Angiogenesis is a fundamental biological process by which new blood vessels are formed from pre-existing microvasculature. It occurs normally during physiologic and reparative processes such as ovulation and wound healing (Folkman, 1992). Angiogenesis is a highly regulated process that is turned on for brief periods of time by the presence of angiogenic factors and then may be completely blocked by specific inhibitors. Defining the mechanisms whereby angiogenesis is triggered might ultimately lead to better ways to regulate the process therapeutically.
Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in a variety of physiological and pathological biological processes. VEGF was initially termed vascular permeability factor (VPF). Due to its ability to enhance vascular permeability, Dvorak and colleagues, 1985) discovered its function, as an inducer of vascular permeability. Furthermore, VEGF induces endothelial cell proliferation, promotes cell migration, mitogenesis, and edema, (Ferrara, 1996). The synthesis of VEGF is regulated by hypoxia-mediated control of gene transcription, alternative mRNA splicing, and post-translationally (at the protein level).
Basic fibroblast growth factor (bFGF) is one of nine structurally-related polypeptides. It was the first angiogenic cytokine originally purified from bovine pituitary gland, based on heparin affinity chromatography (Esch et al., 1985), and sequenced and characterized as an angiogenic factor (Abraham et al. , 1986). Basic FGF stimulates extracellular matrix (EC) proliferation in vitro, induces angiogenesis in vivo, and is frequently present at sites of capillary growth (Schweigerer et al., 1987, Folkman et al., 1987). Araki and colleagues, 1990, were the first group to implicate FGF as a survival factor for endothelial cells. Enhanced activity of protein kinase C was associated with the ability of bFGF to protect endothelial cells against apoptosis induced by growth factor deprivation or ionizing radiation in vivo and in vitro (Araki et al., 1990).
Another important pro-angiogenic factor is interleukin 8 (Il-8). It is secreted by many specific cell types (macrophages and others) and induces proliferation, cell migration and organization into sprout-like structures that mimic stages in the development of rnicrovessels in vivo (Streieter & Polverini 1992) In previous studies it has been demonstrated that 11-8 induces a rapid increase in endothelial cell number that had gradually declined to levels that were at or below levels encountered in control groups (Folkman and Shing, 1992; Risau, 1997). This finding is presumably due to Il-8’s lack of ability to upregulate and sustain endothelial cell survival (Nor et al., 1999).
The role of growth factors in mediating cellular responses to injury in dental pulp still remains unclear. Matsushita et al., (1999) investigated whether VEGF production by pulp cells was regulated by lipopolysaccharides during the pathogenesis of pulpitis. This study suggested that enhanced production of VEGF in human pulp cells induced by lipopolysaccharides is through a CD14-dependent pathway, which requires new protein synthesis and is mediated in part during AP-1 activation. The authors hypothesized that VEGF induces vascular permeability, proliferation, differentiation of pulp cells and also chemotaxis of monocytes and macrophages. Matsushita et al., (2000) attempted to further clarify the role of VEGF in human dental pulp cells and pulp tissue. They investigated the effects of VEGF on the chemotaxis, proliferation, and differentiation of human dental pulp cells. The authors found that VEGF induced a strong chemotactic response in human dental pulps cells in a dose-dependent manner. The results suggested that VEGF was produced by human pulp cells and acted directly upon human dental pulp cells in an autocrine manner. Furthermore, VEGF may promote chemotaxis, proliferation and differentiation via the utilization of kinase insert domain-containing receptor and in part through AP-1 by increasing expression of c-fos. Local angiogenesis at the injury site in the dentin-pulp complex appears to be critical for successful pulpal repair. Roberts-Clark and Smith (2000) have shown that the dentin matrix contains angiogenic factors, and that their release from the matrix after injury could make an important contribution to the overall reparative response of the dentin-pulp complex.
