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. Author manuscript; available in PMC: 2021 May 24.
Published in final edited form as: J Int Acad Periodontol. 2021 Jan 1;23(1):3–10.

TLR2 and TLR4 Differentially Regulate the Osteogenic Capacity of Human Periodontal Ligament Fibroblasts

Sujiwan Seubbuk 1,2,3, Rudee Surarit 4, Danielle Stephens 2, Hatice Hasturk 2, Thomas E Van Dyke 2, Alpdogan Kantarci 2
PMCID: PMC8142849  NIHMSID: NIHMS1698823  PMID: 33512337

Abstract

Aims:

To test that the osteogenic capacity of periodontal ligament (PDL) fibroblasts can be mediated by TLR2 and TLR4 activation.

Materials and Methods:

Human PDL fibroblasts were cultured in osteogenic medium and treated with TLR2 and TLR4 agonists (Pam3CSK4 and monophosphoryl Lipid A (MPLA), respectively). Cell proliferation was measured by MTT and BrdU incorporation. Osteogenic differentiation was measured by alkaline phosphatase (ALP) activity. Nodule formation was measured for osteoblast function. The expression of markers of potential signaling pathways (RUNX2, OCN, BSP and Osterix) was evaluated by quantitative PCR.

Results:

PDL fibroblasts grew at the same rate during the first 5 days in response to both Pam3CSK5 and MPLA. On day 7, cells cultured in the presence of Pam3CSK4 had a significantly higher rate of DNA replication, while cells in MPLA group had a significantly lower DNA replication rate (one-third) compared to the control (p<0.05). Pam3CSK4 induced significantly higher ALP activity and larger calcified nodules. TLR4 activation significantly reduced the expression of RUNX2 and osterix and enhanced OCN. Neither TLR2 nor TLR4 affected BSP expression.

Conclusion:

These data suggest that the activation of TLR2 and TLR4 differentially and perhaps antagonistically modulate osteogenesis by human PDL fibroblasts and have a direct role of TLR-mediated PDL function during periodontal regeneration as a potential target for therapeutics.

Keywords: Periodontal fibroblasts, toll-like receptor, osteogenesis

Introduction

Periodontitis is a chronic inflammatory disease induced by microorganisms (Van Dyke and van Winkelhoff, 2013). Periodontal bacteria such as Porphyromonas gingivalis (Mysak et al., 2014) stimulate the innate immune response through specific toll-like receptors (TLRs). TLRs expressed on immune cells initiate the primary response, play an important role in several cellular functions that are critical for the host defense, and induce secretion of inflammatory cytokines including IL-6, IL-8, IL-1β, and TNF-α (Van Dyke and van Winkelhoff, 2013). For example, the activation of macrophages by P. gingivalis induces TNF-α production through TLR2 and TLR4 (Papadopoulos et al., 2013). Signaling through TLR2 and TLR4 is differentially regulated by cytokine production at different stages of periodontal disease progression. TLR2 mediates TNF-α activation and IL-10 inhibition in response to P. gingivalis infection at a later stage and this process is associated with alveolar bone loss (Lin et al., 2014). TLR2 deficiency was further shown to prevent bone loss when TLR2-deficient mice were treated with P. gingivalis (Burns et al., 2006). While the role of TLR signaling in the innate immune response during periodontal disease initiation and progression is well established, it is not clear how TLRs expressed on other cells of the host regulate cell function. Studies have suggested that TLR3 and TLR4 impact the differentiation of bone marrow stem cells (BMSCs) to osteoblasts and it was demonstrated that other cells of the host that are responsible for structural integrity and repair also sense P. gingivalis and its components through different TLRs (Qi et al., 2014).

