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Osteocalcin Expression in Pulp Inflammation
Basic Research—Biology
Osteocalcin Expression in Pulp Inflammation Ashraf Abd-Elmeguid, BDS, MDSc, PhD,* Marwa Abdeldayem, BDS, DPH, MSc,‡ Loren W. Kline, BSc, MSc, PhD,* Redwan Moqbel, PhD, FRCPPath,§ Harrisios Vliagoftis, MD,† and Donald C. Yu, DMD, CAGS, MScD, FRCD(C)* Abstract Introduction: Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. Although OCN is considered a reparative molecule inside the dental pulp, it is not clear if it is involved in pulpal inflammation. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation. Methods: Pulp tissues in the form of reversible and irreversible pulpitis were collected from the endodontic clinic. Those from impacted teeth were used as controls. Immunohistochemistry was used to localize OCN. Samples were analyzed for OCN and inflammatory mediator expression using multiplex assay. Results: OCN in inflamed tissues was localized in cells and matrix around calcification areas and in cells around blood vessels but not in normal tissues. The plex assay (Bio-Plex 200, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) showed OCN expression in reversible pulpitis significantly higher than in irreversible pulpitis, and both were significantly higher than in the controls. A panel of inflammatory mediators showed an increase in reversible and irreversible pulpitis. Another panel was decreased in both stages compared with the controls. OCN expression in reversible pulpitis was positively correlated to the expression of vascular endothelial growth factor, fibroblast growth factor, macrophage inflammatory protein-1b, monocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin (IL)-17, and soluble IL-2 receptor a and negatively correlated to that of IL-1a, IL-1b, IL-8, granulocyte macrophage colonystimulating factor, and macrophage inflammatory protein-1a. Conclusions: Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment. (J Endod 2013;39:865–872)
Key Words Dental pulp calcification, inflammatory mediators, osteocalcin, pulp inflammation
P
ulpal repair in response to tooth injury caused by caries, trauma, or cavity preparation is a complex process that includes matricellular interactions involving inflammation (1, 2). Cariogenic bacteria soften the dentin as it proceeds toward the dental pulp allowing the leaching out of dentinal matrix composed of many reparative proteins. As caries progress toward the dental pulp, inflammatory cells increase in number and secrete cytokines/chemokines (3, 4). At the same time, reparative molecules are secreted to compensate for the loss in dentinal structure forming reactionary or reparative dentin depending on the presence or absence of odontoblasts (5). Many inflammatory mediators were reported inside the inflamed dental pulp (1, 4, 6–9). However, little is known regarding the relation of these mediators to the reparative molecules involved in reparative/reactionary dentinogenesis. Osteocalcin (OCN) is one of the reparative molecules commonly expressed in response to injury of the dental pulp. It is expressed in response to cavity preparation in which it is associated with collagen fibers and is found embedded inside the tertiary dentin (10). Its gene was up-regulated in acute dental traumatic injury (11). OCN is an osteoblast marker and is considered to be a late differentiation marker of odontoblasts (12, 13). Its role in mineralization is not very clear. However, it is suggested to play an important role in mediating osteoclastic differentiation (14). OCN was found to be related to macrophage protein such as granulocyte macrophage colony-stimulating factor (GM-CSF) in bone-forming cells (15). Macrophages express OCN in their terminal differentiation to osteoblasts (16). Macrophages are the dominant inflammatory cells in pulp inflammation (17). Tumor necrosis factor (TNF) induced the expression of OCN from dental papilla cells (18). Is OCN related to the inflammatory changes inside the dental pulp? When OCN was administered to orthodontically treated teeth, a condition known for its inflammatory nature, it enhanced teeth movement (19), and OCN messenger RNA expression was increased inside the dental pulp of these teeth (20). In a recent study, OCN was expressed in response to the stimulation of pulp cells by a molecule with a known inflammatory role, and this was concomitant with the expression of angiogenic markers (21). Goldberg et al (2) and Cooper et al (1) discussed the inflammatory changes inside the dental pulp as a vital part of dental pulp repair in response to injury. In addition, reparative molecules are involved in the inflammatory process of the pulp (22, 23). It was shown also that fibrosis and calcification are increased in inflamed pulps compared with noninflamed ones (24). Therefore, the objectives of this study were
From the *School of Dentistry and †Division of Pulmonary Medicine, University of Alberta, Edmonton, Alberta, Canada; ‡Department of Orthodontics, Boston University, Boston, Massachusetts; and §Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada. Supported by the Egyptian Bureau of cultural affairs in Canada (grant no. G700000335), the Fund For Dentistry, University of Alberta (grant no. 05-2007), the Canadian Academy of Endodontics endowment fund (grant no. 000000582), and the Alpha Omega Foundation of Canada (grant no. 04-2007). A.A.-E. received a scholarship from the Ministry of Higher Education in Egypt and Medical sciences graduate program scholarship from the faculty of Medicine and Dentistry at the University of Alberta. Address requests for reprints to Dr Donald C. Yu, Endodontic Division, School of Dentistry, Faculty of Medicine and Dentistry, University of Alberta, 5-546 Edm Clinic Health Academy, Edmonton, AB, T6G 1C9, Canada. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.035
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Basic Research—Biology to localize OCN in inflamed pulps, to distinguish the different levels of OCN in 2 stages of pulp inflammation, and to suggest possible interactions of OCN in pulpal inflammation.
