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. Author manuscript; available in PMC: 2023 Nov 1.
Published in final edited form as: J Endod. 2022 Sep 13;48(11):1378–1386.e2. doi: 10.1016/j.joen.2022.08.010

Differential effects of Escherichia coli versus Porphyromonas gingivalis-derived lipopolysaccharides on dental pulp stem cell differentiation in scaffold-free engineered tissues

Kristi Rothermund 1, Tia C Calabrese 1,2, Fatima N Syed-Picard 1,2,3,4
PMCID: PMC9764159  NIHMSID: NIHMS1835940  PMID: 36108879

Abstract

Introduction:

To leverage the therapeutic capabilities of dental pulp stem cells (DPSCs) for regenerative endodontic applications, a better understanding of their innate defense and reparative processes is needed. Lipopolysaccharide (LPS) is a major virulent factor of gram-negative bacteria and contributor to endodontic infections. We have developed three-dimensional scaffold-free DPSC tissues that self-organize into dentin-pulp organoids comprising a mineralized dentin-like tissue on the periphery and an unmineralized pulp-like core. In this study, scaffold-free DPSC constructs were utilized as controllable experimental models to study DPSC response to bacterial challenge.

Methods:

Scaffold-free constructs were engineered using DPSCs isolated from human third molars. To simulate bacterial exposure, DPSC constructs were exposed to either Porphyromonas gingivalis-derived LPS (P. gingivalis-LPS) or Escherichia coli-derived LPS (E. coli-LPS). The effects of LPS on DPSC differentiation, proliferation and apoptosis were evaluated.

Results:

Engineered tissues lacking LPS treatment self-organized into dentin-pulp organoids. LPS treatment did not negatively affect DPSC proliferation or apoptosis in the engineered tissues. Both E. coli-LPS and P. gingivalis-LPS inhibited the upregulation of RUNX2 mRNA expression and reduced the expression of the odontoblast-associated proteins (p<0.05) suggesting that LPS is inhibiting odontoblastic differentiation. However, only E. coli-LPS treatment significantly reduced mineral deposition in the DPSC (p<0.05) constructs indicating that E. coli-LPS, but not P. gingivalis-LPS, reduced functional differentiation of DPSCs and prevented DPSCs from self-organizing into dentin-pulp complex-like structure.

Conclusions:

This study establishes scaffold-free DPSC constructs as models of oral disease. Furthermore, it emphasizes the diversity of LPS derived from different bacterial species and highlights the necessity of utilizing LPS derived from clinically relevant bacteria in basic science investigations.

Keywords: Pulpitis model, organoid, odontoblast differentiation, stem cells, regeneration

Introduction

The dental pulp contains a population of stem/progenitor cells that function in homeostatic and reparative processes (1). A deeper fundamental understanding of dental pulp stem cell (DPSC) behavior under pathological conditions would lead to controllable regenerative management strategies. Gram-negative bacteria are prevalent in infected pulp tissue, and lipopolysaccharide (LPS), the endotoxic lipoprotein found in the outer cell wall of these bacterial species, are considered a major virulent factor of pulpitis (2, 3). Therefore, LPS has been implicated to stimulate defense and repair activities in DPSCs.

DPSC response to LPS has been the aim of several prior research reports (4–15). Interestingly, studies investigating the effects of LPS on odontogenic differentiation of DPSC have yielded contradictory results where some reported that LPS did not affect odontogenic differentiation of DPSCs (11, 14) while others showed that it promoted (9, 10) or inhibited (13, 15) differentiation, respectively. During natural repair processes, DPSCs are thought to migrate to sites of bacterial infiltration and differentiate towards an odontogenic phenotype to deposit reparative dentin (1). Understanding the effects of LPS on DPSC differentiation will provide insight on mechanisms driving reparative dentinogenesis.

The dental pulp is a dynamic three-dimensional (3D) tissue, however, prior research investigating the effects of LPS on DPSCs have predominately utilized traditional two-dimensional (2D) culture conditions (4–15). 2D cell culture assays lack cell-cell or cell-extracellular matrix (ECM) interactions that are essential for recapitulating natural cell behavior. Our research group has previously established and characterized scaffold-free 3D tissue constructs formed by DPSCs (16, 17). These engineered tissues are generated and organized completely by the DPSCs. Furthermore, in these cultures, the DPSCs self-organize into organoids resembling dentin-pulp complexes (16, 17) comprising a central soft central tissue enclosed within an outer mineralized structure. This type of complex tissue patterning cannot be attained in 2D culture systems; in 2D culture systems, DPSCs produce mineralized nodules in a seemingly sporadic manner. Relative to 2D cultures, these engineered 3D tissues more closely recapitulate natural cellular dynamics and mineral tissue patterning of natural structures and, therefore, serve as relevant experimental models to study DPSC behavior.