The periodontal ligament (PDL) is a connective tissue that links teeth and alveolar bone and supports the masticatory system (Beertsen et al., 1997). While their major function is to regenerate the damaged periodontal attachment, PDL fibroblasts can respond to bacterial stimuli during disease progression (Benatti et al., 2007). PDL contains multipotent stem cells that can differentiate into osteoblasts, chondrocytes, and adipocytes (Kim et al., 2012). Cells from human inflamed PDL tissues express mesenchymal stem cell markers and exhibit the capacity to form a mineralized matrix in vitro and in vivo (Liao et al., 2011; Park et al., 2011). Periodontal ligament fibroblasts express higher level of osteopontin, osteocalcin, BMP-2, and BMP-4 compared to gingival fibroblasts (Ivanovski et al., 2001).

Human PDL fibroblasts constitutively express TLR1, TLR2, TLR4 and TLR7 (Scheres et al., 2011). PDL fibroblasts from people with periodontitis express higher TLR1, TLR4, and TLR7 than those from healthy control subjects and also express higher TLR2 than gingival, skin, and lung fibroblasts (Scheres et al., 2011). Anti-TLR4 and anti-TLR-2 treatment of PDL fibroblasts blocks IL-8 production suggesting that PDL fibroblast-induced recruitment of phagocytes during periodontal inflammation may be actively regulated by the TLR signaling (Hatakeyama et al., 2003). It is not known, however, how TLR-induced PDL fibroblast function is associated with new bone formation independent of the activation of TLR by the bacterial sources. Therefore, the aim of this study was to test the hypothesis that TLR2 and TLR4 activation will mediate the osteogenic capacity of the PDL fibroblasts.

Materials and Methods

Cell Culture

Human periodontal ligament (PDL) fibroblasts were purchased from Lonza (Walkersville, MD, USA). Cells were cultured in low glucose Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies Corporation, Grand Island, NY, USA). The medium was supplemented with 10% fetal bovine serum (FBS; Hyclone UK Ltd., Cramlington, Northumberland, UK), and antibiotic/antimycotic solution containing 100 U/ml penicillin G, 100 μg/ml Streptomycin, 0.025 μg/ml Amphotericin B (Life Technologies Corporation, Grand Island, NY, USA). PDL fibroblasts were maintained in a humidified atmosphere at 5% CO2 at 37 °C. Cells were subcultured after reaching confluence with medium changed every 2–3 days. PDL fibroblasts from passages 5 to 9 were used.

To induce osteogenic differentiation, PDL fibroblasts were further cultured in osteogenic medium, which contained 50 μg/ml of ascorbic acid, 10 mM of β-glycerophosphate and 100 nM Dexamethasone (Sigma-Aldrich, St. Louis, MO, USA). Pam3CSK4 (1 μM; InvivoGen, San Diego, CA, USA) was used as a TLR2 ligand. To activate TLR4, monophosphoryl Lipid A (MPLA, 1 μM, InvivoGen, San Diego, CA, USA) was used. Osteogenic medium alone was used in the control group. Each experiment was performed in triplicate.

PDL fibroblast proliferation in response to TLR2 and TLR4 agonist

To test the proliferation and cell viability of PDL fibroblasts, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, which is a quantitative colorimetric used to estimate cell viability and cell proliferation, was performed. The assay is based on the metabolization of MTT (a yellow tetrazole) by mitochondrial succinate dehydrogenase in living cells to a purple formazan product. PDL fibroblasts were seeded in 96-well plates (Costar®, Corning Incorporated, Corning, NY, USA). Cell density was adjusted at 4×103 cells/well. After incubation for 24 hours, the medium was changed to osteogenic medium (see above). On days 1, 3, 5 and 7, cells were incubated with 100 μl of 0.5 mg/ ml MTT (Sigma-Aldrich, St. Louis, MO, USA) for 3 hours. The formazan product was dissolved in 50 μl of dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 10 minutes. The absorbance was measured at 540 nm.

