Fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated periodontal ligament stem cells by regulating the bone morphogenetic protein 2/Smad pathway

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Fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated periodontal ligament stem cells by regulating the bone morphogenetic protein 2/Smad pathway Article in Press: Accepted Manuscript Zongyun Fu , Xusheng Wang , Bin Li and Yanchi Tang Archives of Oral Biology, Article 104927, Copyright © 2020 Highlights Fraxinellone inhibits inflammation of LPS-induced PDLSCs. Fraxinellone promotes differentiation of LPS-stimulated PDLSCs. Fraxinellone regulates BMP2/Smad pathway in LPS-stimulated PDLSCs. Abstract Objective The aim of the present study was to investigate the role of fraxinellone in periodontitis and identify its potential mechanisms. Design Lipopolysaccharide-induced periodontal ligament stem cells (PDLSCs) was employed to simulate the periodontitis in vitro . The levels of inflammatory factors were evaluated. After treatment with fraxinellone, alkaline phosphatase activity was determined. Additionally, calcium nodules staining was evaluated by alizarin red staining and the expression of osteogenesis differentiation-associated proteins was detected using western blot analysis. Moreover, the levels of proteins in bone morphogenetic protein 2 (BMP2)/Smad pathway were measured. Subsequently, BMP2 was silenced by transfection with small hairpin RNA to explore the underlying mechanisms of fraxinellone in lipopolysaccharide-induced PDLSCs. Results Lipopolysaccharide stimulation significantly upregulated the levels of inflammatory factors, which were inhibited by fraxinellone intervention. Moreover, fraxinellone notably promoted osteogenic differentiation and calcification shown by increasing levels of alkaline phosphatase, calcification and osteogenic marker proteins. Furthermore, the expression of BMP2, phosphorylated Smad1 and phosphorylated Smad5 was remarkably upregulated when fraxinellone exposure in lipopolysaccharide-induced PDLSCs. What’s more, BMP2 silencing dramatically restored the effects of fraxinellone on inflammation and osteogenic differentiation of PDLSCs stimulated by lipopolysaccharide. Conclusion These data demonstrated that fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs by regulating the BMP2/Smad pathway, providing experimental supports for the clinical application of fraxinellone in the treatment of periodontitis. 1 Introduction Periodontitis, a widespread infectious human chronic inflammatory disease, is caused by progressive destruction of tissues that surround and support the tooth (Y. ). It has been reported that serious periodontitis infects 9% to 11% of adult population global and the prevalence increases with age, especially in adult population over 50 years of age ( ; ). Existing study has shown that periodontitis is closely implicated in the systemic conditions such as obesity, diabetes, adverse pregnancy outcomes and osteoporosis (J. ). Although great progress has been observed in relation to medical therapies available for patients with periodontitis, there is still no effective treatment for this disease ( ; ). Therefore, it is of clinical importance that novel therapeutic agents associated with periodontal disease are identified for improving oral health and averting systemic diseases. Periodontal ligament stem cells (PDLSCs), a type of mesenchymal stem cell possessing a high proliferative, self-renewal and multi-differentiation abilities, are the predominant functional cells which are widely known in the regeneration and repair of periodontium. PDLSCs can differentiate into osteoblasts, adipocytes and cementoblasts, which is vital for physiological healing of alveolar bone ( ). A growing body of literature has shown that periodontitis suppresses the differentiation potential of periodontal PDLSCs, and treatment using PDLSCs can restore the damaged or diseased periodontal tissues ( ; ). PDLSCs are considered as an ideal cell source for periodontal tissue regeneration and repair ( ; ). Additionally, it is noteworthy that elevated inflammation levels promote the progression of periodontitis ( ). Accumulating evidence shows that endotoxins produced by periodontal pathogens destroys microenvironment homeostasis and disrupts periodontal tissue via inhibition of PDLSCs viability ( ). Lipopolysaccharide, a bacterial endotoxin, is strongly involved in the initiation and