Materials and Methods Collection of Samples and Histologic Examination Thirty-five inflamed pulps were collected from the Endodontic Clinic at the University of Alberta, Edmonton, Alberta, Canada, according to the protocol approved by the University of Alberta Human Research Ethics Board. As described previously (7), pulps of cases with reversible pulpitis (n = 23) referred to the endodontic clinic for prosthetic purposes and pulps with irreversible pulpitis (n = 12) were used in this study. Clinically, teeth with reversible pulpitis are sensitive to cold (not to hot) and have no spontaneous, lingering pain. Teeth with irreversible pulpitis are clinically sensitive to hot with spontaneous lingering pain. Thirty pulps from impacted caries-free teeth were collected, considered as normal pulps, and used as controls. The pulps used for histologic examination were collected in 4% paraformaldehyde and processed for paraffin embedding, and 5-mm sections were stained with hematoxylin-eosin (H&E) and Gomori trichrome stains. H&E-stained tissues were examined under light microscope; cells of the dental pulp tissue were counted under the same magnification (400) in 3 different sections and 3 different high-power fields per section. If the tissue had more than 1 piece per slide (ie, torn), 3 different locations per piece of tissue were used for the examination, and the average of the 3 counts was calculated and tabulated for statistical analysis. Tissues were also examined for calcification areas. When present, images of these areas were taken then were transferred to ImageJ analysis software (National Institutes of Health, Bethesda, MD) to calculate the surface area of calcification (in millimeters). The percentages of these areas to the whole area of the tissue in the image were then calculated. The steps performed were previously described in a previous report (25). The trichrome stain stains collagen fibers (fibrosis) green, preventing the light from being reflected and decreasing its intensity under the microscope. In the presence of denser fibers, the light intensity was measured using ImageJ analysis software. The intensity was tabulated and submitted for statistical analysis. All histologic evaluation and tabulation were performed blindly to eliminate bias. Analysis of Inflammatory and Remodeling Cytokines/ Chemokines Using a Multiplex Assay Tissues were collected in tissue lysis buffer (Cellytic T; SigmaAldrich, St Louis, MO) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich), homogenized, and centrifuged at 14,000g for 15 minutes at 2 C followed by the collection of the supernatant. The protein concentration was measured using the Pierce BCA protein assay kit (Thermo Scientific Pierce Biotechnology, Rockford, IL), and a similar amount of proteins in each sample was sent to Evetechnologies (Calgary, Alberta, Canada) for human multiplex assay (Bio-Plex 200; Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) analysis as described previously (26). The protocol and the 42 analytes measured are listed on the company’s website (www.Evetechnologies.com). Immunohistochemical Localization of OCN and OCN Measurement in Pulp Tissues Samples collected, as previously explained, were examined for the presence of OCN by immunohistochemical staining as performed previously (23). After deparaffinization using serial dilutions of ethanol and xylene, tissues were washed with phosphate-buffered saline (PBS) twice for 5 minutes each followed by permeabilization using 0.5% Triton X100 in PBS for 5 minutes and then blocking unspecific protein binding 866
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by serum-free blocking solution (Dako Canada Inc, Mississauga, ON, Canada) for 10 minutes at room temperature. Tissues were then incubated overnight at 4 C with monoclonal mouse antihuman OCN antibody (10 mg/mL) (R&D Systems, Inc, Minneapolis, MN) or incubated with mouse immunoglobulin G1 (10 mg/mL) (Biolegend, San Diego, CA) as isotype controls. Tissue slides were washed 4 times in PBS (5 minutes each) to eliminate any unspecific binding and then were incubated with alkaline phosphatase–conjugated antimouse secondary antibody (1:100) (Sigma-Aldrich) in a humid chamber for 1 hour at room temperature followed by washing in PBS. The signal was developed using the AP Vector red substrate kit (Vector Laboratories Inc, Burlingame, CA). Counterstaining with hematoxylin was performed next, and slides were kept overnight in the dark. Tissue slides were then examined for OCN staining under light microscope. All antibodies and controls were diluted in antibody diluents with background reducing component (Dako Inc). Samples collected for the plex assay were treated as previously described and sent to Evetechnologies. Data from the plex analysis (OCN) were used together with data from the multiplex analysis (inflammatory cytokines/chemokines) for the same tissues to correlate between the level of OCN and other inflammatory mediators.
Statistical Analysis The Student’s t test was used to compare samples of reversible and irreversible pulpitis in terms of percentage of calcification areas. The analysis of variance test was used to compare cell counts in pulp tissues and collagen amounts as shown by light intensity measured by using ImageJ software. The same test also was used to compare inflammatory mediator expression and OCN expression by human plex assay (Bio-Rad Laboratories Ltd) in normal, reversible, and irreversible pulpitis. Samples of the multiplex assay that were extrapolated beyond the standard range were not assigned a value. A statistical value of P < .05 was considered significant. Correlation analysis using Pearson’s correlation coefficient was used to correlate OCN expression with each mediator expression in inflamed (reversible and irreversible pulpitis) pulps; a P value <.05 was considered significant for either a positive or negative correlation.
Results Presence of Inflammatory Cells and Calcification in Pulpitis Dental pulp cells were counted blindly in slides stained with H&E. Representative pictures of control pulp (Fig. 1A) and pulps with reversible (Fig. 1B) and irreversible pulpitis (Fig. 1C) are shown. The control group and irreversible pulpitis group had lower cell counts compared with the reversible pulpitis group. There was no significant difference between the irreversible stage of pulp inflammation and the noninflamed group (control) (Fig. 1D). Trichrome staining was used to evaluate the degree of fibrosis in our samples. Quantitation of fibrosis showed that the control group (Fig. 1E) was the lowest in collagen staining compared with the reversible (Fig. 1F) and irreversible (Fig. 1G) stages of pulp inflammation; these differences were significant (P < .0001) (Fig. 1H). Pulps from impacted teeth had no evidence of calcification in contrast to 78% of the total number of tissues with reversible pulpitis and 66% of the total number of tissues with irreversible pulpitis. The percentage of calcification areas in tissues with reversible pulpitis was significantly higher than that in irreversible pulpitis (Fig. 1I–L). Presence of Inflammatory Mediators in the Pulp Tissue A human multiplex assay analysis was performed to identify the presence of different inflammatory and remodeling mediators in pulp JOE — Volume 39, Number 7, July 2013
Basic Research—Biology
Figure 1. Cell number and fibrosis of reversible and irreversible pulpitis. (A) H&E staining of the pulp of an impacted molar (control) showing a decreased cell count; (B) the cell number (arrows) is profoundly increased in reversible stages of pulpitis and (C) then tends to decrease again in irreversible stages of pulpitis. Pictures show representative samples for the cell count under the same field microscope magnification (400), which is statistically significant when comparing the cell number of reversible pulpitis with the control (normal) and that of reversible pulpitis to irreversible ones. (D) Although the total cell count in irreversible pulpitis was higher than in the control, it was not statistically significant. (E) Gomori trichrome staining of the pulp from impacted tooth (control). (F) The degree of green (asterisks) (collagen) increased in reversible stages of pulpitis and (G) the irreversible stages of pulpitis (G). (H) Data collected using Image G analysis software showed that the light intensity masked by the green in the samples decreased significantly in reversible and irreversible pulpitis than the control. (I) Pulp tissues from impacted teeth (n = 15) showed no areas of calcification (letter X), whereas (J) reversible (n = 11) and (K) irreversible (n = 7) pulpitis (L) developed calcification areas that were significant compared with the control and each other with the reversible stages having a higher percentage of calcification areas. Data were considered significant at P value <.05.