To further assess cell proliferation, the BrdU incorporation assay was performed. PDL fibroblasts were seeded in 96-well plates (Costar®, Corning Incorporated, Corning, NY, USA) at a density of 4×103 cells/well at 5% CO2 at 37 °C. Cells were treated as above. DNA replication was detected using the BrdU Cell Proliferation Assay (Abcam®, Cambridge, MA, USA) on days 1, 3, 5 and 7. One day before measurement of cell proliferation, the BrdU reagent was added to each well for 24 hours. On days 1, 3, 5 and 7, the BrdU reagent was removed and 100 μl of fixing solution was added. Cells were incubated at room temperature for 30 minutes for permeabilization and washed 3 times with a wash buffer. 100 μl of pre-diluted BrdU detection antibody was incubated in each well at room temperature for 1 hour. 100 μl goat anti-mouse IgG was added and incubated at room temperature for additional 30 minutes. The cells were further washed 3 times. One hundred μl of the substrate was added; cells were further incubated in the dark for 30 minutes at room temperature and 100 μl stop solution was added. Absorbance was read at 450/550 nm on a Beckman visible light spectrophotometer.

Alkaline phosphatase activity in response to TLR2 and TLR4 activation

To measure the osteogenic activity of PDL fibroblasts, cells were cultured in an osteogenic medium and differentiated into an osteoblastic lineage for 7, 14, and 21 days. The osteogenic medium contained DMEM with 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone. Cells were then washed twice with 1× PBS and lysed with 200 μl of lysis buffer containing proteinase inhibitor in CelLytic M (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes at room temperature. Lysate was removed from each well by scraping and transferred into individual microtubes. The lysed cells were centrifuged at 13,000 × g at 4 °C for 3 minutes. The supernatant was collected and stored at −80 °C until subsequent use. Alkaline phosphatase (ALP) concentration was measured using a colorimetric assay (Abcam Inc., Cambridge, MA, USA). For standard wells, 1 mM of the p-nitrophenol phosphate (pNPP) and assay buffer was added. For sample wells, 40 μl of cell lysate was added followed by 40 μl of assay buffer. Ten μl of ALP was added into every standard wells; 50 μl of 5 mM pNPP was added into the sample wells as a substrate for the enzyme from cells. The reaction was stopped by addition of 20 μl of stop solution. Absorbance was measured at 405 nm.

Protein concentration was determined using Novagen BCA Protein Assay Kit (Novagen®, USA). In brief, 25 μl of each BSA standard or protein sample was pipetted into each well of a 96-well plate. 200 μl BCA working reagent (50 parts BCA solution and 1 part 4% Cupric Sulfate) was added to each well. The reaction was incubated at 37°C for 30 minutes. Absorbance was measured at 562 nm on a plate reader. The ALP activity was calculated as nM of p-nitrophenol/mg of protein/minute as described (Sabokbar, et al., 1994).

Nodule Formation by PDL fibroblasts after osteogenic induction

To detect the calcified nodule formation by PDL fibroblasts, cells were incubated with osteogenic medium for 28 days. All groups of induced cells were washed twice with 1× PBS. After fixing the cells with cold absolute methanol at room temperature for 10 minutes, calcified nodules were stained with 1% Alizarin Red S (pH 4.1–4.3) (Sigma-Aldrich, St. Louis, MO, USA). After 30 minutes, the excess dye was removed and plates rinsed with distilled deionized water. Nodules were observed under the light microscope (Virtanen and Isotupa, 1980).

Quantitative polymerase chain reaction (qPCR) for osteogenic gene expression

To evaluate the expression of genes, the PDL fibroblasts were induced with osteogenic medium for 3 days. Cells were washed with 1× PBS twice and kept in TRIzol® (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed into cDNA by High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). qPCR was performed using TaqMan® Fast master mix (Thermo Fisher Scientific, Carlsbad, CA, USA). The expression of target genes was analyzed using RUNX2, OCN, BSP and Osterix assay probes Hs01047973_m1, Hs01587814_g1, Hs00195432_m1 and Hs01866874_s1 (Thermo Fisher Scientific, Carlsbad, CA, USA). Comparative cycle threshold (CT) was further analyzed for gene expression. β-actin expression was used as an endogenous control.