development of a host response caused by infection with gram-negative bacteria. Lipopolysaccharide has been widely used for the construction of experimental models of periodontitis. Therefore, it is crucial to control the inflammatory damage of PDLSCs for the restoration of tissue and to inhibit the progression of periodontitis. Fraxinellone is one of the main active ingredients of the root bark of Dictamnus dasycarpus which is a traditional herb used to treat inflammatory conditions. Fraxinellone has a potential therapeutic effect in inflammatory diseases. For instance, report has demonstrated previously that fraxinellone suppresses inflammatory responses during acute pancreatitis through suppressing activation of inflammasome ( ). Fraxinellone relieves synovial inflammation and osteoclastogenesis in mice with rheumatoid arthritis ( ). However, whether fraxinellone can affect inflammation and osteogenic differentiation of lipopolysaccharide-induced PDLSCs and the potential regulatory mechanisms remains to be elucidated. 2 Materials and methods 2.1 Cell culture and treatment Human PDLSCs were obtained from Shanghai Chuntest biotechnology co. LTD (Shanghai, China). Cells were grown in Dulbecco’s modifed Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37 °C in a 5% CO 2 humidified atmosphere. Cells were incubated with or without 0.1, 1 and 10 μg/mL lipopolysaccharide (Sigma-Aldrich; Merck KGaA) for 12, 24 and 48 h. The biological source of lipopolysaccharide was porphyromonas gingivalis and purified by phenol extraction. To evaluate the effects of fraxinellone on lipopolysaccharide-induced human PDLSCs, 0.1, 1, 10, 50 and 100 μM fraxinellone (Shanghai Tauto Biotech Co. Ltd; Shanghai, China) were employed to pretreat with cells for 1 h, respectively. 2.2 Cell transfection For transfection, short hairpin RNA (shRNA) targeting bone morphogenetic protein 2 (BMP2) (shRNA-BMP2-1 and shRNA-BMP2-2) or its negative control (shRNA-NC) were purchased from GenePharma (Shanghai, China). Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) was utilized for transfect 10 nM above-mentioned vectors into 4 × 10 5 PDLSCs according to standard protocol. At 48 h after post-transfection, successful transfection was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and western blot analysis, respectively. 2.3 Cell viability assay A cell counting kit-8 kit (Shanghai Yi Sheng Biotechnology Co. Ltd., China) was adopted for detect the cell viability after appropriate treatment. Briefly, PDLSCs were seeded in a 96-well plate (5 × 10 3 cells/well). After treatment with lipopolysaccharide or fraxinellone, ten microliters of cell counting kit-8 solution was added to each well, followed by incubation for another 4 h at 37 °C according to standard protocol. The optical density values were detected at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). 2.4 Detection of inflammatory cytokines Protein levels of inflammatory cytokine including tumor necrosis factor-α, interleukin-6 and interleukin-1β in PDLSCs culture supernatants were determined using corresponding enzyme-linked immunosorbent assay (ELISA) kits (WesttangBio-tech, Shanghai, China) in accordance with the manufacturer's guidelines. 2.5 Alkaline phosphatase activity assay Alkaline phosphatase activity was evaluated by an alkaline phosphatase activity kit according to standard protocol (Beyotime, Shanghai, China). Alkaline phosphatase activity was detected on day 14 after osteogenic induction. The absorbance was quantified at 405 nm. 2.6 Alizarin red staining PDLSCs were seeded in 6-well plates (1.5 × 10 5 cells per well) and osteogenesis was examined at 21 days in osteogenic medium with or without lipopolysaccharide or fraxinellone. PDLSCs were fixed in 4% paraformaldehyde for 40 min and subsequently washed with deionized water. 1% alizarin red staining solution (pH4.2, Beijing Solarbio Science & Technology Co., Ltd., China) was employed to stain cells for 15 min. After washing with deionized water three times, the osteogenic differentiation of PDLSCs was detected using an inverted microscope (Leica DM IRM) and the images was captured. 2.7 RT-qPCR analysis Total RNA from PDLSCs cells prepared by using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA). Subsequently, the complementary DNA was synthesized by using a Reverse Transcription kit (Takara, Japan) following manufacturer's recommendation. RT-qPCR was performed using an ABI 7500 Thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc.). All primers used in the present study were synthesized by Sangon Biotech. The 2 -ΔΔCq method was applied to analyze the relative expression of targets genes(15). Glyceraldehyde-phosphate dehydrogenase (GAPDH) was served as an internal control. 