Abstract

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

Stem Cells; Dental Pulp; Periodontal Ligament; Lipopolysaccharides; NF-kappa B

Introduction

Several studies have reported that mesenchymal stem cells (MSCs) have potential uses in tissue engineering and regenerative medicine,¹1. Lina Y, Wijaya A. Adipose-derived stem cells for future regenerative system medicine. Indones Biomed J. 2012;4(2):59-72. https://doi.org/10.18585/inabj.v4i2.164.
https://doi.org/10.18585/inabj.v4i2.164...

2. Meiliana A, Dewi NM, Wijaya A. Stem cell therapy in wound healing and tissue regeneration. Indones Biomed J. 2016;8(2):61-70. https://doi.org/10.18585/inabj.v8i2.191
https://doi.org/10.18585/inabj.v8i2.191... -³3. Meiliana A, Dewi NM, Wijaya A. Mesenchymal stem cells manage endogenous tissue regeneration. Indones Biomed J. 2016;8(2):71-90. https://doi.org/10.18585/inabj.v8i2.211
https://doi.org/10.18585/inabj.v8i2.211... including the field of dentistry.⁴4. Bakopoulou A, About I. Stem cells of dental origin: current research trends and key milestones towards clinical application. Stem Cells Int. 2016;2016:4209891. https://doi.org/10.1155/2016/4209891
https://doi.org/10.1155/2016/4209891... Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) are oral tissue-derived stem cells that possess the properties of MSCs.⁴4. Bakopoulou A, About I. Stem cells of dental origin: current research trends and key milestones towards clinical application. Stem Cells Int. 2016;2016:4209891. https://doi.org/10.1155/2016/4209891
https://doi.org/10.1155/2016/4209891...

5. Feter Y, Afiana NS, Chandra JN, Abdullah K, Shafira J, Sandra F. Dental mesenchymal stem cell: its role in tooth development, types, surface antigens and differentiation potential. Mol Cell Biomed Sci. 2017;1(2):50-7. https://doi.org/10.21705/mcbs.v1i2.15
https://doi.org/10.21705/mcbs.v1i2.15... -⁶6. Sandra F, Sudiono J, Binartha CT, Chouw A, Djamil MS. Growth and osteogenic differentiation of CD117+ dental pulp and periodontal ligament cells. Indones Biomed J. 2017;9(2):78-83. https://doi.org/10.18585/inabj.v9i2.286
https://doi.org/10.18585/inabj.v9i2.286... Under specific culture conditions, DPSCs and PDLSCs can be differentiated into mesenchymal lineages, including osteoblasts.⁷7. D'Aquino R, Graziano A, Sampaolesi M, Laino G, Pirozzi G, De Rosa A, et al. Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation. Cell Death Differ. 2007 Jun;14(6):1162-71. https://doi.org/10.1038/sj.cdd.4402121
https://doi.org/10.1038/sj.cdd.4402121...