tissue and measure their levels. The mean expression in pg/mg protein in normal, reversible, and irreversible pulpitis for each inflammatory mediator and the statistical analysis are shown in Table 1. Inflammatory mediators commonly expressed in the inflamed dental pulp like interleukin (IL)-1a, IL-1b, IL-1ra, IL-4, IL-6, IL-8/CXCL8, and TNF and other inflammatory mediators like monocyte chemoattractant protein-1 (MCP-1/CCL2), MCP-3/CCL7, monocyte-derived chemokine (MDC), macrophage inflammatory protein-1a (MIP-1a/CCL3), JOE — Volume 39, Number 7, July 2013
MIP-1b/CCL4, GM-CSF, and growth-regulated protein not previously reported in the inflamed pulp showed a significant increase in reversible and irreversible stages of pulp inflammation when compared with the control (Table 1, panel A). Another set of inflammatory mediators including IL-7, interferon a, transforming growth factor a, granulocytecolony stimulating factor, and IL-13 showed a decrease in both reversible and irreversible pulpitis when compared with the control (Table 1, panel B). Some mediators like interferon g–induced protein-10 (IP-
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Panel
A
B
C
D JOE — Volume 39, Number 7, July 2013
E
Normal pulps (n = 10–12), mean ± standard error (pg/mg protein)
Reversible pulpitis (n = 9–11), mean ± standard error (pg/mg protein)
GM-CSF GRO IL-1a IL-1b IL-1ra IL-4 IL-6 IL-8 IL-12 p40 MCP-1 MCP-3 MDC MIP-1a MIP-1b TNF-a
0.21 0.15 2.58 1.26 6.04 0.97 2.11 0.55 4.75 1.04 0.90 0.37 0.00 0.00 0.23 0.23 0.00 0.00 16.83 2.15 1.93 1.30 17.41 5.10 6.23 3.54 1.85 1.22 0.00 0.00
7.08 2.08 150.60 59.98 23.48 4.45 17.39 6.19 16.39 4.51 3.08 0.51 15.95 5.53 258.00 101.90 5.09 1.63 255.80 98.34 19.68 4.61 100.80 33.65 37.57 9.88 45.81 17 2.76 1.15
IFN-a G-CSF IL-7 IL-13 TGF-a
38.88 4.34 6.75 1.23 4.23 0.37 2.60 0.39 10.07 1.46
21.81 4.63 1.63 0.90 0.51 0.34 0.50 0.33 4.94 0.74
*
CD40L sIL-2ra IL-15 TNF-b IP-10
3.28 1.13 1.76 0.33 0.00 0.00 0.05 0.05 15.04 3.25
25.57 3.75 17.37 5.89 2.88 0.51 0.58 0.18 379.80 216
‡
Molecule
P value, reversible pulpitis vs normal * † † †
* * †
* * ‡ ‡ †
* †
* † ‡
* * ‡ †
* †
FGF PDGF-AA PDGF-AB/BB RANTES VEGF
488.80 49.10 2.60 0.26 41.54 4.74 27.46 4.29 75.12 7.77
1487 170.80 14.43 3.80 351.6 125.30 490.60 146.40 145.1 15.44
*
Eotaxin flt3ligand Fractalkine
0.00 0.00 103.90 7.95 146.8 14.08
5.66 2.93 78.54 16.24 311.9 78.15
NS NS NS
† ‡ ‡ †
GRO, growth-regulated protein; IFN, interferon; NS, not significant. Minimal expression and ns between all groups: IFN-g, IL-12p70, and IL-17. No expression in all groups: EGF, IL-2, IL-3, 1L-5, IL-9, and IL-10. *P value <.05. † P value <.01. ‡ P value <.001.
Irreversible pulpitis (n = 5–6), mean ± standard error (pg/mg protein) 21.12 6.50 53.09 12.64 31.13 5.33 12.34 1.28 17.33 4.08 3.18 0.46 7.82 4.88 371.40 119.10 32.96 19.40 110.70 31.69 13.10 2.074 56.79 6.53 52.02 12.60 81.36 26.63 3.08 0.42
P value, irreversible pulpitis vs normal
P value, irreversible vs reversible pulpitis
‡
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
* ‡ †
* * * † †
* * * † ‡ †
18.52 2.50 0.99 0.46 0.00 0.00 0.00 0.00 1.25 0.83
* *
12.23 2.76 4.51 0.69 1.52 0.57 0.09 0.09 25.86 6.08
NS NS NS NS NS
164.50 79.46 6.74 1.63 33.28 16.08 77.64 28.93 45.70 30.93
NS NS NS NS NS
11.22 5.31 8.39 2.89 14.82 7.48
†
‡
* ‡
‡
*
NS NS NS NS NS NS NS NS NS NS ‡
NS †
* †
NS * †
Basic Research—Biology
868 TABLE 1. Expression of Inflammatory Mediators in Normal Pulps and Reversible and Irreversible Pulpitis
Basic Research—Biology 10/CXCL10), IL-15, soluble interleukin2 receptor a (sIL-2ra), cluster of differentiation 40 ligand (CD40 L/CD154), and TNF-b showed a significant increase only in reversible pulpitis than the control (Table 1, panel C). Mediators including some angiogenic factors like vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF AA/AB) and regulated upon activation normal T cell expressed and secreted (RANTES/CCL5) showed a marked increase in reversible pulpitis compared with the control,
but irreversible pulpitis samples had levels similar to the levels in the control tissues (Table 1, panel D). Fractalkine/CX3CL1 and flt3ligand expression did not follow any of the previous panels and instead only decreased in irreversible pulpitis and eotaxin/CCL11, which only increased in irreversible pulpitis (Table 1, panel E).
OCN Expression in Pulp Tissue Pulps from impacted teeth did not show evidence of OCN expression by immunohistochemistry (Fig. 2A–C). In the inflamed samples,
Figure 2. Immunohistochemical localization of OCN in inflamed dental pulps. Representative photographs of H&E staining of pulp tissue from (A) impacted molars (control) and tissue sections when stained with (B) mIgG1 (isotype control) or (C) mouse antihuman OCN showing the absence of OCN staining in normal pulps. D, E, and F are representative photographs from inflamed human dental pulp stained with (D) H&E, (E) mIgG, and (F) anti-OCN. Calcification areas are seen in H&E. (F) Intense OCN staining is seen at the borders of calcification areas (arrowhead) (40 magnifications). G, H, and I are pictures of higher magnification of the squared areas in D, E, and F. (I) OCN (red) staining was absent in the calcification area (X) an intense in extracellular matrix (ECM) and in cells surrounding this area (arrow). (J) Inflamed pulp tissue with areas of fibrosis when stained with H&E followed by (K) the negative staining in the isotype control picture and (L) the positive staining of fibers to OCN antibody (arrowhead) and in the matrix surrounding the fibers (asterisk). (M) Another inflamed dental pulp showing blood vessels and pericytes stained with H&E. (N) The isotype control showing negative staining to any part of the tissue. (O) OCN staining is present in the blood vessel wall (arrowhead), cells around the vasculature (arrow), and the ECM (asterisk) of the tissue borders.
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Basic Research—Biology areas of calcification as determined by the dark hematoxylin staining did not show any OCN staining; however, OCN was intensely localized in cells and matrix around these areas (Fig. 2D–I). All inflamed pulps with areas of fibrosis were OCN positive (Fig. 2J–L). Where present, cells in the peripheries of the inflamed tissues showed OCN-positive staining and cells around the blood vessels (Fig. 2N–O). Walls of blood vessels in the inflamed tissues were also OCN immunopositive (Fig. 2O). The presence of OCN in pulp tissue was also measured using a plex assay (Evetechnologies). Reversible and irreversible pulpitis tissues had higher OCN levels compared with the control tissues (Fig. 3A). OCN levels were higher in reversible pulpitis compared with irreversible pulpitis.