2.8 Western blot analysis for protein expression Total proteins were collected using RIPA lysis buffer (Beyotime, Shanghai, China). A bicinchoninic acid kit was applied to determine the concentration of proteins. Equal amounts of protein extracts in lysis buffer were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel transfer to polyvinylidene fluoride membrane, these blots were blocked with 5% skimmed milk and incubated with primary antibodies subsequently. The specimens were then treated with horse-radish peroxidase -conjugated secondary antibody (A0216, Beyotime, Shanghai, China). The levels of protein expression were quantified using an Odyssey infrared imaging system (Lincoln, NE). ImageJ software was employed to evaluate the band density. GAPDH was used as reference protein. 2.9 Statistical analysis Results were reported as means ± standard deviation. All statistical analyses were performed using GraphPad Prism v.5 (GraphPad Software). Quantitative data were compared using Student's t-test between two groups, and the comparisons among multiple groups were performed using analysis of variance followed by Turkey's post hoc test. A P value<0.05 was considered significant. 3 Results 3.1 Lipopolysaccharide stimulation induces the increase in the levels of inflammatory factors in PDLSCs PDLSCs was stimulated by a series of concentrations of lipopolysaccharide for up to 48 h to stimulate the microenvironment of periodontitis in PDLSCs in vitro . As presented in Fig. 1 A, there were no significant effects in response to lipopolysaccharide challenge with 1 µg/ml relative to the control group at all time points. However, a remarkably decrease in cell viability was observed in PDLSCs treated with 20 µg/ml for 24 h compared with the control group. What’s more, 10 and 20 µg/ml lipopolysaccharide exposure for 48 h exerted a notably inhibitory effect on cell viability. Moreover, lipopolysaccharide stimulation (10 and 20 µg/ml) led to significantly upregulated the levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β in PDLSCs ( Fig. 1 B-E ). And 10 μg/ml lipopolysaccharide exposure for 24 h was adopted to mimic the microenvironment of periodontitis in PDLSCs in the following experiments. Fig. 1 Lipopolysaccharide (LPS) stimulation induced the increase in the levels of inflammatory factors in PDLSCs. (A) Cell viability was detected using a cell counting kit-8 kit after induction with a series of concentrations of LPS. (B) The expression of tumor necrosis factor-α (TNF-α) was measured using RT-qPCR after induction with a series of concentrations of LPS. The levels of (C) TNF-α, (D) interleukin-6 (IL-6) and (E) interleukin-1β (IL-1β) were determined using ELISA kits, respectively, after PDLSCs being treated with different doses of LPS. All experiments were repeated three times independently (N = 3). Results were reported as means ± standard deviation. Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. 3.2 Fraxinellone treatment significantly inhibits inflammatory responses in lipopolysaccharide-induced PDLSCs The effect of fraxinellone on viability of PDLSCs was detected using a cell counting kit-8 kit. Statistical evaluation showed that there are no significant differences on cell viability in 0.1, 1, 10 and 50 μM fraxinellone-exposed groups compared with the control group ( Fig. 2 A). Notably reduced viability of PDLSCs was noticed after being treated with 100 μM fraxinellone. Therefore, 50 μM fraxinellone was employed to perform the further experiments. Additionally, the effects of fraxinellone on inflammatory cytokines induced by lipopolysaccharide were determined. Results of Fig. 2 B-D displayed that fraxinellone pretreatment markedly inhibited the concentration of tumor necrosis factor-α, interleukin-6 and interleukin-1β as compared with the lipopolysaccharide group. Together, the above-mentioned data provide evidence that fraxinellone can suppress lipopolysaccharide-induced inflammation in PDLSCs. Fig. 2 Fraxinellone (Frx) treatment significantly inhibited inflammatory responses in lipopolysaccharide (LPS)-induced PDLSCs. (A) Cell viability was examined using a cell counting kit-8 kit after cell being treatment with different doses of Frx. Results