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J Dent Res 82(8): 592-596, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Adhesive Resin Induces Apoptosis and Cell-cycle Arrest of Pulp Cells

M.G. Mantellini1, T.M. Botero1, P. Yaman1, J.B. Dennison1, C.T. Hanks2, and J.E. Nör1,*

1 Department of Cariology, Restorative Sciences, and Endodontics, and
2 Department of Oral Medicine, Oral Pathology, and Oral Oncology, University of Michigan School of Dentistry, 1011 N. University, Rm. 5211, Ann Arbor, MI 48109-1078, USA;

* corresponding author, jenor{at}umich.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The application of an adhesive resin near or directly over the pulp was shown to induce pulp inflammation and lack of dentin regeneration. We hypothesize that the absence of dentin bridging is due to adhesive-resin-induced apoptosis of cells responsible for pulp healing and dentin regeneration. Mouse odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), or macrophages (RAW 264.7) were exposed to SingleBond polymerized for 0–40 seconds. Annexin V and propidium iodide assays demonstrated that SingleBond induced apoptosis of MDPC-23, OD-21, and macrophages. The proportion of apoptotic cells was dependent on the degree of adhesive resin polymerization. Adhesive-resin-induced death of pulp cells was associated with activation of the pro-apoptotic cysteine protease Caspase-3. Interestingly, most cells exposed to adhesive resin that did not undergo apoptosis showed cell-cycle arrest. We conclude that an adhesive resin induces apoptosis and cell-cycle arrest of cells involved in the regeneration of the dentin-pulp complex in vitro.

KEY WORDS: dentin • bonding • pulp capping • odontoblast • macrophage


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adhesive resin systems are used to enhance retention, to reduce microleakage, and to decrease post-operative sensitivity of composite resin restorations. In vivo studies have demonstrated that the application of an adhesive resin directly onto a site of pulp exposure, or to a thin layer of dentin (< 0.5 mm), causes dilatation and congestion of blood vessels, inflammation, and pulp abscesses (Hebling et al., 1999a,b). Importantly, no dentin bridge can be seen in the majority of human teeth treated with direct pulp capping with adhesive resin (Hebling et al., 1999a; Pereira et al., 2000). The lack of dentin bridging might render the pulp more susceptible to inflammation mediated by bacterial contamination if microleakage is observed at the resin-tooth interface (Costa et al., 2000).

Complete polymerization of adhesive resins might be unachievable during direct pulp-capping procedures. Oxygen was shown to prevent complete polymerization of adhesive resin monomers (Rueggeberg and Margeson, 1990; Geurtsen et al., 1999), and hemorrhagic sites tend to have high oxygen tension. Humidity may also prevent complete polymerization of adhesive resin (Gerzina and Hume, 1996), and a site of pulp exposure tends to be humid due to the presence of blood/clot and exudates. Unpolymerized monomers can diffuse directly into the pulp at the exposure site, as well as diffuse through the dentinal tubules, and cause cytotoxic effects in pulp cells (Pashley, 1988; Hanks et al., 1994; Pashley et al., 2000).

The rate of cell division is a tightly regulated process that is intimately associated with growth, differentiation, and tissue turnover (Lodish et al., 1999). However, when cytotoxic stimuli are intense, cells may escape from the cell cycle and undergo a programmed process of cell death called apoptosis. Apoptosis is defined as an active process of cell suicide that is mediated by effector caspases (e.g., Caspase-3) and activation of downstream DNAses (Kerr et al., 1972; Núñez et al., 1998). Apoptotic cells can be identified by flow cytometry as a sub-G1 population after being stained with propidium iodide (Pelliciari et al., 1993). In contrast, necrosis is a passive process of cell death that results in disruption of the cell membrane and release of cell components to the extracellular matrix.

