Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions

Abstract Objectives Vitamin D3 is known to activate osteogenic differentiation of human periodontal ligament stromal cells (hPDLSCs). Recently, inflammatory stimuli were shown to inhibit the transcriptional activity of hPDLSCs, but their effect on vitamin D3‐induced osteogenic differentiation is not known. The present study aimed to investigate whether the effects of 1,25‐dihydroxvitamin D3 (1,25(OH)2D3) and 25‐hydroxvitamin D3 (25(OH)D3) on the osteogenic differentiation of hPDLSCs are also altered under inflammatory conditions. Furthermore, the expression of osteogenesis‐related factors by hPDLSCs under osteogenic conditions was assessed in the presence of inflammatory stimuli. Materials and Methods Primary hPDLSCs of six donors were cultured in osteogenic induction medium containing either 1,25(OH)2D3 (0‐10 nM) or 25(OH)D3 (0‐100 nM) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) or Pam3CSK4 for 7, 14 and 21 days. Osteogenic differentiation of hPDLSCs was evaluated by analysis of mineralization as assessed by Alizarin Red S staining and gene expression levels of osteogenesis‐related factors osteocalcin, osteopontin and runt‐related transcription factor 2 (RUNX2) were analysed with qPCR. Results Treatment with 1,25(OH)2D3 significantly enhanced the osteogenic differentiation of hPDLSCs and their expression of osteocalcin and osteopontin. The 1,25(OH)2D3‐triggered expression of osteogenesis‐related factors was significantly lower in the presence of Pam3CSK4, but not P. gingivalis LPS. None of the inflammatory stimuli had significant effects on the 1,25(OH)2D3‐induced osteogenic differentiation. 25(OH)D3 neither affected gene expression levels nor osteogenic differentiation of hPDLSCs cultured in osteogenic induction medium. Conclusion The results of this study indicate that inflammatory stimuli also diminish the 1,25(OH)2D3‐induced expression of osteogenesis‐related factors in hPDLSCs under osteogenic conditions, while having no effect on the osteogenic differentiation.