were represented as the means ± standard deviation from three independent experiments (N = 3). Statistical significance: *P < 0.05 vs. control. ELISA kits were used to test the levels of (B) tumor necrosis factor-α (TNF-α), (C) interleukin-6 (IL-6) and (D) interleukin-1β (IL-1β) after Frx intervention in LPS-induced PDLSCs. The experiments were generated from three independent repeats (N = 3). Results were presented as means ± standard deviation. Statistical significance: ***P < 0.001 vs. control; ## P < 0.01, ### P < 0.001 vs. LPS. 3.3 Fraxinellone promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs To investigate the effects of fraxinellone on osteogenic differentiation, firstly, the relative activity of alkaline phosphatase, a ubiquitous cellular protein and bone formation marker that is correlated with early osteogenic differentiation was determined by a commercial available kit. As is observable from Fig. 3 A, lipopolysaccharide stimulation significantly reduced the alkaline phosphatase activity relative to the control group, which was reversed after treatment with fraxinellone. We then evaluated the level of calcification, which is phenotypic marker of osteogenic differentiation. Result of alizarin red staining exhibited in Fig. 3 B revealed that fraxinellone treatment remarkably elevated the ability of mineralization in PDLSCs relative to the lipopolysaccharide group. Concurrently, the expression of osteogenic differentiation-associated proteins was assessed using western blot analysis. Runt-related transcription factor 2 (Runx2) is an important gene for osteogenic differentiation and the function of osteoblast, which can regulate the expression of osteocalcin, osteopontin and collagen I ( ). As presented in Fig. 3 C, lipopolysaccharide induction resulted in significant decrease in the expression of alkaline phosphatase, Runx2, osteocalcin, bone sialoprotein, osteopontin and collagen I, whereas fraxinellone treatment restored the inhibitory effects of lipopolysaccharide on expression of above-mentioned proteins. These findings confirm that fraxinellone promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs. Fig. 3 Fraxinellone (Frx) promoted osteogenic differentiation in lipopolysaccharide (LPS)-stimulated PDLSCs. (A) The activity of alkaline phosphatase (ALP) was evaluated using an ALP activity kit. (B) Alizarin red staining was employed to measure the level of calcification. (C) The expression of osteogenic differentiation-related proteins including ALP, osteocalcin (OCN), runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and osteopontin (OPN) was examined using western blot analysis. Values were expressed as mean ± standard deviation (N = 3). ***P < 0.001 vs. control; ## P < 0.01, ### P < 0.001 vs. LPS. 3.4 Fraxinellone treatment activates the BMP2/Smad signaling in lipopolysaccharide-stimulated PDLSCs To study the potential regulatory mechanisms of fraxinellone in inflammation and osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs, the expression of key proteins in BMP2/Smad signaling, a crucial pathway involved in the process of osteogenic differentiation, was detected using western blot analysis. It was found that lipopolysaccharide stimulation conspicuously downregulated the levels of BMP2, phosphorylated Smad1 and phosphorylated Smad5 ( Fig. 4 ). By contrast, the decline of BMP2, phosphorylated Smad1 and phosphorylated Smad5 activities resulted from lipopolysaccharide induction was rescued when fraxinellone treatment. These results implicate that fraxinellone activates the BMP2/Smad signaling in lipopolysaccharide-stimulated PDLSCs. Fig. 4 Fraxinellone (Frx) treatment activated the BMP2/Smad signaling in lipopolysaccharide (LPS)-stimulated PDLSCs. The expression of BMP2, phosphorylated Smad1 (p-Smad1), total Smad1 (t-Smad1), phosphorylated Smad5 (p-Smad5) and total Smad5 (t-Smad5) was detected using western blot analysis. Each experiment was repeated three times independently (N = 3). Data were presented as means ± standard deviation. Statistical significance: **P < 0.01, ***P < 0.001 vs. control; # P < 0.05, ### P < 0.001 vs. LPS. 3.5 BMP2 silencing reverses the effects of fraxinellone on activation of BMP2/Smad signaling in lipopolysaccharide-stimulated PDLSCs To further analysis the regulatory effects of fraxinellone on BMP2/Smad signaling, BMP2 was silenced by transfected with shRNA-BMP2-1 or shRNA-BMP2-2, respectively. As displayed in Fig. 5 A and B , BMP2 level was markedly