In this study, the effects of LPS on DPSCs was investigated using scaffold-free 3D engineered tissues. It has been widely established that LPS structure varies across bacterial species (18), and there have been inconsistencies in the source of LPS utilized in previously published work investigating the effects of this endotoxin on DPSCs (6). Although it is not a prevalent pathogen of pulpitis, the majority of in vitro research investigating the effects of LPS on the odontogenic differentiation of DPSCs utilize Escherichia coli-derived LPS (E. Coli) (5, 9, 10, 12, 14) while others have utilized more relevant LPS sources like Porphyromonas gingivalis (P. gingivalis), which is a major oral pathogen associated with periodontal disease and is present in approximately 50% of primary endodontic infections (2, 19). Here, the effects of P. gingivalis and E. coli-derived LPS on DPSC behavior are directly compared in order to highlight the differential effects of the two LPSs on DPSCs and emphasize the need to utilize LPS derived from clinically relevant bacterial species. The effects of these respective LPSs on DPSC proliferation, apoptosis, and odontogenic differentiation in 3D scaffold-free tissues were evaluated.

Materials and Methods

All chemicals were acquired from Sigma Aldrich (St. Louis, MO) unless otherwise noted.

DPSC isolation and culture

Human teeth were extracted at the University of Pittsburgh, School of Dental Medicine. The teeth were cracked open to access the pulp, which was subsequently minced and then digested for 1 hour at 37° C in 3 mg/ml collagenase (EMD Millipore, Burlington, MA) and 4 mg/ml dispase (Worthington Biochemical) in phosphate buffered saline (PBS; Gibco, Waltham, Massachusetts) similar to previously described (16, 17). The isolated dental pulp cells were plated at an initial density of 5,000 – 10,000 cells/cm2 and cultured in growth medium (GM) consisting of Dulbecco’s Modified Eagle Medium (DMEM; Gibco). 20% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA), and 1X penicillin and streptomycin (P/S; Gibco). The DPSCs were expanded and cryopreserved, and cells from passage 2-5 were used to engineer the scaffold-free constructs.

Flow cytometry

DPSCs were suspended in a solution of 0.5 ml blocking buffer consisting of 0.5% bovine serum albumin (BSA) + 2% FBS in PBS and incubated for 30 minutes on ice. Cells were centrifuged and resuspended in 0.150 ml of blocking buffer with fluorescently conjugated antibodies against CD105, CD146, CD90 or isotype controls; details on antibody product information and usage are listed in Supplementary Table 1. Flow cytometry was performed using LSR II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ) with FACS Diva 8 software (Becton Dickinson); 10, 000 events were assessed for each condition. Data was analyzed and plotted using FlowJo software (Becton Dickinson).

Formation and LPS treatment of scaffold-free DPSC constructs

Scaffold-free DPSC constructs were formed using methods previously described (16, 17). Wells of 6-well plates were coated with 2 ml polydimethylsiloxane (PDMS) using SYLGARD 184 Silicone Elastomer Kit (Dow Corning, Midland, MI). The PDMS layer was coated with 3 μg/cm2 of laminin (Apexbio, Houston, TX) to facilitate DPSC adhesion. DPSCs were plated onto the treated construct dishes at an initial density of 200,000 cells/well in GM. Following 48 hours, the media was changed to an osteo/odontogenic medium (OM1) consisting of GM supplemented with 5 mg/ml ascorbic acid, 10−8 M dexamethasone (Dex), and 5 mM β-glycerophosphate (βGP; MP Biochemical, Irvine, CA). At confluence, the media was changed to OM2, which was similar to OM1 except the FBS concentration was reduced from 20% to 5%. The cells formed cell sheets that contracted into spherical tissues. P.gingivalis-derived LPS (Invivogen, San Diego, CA) or E. coli-derived LPS (011: B4) LPS was added to the culture medium 24 hours after construct formation at 1.0, 5.0, and 10.0 μg/ml similar to previous studies utilizing 2D DPSC cultures (9, 10, 14, 15); fresh LPS was added during each medium change. Throughout the experiment, the medium was changed every 2-3 days. RNA was collected from samples following 2, 7 and 14 days in culture. Additional constructs were fixed for histological analysis at 14 days (Figure 1A).