| INTRODUC TI ON
Vitamin D 3 is a fat-soluble steroid hormone that plays a pivotal role in numerous physiological functions, particularly bone metabolism. 1 Although it can be obtained from several nutritional sources, humans are largely dependent on vitamin D 3 production in the skin. 2 Upon exposure to ultraviolet B radiation, 7-dehydrocholesterol in the epidermis is photolysed to previtamin D 3 and further converted to vitamin D 3 . 3 This biologically inactive form is hydroxylated via the 25-hydroxylase into the most abundant circulating vitamin D 3 metabolite 25-hydroxyvitamin D 3 (25(OH)D 3 ). 4 The conversion into the biologically most active form 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) is facilitated via 1α-hydroxylation. 5 This process occurs predominantly in the kidneys, but can also be observed in extrarenal tissues, such as the periodontium. 5,6 1,25(OH) 2 D 3 exerts its numerous functions via binding to vitamin D receptor (VDR), which is expressed in almost all cells. 7 Apart from its role in bone metabolism, the active vitamin D 3 metabolite possesses strong immunomodulatory, anti-inflammatory and anti-proliferative properties. 8,9 Vitamin D 3 has versatile effects in periodontal ligament stromal cells (hPDLSCs). 10 hPDLSCs fulfill the minimal criteria of mesenchymal stromal cells and play a crucial role in periodontal tissue homeostasis. 11-13 1,25(OH) 2 D 3 has been observed to enhance the osteogenic differentiation of hPDLSCs, to increase their expression of osteogenesis-related factors and to dampen their inflammatory response. 6,14,15 Considering these numerous positive effects, vitamin D 3 deficiency is unsurprisingly associated with an increased risk of periodontal disease. [16][17][18] Periodontitis is a multifactorial chronic disease leading to destruction of all periodontal tissues, namely gingiva, alveolar bone, cementum and periodontal ligament. 19,20 It is initiated by a shift of a symbiotic to a dysbiotic oral microbiota and is driven by an excessive inflammatory response. 21 As summarized in our previous study, there are several reports about the influence of vitamin D 3 supplementation during periodontitis treatment. 22 Surprisingly, supplementation of vitamin D 3 has never been shown to be beneficial during non-surgical periodontal treatment so far. 22 Our previous study focussed on finding a possible explanation for this. As inflammation is still strongly pronounced during the initial phase of periodontitis therapy, we investigated the effects of vitamin D 3 metabolites on the expression of osteogenesisrelated factors in human periodontal ligament stromal cells (hP-DLSCs) under inflammatory conditions. Inflammation was simulated by targeting Toll-like receptors (TLRs), which recognize specific components of pathogens and lead to induction of inflammatory responses. 23 TLR4 and TLR2 agonist Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS) and TLR2/1 agonist Pam3CSK4 are known to strongly enhance the inflammatory response of hP-DLSCs and were therefore chosen for the experiments. [24][25][26] We observed that the 1,25(OH) 2 D 3 -and 25(OH)D 3 -induced expression of osteogenesis-related factors is diminished under inflammatory conditions, suggesting a decreased transcriptional activity of VDR in the presence of inflammatory stimuli. 22 Since the gene expression of osteogenesis-related factors does not always correlate with mineralization, it remained unclear whether inflammatory stimuli also affect the vitamin D 3 -induced osteogenic differentiation. 27 Therefore, the aim of the present study was to elucidate, if the 1,25(OH) 2 D 3 -and 25(OH)D 3 -induced osteogenic differentiation of hPDLSCs is similarly affected by inflammatory conditions. In addition, the effects of inflammatory stimuli on the and CD45. 11 Notably, the isolated cell population is heterogeneous and might additionally contain osteoblasts, fibroblasts and odontoblasts, which express similar surface markers as MSCs. 28 However, the isolated cells still meet the minimal criteria of mesenchymal stromal cells as defined by the position papers of the International Society for Cellular Therapy. 11,29 Each of the subsequent experiments was conducted with hPDLSCs of five different donors within passage 4-6.

| Treatment protocol
hPDLSCs were seeded in 24 well plates at a density of 5 × 10 4 cells/well together with 0.5 ml DMEM supplemented with 1% P/S and 10% FBS for 24 hours. For the following stimulation, osteogenic induction medium was prepared, which was

| Statistical analyses
The statistical analyses were conducted with SPSS 24.0 (IBM). Data were analysed by Friedman test, followed by Wilcoxon test for pairwise comparison. All data are presented as mean ± SEM of five independent experiments with five different donors performed in triplicates.   Figure

| 25(OH)D 3 has no impact on the osteogenic differentiation and expression of osteogenesis-related factors of hPDLSCs under osteogenic conditions
The

| DISCUSS ION
Apart from its fundamental role for bone metabolism, vitamin D 3 is also well known for its anti-inflammatory and immunomodulatory properties. 30 Currently, it is also discussed to play a role in preventing COVID-19 infection, progression and severity. 31,32 Vitamin  Summarizing the data of this study, we could show that the 1,25(OH) 2 D 3 -triggered osteogenic differentiation of hPDLSCs is not affected under inflammatory conditions. However, the effects of 1,25(OH) 2 D 3 seem to be, at least partially, diminished by inflammatory stimuli under osteogenic conditions. Further studies are required to reveal the influence of inflammation on the effectiveness of vitamin D 3 metabolites.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.  Figure 5A, 5B and 5C represent the n-fold expression of the target gene in relation to untreated hPDLSCs. Resulting calcium deposition was analysed by Alizarin Red S staining (D: representative picture of one donor). Quantification of calcium deposits was performed photometrically. Yaxis of Figure 5E represents the optical density measured at 405 nm. All data are presented as mean ±standard error of the mean of five independent experiments