Inhibitors of VEGF have been used also to downregulate vessel growth when unwanted or over-exuberant angiogenesis and/or edema occurs. VEGF-induced effects are mediated through receptor tyrosine kinases, specifically the Flt-1 and Flk-1/KDR receptors (Neufeld et al., 1999). Activation of the Flk-1/ KDR receptor is associated with endothelial cell proliferation, whereas the Flt-1 receptor appears to be more involved in the regulation of cell-cell or cell-matrix interactions. Anti-VEGF antibody or tyrosine kinase inhibitors such as PTK787 inhibitor (Novartis, Basel, Swiss) have been successfully used in phase II and III clinical trials to downregulate pathological angiogenesis.
Previous studies have demonstrated that the application of an adhesive resin directly over the pulp tissue results in enhanced local vascularization and lack of dentin bridge development. The increased vascularization and edema observed after direct pulp capping with adhesive resin might result in irreversible tissue damage. A unique feature of the pulp is that it is confined within rigid, non-expandable dentin walls. Therefore, increases in microvessel density and permeability, which have been associated with increased intra-pulpal pressure (Van Hassel, 1971), might result in pulp necrosis.
However we still do not know which angiogenic factor mediates adhesive resin-induced pulp vascularization. In addition to the effect of adhesive resins on pulp vascularity, it has been also reported that they can induce death of pulp cells in vitro (Costa et al. 1999) and lack of dentin bridging in vivo (Hebling et al., 1999). However, the mechanism of adhesive resin-induced death of pulp cells and its effect on pulp cell cycles are still not completely understood.
Statement of purpose
The specific aims of this study are:
- To evaluate the effect of an adhesive resin (Single Bond), or one its hydrophilic monomers (HEMA), on the viability and cell cycle of odontoblasts-like cells, undifferentiated pulp cells, and macrophages.
- To examine if vascular endothelial growth factor (VEGF) is upregulated in pulp cells exposed to an adhesive resin or HEMA in vitro, and to determine which cells synthesize this factor.
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EFFECTS OF AN ADHESIVE RESIN ON APOPTOSIS AND CELL CYCLE OF DENTAL PULP CELLS AND MACROPHAGES
In vivo studies have demonstrated that direct pulp capping with adhesive resin systems results in pulp inflammatory responses and absence of dentin development of a dentin bridge. The high oxygen tension and humidity observed at the site of pulp exposure might hinder the polymerization of an adhesive resin applied directly to the pulp and enhance its cytotoxicity. Here, we hypothesized that the lack of dentin bridging might be due to adhesive resin-induced apoptosis of cells responsible for dentin regeneration and dental pulp healing. To evaluate the effect of an adhesive resin on viability and cell cycle of these cells, odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21), or macrophages (Raw) were exposed to Single Bond or 2-hydroxyethyl methacrylate (HEMA) and evaluated by Trypan Blue and flow cytometry. Single Bond specimens were light-cured for 0, 10 or 40 s and placed over a 0.4 µm-pore size polycarbonate insert for cell cultures. Alternatively, cells were exposed to 0-1000nM HEMA solubilized in culture medium. We found that unpolymerized (0s) or partially polymerized (10s) Single Bond induced apoptosis of 90-100% MDPC-23, OD-21, and macrophages within 24 h. In contrast, Single Bond polymerized for 40s induced apoptosis of 10%, 10.4%, and 22.6% cells respectively. MDPC-23, OD-21 and macrophages exposed to Single Bond polymerized for 0 s or 10 s showed a significant shift in cell cycle compared to untreated cells, since most cells entered sub-GofG 1 after 6 or 9 h. In contrast, cells exposed to Single Bond polymerized for 40 s did not show significant cell cycle changes. HEMA did not affect viability of the cells evaluated here. We concluded that unpolymerized or partially polymerized adhesive resin induces apoptosis or cell cycle arrest of cells involved in the process of dentin regeneration. This might explain, in part, the lack of dentin bridge observed in teeth treated with direct pulp capping procedure with adhesive resins.