8. Isaka J, Ohazama A, Kobayashi M, Nagashima C, Takiguchi T, Kawasaki H, et al. Participation of periodontal ligament cells with regeneration of alveolar bone. J Periodontol. 2001 Mar;72(3):314-23. https://doi.org/10.1902/jop.2001.72.3.314
https://doi.org/10.1902/jop.2001.72.3.31... -⁹9. Sudiono J, Oka CT, Djamil MS, Sandra F. Regenerative medicine in dental and oral tissues: dental pulp mesenchymal stem cell. Padjadjaran J Dent. 2016;28(1):31-7. https://doi.org/10.24198/pjd.vol28no1.13513
https://doi.org/10.24198/pjd.vol28no1.13... DPSCs and PDLSCs have higher growth potential compared with bone marrow mesenchymal stem cells (BMMSCs),¹⁰10. Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res. 2005 Aug;8(3):191-9. https://doi.org/10.1111/j.1601-6343.2005.00331.x
https://doi.org/10.1111/j.1601-6343.2005... and possess immunomodulatory activity.²2. Meiliana A, Dewi NM, Wijaya A. Stem cell therapy in wound healing and tissue regeneration. Indones Biomed J. 2016;8(2):61-70. https://doi.org/10.18585/inabj.v8i2.191
https://doi.org/10.18585/inabj.v8i2.191... ,³3. Meiliana A, Dewi NM, Wijaya A. Mesenchymal stem cells manage endogenous tissue regeneration. Indones Biomed J. 2016;8(2):71-90. https://doi.org/10.18585/inabj.v8i2.211
https://doi.org/10.18585/inabj.v8i2.211... ,¹¹11. Sandra F, Sudiono J, Feter Y, Afiana NS, Chandra JN, Abdullah K, et al. Investigation on cell surface markers of dental pulp stem cell isolated from impacted third molar based on International Society for Cellular Therapy proposed mesenchymal stem cell markers. Mol Cell Biomed Sci. 2019;3(1):1-6. https://doi.org/10.21705/mcbs.v3i1.34
https://doi.org/10.21705/mcbs.v3i1.34... Hence, DPSCs and PDLSCs are potential candidates for bone tissue engineering and regeneration applications, such as alveolar bone repair.⁴4. Bakopoulou A, About I. Stem cells of dental origin: current research trends and key milestones towards clinical application. Stem Cells Int. 2016;2016:4209891. https://doi.org/10.1155/2016/4209891
https://doi.org/10.1155/2016/4209891...

The process of osteogenesis is influenced by several environmental factors, including inflammatory factors produced by bacteria.¹²12. Li C, Li B, Dong Z, Gao L, He X, Liao L, et al. Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor ?B pathway. Stem Cell Res Ther. 2014 May;5(3):67. https://doi.org/10.1186/scrt456
https://doi.org/10.1186/scrt456... ,¹³13. Chen M, Lin X, Zhang L, Hu X. Effects of nuclear factor-?B signaling pathway on periodontal ligament stem cells under lipopolysaccharide-induced inflammation. Bioengineered. 2022 Mar;13(3):7951-61. https://doi.org/10.1080/21655979.2022.2051690
https://doi.org/10.1080/21655979.2022.20... Lipopolysaccharide (LPS) is the most common inflammatory factor, which is continuously liberated from Gram-negative bacteria colonizing the periodontal tissues, and can cause inflammatory diseases, such as periodontitis.¹⁴14. Nativel B, Couret D, Giraud P, Meilhac O, d'Hellencourt CL, Viranaïcken W, et al. Porphyromonas gingivalis lipopolysaccharides act exclusively through TLR4 with a resilience between mouse and human. Sci Rep. 2017 Nov;7(1):15789. https://doi.org/10.1038/s41598-017-16190-y
https://doi.org/10.1038/s41598-017-16190... LPS activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway and induces inflammatory responses.¹⁵15. Chang J, Zhang C, Tani-Ishii N, Shi S, Wang CY. NF-kappaB activation in human dental pulp stem cells by TNF and LPS. J Dent Res. 2005 Nov;84(11):994-8. https://doi.org/10.1177/154405910508401105
https://doi.org/10.1177/1544059105084011... ,¹⁶16. Hayden MS, West AP, Ghosh S. NF-kappaB and the immune response. Oncogene. 2006 Oct;25(51):6758-80. https://doi.org/10.1038/sj.onc.1209943
https://doi.org/10.1038/sj.onc.1209943... Several studies have reported that LPS-induced NF-κB activity in PDLSCs can be inhibited, enabling undisrupted osteogenesis.¹²12. Li C, Li B, Dong Z, Gao L, He X, Liao L, et al. Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor ?B pathway. Stem Cell Res Ther. 2014 May;5(3):67. https://doi.org/10.1186/scrt456
https://doi.org/10.1186/scrt456... ,¹³13. Chen M, Lin X, Zhang L, Hu X. Effects of nuclear factor-?B signaling pathway on periodontal ligament stem cells under lipopolysaccharide-induced inflammation. Bioengineered. 2022 Mar;13(3):7951-61. https://doi.org/10.1080/21655979.2022.2051690
https://doi.org/10.1080/21655979.2022.20... However, in other types of MSCs, such as BMMSCs, LPS induces NF-κB activity, but does not alter osteogenic differentiation.¹²12. Li C, Li B, Dong Z, Gao L, He X, Liao L, et al. Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor ?B pathway. Stem Cell Res Ther. 2014 May;5(3):67. https://doi.org/10.1186/scrt456
https://doi.org/10.1186/scrt456... In addition, in adipose-derived mesenchymal stem cells (AdMSCs), LPS induced NF-κB activity and stimulated osteogenic differentiation.¹⁷17. Cho HH, Shin KK, Kim YJ, Song JS, Kim JM, Bae YC, et al. NF-kappaB activation stimulates osteogenic differentiation of mesenchymal stem cells derived from human adipose tissue by increasing TAZ expression. J Cell Physiol. 2010 Apr;223(1):168-77. https://doi.org/10.1002/jcp.22024
https://doi.org/10.1002/jcp.22024... Therefore, NF-κB inhibition affects the osteogenic potency of different types of MSCs differently. The aim this study was to evaluate the effect of LPS-induced NF-κB activity, and its inhibition using a specific inhibitor, Bay 11-7082, in DPSCs and PDLSCs.