Correlation between OCN Expression and Other Inflammatory Mediators To improve our understanding of the pathophysiology of pulpitis, we examined the correlation between the levels of OCN and other inflammatory mediators in pulp tissue. There was no correlation between these measurements in normal pulps. The correlation analyses in the reversible stages of pulp inflammation showed the presence of a positive correlation between OCN, FGF, VEGF, MCP-1, MDC, MIP-1b, and sIL-2ra. It was also found that OCN is negatively correlated to IL-1b, IL-8, IL-1a, and MIP-1a. Only MIP-1b and sIL-2ra maintained their positive correlation to OCN in the irreversible stages of pulp inflammation. IL-1a and IL-1b continued to negatively correlate to OCN in irreversible stages of pulp inflammation. Samples of reversible pulpitis were added to those of irreversible pulpitis to compare between the expression of OCN and the inflammatory mediators in both stages of pulp inflammation together (generalized pulp inflammation) (Fig. 3B and C). In addition to those proteins that showed positive correlation in reversible pulpitis, CD40L was found to be positively associated to OCN in generalized pulp inflammation; however, GM-CSF and IL-1ra were negatively correlated to OCN expression.
Discussion The current study has shown that OCN expression increased in inflamed pulps compared with normal pulps. The data of the present study regarding OCN expression in fibrotic areas are in line with what was previously shown in a rat model of tooth injury (10). Our data indicate that OCN is expressed in reversible more than in irreversible pulpitis, and pulp calcification in reversible pulpitis is more pronounced than in irreversible pulpitis. This would suggest the importance of OCN in mineralization and is consistent with a previous report regarding the development of pulp fibrosis and calcification in chronic pulpitis without irreversible changes (27). OCN was expressed in the pulp cells related to calcification areas although it was not expressed in these areas. This is in agreement with Ninomiya et al (28), who did not localize OCN in human pulp stones. OCN localization in cells around calcification areas can be explained by the fact that it is considered a marker of osteoblast differentiation (15, 16). OCN was also expressed by human pulp cells after the use of osteogenic induction medium (12). In response to injury, pulp cells migrate, proliferate, and differentiate into odontoblast-like cells to lay down new reparative dentin (5). The mechanism of pulp cell response to injury explains the immunolocalization of OCN at the peripheries of inflamed pulp tissues because OCN is considered one of the late differentiation markers of odontoblasts (12, 13). The extraction of pulp tissues in our experiment was performed by a size #10 K-file, which removed the pulp tissue without the odontoblastic cell layer (hence, the lack of immunolocalization of OCN in the control group). However, there was a minimal expression of OCN in normal pulps from impacted teeth in the human plex assay; this is probably related to the difference in sensitivity between the 2 assays. This assay helped identify the difference between OCN expression in reversible versus irreversible stages of pulp inflammation, which may be explained by the difference in cell number between both stages (ie, fewer cells are present to produce OCN in irreversible stages of pulp inflammation). It can be speculated that a specific cell that increases in number in
Figure 3. The expression of OCN in reversible and irreversible pulpitis and representative cytokines positive and negative correlation to its expression. (A) Values of OCN expression in pg/mg protein (dots) are presented in control pulps from impacted teeth (normal pulp, n = 12) and in reversible (n = 11) and irreversible (n = 6) pulpitis. The line between the dots represents the mean. *P <.05 was considered significant. (B) A positive correlation (Pearson coefficient) exists between the expression of OCN and the expression of FGF (P = .0053) in pulpitis cases. (C) A negative correlation (Pearson coefficient) exists between the expression of OCN and IL-1b (P = .0412) in pulpitis cases. The values are in pg cytokine/mg total protein in the sample. A P value <.05 was considered significant.
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Basic Research—Biology inflamed pulps is expressing OCN. This speculation is based on the fact that macrophage cells are dominant in pulp inflammation (17) and express OCN in their transdifferentiation to osteoblasts (16). Further in vitro experiments are needed to support such speculation. In a pulp capping model of pulp repair, the amount of dentinal bridge formation in the severe type of pulp inflammation is negligible when compared with mild, moderate, or no pulp inflammation (29), which questions the presence of OCN in the irreversible stage of pulp inflammation. In an attempt to identify a possible role for OCN in pulp inflammation, the inflammatory mediators inside the same inflamed pulps were measured to be able to correlate between their presence and the presence of OCN. The human multiplex assay analyses revealed the expression of inflammatory mediators corresponding to pulp inflammation. Some of these mediators have been previously described in dental pulp inflammation (1, 4, 6–9, 23, 30). The expression of inflammatory mediators was discussed in reports, which depended on the caries level in dentin or whether the pulp comes from a carious diseased or nondiseased tooth. We believe that evaluating a large panel of inflammatory mediators in a clinically determined stage of pulp inflammation would provide a better understanding of what is happening at the molecular level in clinically diagnosed pulpitis. Further clinical correlation experiments between the detailed clinical diagnostic criteria and the expression of these mediators of inflammation would potentially help in the identification of pulp inflammatory markers and accordingly the development of a new therapeutic intervention at a specific clinical stage of pulp inflammation. In the current study, markers of angiogenesis (ie, VEGF, PDGF, and FGF) have shown an increase in reversible stages of dental pulp inflammation, and this increase was positively correlated to the expression of OCN. VEGF and FGF are crucial elements for dental pulp repair in response to injury (31). In an in vivo model of angiogenesis, OCN induced a similar pattern of healing to that of FGF (32). Interestingly, FGF and VEGF were shown to induce OCN gene expression in preosteoblast cells (33, 34). OCN was also expressed in reversible pulpitis corresponding to the expression of the proinflammatory mediators MCP-1, MIP-1b, sIL-2ra, and MDC. OCN expression in reversible pulpitis was found to be inversely proportional to the expression of IL-1a, IL-1b, IL-8, and MIP-1a, and this negative correlation continued with the progression of pulpal inflammation in irreversible pulpitis together with the expression of IL-1a and IL-1b. These data are supported by the finding that MCP-1 is positively correlated to OCN in the human serum of lean individuals (35) and by reports (36, 37) that showed the inhibition of OCN production from osteoblast-like cells after their stimulation with IL-1a or IL-1b and from bone marrow stromal cells after their stimulation with MIP-1a (38). Considering these facts collectively, it can be speculated that OCN in reversible pulpitis plays a role in regulating dental pulp repair. This role is decreased with the progression of inflammation and the expression of catabolic molecules like IL-1a and IL-1b. However, further ex vivo experiments are needed to confirm this speculation. In conclusion, the expression of OCN was shown inside the inflamed dental pulp, and inflammatory mediators were found related to this expression especially in the reversible stages of pulpal inflammation. OCN is positively correlated to angiogenic markers such as FGF and VEGF and is negatively correlated to catabolic molecules frequently expressed in deep carious lesions such as IL-1. A clearer understanding of how these reparative molecules act inside the inflamed dental pulp and questions regarding cell-matrix, cell-cell, and molecular interactions need to be addressed before these reparative molecules are used clinically. JOE — Volume 39, Number 7, July 2013
Acknowledgments The authors would like to thank Dr Arwa Siyam for her help with the preparation of tissue sections and Mr Andrew Kim for his reading of the histologic studies blindly. The authors deny any conflicts of interest related to this study.