Adhesive resin systems are used to reduce microleakage, to enhance retention, and to decrease post-operative sensitivity of composite resin restorations. In vivo studies have demonstrated that the direct application of adhesive resins to the site of pulp exposure, or to a thin layer of dentin ( 0.5mm) induces dilatation and congestion of blood vessels, inflammation, and pulp abscesses accompanied or not with tooth sensitivity (Hebling et al., 1999). Importantly, no dentin bridge can be seen in the majority of teeth treated with direct pulp capping with adhesive resin (Pereira et al.,2000). The observed lack of dentin bridge might render the pulp more susceptible to inflammation and/or necrosis mediated by bacterial contamination if microleakage is observed at the resin-tooth interface (Costa et al., 2000).
Complete polymerization of adhesive resins is largely unachievable during direct pulp capping procedures due to the high humidity and oxygen tension of the pulp tissue (Paul et al., 1999). Studies have demonstrated that oxygen inhibits or prevents complete polymerization of adhesive resin monomers (Rueggeberg et al., 1990). In carious teeth, in which the remaining dentin thickness is 0.5mm, it has been demonstrated that humidity may prevent complete polymerization of adhesive resin (Hamid et al., 1990; Gerzina etal., 1995, 1996). Once dentin has been cut for a cavity preparation in a vital tooth, the water content within the intratubular compartment is free to move outward. Even in the absence of fluid shifts, the water-filled tubules provide diffusion channels for noxious substances, such as unpolymerized dentin bonding agent, which might diffuse inward toward the pulp (Hanks et al., 1992, 1994; Pashley, 2000). The diffusion of unpolymerized dentin bonding agents becomes significant clinically when these materials permeate across dentin of a fresh cavity preparation and the concentrations are high enough to produce a biological effect in dental pulps (Pashley et al., 1988, 1996).
All cells have the ability to die through activation of an internally encoded suicide program, which initiates a characteristic form of cell death called “apoptosis”, (Thompson et al., 1995). On the contrary necrosis is a pathological form of cell death that results in disruption of cell membrane, release of cell components to the extracellular matrix, and inflammation. The rate of cell division is a tightly regulated process that is intimately associated with tissue turnover, differentiation, and wound healing. Cell proliferation involves two easily recognized and coordinated events: DNA duplication and physical cell division into two daughter cells. Cytotoxicity is frequently translated in changes on cell cycle patterns (Darnell et al., 1990). When cytotoxic stimuli are intense, cells may escape from the cell cycle and undergo programmed cell death (apoptosis). Apoptotic cells are identifiable by flow cytometry as a sub G0-G1 population after staining with propidium iodide (Nor et al., 2002).
Dentin regeneration is a process characterized by the mineralization of a dentin bridge at the site of pulp exposure (Pashley et al., 1988). This process is mediated byodontoblasts, which are terminally differentiated cells that do not proliferate in vivo(Schroder, 1985). Undifferentiated stromal pulp cells are believed to be responsible for the replacement of dead odontoblasts (Fitzgerald 1990; Smith 1995; Tziafas 1995). Macrophages are considered important orchestrators of wound healing responses (Polverini, 1995 ). It has been previously reported that direct pulp capping with adhesive resin does not result in dentin bridge formation even in absence of bacterial contamination (Hebling et al., 1999) Here, we hypothesized that the lack of dentin bridge might be explained in part by adhesive resin-induced apoptosis of cells that are involved in dentin bridge organization (i.e. odontoblasts or undiffe~entiated pulp cells) and wound healing (i.e. macrophages). The purpose of this study was to evaluate the effect of an adhesive resin (Single Bond), or one of its hydrophilic monomers (HEMA), on the viability and cell cycle of odontoblast-like cells, undifferentiated pulp cells, and macrophages.