decreased after transfection compared with the shRNA-NC group. And PDLSCs transfected with shRNA-BMP-2 presented the lower expression of BMP2, therefore it was selected for perform the following experiments. Subsequently, western blot analysis was utilized to evaluate the expression of key proteins in BMP2/Smad signaling. As it is displayed in Fig. 5 C, BMP2-downregulation conspicuously restored the activated effects of fraxinellone on the expression of BMP2, phosphorylated Smad1 and phosphorylated Smad5. These observations reveal that silencing of BMP2 could reverse the effects of fraxinellone on activation of BMP2/Smad signaling in lipopolysaccharide-stimulated PDLSCs. Fig. 5 BMP2 silencing reversed the effects of fraxinellone (Frx) on activation of BMP2/Smad signaling in lipopolysaccharide (LPS)-stimulated PDLSCs. (A) RT-qPCR and (B) western blot analysis were used to assess the expression of BMP2 after transfection. The experiments were generated from three independent repeats (N = 3). Results were exhibited as means ± standard deviation. Statistical significance: **P < 0.01, ***P < 0.001 vs. shRNA-NC. (C) The expression of BMP2, phosphorylated Smad1 (p-Smad1), total Smad1 (t-Smad1), phosphorylated Smad5 (p-Smad5) and total Smad5 (t-Smad5) was determined using western blot analysis after BMP2 silencing. All experiments were repeated three times independently (N = 3). Data were presented as means ± standard deviation. Statistical significance: ***P < 0.001 vs. control; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. LPS; △ P < 0.05, △△ P < 0.01 vs. LPS +50 μM Frx + shRNA-NC. 3.6 BMP2-downregulation restores the effects of fraxinellone on inflammation and osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs Subsequently, the levels of inflammation and osteogenic differentiation after BMP2 silencing were evaluated to clarify the regulatory mechanisms underlying fraxinellone functioned in lipopolysaccharide-stimulated PDLSCs. As exhibited in Fig. 6 A-C , BMP2 silencing significantly elevated the levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β relative to the lipopolysaccharide+50 μM fraxinellone + shRNA-NC group. Additionally, the level of alkaline phosphatase activity and mineralization ability in PDLSCs was remarkably decreased when cells transfection with shRNA-BMP2-1, accompanied by downregulated expression of alkaline phosphatase, osteocalcin, Runx2, bone sialoprotein, osteopontin and collagen I ( Fig. 6 D-F ). Through the above findings we prove that fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs by regulating the BMP2/Smad pathway. Fig. 6 BMP2-downregulation restored the effects of fraxinellone (Frx) on inflammation and osteogenic differentiation in lipopolysaccharide (LPS)-stimulated PDLSCs. The levels of (A) tumor necrosis factor-α (TNF-α), (B) interleukin-6 (IL-6) and (C) interleukin-1β (IL-1β) were detected using ELISA kits. (D) An alkaline phosphatase (ALP) activity kit was adopted for measuring the activity of ALP. (E) Alizarin red staining was used to test the level of calcification. (F) The expression of osteogenic differentiation-related proteins including ALP, osteocalcin (OCN), runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and osteopontin (OPN) was examined using western blot analysis. Results were represented as the means ± standard deviation from three independent experiments (N = 3). Statistical significance: ***P < 0.001 vs. control; ## P < 0.01, ### P < 0.001 vs. LPS; △ P < 0.05, △△ P < 0.01 vs. LPS +50 μM Frx + shRNA-NC. 4 Discussion Periodontitis is considered as a complex progressive inflammatory disease that affects the tooth-supporting tissues. It is more prevalent in adults, especially in adult population over 50 years of age. PDLSCs were recently used to regenerate lost tooth-supporting apparatus because of its features of high-proliferation, self-renewal and multi-differentiation abilities. Therefore, identification of a novel agent that can protect PDLSCs from inflammation-related injury is crucial. The present study used lipopolysaccharide as the inflammation stimulator for the construction of the cell injury model. Lipopolysaccharide, a major stimulator of inflammation, rapidly promotes the generation of pro-inflammatory cytokines or inflammatory mediators. It has been reported that the inflammatory microenvironments result in the resorption of alveolar bone and the destruction of the other periodontium, which takes an important part in