Pulp healing involves the activation of odontoblasts and the mineralization of a dentin bridge at the site of pulp exposure (Schroder, 1985; Pashley, 1988). Undifferentiated pulp cells are believed to be responsible for the replacement of dead odontoblasts (Smith et al., 1995). Macrophages are considered important orchestrators of wound healing throughout the body (Polverini, 1997). It has been previously reported that adhesive resins induce death of pulp cells in vitro (Costa et al., 1999). However, the mechanism of adhesive-resin-induced death of pulp cells and its effect on the cell cycle are not fully understood. The purpose of this study was to evaluate the effect of an adhesive resin on the viability and cell cycle of odontoblast-like cells, undifferentiated pulp cells, and macrophages in vitro.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
Mouse odontoblast-like cells (MDPC-23) and undifferentiated mouse pulp cells (OD-21) (Hanks et al., 1998), or mouse macrophages (RAW 264.7; ATCC, Manassas, VA, USA), were cultured cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% Fetal Bovine Serum (Gibco), 250 µg/mL L-glutamine, 125 units/mL Penicillin, and 125 µg/mL Streptomycin in a humidified CO2 incubator at 37°C.

Adhesive Resin Specimens
SingleBond (3M/ESPE, Minneapolis, MN, USA) discs measuring 5 mm x 2 mm were prepared under sterile conditions with a pre-fabricated mold. The specimens were light-cured with an Optilux 401 unit (Demetron; Kerr, Danbury, CT, USA) calibrated at 800 mW/cm2. The light-curing times were as follows: 0 sec, unpolymerized adhesive resin; 10 sec, partially polymerized; or 40 sec, polymerized adhesive resin. Specimens were weighed in an electronic balance, and only those weighing 11 ± 1 mg were used. SingleBond specimens were placed over permeable membrane inserts (0.4-µm pore size) measuring 24 mm in diameter (Corning, New York, NY, USA) to prevent direct physical interaction between adhesive resin and cells.

Flow Cytometry
Propidium iodide staining followed by flow cytometry was used to evaluate the effects of SingleBond on cell apoptosis and cell cycle, as described (Nör et al., 2002). MDPC-23, OD-21, or macrophages (2.5 x 105 cells/well) were seeded in six-well plates (Corning) containing 2 mL of culture medium, and allowed to attach overnight. Cells were exposed for 0–24 hrs to a SingleBond disc placed over the membrane insert, in triplicate wells per condition. At the end of the treatment period, both attached and floating cells were harvested, centrifuged, and re-suspended in an aqueous solution containing 50 µL/mL propidium iodide (Sigma, St Louis, MO, USA), 0.1% sodium citrate, 0.1% Triton X, and 100 µg/mL RNAse A. Samples were incubated in the dark for 30 min at 4°C, and the proportion of apoptotic cells was quantified by flow cytometry (EPICS; Beckman Coulter, Miami, FL, USA). Cell cycle was evaluated with "MPlus software" (Phoenix Plus Systems, San Diego, CA, USA). Three independent experiments were performed per cell type and treatment protocol.

Annexin V Assay
MDPC-23, OD-21, or macrophages were exposed to a partially polymerized SingleBond disc for 0–6 hrs, as described above. Both attached and floating cells were harvested, washed, and incubated in 25 ng/mL of annexin V (ApoAlert Apoptosis detection kit; BD Biosciences, Palo Alto, CA, USA) in the dark for 15 min at room temperature. The proportion of annexin-V-positive cells was analyzed by flow cytometry (EPICS) at 488-nm wavelength. Data were obtained from triplicate wells per condition, and were reproducible in three independent experiments.

Fluorometric Assay of Caspase-3 Activity
The involvement of Caspase-3 on adhesive-resin-induced apoptosis was evaluated with a fluorometric assay (Nör et al., 2000). Cells were exposed to partially polymerized SingleBond discs for 4 hrs, as described above. Both attached and floating cells were retrieved and subjected to lysis in 50 mM HEPES, 1 mM DTT, 0.1 mM EDTA, and 0.1% CHAPS (pH = 7.4). Cell extracts (20 µg protein/well) were re-suspended in assay buffer (100 mM NaCl, 50 mM HEPES, 10 mM DTT, 1 mM EDTA, 10% glycerol, 0.1% CHAPS) in a 96-well plate. The reactions were carried out at 37°C with 10 mM Ac-DEVD-AMC (Alexis Biochemicals, San Diego, CA, USA). DEVDase activity was monitored at excitation and emission wavelengths of 360 nm and 460 nm, respectively, in a fluorometer (GENios; TECAN, Grödig, Austria). Purified human recombinant Caspase-3 (Alexis) was used as a positive control, and Ac-DEVD-CHO (Alexis) was used to examine non-specific Caspase-3 activity. Data were obtained from triplicate wells per cell and condition, from three independent experiments.