Figure 1:

Figure 1:

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Experiment timeline and formation of scaffold-free DPSC constructs. (A) Timeline of DPSC construct formation. LPS treatment, and experimental endpoints. (B) Flow cytometry analysis confirms that DPSCs express cell surface markers characteristic of CD90, CD105, and CD146. Plots show isotype control histograms (blue) and histograms of respective cell surface protein expression (red). (C) DPSCs were plated onto construct dishes and grew to confluence. (D) DPSCs formed a cell sheet that naturally started detaching from the substrate (yellow arrow). (E) The DPSC sheet contracted to form a spherical construct. (E) Scale bars: (B) = 100 μm, (C) = 0.5 cm, (D) = 4 mm

Histological analysis

DPSC constructs were fixed in 10% phosphate buffered formalin and embedded in paraffin: sections were acquired at thickness of 5 μm. Sections were stained with hematoxylin and eosin (H&E) (Gill hematoxylin and eosin Y alcoholic solutions), Alizarin Red (2% w/v in water), or Von Kossa (Scytek Laboratories Calcium Stain Kit).

Tissue sections were prepared for immunostaining by incubating hydrated slides in heat-induced epitope retrieval solution (Sodium citrate 10 mM, 0.05% Tween-20, pH 6.0) at 60° overnight and then permeabilized in 0.1% Triton-X for 10 minutes at room temperature. The sections were blocked in 5% goat serum for 2 hours at room temperature. Immunostaining was performed using antibodies against dentin matrix protein 1 (DMP1), proliferating cell nuclear antigen (PCNA), nestin, and cleaved caspase 3 (CC3). Fluorescently tagged secondary antibodies, Alexa Fluor 546 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit, were used to detect the signal. Antibody product details and usage are listed in Supplementary Table 1. DAPI staining was performed to detect nuclei. Brightfield images were acquired using a Nikon (Tokyo, Japan) TE2000-E inverted microscope, and fluorescent images were taken using a Nikon Eclipse Ti inverted microscope.

Quantitative image analysis

Quantitative image analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD). To quantify mineralization of the constructs, the background was digitally removed from micrographs of alizarin red-stained histological sections and then the images were converted to gray scale. To determine the area of the entire tissue section, a binary image was generated with threshold values of 0-235 and the number of white pixels was recorded. The area of positive alizarin red staining was determined using a separate binary image with threshold 0-175. The percent area of mineralized tissue was calculated by normalizing the area of positive alizarin red staining to the area of the entire tissue section. Sections were acquired from the center of separate constructs and averaged with n = 2 organoids for control, 5 μg/ml P. gingivalis-LPS, 1 and 5 μg/ml E. coli-LPS, and n = 3 organoids for 1 and 10 μg/ml P. gingivalis-LPS and 10 μg/ml E. coli LPS.

DMP1, nestin, and PCNA intensity was quantified on micrographs taken on the same microscope with uniform exposure times and magnifications. Following background subtraction, the mean gray intensity of fluorescent signal was averaged from 5 randomly selected regions of interest within 1-2 images acquired of a representative organoid and plotted.

Quantitative real-time PCR (qPCR)

RNA was isolated from the constructs 2d, 7d, and 14d following construct formation using the QiaShredder and RNeasy Mini Kits (Qiagen, Hilden, Germany). qPCR was performed with the TaqMan RNA to Ct One Step Kit (Applied Biosystems, Waltham, MA) using primers of human RUNX2 and housekeeping gene 18s (TaqMan Gene Expression Assays, Applied Biosystems). Relative gene expression was calculated using the 2−ΔΔCt method.

Statistical Analyses

Data is presented as averages +/− standard deviation. Statistical tests were performed using SPSS software (IBM, Armonk, NY). Quantitative comparisons of percent area of mineralized tissue were determined using a Kruskall-Wallis test with a Dunn’s post-hoc analysis. Differences in immunofluorescent signal intensity were determined using a one-way ANOVA with a Tukey’s HSD post-hoc analysis. Differences were considered significant at p < 0.05.

Results

Formation of Scaffold-free DPSC constructs

Flow cytometry analysis confirmed that the DPSCs utilized in these experiments expressed mesenchymal cell surface markers including CD90 (87.4% positive), CD105 (98.8% positive), and CD146 (81.5% positive) (Figure 1B). Following plating onto the construct dishes, the DPSCs became confluent and deposited endogenous ECM to produce tissue sheets that naturally detached from the substrate and contracted into spherical constructs ranging from 750 μm – 1 mm in diameter. (Figure 1C–E).