Data analysis

The data were analyzed for the differences among groups using one-way ANOVA with Bonferroni’s correction for multiple comparisons. Statistical significance was determined at p<0.05.

Results

PDL fibroblast proliferation in response to TLR2 and TLR4 activation

When PDL fibroblasts were cultured in osteogenic medium, they grew at the same rate in response to both Pam3CSK5 and MPLA with no loss of viability demonstrating that TLR2 or TLR4 activation does not interfere with cell viability over the assay period (Figure 1A). BrdU results (Figure 1B) showed that the proliferation capacity of PDL fibroblasts in all groups declined by day 5. On day 7, the TLR2 agonist (Pam3CSK4) sustained a higher rate of DNA replication of PDL fibroblasts (2-fold) compared to the control (p<0.05), while the TLR4 agonist (MPLA) induced a significantly lower DNA replication rate (one-third) compared to the control (p<0.05) suggesting a differential proliferative response to different TLR agonist activation by the PDL fibroblasts.

Figure 1.

Figure 1.

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(A) MTT assay shows the relative number of PDL fibroblasts under various conditions and (B) BrdU assay shows the relative DNA replication of PDL fibroblasts when cultured in osteogenic medium alone, plus TLR2 agonist (Pam3CSK4) or plus TLR4 agonist (MPLA). *Statistically significant difference (p<0.05) when compared to control on the same day.

Osteogenic capacity of PDL fibroblasts in response to TLR2 and TLR4 activation

Next, we measured whether TLR2 and TLR4 activation was associated with the osteogenic capacity of the PDL fibroblasts. When the cells were cultured in osteogenic medium for 14 days, the TLR2 agonist significantly induced ALP activity by approximately 0.6 nM pNP/mg of protein/min when compared to the control and the TLR4 agonist (Figure 2A). In parallel, PDL fibroblasts cultured with TLR2 agonist produced larger calcified nodules than those in the control and TLR4 activation groups (Figure 2B). While there was no statistical significance among groups, TLR4 activation tended to induce a higher number of calcified nodules than TLR2 activation without affecting the size of the nodules (Figure 2C).

Figure 2.

Figure 2.

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(A) Alkaline phosphatase activity of PDL fibroblasts after culture in osteogenic medium alone, plus TLR2 agonist (Pam3CSK4) or plus TLR4 agonist (MPLA) for 14 days. (B) Calcified nodules and (C) the number of calcified nodules after PDL fibroblasts were cultured in osteogenic medium alone, plus TLR2 agonist (Pam3CSK4) or plus TLR4 agonist (MPLA) for 28 days. *Statistically significant difference (p<0.05) when compared to control.

Osteogenic gene expression by the PDL fibroblasts in response to TLR2 and TLR4 activation

We focused on the expression of the osteogenic genes in the PDL fibroblasts when the cells were treated with TLR2 and TLR4 agonists. Activation of TLR4 significantly reduced the expression of RUNX2 and Osterix (Figure 3). In contrast, the activation of TLR2 did not affect the levels of RUNX2 and Osterix expression. TLR4 activation significantly induced OCN expression (p<0.05), while TLR2 activation did not have an impact. BSP expression was not affected by either TLR2 or TLR4 activation (Figure 4).

Figure 3.

Figure 3.

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mRNA expression of RUNX2 (A) and Osterix (B) by qRT-PCR in PDL fibroblasts after they were cultured in osteogenic medium alone, plus TLR2 agonist (Pam3CSK4) or plus TLR4 agonist (MPLA) for 3 days. * Statistically significant difference (p<0.05) when compared to control.

Figure 4.

Figure 4.

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mRNA expression of OCN (A) and BSP (B) by qRT-PCR in PDL fibroblasts after they were cultured in osteogenic medium alone, plus TLR2 agonist (Pam3CSK4) or plus TLR4 agonist (MPLA) for 3 days. * Statistically significant difference (p<0.05) when compared to control.