Statistical Analyses
The statistical analyses of the data were performed by t tests or one-way ANOVA followed by a multiple-comparison Tukey’s test, with the use of SigmaStat 2.0 software (SPSS, Chicago, IL, USA). Statistical significance was determined at p <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Apoptosis was Dependent on Degree of Adhesive Resin Polymerization
To study the effect of the degree of adhesive resin polymerization on induction of apoptosis, we exposed MDPC-23, OD-21, or macrophages to unpolymerized, partially polymerized, or polymerized SingleBond. We observed that nearly 100% MDPC-23, OD-21, or macrophages were apoptotic after a 12-hour exposure to unpolymerized SingleBond, while the untreated controls showed less than 10% apoptotic cells (Fig. 1Go). Exposure to partially polymerized SingleBond induced apoptosis of approximately 40–50% MDPC-23 and OD-21 after 12 hrs, while exposure to adhesive resin polymerized for 40 sec did not induce apoptosis of these cells (Fig. 1Go). Macrophages seemed to be more susceptible to apoptosis upon exposure to partially polymerized or polymerized adhesive resin than MDPC-23 or OD-21 cells (Fig. 1Go).



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  		Figure 1. Adhesive resin induces apoptosis of pulp cells and macrophages. Percentage of apoptotic mouse odontoblast-like cells (MDPC-23) (a), undifferentiated pulp cells (OD-21) (b), or macrophages (RAW 264.7) (c) after a 12- or 24-hour exposure to unpolymerized, partially polymerized (light-cured for 10 sec), or polymerized (light-cured for 40 sec) SingleBond, respectively. Apoptotic cells were identified as a sub-G1 population after being stained with propidium iodide and sorted by flow cytometry. Asterisk indicates statistical significance at p <= 0.05, as compared with untreated controls (white bars). Data represent mean values (± SD) of triplicate samples per condition and cell type, and each sample consisted of approximately 10,000 cells.

 
The process of cell death induced by unpolymerized or partially polymerized adhesive resin was characterized by rounding and cell detachment (Figs. 2d–Go2iGo), which are typical features of apoptotic cell death (Kerr et al., 1972). The morphological changes indicative of the apoptotic process were correlated with a significant increase in the proportion of cells in Sub-G1 (Figs. 2p–Go2uGo). In contrast, MDPC-23 and OD-21 exposed to polymerized adhesive resin showed similar morphology and flow cytometry pattern, as compared with untreated controls (Fig. 2Go).



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  		Figure 2. Micromorphology and flow cytometry profile of pulp cells and macrophages exposed to an adhesive resin. MDPC-23, OD-21, or macrophages were exposed for 24 hrs to SingleBond light-cured for 0 sec (unpolymerized), 10 sec (partially polymerized), or 40 sec (polymerized), or untreated controls. (a–l) Phase-contrast photomicrographs of representative fields at 200x. (m–z) Flow cytometry profile of approximately 10,000 cells/condition stained with propidium iodide. The proportion of apoptotic cells per condition ("A") is depicted in the top left-hand corner of each graph.

 
The humidity present in sites of pulp exposure or in deep dentin might prevent complete polymerization of adhesive resins. Therefore, the response of cells to partially polymerized adhesive resin was studied in more detail. We observed that all cell types studied here presented an increase in the proportion of apoptotic cells within a few hours of exposure to partially polymerized SingleBond (Appendix, www.dentalresearch.org). Annexin-V staining corroborated the propidium iodide data and confirmed that an adhesive resin induces apoptosis (not necrosis) of pulp cells and macrophages. We observed that partially polymerized SingleBond induced a time-dependent translocation of phosphatidylserine to the outer leaflet of the cell membrane, which is an early phenomenon of apoptotic death (Figs. 3a–Go3cGo).



