G on hPDLSCs proliferation and osteogenic differentiation and suppress LPS-G-induced upregulation of inflammatory gene expressions.


Introduction
Periodontitis is a chronic infectious disease of tooth-supporting tissues caused by dental plaque microorganisms (predominantly Porphyromonas gingivalis and Treponema denticola) [1]. It often leads to inflammation and destruction of periodontal structures [2]. Removing pathogenic factors and controlling inflammation are the basic treatments for periodontitis, and periodontal regeneration depends mainly on periodontal surgery.
As one of the most promising types of seed cells in periodontal tissue regeneration, human periodontal ligament stem cells (hPDLSCs) are mesenchymal stem cells that have the self-renewal and multi-directional differentiation abilities. Their osteogenic differentiation ability may represent a breakthrough in the treatment of periodontal supporting tissue defects. Evidence suggests that the co-culture of hPDLSCs and scaffolds significantly accelerates osteogenic differentiation and mineralization. At the same time, titanium surface structure affects the adhesion growth and osteogenesis of hPDLSCs in dental implants [3][4][5]. However, some crucial issues including the influence of inflammation on hPDLSCs, growth-promoting factors of hPDLSCs, and host state factors remain unknown in the complex periodontal microenvironment.
Vitamin D (VitD) is a fat-soluble vitamin that acts through its active metabolite 1α,25-dihydroxyvitamin D 3 [1,25(OH) 2 VitD 3 ], and its deficiency is closely related to the development of periodontitis [6,7]. Evidence suggests that VitD can exert anti-inflammatory effects by inhibiting pro-inflammatory factors and other pathways [8]. Thus, appropriate VitD supplementation can assist basic periodontal treatment. As an endotoxin, lipopolysaccharide (LPS) is a component of the cell wall of gram-negative bacteria. It is a major causative factor for periodontitis and can regulate the expressions of inflammatory cytokines through the transcription factor nuclear factor κB (NF-κB) [9].
In the present study, LPS was used to construct an in vitro periodontitis model of hPDLSCs to explore the effect of VitD on osteogenic activityand whether VitD could exert anti-inflammatory effects to ensure the osteogenic differentiation of hPDLSCs.

Isolation and culture of hPDLSCs
All methods were carried out in accordance with relevant guidelines and regulations. This study was approved by the Medical Research Ethics Committee of General Hospital of Ningxia Medical University (Protocol No. 2018-345) and informed consent was obtained from the patients. Premolars were extracted from healthy individuals for orthodontic reasons. The periodontal ligament was scraped after washing and then digested with collagenase I (3 mg/mL; Sigma, Germany) for 30 min. Cell suspension was inoculated into a low-sugar DMEM (Vivacell, China) containing 10% fetal bovine serum (FBS; Gibco, USA) and cultured in an incubator at 37 • C and 5% CO 2 . Cells at the 3rd and 4th passages were used for subsequent experiments.

Analysis of hPDLSCs characteristics
The cells were digested with trypsin to make a cell suspension, and incubated with fluorescent antibodies in dark for 20 min, and a flow cytometry analyzer (FlowSight, Merck Millipore, Germany) was used to analyze stem cell surface markers and determine the characteristics of hPDLSCs. The antibodies included CD14-PE, CD45-PE, CD90-PE, and CD105-FITC (BD Biosciences, USA) and STRO-1-APC (Invitrogen, USA).

Multidirectional differentiation of hPDLSCs
HPDLCs were inoculated in 12-well plates containing different types of culture medium for 14 days to assess their multidirectional differentiation capacity. The osteogenic ability was identified using an osteogenic induction medium (OM) containing 10% FBS, 10 nmol/L dexamethasone, 10 mmol/L β-glycerophosphate, and 50 μg/mL ascorbic acid, and the formation of mineralized nodules was observed by alizarin red S staining (Solarbio, China). The adipogenic ability was identified using an adipogenic induction medium containing 10% FBS, 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, and 100 μmol/L indomethacin, and the formation of lipid droplets was observed by Oil red O staining (Solarbio, China).