Effects of LPS treatment on histological structure and mineral patterning of DPSC constructs

H & E staining showed that scaffold-free DPSC constructs are solid and cellular tissues (Figure 2). Alizarin red staining showed that control scaffold-free DPSC constructs exhibit a mineral pattern characteristic of a dentin-pulp complex where the periphery of the construct becomes mineralized while the center remains unmineralized (Figure 2A). Culturing DPSC organoids with P. gingivalis-LPS did not affect mineral deposition (Figure 2B and D). However, E. coli-LPS exposure caused a dose-dependent decrease in mineralization where a significant reduction in mineralization was detected between the control conditions and 10 μg/ml treatment (Figure 2 C and D). Von Kossa staining of the DPSC organoids showed similar results (Supplemental Figure 1).

Figure 2:

Figure 2:

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Effects of LPS treatment on tissue structure and mineral deposition in scaffold-free DPSC constructs. H & E and alizarin red staining of control (A) control DPSC constructs lacking LPS treatment, (B) DPSC constructs treated with different concentrations of P. gingivalis-LPS, and (C) DPSC constructs treated with different concentrations of E. coli-LPS. (D) Quantitative image analysis comparing mineralized tissue area in DPSC constructs +/− LPS treatment. P. gingivalis-LPS and E. coli-LPS constructs were compared to the same control samples (*p<0.05). Scale bars = 200 μm

Effects of LPS treatment on RUNX2 mRNA expression in DPSC constructs

RUNX2 mRNA expression, a key transcription factor that regulates odontoblastic differentiation(20), increased in control samples overtime (Figure 3A). At the 2-day time point, the 5 μg/ml concentration of both P. gingivalis and E. coli-derived LPS caused a slight increase in RUNX2 expression. At the 7-day time point, P. gingivalis and E. coli-LPS did not strongly affect RUNX2 mRNA expression relative to untreated controlled. At day 14, treatment of either P. gingivalis-LPS or E. coli-LPS substantially reduced RUNX2 mRNA expression in the scaffold-free DPSC constructs relative to the untreated controls.

Figure 3:

Figure 3:

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Effects of LPS on the expression of odontoblast-related mRNA and proteins in scaffold-free DPSC constructs. (A) P. gingivalis-LPS and E. coli-LPS treatment on RUNX2 mRNA expression in DPSC constructs. Data presented as fold change relative to 2d untreated control. P. gingivalis-LPS and E. coli-LPS constructs were compared to the same control samples. (B) Immunofluorescent staining to detect DMP1 (yellow) expression of the DPSC +/− P. gingivalis or E. coli-LPS treatment (B’) Quantitative image analysis comparing fluorescent intensity of DMP1 signal in DPSC constructs +/− LPS; LPS treated samples are compared to the same control samples (*p < 0.05). (C) Immunofluorescent staining to detect nestin (yellow) expression of the DPSC +/− P. gingivalis or E. coli-LPS treatment (C’) Quantitative image analysis comparing fluorescent intensity of nestin signal in DPSC constructs +/− LPS; LPS treated samples are compared to the same control samples (*p < 0.05). (B) and (C) show images of the periphery of the DPSC constructs, nuclei are stained with DAPI (blue), and scale bars = 30 μm.

Effects of LPS treatment on dentin-related protein expression in DPSC constructs

Localized expression of the odontoblast-related proteins DMP1 and nestin was detected on the periphery of the control constructs (Figure 3 B and C) indicating that the cells in this region of the tissues are differentiating down an odontoblast-like lineage to self-organize into dentin-pulp organoids. DMP1 and nestin expression was also localized to the periphery of DPSC constructs exposed to LPS (Figure 3 B and C). One μg/ml P. gingivalis-LPS treatment did not alter DMP1 expression relative to control samples; however, 5 and 10 μg/ml of P. gingivalis-LPS caused a significant reduction in expression (Figure 3B and B’). E. coli-LPS treatment significantly reduced DMP1 expression in DPSC organoids at all concentrations relative to the untreated control (Figure 3B and B’). A reduction in nestin expression was detected in DPSC organoids exposed to both P. gingivalis and E. coli-derived LPS (Figure 3C and C’). Quantitative image analysis confirmed that culturing DPSC constructs with 1 and 10 μg/ml P. gingivalis-LPS significantly reduced nestin signal intensity relative to untreated controls (Figure 3C’). Although significant differences in nestin expression among control and E. coli-LPS treated constructs was not detected, likely due to the substantial variance in nestin signal intensity of the control sample, a trend in reduced nestin expression in E. Coli-LPS treated samples relative to controls can be observed (Figure 3C’).