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  		Figure 3. Translocation of phosphatidylserine and activation of Caspase-3 constitute early stages of adhesive resin-induced apoptosis of pulp cells and macrophages. Percentage of annexin-V-positive MDPC-23 (a), OD-21 (b), or macrophages (c) after a zero- to six-hour exposure to partially polymerized (light-cured for 10 sec) SingleBond. Asterisk indicates statistical significance at p <= 0.05, as compared with untreated controls. Caspase-3 activity was determined in lysates of MDPC-23 (d), OD-21 (e), or macrophages (f) after a four-hour exposure to partially polymerized SingleBond ({blacktriangleup}), or untreated controls (•). Positive control was 1 ng of purified recombinant Caspase-3 (). The specificity of Caspase-3 activation was determined by the addition of the inhibitor Ac-DEVD-CHO to the reaction ({circ}). Data represent mean values (± SD) of triplicate samples per condition and cell type.

 
Adhesive-resin-induced Apoptosis is Associated with Caspase-3 Activation
To understand if adhesive-resin-induced apoptosis of pulp cells and macrophages is mediated by Caspase-3 activation, we exposed the cells to partially polymerized adhesive resin and performed a fluorometric assay. We observed a significant increase in Caspase-3 activity in cells exposed to the adhesive resin (Figs. 3d–Go3fGo). In contrast, untreated cells essentially did not show active Caspase-3 (Fig. 3Go). The Caspase-3 inhibitor Ac-DEVD-CHO completely blocked the fluorogenic activity in cells exposed to the adhesive resin (Fig. 3Go), which demonstrates the specificity of the enzymatic activity in these lysates.

Adhesive Resin Induces Cell-cycle Arrest
To study the effect of partially polymerized SingleBond on cell cycle, we exposed MDPC-23, OD-21, or macrophages to the same conditions described above and performed flow cytometry. Untreated MDPC-23, OD-21, and macrophages showed cell-cycle patterns of healthy, proliferating cells in culture (Fig. 4Go). We observed that exposure to partially polymerized adhesive resin induces a decrease in the proportion of MDPC-23, OD-21, or macrophages in the S phase (DNA synthesis) of the cell cycle, as compared with controls (Fig. 4Go). This was correlated with G2 cell-cycle arrest in MDPC-23 and OD-21, and G1/G2 cell-cycle arrest in macrophages.



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  		Figure 4. Cell-cycle analysis of viable pulp cells and macrophages. Flow cytometry profile of MDPC-23, OD-21, or macrophages exposed to partially polymerized SingleBond (light-cured for 10 sec) and stained with propidium iodide. (a) Percentage of viable cells at each phase of the cell cycle (G1, S, G2). Oblique line bars depict untreated control cells, and solid black bars depict cells exposed for 12 hrs to the adhesive resin (b–g). Cell-cycle profiles of cells exposed for 12 hrs to partially polymerized SingleBond and untreated controls. Data represent mean values (± SD) of triplicate samples per condition and cell type, and each sample consisted of approximately 10,000 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The apparent lack of cytotoxicity of adhesive resins in vivo has led to the suggestion that they could be utilized for direct pulp-capping procedures (Cox et al., 1998). Some in vivo studies have shown that pulp repair correlated with dentin bridging when adhesive resins were applied on micro-mechanical pulp exposures in non-human primate teeth (Kitasako et al., 1999). In contrast, the histological evaluation of human dental pulps capped with adhesive resins has demonstrated severe inflammatory responses and lack of dentin regeneration (Hebling et al., 1999a; Costa et al., 2000; Pereira et al., 2000).

Previous reports have demonstrated the cytotoxic effects of adhesive resins in vitro and in vivo (Hanks et al., 1992; Hebling et al., 1999a). In preliminary experiments, we observed that soon after exposure to an adhesive resin, most pulp cells showed a process of rounding and detachment from the culture surface. This observation led us to hypothesize that the mode of cell death induced by the adhesive resin was apoptosis, instead of necrosis. Our studies confirmed this hypothesis. Analysis of cells stained with propidium iodide demonstrated the establishment of a sub-G1 population of cells within a few hours of exposure to unpolymerized or partially polymerized adhesive resin, which demonstrates that these cells were undergoing apoptosis (Pellicciari et al., 1993). The annexin V experiments corroborated the observations described above. This methodology is based on the fact that phosphatidylserine is translocated from the internal (cytoplasmic) to the external surface of the cell membrane at early stages of the apoptotic process (Martin et al., 1995). Annexin V has a strong and specific binding affinity to phosphatidylserine available at the external surface of the cell, and has been used to detect apoptosis. We observed a significant increase in annexin-V-positive (apoptotic) MDPC-23 and OD-21 cells after a two-hour exposure to partially polymerized adhesive resin, and macrophages were apoptotic within one hour.