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
hPDLSCs were inoculated in 6-well plates and cultured using each set of conditions for 72 h. Total RNA was extracted using the Total RNA Extraction Kit (Omega, USA) and reverse-transcribed into cDNA (Takara, Japan). qRT-PCR was performed using the TB Green kit (Takara, Japan) on the qTOWER® 3 real-time qPCR system (Analytik Jena, Germany). Specific primers were synthesized by Tsingke, China, and the sequences are shown in Table 1. All data were analyzed by the 2 -ΔΔCT method, with GAPDH as the reference gene.

Enzyme-linked immunosorbent assay (ELISA)
The IL-1β level in cells was detected after 72 h conditional culture. The cells were digested by trypsin to make a cell suspension. Supernatants were harvested to detect intracellular IL-1β level after repeated liquid nitrogen freezing and thawing according to the instructions of the kit (Meimian, China). qRT-PCR of the gene expression levels of ALP, Runx2, and OPN after 72 h of culture. The graphs represent the mean ± standard deviation (n = 3-8). * P < 0.05, * * P < 0.01, * ** P < 0.001, * ** * P < 0.0001, compared with the LPS-G group.

Statistical analysis
Data were analyzed and plotted using the GraphPad 8.0 software and compared by t test and one-way ANOVA. A P value of less than 0.05 was considered statistically significant.

1,25(OH)2VitD3 reversed the inhibitory effects of LPS-G on the proliferation and osteogenic differentiation of hPDLSCs
The effects of both 1,25(OH) 2 VitD 3 and LPS-G on the proliferation of hPDLSCs were evaluated by CCK-8 assay. As shown in Figs. 2A, 1,25 (OH) 2 VitD 3 treatment slightly improved cell viability, which was not significantly different among all four groups at 24 h; after 24 h, 1,25 (OH) 2 VitD 3 showed a certain inhibitory effect on cell proliferation. In contrast, the viability of hPDLSCs in the LPS-G group was significantly lower than that in the other groups, and the viability reached the trough level at 72 h. Furthermore, the presence of 1,25(OH) 2 VitD 3 reversed the effect of LPS-G and improved cell viability.
Next, we investigated the effects of 1,25(OH) 2 VitD 3 and LPS-G on osteogenic differentiation of hPDLSCs. Western blotting showed that the expressions of osteogenic markers ALP and Runx2 significantly increased in the 1,25(OH) 2 VitD 3 group, although the change of OPN was not obvious. LPS-G exerted obvious inhibitory effect on the expressions of these proteins, especially for Runx2 protein, and such inhibition was reversed to varying degrees in the presence of 1,25(OH)2VitD3 (Fig. 2B,  C). qRT-PCR also showed that the presence of 1,25(OH)2VitD3 significantly increased the mRNA expression levels of ALP, Runx2, and OPN (Fig. 2D).
In addition, the confocal microscopy displayed the localization and expressions of ALP and Runx2 in hPDLSCs (Figs. 3 and 4). 1,25 (OH) 2 VitD 3 and LPS-G had no significant effect on the morphology of hPDLSCs. In the Con group, ALP immunofluorescence signals were present in both intranuclear and extranuclear areas, while Runx2 was mainly expressed within the nuclei. 1,25(OH) 2 VitD 3 treatment significantly enhanced the intensities of intranuclear and extranuclear ALP signals and intracytoplasmic Runx2 fluorescence signals, which was quite different from the weakened intensities of ALP and Runx2 signals after LPS-G treatment. Consistent with previous experiments, the ALP and Runx2 signals were enhanced to a certain extent in cells co-cultured with LPS-G and 1,25(OH) 2 VitD 3 , which further confirmed that 1,25 (OH) 2 VitD 3 could reverse the inhibitory effect of LPS-G on the osteogenic differentiation of hPDLSCs.
Furthermore, we analyzed the localization and expression of Casp1 using immunofluorescence (Fig. 6). The Casp1 fluorescence signal was mainly present within the nuclei and was rarely expressed in the cytoplasm. In contrast to the significantly enhanced Casp1 signals inside and outside the nuclei in the LPS-G group, the Casp1 signals were weakened in the 1,25(OH) 2 VitD 3 treatment groups. The inhibition of LPS-G-