Effect of LPS on DPSC proliferation and apoptosis

Immunostaining against CC3 and PCNA was performed to evaluate the effects of LPS on cell apoptosis and proliferation, respectively (Figure 4). Only background levels of CC3 were detected in both control and LPS treated DPSC constructs indicating that E. coli and P. gingivalis-derived LPSs did not induce apoptotic cell death (Figure 4A). The DPSCs were highly proliferative in the control and LPS treated scaffold-free engineered tissues as detected by strong, positive nuclear PCNA expression throughout the constructs (Figure 4B). Quantitative image analysis showed that treating DPSC constructs with 10 μg/ml P. gingivalis-LPS significantly increased PCNA expression relative to control samples (Figure 4C). Interestingly, DPSC constructs subjected to 1 μg/ml E. coli-LPS had significantly increased PCNA signal relative to control samples, however, a statistically different signal was not detected between control samples and constructs treated with the higher 5 and 10 μg/ml concentrations (Figure 4C).

Figure 4:

Figure 4:

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Effects of LPS on cell apoptosis and proliferation in scaffold-free DPSC constructs. (A) Immunofluorescent staining to detect CC3 (yellow) as a marker of apoptosis in DPSC constructs +/− LPS treatment, minimal expression was detected in all groups. (B) Immunofluorescent staining against PCNA (yellow) as a marker of cell proliferation in DPSC constructs +/− LPS treatment. (C) Quantitative image analysis comparing fluorescent intensity of PCNA signal in DPSC constructs +/− LPS treatment; LPS treated samples are compared to the same control samples (*p < 0.05). Nuclei are stained with DAPI (blue) in (A) and (B), scale bars = 50 μm.

Discussion

The dental pulp is capable of providing a biological defense against bacterial infection, and DPSCs are considered critical players in driving these activities (4, 21). Recognition of bacterial byproducts by host cells is one of the first events to trigger reparative processes (4, 21). However, DPSC response to the bacterial endotoxin LPS has not been fully and clearly elucidated. In this study, the effects of LPS on odontoblastic differentiation of DPSCs was investigated using scaffold-free engineered tissues. These engineered systems serve as relevant experimental models that more closely emulate native tissues than traditional 2D culture systems. Furthermore, these engineered tissues support DPSCs to self-organize into a structure that resembles a dentin-pulp complex (16, 17). This is the first report to establish scaffold-free DPSC constructs as experimental models of infection.

The scaffold-free tissue engineering methods utilized in the current study entailed culturing DPSCs to generate cell sheets that subsequently detached from the substrate and contracted into 3D tissues. There are multiple scaffold-free tissue engineering approaches including cell sheet or aggregate engineering (22). The technique utilized in this study could be considered as an extension of cell sheet engineering since the constructs originate at sheets that then coalesce into a more substantial 3D structure. Cell sheets are formed by 2D cultured cells that proliferate and deposit endogenous ECM. Aggregates are created by culturing cells in non-adherent conditions necessitating that the cells initially form direct cell-cell interactions to generate spherical cell masses and subsequently deposit ECM. Complex geometries can be created using cell aggregates by forming these structures in 3D printed molds or utilizing the aggregates as building blocks for 3D printing techniques (23, 24). Multiple scaffold-free tissue engineered constructs have been employed as model systems to specifically study dental tissue development and repair (16, 17, 25–27). Cell sheets require lower initial cell densities since the cells readily proliferate in the culture system prior to 3D tissue formation, and these engineered tissues can be formed using standard tissue culture supplies. Therefore, the simplicity of these scaffold-free constructs may provide benefits for use as high-throughput experimental models.