The degree of cure of the polymer network is correlated with the elution of leachable substances from composites (Ferracane, 1994). Components of adhesive resins (e.g., TEGDMA and HEMA) were shown to be soluble in aqueous solutions, and cytotoxic to immortalized 3T3-fibroblast cultures (Geurtsen et al., 1999). A recent report demonstrated that when the adhesive resin is not completely polymerized as a consequence of low light intensity, its cytotoxic effects on pulp cells are increased in vitro (Chen et al., 2001). Cytotoxic responses were observed when adhesive resins were applied directly to human pulps in vivo (Hebling et al., 1999a), which might be attributable in part to the incompleteness of adhesive resin polymerization and consequent release of cytotoxic components at the site of pulp exposure. These observations led us to evaluate the effect of adhesive resin polymerization in the induction of apoptosis. We observed a dramatic difference in the responses of the cells to the three conditions of polymerization (i.e., light-curing for 0, 10, or 40 sec). While unpolymerized and partially polymerized adhesive resin induced apoptosis very rapidly in all cell types evaluated here, polymerized adhesive resin induced significant apoptosis of only macrophages. These findings might be explained by the lower leaching of toxic elements from polymerized as compared with unpolymerized adhesive resins, and underline the importance of thorough polymerization of the adhesive resin before placement of the composite resin.

The cysteine protease Caspase-3 is one of the key executioners of apoptosis (Núñez et al., 1998). Caspase-3 is involved in the proteolytic cleavage of key downstream proteins, such as poly(ADP-ribose) polymerase (PARP), which ultimately result in DNA fragmentation and apoptotic death. Our findings demonstrated that all pulp cells and macrophages presented significant activation of Caspase-3 four hours after exposure to partially polymerized adhesive resin. Analysis of these data provides insights into the molecular mechanisms of the effects of an adhesive resin on pulp cells and macrophages. Furthermore, it corroborates our finding that adhesive resins cause apoptosis (i.e. programmed cell death), and not simply necrosis of cells.

The requirement of a minimum number of cell cycles prior to the inductive signaling for differentiation suggests that a certain level of competence is required before the cell can respond. Such cell competence might be especially important when a mature tissue needs to be regenerated (Tziafas et al., 2000). In addition to the direct induction of apoptosis, we observed here that most cells that were not apoptotic were arrested either at the G2 phase of the cell cycle (MDPC-23 and OD-21), or at both the G1 and G2 phases (macrophages), with fewer cells in the S phase. These results demonstrate that the majority of cells that did not die by exposure to the adhesive resin are quiescent. We speculate that quiescent cells would make minimal contributions to dentin regeneration.

In summary, we have shown here that an adhesive resin induces apoptosis or cell-cycle arrest of cells that are major players in pulp healing and dentin regeneration. This might explain, in part, the lack of dentin bridging observed in teeth treated with direct pulp capping with an adhesive resin. We believe that understanding the mechanisms of cytotoxicity of dental materials is necessary for the selection of a strategy for protection of the dentin-pulp complex that allows for pulp healing and dentin regeneration.


   ACKNOWLEDGMENTS
 
We thank Wenying Song for excellent support and technical assistance to this project, and Ann Marie Deslauries for her expertise in flow cytometry. This research was supported in part by a grant from the Delta Dental Fund (to M.G.M.), and by start-up funds from the Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan School of Dentistry (to J.E.N.). This paper is based on a thesis submitted to the graduate faculty, University of Michigan, in partial fulfillment of the requirements for a Master’s of Science degree in Operative Dentistry (M.G.M).


   FOOTNOTES
 
A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

Received September 4, 2002; Last revision April 22, 2003; Accepted May 22, 2003


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 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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