the pathogenesis of periodontal disease ( ). Existing studies have shown that lipopolysaccharide stimulation destroys microenvironment homeostasis and disrupts periodontal tissue via promoting pro-inflammatory cytokine production and suppressing osteoblastic differentiation in human PDLSCs ( ; ). PDLSCs, a type of stem/progenitor cells, possesses highly efficient repair capability in alveolar bone regeneration ( ). Therefore, medicament which could inhibit inflammation and promote osteogenic differentiation of PDLSCs may act as a promising candidate to restore the function of PDLSCs in periodontitis. Historically, the phytochemical and pharmacological activities of natural compounds from traditional medicines plays significant roles in the treatment of periodontitis ( ; ). fraxinellone is a traditional herb used to treat inflammatory conditions. Emerging evidence supports the notion that fraxinellone can block lipopolysaccharide-induced inflammatory responses in RAW 264.7 macrophages cells ( ). Previous studies have highlighted the importance of fraxinellone in therapeutic effect of animal models with inflammatory diseases ( ; ). What’s more, fraxinellone can attenuate synovial inflammation and osteoclastogenesis in mice ( ). The present study revealed that fraxinellone inhibited the levels of tumor necrosis factor-α, interleukin-6 and interleukin-1β induced by lipopolysaccharide in PDLSCs. Moreover, the ability of osteogenic differentiation was promoted when fraxinellone intervention. Collectively, these data suggested that fraxinellone can suppress inflammation and enhance osteogenic differentiation in lipopolysaccharide-induced PDLSCs, showing the potential of treating periodontitis. BMP2 is a key inducer of osteogenic differentiation. BMP2 signals have been reported to be mediated by activation of Smad family member upon ligand binding ( ). In the Smad-dependent pathway, BMP2 binds to heteromeric receptor complexes, promoting the phosphorylation of specific Smads, including Smad1 and Smad5 ( ). Significant upregulation of BMP2 expression was observed in the current study after fraxinellone treatment in lipopolysaccharide-stimulated PDLSCs. A growing body of literature has shown that BMP2/Smad signaling is closely implicated in regulation of osteogenic differentiation in human PDLSCs ( ; ). It has been well reported that myricetin promotes osteogenic differentiation of PDLSCs partly via BMP2/Smad signaling pathway ( ). Therefore, whether fraxinellone regulates BMP2/Smad signaling to inhibit inflammation and promote osteogenic differentiation became the focus of our research. This study revealed that fraxinellone markedly enhanced the expression of phosphorylated Smad1 and phosphorylated Smad5. By contrast, the effects of fraxinellone on inflammation and osteogenic differentiation were reversed after BMP2 silencing. These data provide evidence that fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs through activating the BMP2/Smad pathway. In summary, for the first time, we demonstrated that fraxinellone alleviates inflammation and promotes osteogenic differentiation in lipopolysaccharide-stimulated PDLSCs through activating the BMP2/Smad pathway. Our findings provides experimental supports for the clinical application of fraxinellone in the treatment of periodontitis. However, the lack of animal experiments is a limitation of the present study, which will be investigated in future research. Authors’ contributions All authors read and approval the final manuscript. CRediT authorship contribution statement Zongyun Fu: Conceptualization, Methodology, Investigation, Writing - review & editing. Xusheng Wang: Conceptualization, Methodology, Investigation, Formal analysis, Writing - review & editing. Bin Li: Methodology, Investigation, Formal analysis, Writing - original draft. Yanchi Tang: Methodology, Writing - original draft. Declaration of Competing Interest The authors reported no declarations of interest. Funding Not applicable. Appendix A Supplementary data The following are Supplementary data to this article: Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file Attached file References Bella P., Istvan G.: The comprehensive periodontal, resorative end prosthodontic therapy of chronic periodontitis Case presentation. Fogorv Sz 2016; 109: pp. 125-135. Bruderer M., Richards R.G., Alini M., Stoddart M.J.: Role and regulation of RUNX2 in osteogenesis. 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