Discussion
It has been found that periodontitis not only affects oral function and periodontal aesthetics but also relates to cardiovascular diseases, diabetes, respiratory diseases, and other conditions [10][11][12]. Its relationship with overall health has also received considerable attention. Periodontal tissue engineering with hPDLSCs as a starting point has brought hope for the fundamental repair of periodontal bone defects and regeneration of periodontal tissues. hPDLSCs themselves exist in the normal periodontal ligament. Under suitable induction conditions, they can exert the multi-directional differentiation abilities including osteogenesis, adipogenesis, and chondrogenesis. In the present study, hPDLSCs were isolated from normal human periodontal ligament and then cultured, in an attempt to validate their potentials for osteogenesis and adipogenesis in vitro.
However, the local inflammatory microenvironment of the affected tooth poses challenges to the repair and regeneration of periodontal tissues. Periodontitis results from an imbalance between bacterial invasion and host defense, during which host susceptibility, dysbacteriosis, and immune response play important roles. Bacteria trigger the body's immune response locally around the affected tooth in susceptible patients, leading to the massive release of pro-inflammatory factors and chemokines such as IL-1β, tumor necrosis factor α (TNF-α), and C-C motif chemokine ligand 5 (CCL5) [13] and inducing innate immune cells (including NK cells, macrophages, and dendritic cells) to rapidly participate in the immune response [14]. Subsequently, the adaptive immune system is involved in resisting bacterial invasion and protecting periodontal tissue, which unfortunately further promotes the inflammatory response [15][16][17]. However, bacteria can suppress the immune response to protect themselves. For example, Porphyromonas gingivalis can resist the killing effect of neutrophils [18]. With the stimulation and maintenance of inflammation, bacterial immune evasion further promotes the destruction of periodontal tissues by dysbacteriosis and inflammatory response. The massive proliferation of helper T cell 17 (Th17) in periodontitis [19] promotes the release of pro-inflammatory factors such as prostaglandin E2 (PGE2) and IL-8 and leads to a decrease in the osteogenic capacity of mesenchymal stem cells (MSCs) [20]. The inflammatory microenvironment and host responses disrupt signaling networks, leading to a decrease in the osteogenic differentiation capacity of hPDLSCs [21,22]. On the other hand, the damaged hPDLSCs can also promote osteoclast maturation, leading to periodontal bone resorption [23]. In the present study, after the periodontitis model was constructed using LPS-G, the expressions of inflammatory genes IL-1β and Casp1 significantly increased, and the expressions of osteogenic markers ALP, Runx2, and OPN significantly decreased, suggesting that the osteogenic differentiation ability of hPDLSCs was remarkably affected.
There is increasing evidence that VitD is an important part of the body's defenses against bacterial and viral invasion. 1,25(OH)2VitD3mediated downstream signaling is an important pathway for innate immune responses that regulate the gene expressions of patternrecognition receptors (PRRs), cytokines, and chemokines. The value of VitD supplementation in the treatment of bacterial infections has increasingly been recognized [24][25][26]. Studies have also confirmed the immunoprotective effect of VitD against viruses including human immunodeficiency virus (HIV) and respiratory viruses [27,28]. For instance, VitD supplementation can significantly reduce the incidence of acute respiratory infections, which also highlights the clinical value of VitD as an adjuvant treatment for viral infections. In our in vitro experiments, 1,25(OH) 2 VitD 3 reversed the inhibition of osteogenic markers by LPS-G and downregulated the expressions of inflammatory genes, which again confirmed the importance of 1,25(OH) 2 VitD 3 in promoting osteogenic differentiation of hPDLSCs and resisting inflammation.
Therefore, it is assumed that hPDLSC may be used in combination with VitD supplementation in the treatment of periodontitis. Due to the high prevalence of vitamin D deficiency worldwide and its important role in disease treatment [29,30], VitD supplementation is urgently recommended.

Funding
Key Research and Development Program of Ningxia, China (grant no. 2019BEG03035).

CRediT authorship contribution statement
Jingjiao Wang and Chenglei Zhang have made contributions to the writing of the manuscript, the design of the figures. Xiaoqian Guo and Changyi Yang participated in literature survey. All authors have approved the submitted version of the article and have agreed to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work. All authors read and approved the final manuscript.

Data Availability
Data will be made available on request. The data used to support the findings of the present study are available from the corresponding authors upon request.