Methodology

Cells Thawing and Culture

Cryopreserved passage five DPSCs and PDLSCs reported in previous research⁶6. Sandra F, Sudiono J, Binartha CT, Chouw A, Djamil MS. Growth and osteogenic differentiation of CD117+ dental pulp and periodontal ligament cells. Indones Biomed J. 2017;9(2):78-83. https://doi.org/10.18585/inabj.v9i2.286
https://doi.org/10.18585/inabj.v9i2.286... ,¹¹11. Sandra F, Sudiono J, Feter Y, Afiana NS, Chandra JN, Abdullah K, et al. Investigation on cell surface markers of dental pulp stem cell isolated from impacted third molar based on International Society for Cellular Therapy proposed mesenchymal stem cell markers. Mol Cell Biomed Sci. 2019;3(1):1-6. https://doi.org/10.21705/mcbs.v3i1.34
https://doi.org/10.21705/mcbs.v3i1.34... were thawed and cultured in MesenCult MSC Basal Medium (StemCell Technologies, Vancouver, Canada) supplemented with MesenCult MSC Stimulatory Supplement (StemCell Technologies), 200 U/mL penicillin, 200 µg/mL streptomycin, and 0.5 µg/mL amphotericin (Gibco, Grand Island, NY, USA). DPSCs and PDLSCs were harvested after reaching confluency and used in this study. This study was conducted in accordance with the Declaration of Helsinki. Approval was obtained from the Ethics Committee of Faculty of Dentistry Universitas Trisakti, Indonesia (No. #167/KE/FKG/11/2014). Written informed consent was obtained for the collection of human samples.

Flow Cytometric Analysis

Flow cytometric analysis was conducted using a BD Stemflow hMSC Analysis Kit (BD Biosciences, Franklin Lakes, USA) to confirm whether DPSCs and PDLSCs had MSC markers as previously described.¹¹11. Sandra F, Sudiono J, Feter Y, Afiana NS, Chandra JN, Abdullah K, et al. Investigation on cell surface markers of dental pulp stem cell isolated from impacted third molar based on International Society for Cellular Therapy proposed mesenchymal stem cell markers. Mol Cell Biomed Sci. 2019;3(1):1-6. https://doi.org/10.21705/mcbs.v3i1.34
https://doi.org/10.21705/mcbs.v3i1.34... DPSCs (1 × 10⁷ cells) and PDLSCs (1 × 10⁷ cells) were incubated with/without marker-specific antibodies as well as their isotypes for positive (CD90, CD105, and CD73) and negative (CD45, CD34, CD11b, CD19, and HLA-DR) markers. FACSCanto II flow cytometer (BD Biosciences) was used to analyze labeled DPSCs and PDLSCs using the FACSDiva software (BD Biosciences). The characteristics of DPSCs and PDLSCs were confirmed using the minimal surface marker criteria for defining MSCs, proposed by the International Society for Cellular Therapy (ISCT).¹⁸18. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7. https://doi.org/10.1080/14653240600855905
https://doi.org/10.1080/1465324060085590...