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35. Sukumar D, Partridge NC, Wang X, et al. The high serum monocyte chemoattractant protein-1 in obesity is influenced by high parathyroid hormone and not adiposity. J Clin Endocrinol Metab 2011;96:1852–8. 36. Beresford JN, Gallagher JA, Gowen M, et al. The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro. Biochim Biophys Acta 1984;801:58–65. 37. Taichman RS, Hauschka PV. Effects of interleukin-1 beta and tumor necrosis factoralpha on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro. Inflammation 1992;16:587–601. 38. Vallet S, Pozzi S, Patel K, et al. A novel role for CCL3 (MIP-1alpha) in myelomainduced bone disease via osteocalcin downregulation and inhibition of osteoblast function. Leukemia 2011;25:1174–81.
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Osteocalcin Expression in Pulp Inflammation Ashraf Abd-Elmeguid, BDS, MDSc, PhD,* Marwa Abdeldayem, BDS, DPH, MSc,‡ Loren W. Kline, BSc, MSc, PhD,* Redwan Moqbel, PhD, FRCPPath,§ Harrisios Vliagoftis, MD,† and Donald C. Yu, DMD, CAGS, MScD, FRCD(C)* Abstract Introduction: Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. Although OCN is considered a reparative molecule inside the dental pulp, it is not clear if it is involved in pulpal inflammation. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation. Methods: Pulp tissues in the form of reversible and irreversible pulpitis were collected from the endodontic clinic. Those from impacted teeth were used as controls. Immunohistochemistry was used to localize OCN. Samples were analyzed for OCN and inflammatory mediator expression using multiplex assay. Results: OCN in inflamed tissues was localized in cells and matrix around calcification areas and in cells around blood vessels but not in normal tissues. The plex assay (Bio-Plex 200, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) showed OCN expression in reversible pulpitis significantly higher than in irreversible pulpitis, and both were significantly higher than in the controls. A panel of inflammatory mediators showed an increase in reversible and irreversible pulpitis. Another panel was decreased in both stages compared with the controls. OCN expression in reversible pulpitis was positively correlated to the expression of vascular endothelial growth factor, fibroblast growth factor, macrophage inflammatory protein-1b, monocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin (IL)-17, and soluble IL-2 receptor a and negatively correlated to that of IL-1a, IL-1b, IL-8, granulocyte macrophage colonystimulating factor, and macrophage inflammatory protein-1a. Conclusions: Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment. (J Endod 2013;39:865–872)
Key Words Dental pulp calcification, inflammatory mediators, osteocalcin, pulp inflammation
P
ulpal repair in response to tooth injury caused by caries, trauma, or cavity preparation is a complex process that includes matricellular interactions involving inflammation (1, 2). Cariogenic bacteria soften the dentin as it proceeds toward the dental pulp allowing the leaching out of dentinal matrix composed of many reparative proteins. As caries progress toward the dental pulp, inflammatory cells increase in number and secrete cytokines/chemokines (3, 4). At the same time, reparative molecules are secreted to compensate for the loss in dentinal structure forming reactionary or reparative dentin depending on the presence or absence of odontoblasts (5). Many inflammatory mediators were reported inside the inflamed dental pulp (1, 4, 6–9). However, little is known regarding the relation of these mediators to the reparative molecules involved in reparative/reactionary dentinogenesis. Osteocalcin (OCN) is one of the reparative molecules commonly expressed in response to injury of the dental pulp. It is expressed in response to cavity preparation in which it is associated with collagen fibers and is found embedded inside the tertiary dentin (10). Its gene was up-regulated in acute dental traumatic injury (11). OCN is an osteoblast marker and is considered to be a late differentiation marker of odontoblasts (12, 13). Its role in mineralization is not very clear. However, it is suggested to play an important role in mediating osteoclastic differentiation (14). OCN was found to be related to macrophage protein such as granulocyte macrophage colony-stimulating factor (GM-CSF) in bone-forming cells (15). Macrophages express OCN in their terminal differentiation to osteoblasts (16). Macrophages are the dominant inflammatory cells in pulp inflammation (17). Tumor necrosis factor (TNF) induced the expression of OCN from dental papilla cells (18). Is OCN related to the inflammatory changes inside the dental pulp? When OCN was administered to orthodontically treated teeth, a condition known for its inflammatory nature, it enhanced teeth movement (19), and OCN messenger RNA expression was increased inside the dental pulp of these teeth (20). In a recent study, OCN was expressed in response to the stimulation of pulp cells by a molecule with a known inflammatory role, and this was concomitant with the expression of angiogenic markers (21). Goldberg et al (2) and Cooper et al (1) discussed the inflammatory changes inside the dental pulp as a vital part of dental pulp repair in response to injury. In addition, reparative molecules are involved in the inflammatory process of the pulp (22, 23). It was shown also that fibrosis and calcification are increased in inflamed pulps compared with noninflamed ones (24). Therefore, the objectives of this study were
From the *School of Dentistry and †Division of Pulmonary Medicine, University of Alberta, Edmonton, Alberta, Canada; ‡Department of Orthodontics, Boston University, Boston, Massachusetts; and §Department of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada. Supported by the Egyptian Bureau of cultural affairs in Canada (grant no. G700000335), the Fund For Dentistry, University of Alberta (grant no. 05-2007), the Canadian Academy of Endodontics endowment fund (grant no. 000000582), and the Alpha Omega Foundation of Canada (grant no. 04-2007). A.A.-E. received a scholarship from the Ministry of Higher Education in Egypt and Medical sciences graduate program scholarship from the faculty of Medicine and Dentistry at the University of Alberta. Address requests for reprints to Dr Donald C. Yu, Endodontic Division, School of Dentistry, Faculty of Medicine and Dentistry, University of Alberta, 5-546 Edm Clinic Health Academy, Edmonton, AB, T6G 1C9, Canada. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.12.035
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Materials and Methods Collection of Samples and Histologic Examination Thirty-five inflamed pulps were collected from the Endodontic Clinic at the University of Alberta, Edmonton, Alberta, Canada, according to the protocol approved by the University of Alberta Human Research Ethics Board. As described previously (7), pulps of cases with reversible pulpitis (n = 23) referred to the endodontic clinic for prosthetic purposes and pulps with irreversible pulpitis (n = 12) were used in this study. Clinically, teeth with reversible pulpitis are sensitive to cold (not to hot) and have no spontaneous, lingering pain. Teeth with irreversible pulpitis are clinically sensitive to hot with spontaneous lingering pain. Thirty pulps from impacted caries-free teeth were collected, considered as normal pulps, and used as controls. The pulps used for histologic examination were collected in 4% paraformaldehyde and processed for paraffin embedding, and 5-mm sections were stained with hematoxylin-eosin (H&E) and Gomori trichrome stains. H&E-stained tissues were examined under light microscope; cells of the dental pulp tissue were