Both E. coli and P. gingivalis-derived LPSs reduce RUNX2 mRNA expression and the expression of dentin-related proteins in scaffold-free DPSC constructs, which suggests that LPS decreases the odontoblastic differentiation potential of DPSCs. However, only E. coli-LPS reduced the capacity of DPSCs to mineralize the ECM indicating that only E. coli-LPS prevented terminal differentiation of DPSCs towards an odontoblastic phenotype. LPS is an amphiphilic molecule comprising three regions that include a lipid A portion, a polysaccharide moiety, and an O-antigen (18). It has been well established that lipid A is the immunostimulatory region of LPS, and the structure of lipid A, which varies across bacterial species, determines the potency of the LPS (18). E. coli and P. gingivalis-derived LPSs have been shown to differ in structure and function, and E. coli-LPS has been described as more virulent relative to P. gingivalis-LPS (28). Potentially, both LPSs reduced the odontoblastic differentiation of DPSCs in the scaffold-free tissues as detected by decreases in RUNX2, DMP1, and nestin expression, but only E. coli-LPS was potent enough at the tested concentrations to induce functional differences in mineral deposition. Alternatively, the differences detected between E. coli and P. gingivalis-derived LPSs on mineral deposition could be a consequence of the LPSs binding different toll-like receptors and/or activating unique signaling pathways, as proposed in other studies (29, 30).

In native structures, the onset of bacterial insult stimulates the formation of reparative dentin to block further bacterial infiltration into the pulp tissue (1). Reparative dentinogenesis is thought to be driven by DPSCs, therefore the potential reduction in odontoblastic differentiation by DPSCs in the scaffold-free cultures, seen here, is an unexpected result. DPSCs naturally reside in a multifaceted microenvironment, which is increasingly complex under true infectious conditions. ECM molecules released from the degradation of mineralized tissues by acidic bacterial byproducts are known to also trigger reparative processes in DPSCs (1). Therefore, DPSC constructs may exhibit an alternate response to LPS delivered in combination with these ECM proteins. Furthermore, additional cell types reside in the pulp tissue including mature odontoblasts, fibroblasts, and inflammatory cells, which, in response to LPS, may provide additional paracrine signals to DPSCs to orchestrate the formation of reparative dentin. In future studies, a more complex engineered tissue could be developed that incorporates additional cell types found in the natural dentin-pulp complex. Alternatively, DPSCs could have reduced odontoblastic differentiation potential in response to LPS alone because DPSCs are instead triggered to modulate host inflammatory response. LPS has been shown to induce the expression of inflammatory cytokines by DPSCs in 2D culture (4, 7, 14); similar studies are needed to evaluate LPS-induced inflammatory cytokines production by DPSC cultured as scaffold-free engineered constructs.

P. gingivalis-LPS induced a dose-dependent increase in DPSC proliferation within the scaffold-free constructs. LPS stimulation has been reported to promote cell proliferation in 2D cultures of many cell types (31–33) indicating that the proliferative response of DPSCs to LPS is similar in 2D and 3D culture systems. It has been suggested that this increased proliferation may be associated with reduced stem cell differentiation (31). Similar to P. gingivalis-LPS, DPSCs exhibited increased proliferation when exposed to E. coli-LPS at the 1 and 5 μg/ml concentrations, however, this effect was diminished at 10 μg/ml E. coli-LPS. Potentially, this higher concentration of E. coli-LPS induces different adaptive mechanisms than P. gingivalis-LPS or the lower concentrations of E. coli-LPS. Since E. coli-LPS is considered more virulent than P. gingivalis-LPS, potentially a similar response would be detected in DPSC constructs exposed to further increased P. gingivalis-LPS concentrations.

The present work shows that P. gingivalis and E. coli-LPS did not induce apoptosis in DPSCs cultured in scaffold-free constructs. This result corroborates what has been previously reported by others regarding the effects of LPS on apoptosis and viability of DPSCs cultured in 2D (9, 14, 34). However, Yang et al. showed a contradictory result of LPS induced apoptosis in DPSCs (35) . The DPSCs used by Yang et al. were at a much later passage than what was used in the present work and what was reported by Widbillar et al and He et al.; potentially when approaching senescence, DPSCs apoptose in response to LPS.

This work establishes the use of scaffold-free DPSC tissues as models of pulpitis. These scaffold-free engineered tissues organize into dentin-pulp organoids with mineral patterning that emulates native tissues. Both E. coli and P. gingivalis-derived LPSs reduce the odontogenic differentiation potential of DPSCs in these systems, but only E. coli-LPS affected functional differentiation by reducing mineral deposition. This emphasizes that LPS origin can uniquely dictate the type or severity of DPSC response.

Supplementary Material

1

Supplemental Figure 1: Von kossa staining to detect mineral deposition in control DPSC constructs lacking LPS treatment and DPSC constructs treated with different concentrations of either P. gingivalis-LPS or E. coli-LPS. Mineral (black), nuclei (red), cytoplasm (pink), scale = 200 μm.