Discussion

Toll-like receptors are expressed on various cells of the mammals and are involved in a wide array of functions during infection (Akira et al., 2001). A previous study demonstrated the expression of TLR2 and TLR4 on human PDL fibroblasts and showed that the TLRs were induced by P. gingivalis and its LPS leading to a stimulation of cytokine production (Sun et al., 2010). While TLR expression has been shown on non-immune cells of the periodontium such as the PDL fibroblasts, the impact of the TLRs on PDL function is not clear. Our work is the first study to evaluate a direct activation of TLR2 and TLR4 activation on the PDL fibroblast proliferation and osteogenic capacity. The data demonstrate that DNA replication and ALP activity of PDL fibroblasts was increased by TLR2 agonist together with induction of larger mineralized nodules. The results may also suggest that the TLR2-agonist-induced cell proliferation reach a particular density at which these cells are ready to differentiate but cannot proliferate further. Conversely, the expression of RUNX2 and Osterix was decreased by the TLR4 agonist while the TLR4 agonist significantly upregulated the expression of OCN.

RUNX2 is expressed on osteoblast lineage cells and hypertrophic chondrocytes, odontoblasts, and ameloblasts (Liu and Lee, 2013). During bone formation, RUNX2 is an important transcription factor for osteoblast differentiation, matrix production, and mineralization (Liu and Lee, 2013). It induces the differentiation of mesenchymal cells into osteoblast-like cells which direct the formation of osteoid material leading to bone formation (Komori, 2010). Osterix, the downstream gene of RUNX2, is an early marker when mesenchymal cells enter the osteoblast lineage, and its signal becomes stronger as osteoblast differentiation occurs (Tang et al., 2011). It has been shown that the TLR4 expression on MC3T3-E1 cells promoted by LPS suppressed the expression of ALP, OCN, and RUNX2 mRNA resulting in an inhibition of the osteoblast differentiation (Liu et al., 2016). The suppression of RUNX2 and osterix expression was demonstrated in osteoblasts by LPS (Bandow et al., 2010), which corresponded to our result.

In contrast to the RUNX2, OCN is expressed in mature osteoblasts. In the diaphysis at 1 week of age and the metaphysis at 4 weeks of age, osteoblasts, which expressed RUNX2 and osteopontin strongly, weakly expressed OCN. In the diaphysis at 4 weeks of age, RUNX2 and osteopontin were barely detectable in osteoblasts, but osteocalcin was strongly expressed in the osteoblasts (Maruyama et al., 2007). In addition, the study of Ducy et al. (1996) found the increase in bone formation without impairing bone resorption in osteocalcin-deficient mice suggesting OCN as a negative regulator of bone formation. These two studies, while performed on osteoblasts, support our results with TLR4 activation in that the upregulation of OCN with the reduction of RUNX2 affects the osteogenic differentiation of PDL fibroblasts. These data collectively suggest that PDL fibroblasts are actively involved in osteogenesis through TLR-mediated signaling.