counted under the same magnification (400) in 3 different sections and 3 different high-power fields per section. If the tissue had more than 1 piece per slide (ie, torn), 3 different locations per piece of tissue were used for the examination, and the average of the 3 counts was calculated and tabulated for statistical analysis. Tissues were also examined for calcification areas. When present, images of these areas were taken then were transferred to ImageJ analysis software (National Institutes of Health, Bethesda, MD) to calculate the surface area of calcification (in millimeters). The percentages of these areas to the whole area of the tissue in the image were then calculated. The steps performed were previously described in a previous report (25). The trichrome stain stains collagen fibers (fibrosis) green, preventing the light from being reflected and decreasing its intensity under the microscope. In the presence of denser fibers, the light intensity was measured using ImageJ analysis software. The intensity was tabulated and submitted for statistical analysis. All histologic evaluation and tabulation were performed blindly to eliminate bias. Analysis of Inflammatory and Remodeling Cytokines/ Chemokines Using a Multiplex Assay Tissues were collected in tissue lysis buffer (Cellytic T; SigmaAldrich, St Louis, MO) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich), homogenized, and centrifuged at 14,000g for 15 minutes at 2 C followed by the collection of the supernatant. The protein concentration was measured using the Pierce BCA protein assay kit (Thermo Scientific Pierce Biotechnology, Rockford, IL), and a similar amount of proteins in each sample was sent to Evetechnologies (Calgary, Alberta, Canada) for human multiplex assay (Bio-Plex 200; Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) analysis as described previously (26). The protocol and the 42 analytes measured are listed on the company’s website (www.Evetechnologies.com). Immunohistochemical Localization of OCN and OCN Measurement in Pulp Tissues Samples collected, as previously explained, were examined for the presence of OCN by immunohistochemical staining as performed previously (23). After deparaffinization using serial dilutions of ethanol and xylene, tissues were washed with phosphate-buffered saline (PBS) twice for 5 minutes each followed by permeabilization using 0.5% Triton X100 in PBS for 5 minutes and then blocking unspecific protein binding 866
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by serum-free blocking solution (Dako Canada Inc, Mississauga, ON, Canada) for 10 minutes at room temperature. Tissues were then incubated overnight at 4 C with monoclonal mouse antihuman OCN antibody (10 mg/mL) (R&D Systems, Inc, Minneapolis, MN) or incubated with mouse immunoglobulin G1 (10 mg/mL) (Biolegend, San Diego, CA) as isotype controls. Tissue slides were washed 4 times in PBS (5 minutes each) to eliminate any unspecific binding and then were incubated with alkaline phosphatase–conjugated antimouse secondary antibody (1:100) (Sigma-Aldrich) in a humid chamber for 1 hour at room temperature followed by washing in PBS. The signal was developed using the AP Vector red substrate kit (Vector Laboratories Inc, Burlingame, CA). Counterstaining with hematoxylin was performed next, and slides were kept overnight in the dark. Tissue slides were then examined for OCN staining under light microscope. All antibodies and controls were diluted in antibody diluents with background reducing component (Dako Inc). Samples collected for the plex assay were treated as previously described and sent to Evetechnologies. Data from the plex analysis (OCN) were used together with data from the multiplex analysis (inflammatory cytokines/chemokines) for the same tissues to correlate between the level of OCN and other inflammatory mediators.
Statistical Analysis The Student’s t test was used to compare samples of reversible and irreversible pulpitis in terms of percentage of calcification areas. The analysis of variance test was used to compare cell counts in pulp tissues and collagen amounts as shown by light intensity measured by using ImageJ software. The same test also was used to compare inflammatory mediator expression and OCN expression by human plex assay (Bio-Rad Laboratories Ltd) in normal, reversible, and irreversible pulpitis. Samples of the multiplex assay that were extrapolated beyond the standard range were not assigned a value. A statistical value of P < .05 was considered significant. Correlation analysis using Pearson’s correlation coefficient was used to correlate OCN expression with each mediator expression in inflamed (reversible and irreversible pulpitis) pulps; a P value <.05 was considered significant for either a positive or negative correlation.
Results Presence of Inflammatory Cells and Calcification in Pulpitis Dental pulp cells were counted blindly in slides stained with H&E. Representative pictures of control pulp (Fig. 1A) and pulps with reversible (Fig. 1B) and irreversible pulpitis (Fig. 1C) are shown. The control group and irreversible pulpitis group had lower cell counts compared with the reversible pulpitis group. There was no significant difference between the irreversible stage of pulp inflammation and the noninflamed group (control) (Fig. 1D). Trichrome staining was used to evaluate the degree of fibrosis in our samples. Quantitation of fibrosis showed that the control group (Fig. 1E) was the lowest in collagen staining compared with the reversible (Fig. 1F) and irreversible (Fig. 1G) stages of pulp inflammation; these differences were significant (P < .0001) (Fig. 1H). Pulps from impacted teeth had no evidence of calcification in contrast to 78% of the total number of tissues with reversible pulpitis and 66% of the total number of tissues with irreversible pulpitis. The percentage of calcification areas in tissues with reversible pulpitis was significantly higher than that in irreversible pulpitis (Fig. 1I–L). Presence of Inflammatory Mediators in the Pulp Tissue A human multiplex assay analysis was performed to identify the presence of different inflammatory and remodeling mediators in pulp JOE — Volume 39, Number 7, July 2013
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Figure 1. Cell number and fibrosis of reversible and irreversible pulpitis. (A) H&E staining of the pulp of an impacted molar (control) showing a decreased cell count; (B) the cell number (arrows) is profoundly increased in reversible stages of pulpitis and (C) then tends to decrease again in irreversible stages of pulpitis. Pictures show representative samples for the cell count under the same field microscope magnification (400), which is statistically significant when comparing the cell number of reversible pulpitis with the control (normal) and that of reversible pulpitis to irreversible ones. (D) Although the total cell count in irreversible pulpitis was higher than in the control, it was not statistically significant. (E) Gomori trichrome staining of the pulp from impacted tooth (control). (F) The degree of green (asterisks) (collagen) increased in reversible stages of pulpitis and (G) the irreversible stages of pulpitis (G). (H) Data collected using Image G analysis software showed that the light intensity masked by the green in the samples decreased significantly in reversible and irreversible pulpitis than the control. (I) Pulp tissues from impacted teeth (n = 15) showed no areas of calcification (letter X), whereas (J) reversible (n = 11) and (K) irreversible (n = 7) pulpitis (L) developed calcification areas that were significant compared with the control and each other with the reversible stages having a higher percentage of calcification areas. Data were considered significant at P value <.05.