In vitro Osteogenic Functional Assay

In vitro osteogenic functional assay was performed as previously described.⁶6. Sandra F, Sudiono J, Binartha CT, Chouw A, Djamil MS. Growth and osteogenic differentiation of CD117+ dental pulp and periodontal ligament cells. Indones Biomed J. 2017;9(2):78-83. https://doi.org/10.18585/inabj.v9i2.286
https://doi.org/10.18585/inabj.v9i2.286... DPSCs (8 × 10⁴ cells) and PDLSCs (8 × 10⁴ cells) were cultured using osteogenic medium containing 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, USA), 100 nM dexamethasone (Sigma-Aldrich), and 50 µg/mL L-ascorbic acid (Sigma-Aldrich) on a 6-well plate. DPSCs and PDLSCs were pretreated with/without 100 µM NF-κB inhibitor Bay 11-7082 (Sigma-Aldrich) for 30 min and supplemented with/without 10 µg/mL Porphyromonas gingivalis LPS (Wako, Osaka, Japan) for 1, 2, or 3 weeks. After removing the medium, the plates were washed twice with PBS and fixed for 2 min in 4% paraformaldehyde (Wako) in phosphate buffer solution (PBS). This was followed by treatment with glycerol (Bio-Rad, Hercules, USA) at room temperature for 5 min. The cells were washed thrice with distilled water after removal of the fixative. The cells were then stained with 2% alizarin red solution (Sigma-Aldrich) for 20 min. After removing the alizarin red stain, the plates were washed thrice with distilled water. The cells were finally observed and documented under an inverted light microscope (Zeiss, Jena, Germany). The experiment was performed twice in triplicate.

NF-κB Activity Assay

After pretreatment with Bay 11-7082 for 30 min and LPS supplementation for three weeks, NF-κB activity in DPSCs (2 × 10⁶ cells) and PDLSCs (2 × 10⁶ cells) was determined using NF-κB p65 Transcription Factor Assay Kit (Abcam, Cambridge, UK) in accordance with the manufacturer’s protocol. Nuclear extraction of the treated DPSCs and PDLSCs was performed using the Nuclear Extraction Kit (Abcam) in accordance with the manufacturer’s instructions, before determining NF-κB activity. The nuclear extracts containing NF-κB were loaded into 96-well plates containing dsDNA with NF-κB response element sequence, followed by the sequential addition of rabbit anti-NF-κB primary antibody and HRP-linked goat antirabbit IgG secondary antibody. Results were measured at OD₄₅₀ nm using a spectrophotometer (Bio-Rad). The experiment was performed twice in triplicate.

Alkaline Phosphatase (ALP) Activity Assay

Following pretreatment with Bay 11-7082 for 30 min and LPS supplementation with/without Bay 11-7082 for three weeks, ALP activity in DPSCs and PDLSCs was evaluated using the colorimetric Alkaline Phosphatase Assay Kit (Abcam) in accordance with the manufacturer’s protocol. Briefly, homogenized DPSCs or PDLSCs (1 × 10⁵ cells) and p-nitrophenyl phosphate (pNPP) were loaded into 96-well plates. The plates were incubated in the dark. This was followed by the addition of the stopping solution, and measurement at OD₄₀₅ nm using a spectrophotometer (Bio-Rad). The activity of ALP (U/L) was calculated. The experiment was performed twice in triplicate.