Materials and Methods
An in vitro system was designed to study if dental pulp cell viability is affected by exposure to 2-hydroxyethyl-methacrylate (HEMA) or to an adhesive resin. Mouse odontoblast-like cells (MDPC-23, gift from C.T. Hanks), undifferentiated mouse pulp cells (OD-21, gift from C.T. Hanks), or mouse macrophages (Raw, ATCC, Manassas, VA) were cultured at 37°C in a humidified C02 incubator. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% Fetal Bovine Serum FBS, (Gibco), 250 µg/ml L-glutamine (Gibco), 125 units/ml Penicillin (Gibco), and 125 µg/ml Streptomycin (Gibco). Cells were plated in 12-well-plates, and 0, 10, 100, or 1000 nM of HEMA (batch # 868-77-9, Aldrich Chemical Company, St. Louis, MO) and were added to the culture medium in triplicate wells per condition. Experiments using the adhesive resin Single Bond (3M, Minneapolis, MN) were performed by culturing the cells in 12-well plates and placing the specimens over permeable membrane inserts (0.4 ).!m pore size, Coming, New York, NY) to prevent direct physical interaction between adhesive resin and cells.
Single Bond discs measunng 5 mm x 2 mm were prepared under sterile conditions with a prefabricated mold. The specimens were polymerized with an Optilux 401 light-curing unit (Demetron, Kerr, Danbury, CT) calibrated at 800 mW/cm2. The light-curing times were as follows: 10s, partially polymerized adhesive resin; and 40s, polymerized adhesive resin. All specimens were weighed in a calibrated electric balance and only those weighing 11 ± 1 mg were utilized. Unpolymerized (0 slight-curing time) specimens (11 ± 1 mg) were used as positive control, and untreated cells were used as negative controls.
Trypan Blue Exclusion Assays
Cell viability was assessed using trypan blue exclusion assay technique, which measures integrity of the cell membrane (Freshney). After a 0-24 h exposure either to HEMA or Single Bond, MDPC-23, OD-21, or Raw macrophages were retrieved with a solution of 0.25% trypsin supplemented with 1 mM EDTA (Gibco). Aliquots of harvested cells from each well were resuspended in 0.4 % trypan blue dye. The number of necrotic cells and the total number of cells was determined from triplicate wells for each condition by means of a hemacytometer with an optical microscope at 100x.
Flow Cytometry Assays
Flow cytometry was used to evaluate the effects of Single Bond or HEMA on MDPC23, OD21, or macrophage apoptosis and cell cycle. The same experimental conditions described above were used here, except that cells analyzed by flow cytometry were cultured in 6-well plates and treated with twice the volume of HEMA or by using 2 Single Bond discs in each well. After a 0-24 h exposure to Single Bond or HEMA, both attached and floating cells were collected for flow cytometry. Harvested cells were centrifuged at 800-rpm, at 4°C for 5 minutes in 12 x 75 mm tubes. The culture medium was aspirated; and cell pellets were resuspended with 2 ml of ice-cold phosphate-buffered saline (PBS). Samples were centrifuged again, PBS was aspirated, and cell pellets were resuspended in 50 11Vml propidium iodide (Sigma, St Louis, MO), 0.1% sodium citrate (Sigma), 0.1% Triton X (Sigma), 1 00!-lg/ml Rnase A (Sigma), in Milli-Q-water. Immediately after the propidium iodide solution was added, the samples were incubated for 30 minutes in the dark at 4°C, before flow cytometric analysis.
The statistical analyses of the data were performed by Student’s t-test and one-way ANOVA with Sigmastat 2.0 software (SPSS, Chicago, IL); and to isolate the groups that differ from others we used Tukey’s tests multiple comparisons. Statistical significance was determined at the p 0.05 level.