tissue and measure their levels. The mean expression in pg/mg protein in normal, reversible, and irreversible pulpitis for each inflammatory mediator and the statistical analysis are shown in Table 1. Inflammatory mediators commonly expressed in the inflamed dental pulp like interleukin (IL)-1a, IL-1b, IL-1ra, IL-4, IL-6, IL-8/CXCL8, and TNF and other inflammatory mediators like monocyte chemoattractant protein-1 (MCP-1/CCL2), MCP-3/CCL7, monocyte-derived chemokine (MDC), macrophage inflammatory protein-1a (MIP-1a/CCL3), JOE — Volume 39, Number 7, July 2013
MIP-1b/CCL4, GM-CSF, and growth-regulated protein not previously reported in the inflamed pulp showed a significant increase in reversible and irreversible stages of pulp inflammation when compared with the control (Table 1, panel A). Another set of inflammatory mediators including IL-7, interferon a, transforming growth factor a, granulocytecolony stimulating factor, and IL-13 showed a decrease in both reversible and irreversible pulpitis when compared with the control (Table 1, panel B). Some mediators like interferon g–induced protein-10 (IP-
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Panel
A
B
C
D JOE — Volume 39, Number 7, July 2013
E
Normal pulps (n = 10–12), mean ± standard error (pg/mg protein)
Reversible pulpitis (n = 9–11), mean ± standard error (pg/mg protein)
GM-CSF GRO IL-1a IL-1b IL-1ra IL-4 IL-6 IL-8 IL-12 p40 MCP-1 MCP-3 MDC MIP-1a MIP-1b TNF-a
0.21 0.15 2.58 1.26 6.04 0.97 2.11 0.55 4.75 1.04 0.90 0.37 0.00 0.00 0.23 0.23 0.00 0.00 16.83 2.15 1.93 1.30 17.41 5.10 6.23 3.54 1.85 1.22 0.00 0.00
7.08 2.08 150.60 59.98 23.48 4.45 17.39 6.19 16.39 4.51 3.08 0.51 15.95 5.53 258.00 101.90 5.09 1.63 255.80 98.34 19.68 4.61 100.80 33.65 37.57 9.88 45.81 17 2.76 1.15
IFN-a G-CSF IL-7 IL-13 TGF-a
38.88 4.34 6.75 1.23 4.23 0.37 2.60 0.39 10.07 1.46
21.81 4.63 1.63 0.90 0.51 0.34 0.50 0.33 4.94 0.74
*
CD40L sIL-2ra IL-15 TNF-b IP-10
3.28 1.13 1.76 0.33 0.00 0.00 0.05 0.05 15.04 3.25
25.57 3.75 17.37 5.89 2.88 0.51 0.58 0.18 379.80 216
‡
Molecule
P value, reversible pulpitis vs normal * † † †
* * †
* * ‡ ‡ †
* †
* † ‡
* * ‡ †
* †
FGF PDGF-AA PDGF-AB/BB RANTES VEGF
488.80 49.10 2.60 0.26 41.54 4.74 27.46 4.29 75.12 7.77
1487 170.80 14.43 3.80 351.6 125.30 490.60 146.40 145.1 15.44
*
Eotaxin flt3ligand Fractalkine
0.00 0.00 103.90 7.95 146.8 14.08
5.66 2.93 78.54 16.24 311.9 78.15
NS NS NS
† ‡ ‡ †
GRO, growth-regulated protein; IFN, interferon; NS, not significant. Minimal expression and ns between all groups: IFN-g, IL-12p70, and IL-17. No expression in all groups: EGF, IL-2, IL-3, 1L-5, IL-9, and IL-10. *P value <.05. † P value <.01. ‡ P value <.001.
Irreversible pulpitis (n = 5–6), mean ± standard error (pg/mg protein) 21.12 6.50 53.09 12.64 31.13 5.33 12.34 1.28 17.33 4.08 3.18 0.46 7.82 4.88 371.40 119.10 32.96 19.40 110.70 31.69 13.10 2.074 56.79 6.53 52.02 12.60 81.36 26.63 3.08 0.42
P value, irreversible pulpitis vs normal
P value, irreversible vs reversible pulpitis
‡
NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS
* ‡ †
* * * † †
* * * † ‡ †
18.52 2.50 0.99 0.46 0.00 0.00 0.00 0.00 1.25 0.83
* *
12.23 2.76 4.51 0.69 1.52 0.57 0.09 0.09 25.86 6.08
NS NS NS NS NS
164.50 79.46 6.74 1.63 33.28 16.08 77.64 28.93 45.70 30.93
NS NS NS NS NS
11.22 5.31 8.39 2.89 14.82 7.48
†
‡
* ‡
‡
*
NS NS NS NS NS NS NS NS NS NS ‡
NS †
* †
NS * †
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868 TABLE 1. Expression of Inflammatory Mediators in Normal Pulps and Reversible and Irreversible Pulpitis
Basic Research—Biology 10/CXCL10), IL-15, soluble interleukin2 receptor a (sIL-2ra), cluster of differentiation 40 ligand (CD40 L/CD154), and TNF-b showed a significant increase only in reversible pulpitis than the control (Table 1, panel C). Mediators including some angiogenic factors like vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF AA/AB) and regulated upon activation normal T cell expressed and secreted (RANTES/CCL5) showed a marked increase in reversible pulpitis compared with the control,
but irreversible pulpitis samples had levels similar to the levels in the control tissues (Table 1, panel D). Fractalkine/CX3CL1 and flt3ligand expression did not follow any of the previous panels and instead only decreased in irreversible pulpitis and eotaxin/CCL11, which only increased in irreversible pulpitis (Table 1, panel E).
OCN Expression in Pulp Tissue Pulps from impacted teeth did not show evidence of OCN expression by immunohistochemistry (Fig. 2A–C). In the inflamed samples,
Figure 2. Immunohistochemical localization of OCN in inflamed dental pulps. Representative photographs of H&E staining of pulp tissue from (A) impacted molars (control) and tissue sections when stained with (B) mIgG1 (isotype control) or (C) mouse antihuman OCN showing the absence of OCN staining in normal pulps. D, E, and F are representative photographs from inflamed human dental pulp stained with (D) H&E, (E) mIgG, and (F) anti-OCN. Calcification areas are seen in H&E. (F) Intense OCN staining is seen at the borders of calcification areas (arrowhead) (40 magnifications). G, H, and I are pictures of higher magnification of the squared areas in D, E, and F. (I) OCN (red) staining was absent in the calcification area (X) an intense in extracellular matrix (ECM) and in cells surrounding this area (arrow). (J) Inflamed pulp tissue with areas of fibrosis when stained with H&E followed by (K) the negative staining in the isotype control picture and (L) the positive staining of fibers to OCN antibody (arrowhead) and in the matrix surrounding the fibers (asterisk). (M) Another inflamed dental pulp showing blood vessels and pericytes stained with H&E. (N) The isotype control showing negative staining to any part of the tissue. (O) OCN staining is present in the blood vessel wall (arrowhead), cells around the vasculature (arrow), and the ECM (asterisk) of the tissue borders.
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Basic Research—Biology areas of calcification as determined by the dark hematoxylin staining did not show any OCN staining; however, OCN was intensely localized in cells and matrix around these areas (Fig. 2D–I). All inflamed pulps with areas of fibrosis were OCN positive (Fig. 2J–L). Where present, cells in the peripheries of the inflamed tissues showed OCN-positive staining and cells around the blood vessels (Fig. 2N–O). Walls of blood vessels in the inflamed tissues were also OCN immunopositive (Fig. 2O). The presence of OCN in pulp tissue was also measured using a plex assay (Evetechnologies). Reversible and irreversible pulpitis tissues had higher OCN levels compared with the control tissues (Fig. 3A). OCN levels were higher in reversible pulpitis compared with irreversible pulpitis.