Statistical Analysis

IBM SPSS Statistics version 26.0 (SPSS IBM, Armonk, USA) was used to conduct the statistical analyses. The Shapiro–Wilk test was used as a normality test. Comparison of NF-κB and ALP activities of DPSCs and PDLSCs in different treatment groups was accomplished using two-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD). p-values < 0.05 were considered statistically significant.

Results

Phenotypic Characterization of DPSCs and PDLSCs

High expression of CD90, CD105, and CD73 (>95%) was exhibited by DPSCs and PDLSCs, whereas expression of negative markers were < 2% (Figures 1 and 2). The characteristics of these surface biomarkers matched the standard criteria defining MSCs proposed by the ISCT, suggesting that the cultured DPSCs and PDLSCs had the properties of MSCs.

LPS Inhibited Osteogenic Differentiation of DPSCs and PDLSCs

Bone nodules, in the form of alizarin positive-red mineralized deposits, were observed in DPSCs on the third-week culture and in PDLSCs on the second-week culture under an inverted light microscope. No bone nodules were observed in 10 µg/mL LPS-supplemented DPSCs and PDLSCs after 1, 2, and 3 weeks (Figure 3).

LPS-Induced NF-κB Activity in DPSCs and PDLSCs

NF-κB activities of untreated DPSCs and PDLSCs were 0.236 ± 0.005 AU and 0.253 ± 0.008 AU, respectively. Following three weeks of LPS supplementation, NF-κB activities of DPSCs and PDLSCs were 0.580 ± 0.029 AU and 0.667 ± 0.051 AU. NF-κB activities of LPS-supplemented DPSCs and PDLSCs following pretreatment with Bay 11-7082 were 0.349 ± 0.037 and 0.420 ± 0.022 AU (Figure 4).

No significant interaction between the types of stem cells and treatments on NF-κB activity was indicated by two-way ANOVA (p = 0.148). NF-κB activity significantly differed in different treatment groups (p = 0.000). The 3-week-LPS-supplemented NF-κB activities of DPSCs and PDLSCs were significantly higher than those of untreated DPSCs and PDLSCs (p = 0.000) as well as those of Bay 11-7082-pretreated LPS-supplemented DPSCs and PDLSCs (p = 0.000). The NF-κB activities of untreated DPSCs and PDLSCs were significantly lower than those of Bay 11-7082-pretreated LPS-supplemented DPSCs and PDLSCs (p = 0.000). These results demonstrated that LPS-induced NF-κB activation in DPSCs and PDLSCs, and that Bay 11-7082 partially inhibited the LPS-induced NF-κB pathway.

LPS Reduced ALP Activity and Inhibited Bone Nodule Formation in DPSCs and PDLSCs

Two-way ANOVA did not indicate a significant interaction between stem cells and treatments on ALP activity (p = 0.148). Significant differences in ALP activity were observed in different treatment groups (p = 0.000). ALP activities of untreated DPSCs and PDLSCs were 60.893 ± 6.516 U/mL and 70.637 ± 4.902 U/mL, respectively. The ALP activities of DPSCs (5.333 ± 0.323 U/mL) and PDLSCs (6.277 ± 2.026 U/mL) were significantly lower than those of untreated DPSCs and PDLSCs after three weeks of LPS supplementation (p = 0.000) (Figure 5). Lower ALP activity was associated with the absence of bone nodule formation in LPS-supplemented DPSCs and PDLSCs (Figure 6). Pretreatment with Bay 11-7082 resulted in significantly higher ALP activities of LPS-supplemented DPSCs (44.677 ± 5.193 U/mL) and PDLSCs (55.530 ± 4.478 U/mL) compared with those supplemented with LPS (p = 0.000), but significantly lower than those of untreated (p = 0.000). These results showed that Bay 11-7082 was responsible for the partial maintenance of ALP activity in DPSCs and PDLSCs (Figure 5). Moreover, pretreatment with Bay 11-7082 partially maintained the osteogenic potency of LPS-supplemented DPSCs and PDLSCs (Figure 6).