Degree of Adhesive Resin Polymerization Affects Cell Viability
To examine the effect of the degree of polymerization of an adhesive resin on pulp cells and macrophages viability, we exposed these cells to discs of Single Bond resin that were light-cured for 10s (partially polymerized), or 40s (polymerized), or a drop of 0s (unpolymerized). We observed that unpolymerized and partially polymerized Single Bond rapidly induced necrosis of MDPC-23, OD-21, and Raw Macrophages (Figure 2.1). Nearly 100% of all cell types evaluated were considered dead (positive for trypan blue) 3 hours after exposure to unpolymerized Single Bond, while around 60% of MDPC-23 and OD21, and 80% of macrophages were viable after a 3-hour exposure to partially polymerized Single Bond. After 9 hours, only 20% of MDPC-23 and OD-21, and essentially no macrophages were found viable upon exposure to partially polymerized Single Bond (Figure 2.1) In contrast, when these cells were exposed to polymerized Single Bond their viability was undistinguishable from untreated controls (p>0.05) at all time periods evaluated. These data demonstrate a direct correlation between degree of polymerization of the bonding agent and the viability of dental pulp cells and macrophages.
Adhesive Resin Induces Apoptosis of Pulp Cells and Macrophages
To understand the mechanism of cell death induced by an adhesive resin, we exposed MDPC-23, OD-21 , and macrophages to Single Bond as above and performed flow cytometry after propidium iodide staining. Unpolymerized Single Bond is a strong inducer of apoptosis since nearly 100% of MDPC-23, OD-21 or Macrophages were apoptotic after a 12 or 24-hour exposure (p<O.OOl) (Figure 2.2). Exposure to partially polymerized Single Bond induced apoptosis of approximately 50% MDPC-23 (p<0.05) and 50% OD-21 (p<0.05) after 12 hours (Figure 2.2). This effect was intensified at 24 hours, when about 70% MDPC-23 (p<0.05) and 80% OD-21 (p<0.05) were found apoptotic (Figure 2.2). Macrophages seemed to be more prone to partially polymerized Single Bond-induced apoptosis, since nearly 90% of the cells were found to be apoptotic at both time periods (p<O.OOl) (Figure 2.2). We observed that the process of pulp cell and macrophage death induced by an adhesive resin is characterized by typical morphological changes of apoptosis, such as nuclear condensation, rounding, and detachment of the cells (Figure 2.3).
Since the condition of partial polymerization seems the most relevant to the clinical use of an adhesive resin in deep cavity preparations, with or without pulp exposure, we decided to evaluate this condition in more detail with a time course experiment. Macrophages were again the cells that were demonstrated to be more sensitive (p<0.05) to partially polymerized Single Bond induced apoptosis (Figure 2.4).
We observed a significant number of apoptotic macrophages after a 6-hour exposure, and by 12 hours most cells were apoptotic. In contrast, MDPC-23 presented a significant number of apoptotic cells after 9 hours (p<0.05) (Figure 2.5) and OD-21 only after 12 hours (p<0.05) (Figure 2.6) as compared to untreated controls (Figure 2.4). We show here photomicrographs of a time course experiment with MDPC-23 as a demonstration that the morphological changes indicative of apoptosis are temporally correlated with the patterns of apoptosis depicted by the flow cytometric analyses (Figure 2.4) The morphological changes observed in these cells are closely correlated with the patterns of apoptosis depicted through the flow cytometric analysis (Figure 2.5 and 2.6)
Adhesive Resin Affects the Cell Cycle of Pulp Cells and Macrophages
To study the effect of an adhesive resin at several degrees of polymerization on pulp cell and macrophage cell cycle, we performed a similar experiment as described above and performed cell cycle analysis. The three cell lines showed a normal cell cycle pattern while not exposed to the Single Bond (Figure 2.7). They presented few apoptotic cells, and the majority of the cells were in G1 phase of cell cycle. In contrast, a dramatic shift in cell cycle profile was observed after a 12-hour exposure of every cell studied here to unpolymerized Single Bond (Figure 2.7). Under this condition, essentially all cells escaped from cell cycle and were now grouped as a sub-Go/G1 population. When cells were exposed to partially polymerized Single Bond, the effect on cell cycle was not as intense as the one observed with unpolymerized Single Bond, but followed a similar trend (Figure 2.7). The proportion of cells at sub-Go/G1 was increased as compared to untreated controls, and this was concomitant with a smaller the proportion of cells in the cell cycle (Figure 2. 7). Macrophages were the cells that were more sensitive to the cytotoxic effect of the adhesive resin, since approximately 90% of the cells were in sub Go/G1 phase, as compared to 45% MDPC-23, and 50% OD-21. The exposure to polymerized Single Bond essentially did not affect the cell cycle patterns of MDPC-23 and OD-21, as compared to controls (Figure 2. 7). In contrast, macrophages exposed to polymerized Single Bond showed more sub-G0/G1 cells compared to controls, and a decrease in the proportion of cells in S phase that was accompanied by a G2 cell cycle arrest (Figure 2.7) The trends of cell cycle profile observed at 24 hours were similar to the ones observed at 12 hours, but the cytotoxic effects were more pronounced (Figure 2.8).