Correlation between OCN Expression and Other Inflammatory Mediators To improve our understanding of the pathophysiology of pulpitis, we examined the correlation between the levels of OCN and other inflammatory mediators in pulp tissue. There was no correlation between these measurements in normal pulps. The correlation analyses in the reversible stages of pulp inflammation showed the presence of a positive correlation between OCN, FGF, VEGF, MCP-1, MDC, MIP-1b, and sIL-2ra. It was also found that OCN is negatively correlated to IL-1b, IL-8, IL-1a, and MIP-1a. Only MIP-1b and sIL-2ra maintained their positive correlation to OCN in the irreversible stages of pulp inflammation. IL-1a and IL-1b continued to negatively correlate to OCN in irreversible stages of pulp inflammation. Samples of reversible pulpitis were added to those of irreversible pulpitis to compare between the expression of OCN and the inflammatory mediators in both stages of pulp inflammation together (generalized pulp inflammation) (Fig. 3B and C). In addition to those proteins that showed positive correlation in reversible pulpitis, CD40L was found to be positively associated to OCN in generalized pulp inflammation; however, GM-CSF and IL-1ra were negatively correlated to OCN expression.
Discussion The current study has shown that OCN expression increased in inflamed pulps compared with normal pulps. The data of the present study regarding OCN expression in fibrotic areas are in line with what was previously shown in a rat model of tooth injury (10). Our data indicate that OCN is expressed in reversible more than in irreversible pulpitis, and pulp calcification in reversible pulpitis is more pronounced than in irreversible pulpitis. This would suggest the importance of OCN in mineralization and is consistent with a previous report regarding the development of pulp fibrosis and calcification in chronic pulpitis without irreversible changes (27). OCN was expressed in the pulp cells related to calcification areas although it was not expressed in these areas. This is in agreement with Ninomiya et al (28), who did not localize OCN in human pulp stones. OCN localization in cells around calcification areas can be explained by the fact that it is considered a marker of osteoblast differentiation (15, 16). OCN was also expressed by human pulp cells after the use of osteogenic induction medium (12). In response to injury, pulp cells migrate, proliferate, and differentiate into odontoblast-like cells to lay down new reparative dentin (5). The mechanism of pulp cell response to injury explains the immunolocalization of OCN at the peripheries of inflamed pulp tissues because OCN is considered one of the late differentiation markers of odontoblasts (12, 13). The extraction of pulp tissues in our experiment was performed by a size #10 K-file, which removed the pulp tissue without the odontoblastic cell layer (hence, the lack of immunolocalization of OCN in the control group). However, there was a minimal expression of OCN in normal pulps from impacted teeth in the human plex assay; this is probably related to the difference in sensitivity between the 2 assays. This assay helped identify the difference between OCN expression in reversible versus irreversible stages of pulp inflammation, which may be explained by the difference in cell number between both stages (ie, fewer cells are present to produce OCN in irreversible stages of pulp inflammation). It can be speculated that a specific cell that increases in number in
Figure 3. The expression of OCN in reversible and irreversible pulpitis and representative cytokines positive and negative correlation to its expression. (A) Values of OCN expression in pg/mg protein (dots) are presented in control pulps from impacted teeth (normal pulp, n = 12) and in reversible (n = 11) and irreversible (n = 6) pulpitis. The line between the dots represents the mean. *P <.05 was considered significant. (B) A positive correlation (Pearson coefficient) exists between the expression of OCN and the expression of FGF (P = .0053) in pulpitis cases. (C) A negative correlation (Pearson coefficient) exists between the expression of OCN and IL-1b (P = .0412) in pulpitis cases. The values are in pg cytokine/mg total protein in the sample. A P value <.05 was considered significant.
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Basic Research—Biology inflamed pulps is expressing OCN. This speculation is based on the fact that macrophage cells are dominant in pulp inflammation (17) and express OCN in their transdifferentiation to osteoblasts (16). Further in vitro experiments are needed to support such speculation. In a pulp capping model of pulp repair, the amount of dentinal bridge formation in the severe type of pulp inflammation is negligible when compared with mild, moderate, or no pulp inflammation (29), which questions the presence of OCN in the irreversible stage of pulp inflammation. In an attempt to identify a possible role for OCN in pulp inflammation, the inflammatory mediators inside the same inflamed pulps were measured to be able to correlate between their presence and the presence of OCN. The human multiplex assay analyses revealed the expression of inflammatory mediators corresponding to pulp inflammation. Some of these mediators have been previously described in dental pulp inflammation (1, 4, 6–9, 23, 30). The expression of inflammatory mediators was discussed in reports, which depended on the caries level in dentin or whether the pulp comes from a carious diseased or nondiseased tooth. We believe that evaluating a large panel of inflammatory mediators in a clinically determined stage of pulp inflammation would provide a better understanding of what is happening at the molecular level in clinically diagnosed pulpitis. Further clinical correlation experiments between the detailed clinical diagnostic criteria and the expression of these mediators of inflammation would potentially help in the identification of pulp inflammatory markers and accordingly the development of a new therapeutic intervention at a specific clinical stage of pulp inflammation. In the current study, markers of angiogenesis (ie, VEGF, PDGF, and FGF) have shown an increase in reversible stages of dental pulp inflammation, and this increase was positively correlated to the expression of OCN. VEGF and FGF are crucial elements for dental pulp repair in response to injury (31). In an in vivo model of angiogenesis, OCN induced a similar pattern of healing to that of FGF (32). Interestingly, FGF and VEGF were shown to induce OCN gene expression in preosteoblast cells (33, 34). OCN was also expressed in reversible pulpitis corresponding to the expression of the proinflammatory mediators MCP-1, MIP-1b, sIL-2ra, and MDC. OCN expression in reversible pulpitis was found to be inversely proportional to the expression of IL-1a, IL-1b, IL-8, and MIP-1a, and this negative correlation continued with the progression of pulpal inflammation in irreversible pulpitis together with the expression of IL-1a and IL-1b. These data are supported by the finding that MCP-1 is positively correlated to OCN in the human serum of lean individuals (35) and by reports (36, 37) that showed the inhibition of OCN production from osteoblast-like cells after their stimulation with IL-1a or IL-1b and from bone marrow stromal cells after their stimulation with MIP-1a (38). Considering these facts collectively, it can be speculated that OCN in reversible pulpitis plays a role in regulating dental pulp repair. This role is decreased with the progression of inflammation and the expression of catabolic molecules like IL-1a and IL-1b. However, further ex vivo experiments are needed to confirm this speculation. In conclusion, the expression of OCN was shown inside the inflamed dental pulp, and inflammatory mediators were found related to this expression especially in the reversible stages of pulpal inflammation. OCN is positively correlated to angiogenic markers such as FGF and VEGF and is negatively correlated to catabolic molecules frequently expressed in deep carious lesions such as IL-1. A clearer understanding of how these reparative molecules act inside the inflamed dental pulp and questions regarding cell-matrix, cell-cell, and molecular interactions need to be addressed before these reparative molecules are used clinically. JOE — Volume 39, Number 7, July 2013
Acknowledgments The authors would like to thank Dr Arwa Siyam for her help with the preparation of tissue sections and Mr Andrew Kim for his reading of the histologic studies blindly. The authors deny any conflicts of interest related to this study.
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