Discussion

LPS-induced NF-κB activation, was reported to play an important role in inflammatory responses and bone loss in periodontitis.¹²12. Li C, Li B, Dong Z, Gao L, He X, Liao L, et al. Lipopolysaccharide differentially affects the osteogenic differentiation of periodontal ligament stem cells and bone marrow mesenchymal stem cells through Toll-like receptor 4 mediated nuclear factor ?B pathway. Stem Cell Res Ther. 2014 May;5(3):67. https://doi.org/10.1186/scrt456
https://doi.org/10.1186/scrt456... This study demonstrated that P. gingivalis-derived LPS not only induced NF-κB activity but also inhibited bone nodule formation in DPSCs and PDLSCs. These findings are consistent with a previously conducted study that demonstrated that LPS-induced NF-κB activity impaired the osteogenic potency of gingival-derived mesenchymal stem cells (GMSCs).¹⁹19. Zhao Y, Cai B, Zhu W, Shi J, Wang Y, Si M. IL-1 receptor antagonist protects the osteogenesis capability of gingival-derived stem/progenitor cells under inflammatory microenvironment induced by porphyromonas gingivalis lipopolysaccharides. Stem Cells Int. 2021 Jan;2021:6638575. https://doi.org/10.1155/2021/6638575
https://doi.org/10.1155/2021/6638575... LPS supplementation also inhibited osteogenic differentiation in dental follicle stem cells (DFSCs).²⁰20. Um S, Lee JH, Seo BM. TGF-ß2 downregulates osteogenesis under inflammatory conditions in dental follicle stem cells. Int J Oral Sci. 2018 Oct;10(3):29. https://doi.org/10.1038/s41368-018-0028-8
https://doi.org/10.1038/s41368-018-0028-...

The activated NF-κB targeted the κB site and inhibit Smad in regulating Runx2,²¹21. Novack DV. Role of NF-?B in the skeleton. Cell Res. 2011 Jan;21(1):169-82. https://doi.org/10.1038/cr.2010.159
https://doi.org/10.1038/cr.2010.159... thereby inhibiting ALP production.²²22. Kim YJ, Lee MH, Wozney JM, Cho JY, Ryoo HM. Bone morphogenetic protein-2-induced alkaline phosphatase expression is stimulated by Dlx5 and repressed by Msx2. J Biol Chem. 2004 Dec;279(49):50773-80. https://doi.org/10.1074/jbc.M404145200
https://doi.org/10.1074/jbc.M404145200... In this study, bone nodule formation was clearly observed after 3 weeks of culturing with DPSCs and PDLSCs. In addition, ALP activity, which was observed in the 3-week culture, was reduced by LPS supplementation. Thus, NF-κB activity, which was induced by LPS, could reduce ALP activity in DPSCs and PDLSCs, leading to inhibition of bone nodule formation. This finding corroborates a previous study that revealed that LPS-induced NF-κB activity downregulated ALP mRNA and protein expressions in GMSCs.¹⁹19. Zhao Y, Cai B, Zhu W, Shi J, Wang Y, Si M. IL-1 receptor antagonist protects the osteogenesis capability of gingival-derived stem/progenitor cells under inflammatory microenvironment induced by porphyromonas gingivalis lipopolysaccharides. Stem Cells Int. 2021 Jan;2021:6638575. https://doi.org/10.1155/2021/6638575
https://doi.org/10.1155/2021/6638575... Furthermore, ALP activity was reported to be reduced by LPS in DFSCs.²⁰20. Um S, Lee JH, Seo BM. TGF-ß2 downregulates osteogenesis under inflammatory conditions in dental follicle stem cells. Int J Oral Sci. 2018 Oct;10(3):29. https://doi.org/10.1038/s41368-018-0028-8
https://doi.org/10.1038/s41368-018-0028-...