2

Supplemental Table 1: Product information and usage of antibodies utilized in this study.

Acknowledgements

This research was funded by the University of Pittsburgh, School of Dental Medicine, and the National Institute of Dental & Craniofacial Research of the National Institutes of Health under Award Number R00 DE025088 to FNS. The authors deny any conflicts of interest.

Footnotes

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References

  • 1.Goldberg M, Smith AJ. Cells and Extracellular Matrices of Dentin and Pulp: A Biological Basis for Repair and Tissue Engineering. Crit Rev Oral Biol Med 2004;15:13–27. [DOI] [PubMed] [Google Scholar]
  • 2.Siqueira JF Jr., Rocas IN, Silva MG. Prevalence and clonal analysis of Porphyromonas gingivalis in primary endodontic infections. J Endod 2008;34:1332–6. [DOI] [PubMed] [Google Scholar]
  • 3.Warfvinge J, Dahlen G, Bergenholtz G. Dental pulp response to bacterial cell wall material. J Dent Res 1985;64:1046–1050. [DOI] [PubMed] [Google Scholar]
  • 4.Bindal P, Ramasamy TS, Kasim NHA, et al. Immune responses of human dental pulp stem cells in lipopolysaccharide-induced microenvironment. Cell Biol Int 2018;42:832–840. [DOI] [PubMed] [Google Scholar]
  • 5.Botero TM, Son JS, Vodopyanov D, et al. MAPK signaling is required for LPS-induced VEGF in pulp stem cells. J Dent Res 2010;89:264–9. [DOI] [PubMed] [Google Scholar]
  • 6.Brodzikowska A, Ciechanowska M, Kopka M, et al. Role of Lipopolysaccharide, Derived from Various Bacterial Species, in Pulpitis-A Systematic Review. Biomolecules 2022;12:138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chang J, Zhang C, Tani-Ishii N, et al. NF-kappaB activation in human dental pulp stem cells by TNF and LPS. J Dent Res 2005;84:994–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.He W, Qu T, Yu Q, et al. LPS induces IL-8 expression through TLR4, MyD88, NF-kappaB and MAPK pathways in human dental pulp stem cells. Int Endod J 2013;46:128–136. [DOI] [PubMed] [Google Scholar]
  • 9.He W, Wang Z, Luo Z, et al. LPS promote the odontoblastic differentiation of human dental pulp stem cells via MAPK signaling pathway. J Cell Physiol 2015;230:554–561. [DOI] [PubMed] [Google Scholar]
  • 10.Huang Y, Jiang H, Gong Q, et al. Lipopolysaccharide stimulation improves the odontoblastic differentiation of human dental pulp cells. Mol Med Rep 2015;11:3547–3552. [DOI] [PubMed] [Google Scholar]
  • 11.Kim JH, Woo SM, Choi NK, et al. Effect of Platelet-rich Fibrin on Odontoblastic Differentiation in Human Dental Pulp Cells Exposed to Lipopolysaccharide. J Endod 2017;43:433–8. [DOI] [PubMed] [Google Scholar]
  • 12.Li D, Fu L, Zhang Y, et al. The effects of LPS on adhesion and migration of human dental pulp stem cells in vitro. J Dent Res 2014;42:1327–1334. [DOI] [PubMed] [Google Scholar]
  • 13.Liu ZN, Jiang T, Wang XZ, et al. Fluocinolone acetonide partially restores the mineralization of LPS-stimulated dental pulp cells through inhibition of NF-kappa B pathway and activation of AP-1 pathway. Br J Pharmacol 2013;170:1262–1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Widbiller M, Eidt A, Wolflick M, et al. Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells. Int Endod J 2018;51:877–888. [DOI] [PubMed] [Google Scholar]
  • 15.Yamagishi VT, Torneck CD, Friedman S, et al. Blockade of TLR2 inhibits Porphyromonas gingivalis suppression of mineralized matrix formation by human dental pulp stem cells. J Endod 2011;37:812–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Syed-Picard FN, Jayaraman T, Lam RS, et al. Osteoinductivity of calcium phosphate mediated by connexin 43. Biomaterials 2013;34:3763–3774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Syed-Picard FN, Ray HL Jr., Kumta PN, et al. Scaffoldless tissue-engineered dental pulp cell constructs for endodontic therapy. J Dent Res 2014;93:250–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Mazgaeen L, Gurung P. Recent Advances in Lipopolysaccharide Recognition Systems. Int J Mol Sci 2020:21:379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Gomes BP, Montagner F, Jacinto RC, et al. Polymerase chain reaction of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in primary endodontic infections. J Endod 2007;33:1049–1052. [DOI] [PubMed] [Google Scholar]