When the actions of TLR2 and TLR4 agonists on PDL fibroblasts were compared, the TLR2 agonist showed a positive regulation of PDL fibroblast proliferation and osteogenesis, but the TLR4 agonist exhibited negative regulation. TLR4 may impact the upstream level of RUNX2 at an early stage of osteogenesis, which leads to less differentiating preosteoblasts/osteoblasts. In the case of TLR4, RUNX2 expression was reduced while OCN expression was increased without any impact on ALP. TLR4 activation did not block nodule formation. The data also suggested that TLR2 may impact at a later stage after the cells differentiate into osteoblasts possibly increasing their proliferation and ALP activity. Hence, bigger calcified nodules were seen in TLR2 activation. This difference in TLR 2 and TLR4 activation could be due to differential regulation of the PDL fibroblast function by the immune system of the host during microbial infection. When TLR4 was activated over TLR2, inflammation from pathogenic bacteria induced more damage of periodontal tissue and less osteogenesis (Chen et al., 2013). Our findings suggest a pathological involvement of the TLR-specific activation where a smaller size of calcified nodules was presented in response to TLR4 activation in line with these observations. Indeed, a more recent study in mice showed that the P. gingivalis-induced periodontal bone resorption is dependent on TLR4 (Lin et al., 2017) suggesting a differential role for TLR2 and TLR4 on periodontal ligament cells in periodontal diseases. This function is not limited to the PDL; epithelial cells from periodontitis patients express significantly higher TLR4 in health whereas those from health and periodontitis express equivalent level of TLR2 (AlQallaf et al., 2018) suggesting a more ubiquitous role for the TLRs. Collectively, our study was the first work suggesting a possible role of TLR2 and TLR4 expression on PDL fibroblasts and differential involvement in bone formation-a vital process in healing of periodontal defects. Further analyses are needed to validate the signaling pathways and post-receptor activation to elucidate their function in periodontal regeneration and in treatment of periodontitis.

Conclusion

The data collectively demonstrate that activation of TLR2 and TLR4 modulated osteogenesis by human periodontal ligament fibroblast differently at different stages of function.

Acknowledgement:

This work was supported by NIH/NIDCR grants DE020906, DE025020 and DE15566 and Mahidol Medical Scholars Program.