NF-κB signaling can be blocked by several substances and natural products,²³23. Sandra F, Kukita T, Tang QY, Iijima T. Caffeic acid inhibits NF?B activation of osteoclastogenesis signaling pathway. Indones Biomed J. 2011;3(3):216-22. https://doi.org/10.18585/inabj.v3i3.153
https://doi.org/10.18585/inabj.v3i3.153... ,²⁴24. Sandra F, Kukita T, Muta T, Iijima T. Caffeic acid inhibited receptor activator of nuclear factor ?B ligand (RANKL)-tumor necrosis factor (TNF) a-TNF receptor associated factor (TRAF) 6 induced osteoclastogenesis pathway. Indones Biomed J. 2013;5(3):173-8. https://doi.org/10.18585/inabj.v5i3.68
https://doi.org/10.18585/inabj.v5i3.68... one of which is Bay 11-7082, which inhibits NF-κB activity in various types of stem cells, including BMMSCs,²⁵25. Huang RL, Yuan Y, Zou GM, Liu G, Tu J, Li Q. LPS-stimulated inflammatory environment inhibits BMP-2-induced osteoblastic differentiation through crosstalk between TLR4/MyD88/NF-?B and BMP/Smad signaling. Stem Cells Dev. 2014 Feb;23(3):277-89. https://doi.org/10.1089/scd.2013.0345
https://doi.org/10.1089/scd.2013.0345... ,²⁶26. Zhang P, Liu Y, Jin C, Zhang M, Tang F, Zhou Y. Histone acetyltransferase GCN5 regulates osteogenic differentiation of mesenchymal stem cells by inhibiting NF-?B. J Bone Miner Res. 2016 Feb;31(2):391-402. https://doi.org/10.1002/jbmr.2704
https://doi.org/10.1002/jbmr.2704... AdMSCs,²⁶26. Zhang P, Liu Y, Jin C, Zhang M, Tang F, Zhou Y. Histone acetyltransferase GCN5 regulates osteogenic differentiation of mesenchymal stem cells by inhibiting NF-?B. J Bone Miner Res. 2016 Feb;31(2):391-402. https://doi.org/10.1002/jbmr.2704
https://doi.org/10.1002/jbmr.2704... and neural stem cells (NSCs).²⁷27. Xie Z, Han P, Cui Z, Wang B, Zhong Z, Sun Y, et al. Pretreatment of mouse neural stem cells with carbon monoxide-releasing molecule-2 interferes with NF-?B p65 signaling and suppresses iron overload-induced apoptosis. Cell Mol Neurobiol. 2016 Nov;36(8):1343-51. https://doi.org/10.1007/s10571-016-0333-8
https://doi.org/10.1007/s10571-016-0333-... This study highlighted the role of Bay 11-7082 and its mechanism in maintaining osteogenic differentiation in LPS-stimulated DPSCs and PDLSCs. Bay 11-7082 supplementation led to the suppression of NF-κB activity, which was partially responsible for maintaining ALP activity and osteogenic potency in DPSCs and PDLSCs.

LPS could induce an inflammatory signaling pathway via NF-κB and other molecules, such as AP-1.²⁸28. Yang Y, Ren D, Zhao D, Zhang B, Ye R. MicroRNA-203 mediates Porphyromonas gingivalis LPS-induced inflammation and differentiation of periodontal ligament cells. Oral Dis. 2023 May;29(4):1715-25. https://doi.org/10.1111/odi.14132
https://doi.org/10.1111/odi.14132... Therefore, Bay 11-7082 was only able to partially suppress the inflammatory signaling pathway via NF-κB; however, AP-1 could still inhibit the osteogenic differentiation of DPSCs and PDLSCs. Consequently, further investigation of other inhibitors is necessary to enable complete suppression of the LPS-induced inflammatory signaling pathway, so that osteogenic differentiation of DPSCs and PDLSCs could be undisrupted.

Conclusion

Inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

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