  • 20.Chen S, Gluhak-Heinrich J, Wang YH, et al. Runx2, Osx, and Dspp in Tooth Development. J Dent Res 2009;88:904–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Farges JC, Alliot-Licht B, Renard E, et al. Dental Pulp Defence and Repair Mechanisms in Dental Caries. Mediators Inflamm 2015;2015:230251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.DuRaine GD, Brown WE, Hu JC, et al. Emergence of scaffold-free approaches for tissue engineering musculoskeletal cartilages. Ann Biomed Eng 2015;43:543–554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Mironov V, Visconti RP, Kasyanov V, et al. Organ printing: tissue spheroids as building blocks. Biomaterials 2009;30:2164–2174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Strobel HA, Calamari EL, Alphonse B, et al. Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds. J Vis Exp 2018:134:56618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Basu A, Rothermund K, Ahmed MN, et al. Self-Assembly of an Organized Cementum-Periodontal Ligament-Like Complex Using Scaffold-Free Tissue Engineering. Front Physiol 2019;10:422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Li FC, Hussein H, Magalhaes M, et al. Deciphering Stem Cell From Apical Papilla-Macrophage Choreography Using a Novel 3-dimensional Organoid System. J Endod 2022;48:1063–1072 e1067. [DOI] [PubMed] [Google Scholar]
  • 27.Yu J, Deng Z, Shi J, et al. Differentiation of dental pulp stem cells into regular-shaped dentin-pulp complex induced by tooth germ cell conditioned medium. Tissue Eng 2006;12:3097–3105. [DOI] [PubMed] [Google Scholar]
  • 28.Martin M, Katz J, Vogel SN, et al. Differential induction of endotoxin tolerance by lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli. J Immunol 2001;167:5278–5285. [DOI] [PubMed] [Google Scholar]
  • 29.Darveau RP, Pham TTT, Lemley K, et al. Porphyromonas gingivalis lipopolysaccharide contains multiple lipid a species that functionally interact with both toll-like receptors 2 and 4. Infect Immun 2004;72:5041–5051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Zhang D, Chen L, Li S, et al. Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1 beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS. Innate Immun 2008;14:99–107. [DOI] [PubMed] [Google Scholar]
  • 31.Li XY, Yang X, Zhao QD, et al. Lipopolysaccharide promotes tumorigenicity of hepatic progenitor cells by promoting proliferation and blocking normal differentiation. Cancer Lett 2017;386:35–46. [DOI] [PubMed] [Google Scholar]
  • 32.Liu G, Deng J, Zhang Q, et al. Porphyromonas gingivalis Lipopolysaccharide Stimulation of Vascular Smooth Muscle Cells Activates Proliferation and Calcification. J Periodontol 2016;87:828–836. [DOI] [PubMed] [Google Scholar]
  • 33.Yang H, Kaneko M, He C, et al. Effect of a lipopolysaccharide from E. coli on the proliferation of fibroblasts and keratinocytes in vitro. Phytother Res 2002;16:43–7. [DOI] [PubMed] [Google Scholar]
  • 34.Hadjichristou C, Papachristou E, Bonovolias I, et al. Three-dimensional tissue engineering-based Dentin/Pulp tissue analogue as advanced biocompatibility evaluation tool of dental restorative materials. Dent Mater 2020;36:229–248. [DOI] [PubMed] [Google Scholar]
  • 35.Yang H, Zhu YT, Cheng R, et al. Lipopolysaccharide-induced dental pulp cell apoptosis and the expression of Bax and Bcl-2 in vitro. Braz J Med Biol Res 2010;43:1027–1033. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure 1: Von kossa staining to detect mineral deposition in control DPSC constructs lacking LPS treatment and DPSC constructs treated with different concentrations of either P. gingivalis-LPS or E. coli-LPS. Mineral (black), nuclei (red), cytoplasm (pink), scale = 200 μm.

NIHMS1835940-supplement-1.tif (7.9MB, tif)
2

Supplemental Table 1: Product information and usage of antibodies utilized in this study.

NIHMS1835940-supplement-2.tif (264.6KB, tif)

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