References

  1. Akira S, Takeda K and Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nature Immunology 2001; 2:675–680. [DOI] [PubMed] [Google Scholar]
  2. AlQallaf H, Hamada Y, Blanchard S, Shin D, Gregory R and Srinivasan M. Differential profiles of soluble and cellular toll like receptor (TLR)-2 and 4 in chronic periodontitis. PLoS One 2018; 13:e0200231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bandow K, Maeda A, Kakimoto K, et al. Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation. Biochemical and Biophysical Research Communications 2010; 402:755–761. [DOI] [PubMed] [Google Scholar]
  4. Beertsen W, McCulloch CA and Sodek J. The periodontal ligament: a unique, multifunctional connective tissue. Periodontology 2000 1997; 13:20–40. [DOI] [PubMed] [Google Scholar]
  5. Benatti BB, Silvério KG, Casati MZ, Sallum EA and Nociti FH Jr. Physiological features of periodontal regeneration and approaches for periodontal tissue engineering utilizing periodontal ligament cells. Journal of Bioscience and Bioengineering 2007; 103:1–6. [DOI] [PubMed] [Google Scholar]
  6. Burns E, Bachrach G, Shapira L and Nussbaum G. Cutting Edge: TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption. Journal of Immunology 2006; 177:8296–8300. [DOI] [PubMed] [Google Scholar]
  7. Chen X, Hu C, Wang G, et al. Nuclear factor-kappaB modulates osteogenesis of periodontal ligament stem cells through competition with beta-catenin signaling in inflammatory microenvironments. Cell Death and Disease 2013; 4:e510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ducy P, Desbois C, Boyce B, et al. Increased bone formation in osteocalcin-deficient mice. Nature 1996; 382:448–452. [DOI] [PubMed] [Google Scholar]
  9. Hatakeyama J, Tamai R, Sugiyama A, Akashi S, Sugawara S and Takada H. Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell-surface components through the CD14/Toll-like receptor system. Oral Microbiology and Immunology 2003; 18:14–23. [DOI] [PubMed] [Google Scholar]
  10. Ivanovski S, Li H, Haase HR and Bartold PM. Expression of bone associated macromolecules by gingival and periodontal ligament fibroblasts. Journal of Periodontal Research 2001; 36:131–141. [DOI] [PubMed] [Google Scholar]
  11. Kim SS, Kwon DW, Im I, et al. Differentiation and characteristics of undifferentiated mesenchymal stem cells originating from adult premolar periodontal ligaments. The Korean Journal of Orthodontics 2012; 42:307–317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Komori T Regulation of osteoblast differentiation by Runx2. Advances in Experimental Medicine and Biology 2010; 658:43–49. [DOI] [PubMed] [Google Scholar]
  13. Liao J, Al Shahrani M, Al-Habib M, Tanaka T and Huang GT. Cells isolated from inflamed periapical tissue express mesenchymal stem cell markers and are highly osteogenic. Journal of Endodontics 2011; 37:1217–1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lin J, Bi L, Yu X, et al. Porphyromonas gingivalis exacerbates ligature-induced, RANKL-dependent alveolar bone resorption via differential regulation of Toll-like receptor 2 (TLR2) and TLR4. Infection and Immunology 2014; 82:4127–4134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lin M, Hu Y, Wang Y, Kawai T, Wang Z and Han X. Different engagement of TLR2 and TLR4 in Porphyromonas gingivalis vs. ligature-induced periodontal bone loss. Brazilian Oral Reesarch 2017; 31:e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu TM and Lee EH. Transcriptional regulatory cascades in Runx2-dependent bone development. Tissue Engineering Part B: Reviews 2013; 19:254–263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu YH, Huang D, Li ZJ, et al. Toll-like receptor4-dependence of the lipopolysaccharide-mediated inhibition of osteoblast differentiation. Genetics and Molecular Research 2016; 15:1–11. [DOI] [PubMed] [Google Scholar]
  18. Maruyama Z, Yoshida CA, Furuichi T, et al. Runx2 determines bone maturity and turnover rate in postnatal bone development and is involved in bone loss in estrogen deficiency. Developmental Dynamics 2007; 236:1876–1890. [DOI] [PubMed] [Google Scholar]
  19. Mysak J, Podzimek S, Sommerova P, et al. Porphyromonas gingivalis: major periodontopathic pathogen overview. Journal of Immunology Research 2014; 2014:476068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Papadopoulos G, Weinberg EO, Massari P, et al. Macrophage-specific TLR2 signaling mediates pathogen-induced TNF-dependent inflammatory oral bone loss. Journal of Immunology 2013; 190:1148–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Park JC, Kim JM, Jung IH, et al. Isolation and characterization of human periodontal ligament (PDL) stem cells (PDLSCs) from the inflamed PDL tissue: in vitro and in vivo evaluations. Journal of Clinical Periodontology 2011; 38:721–731. [DOI] [PubMed] [Google Scholar]
  22. Qi C, Xiaofeng X and Xiaoguang W. Effects of Toll-Like Receptors 3 and 4 in the osteogenesis of stem cells. Stem Cells International 2014; 2014:917168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sabokbar A, Millett PJ, Myer B and Rushton N. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro. Journal of Bone and Mineral Research 1994; 27:57–67. [DOI] [PubMed] [Google Scholar]
  24. Scheres N, Laine ML, Sipos PM, et al. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis. Journal of Periodontal Research 2011; 46:407–416. [DOI] [PubMed] [Google Scholar]
  25. Sun Y, Shu R, Li CL and Zhang MZ. Gram-negative periodontal bacteria induce the activation of Toll-like receptors 2 and 4, and cytokine production in human periodontal ligament cells. Journal of Periodontology 2010; 81:1488–1496. [DOI] [PubMed] [Google Scholar]
  26. Tang W, Li Y, Osimiri L and Zhang C. Osteoblast-specific transcription factor Osterix (Osx) is an upstream regulator of Satb2 during bone formation. The Journal of Biological Chemistry 2011; 286:32995–33002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Dyke TE and van Winkelhoff AJ. Infection and inflammatory mechanisms. Journal of Clinical Periodontology 2013; 40:S1–S7. [DOI] [PubMed] [Google Scholar]
  28. Virtanen P and Isotupa K. Staining properties of alizarin red S for growing bone in vitro. Acta Anatomica 1980; 108:202–207. [DOI] [PubMed] [Google Scholar]

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