We performed a time course experiment to examine in more detail the changes in cell cycle profiles induced by a partially polymerized adhesive resin. These experiments demonstrated that partially polymerized Single Bond induced an increase in the proportion of sub-G0/G1 MDPC-23 after 9 hours (Figure 2.9). At 12 hours, a transient phase of G2 cell cycle arrest was observed (Figure 2.9). Both, at 12 and 24 hours exposure times to partially polymerized Single Bond, the proportion of MDPC-23 cells in S phase was reduced in comparison to controls (Figure 2.9). Taken together, these data suggests that the MDPC-23 exposed to partially polymerized Single Bond were either undergoing apoptosis or were quiescent, non-proliferating cells. A similar trend was observed with OD-21, (Figure 2.1 0) but these cells started to show changes in cell cycle at 9 hours and at 24 hours the proportion of cells in sub-G0/G1 was increased as compared to MDPC-23. Among the three cell types evaluated, macrophages were the ones that showed the most significant changes in cell cycle upon exposure to partially polymerized Single Bond (Figure 2.11). After a 12 or 24 hour-exposure to partially polymerized Single Bond, the majority of the cells were in sub-G0/G1. Untreated MDPC-23, OD-21, and macrophages presented fairly constant cell cycle profiles during the time of the experiments (Figures 2.9, 2.10, 2.11).
Percentages ofMDPC-23, OD-21 and macrophages at each cell cycle phase (Gl, S and G2) are depicted at (Figure 2.12). A G2 cell cycle arrest at 12 hours was observed on MDPC-23, OD-21 at 12 hours (p<0.05), whereas macrophages showed a 02 cell cycle arrest that was not statistically significant different as compared to untreated. A similar trend, with a 02 cell cycle arrest was observed at 24 hours in OD-21 and macrophages group but not in MDPC-23 (p>0.05). Taken together, these findings suggest that cells exposed to partially polymerized Single Bond were either undergoing apoptosis or were quiescent, non-proliferating cells; which is in consensus with decreased cell proportion in S phase at 12 and 24 hours (p>0.05)(Figure 2.12)
HEMA did not Affect Cell Viability or Morphology
We decided to evaluate HEMA’s effect on viability of MDPC-23, OD-21 and macrophages, because this is a low-molecular weight hydrophilic monomer that is frequently present in adhesive resin systems. We observed that exposure of these cells to 10-1000 nM HEMA for 24 hours did not have a significant effect on the viability of the cells evaluated (Table 2.1 ), nor in cell morphology (Figure 2.13)
The use of adhesive resins has increased significantly over the last years as a consequence of the increase in the indication of composite resin restorations. The apparent lack of cytotoxicity of these agents led to the suggestion that they could be utilized for direct pulp capping procedures (Cox, 1998; Kanca, 1990; Murray, 2000). Some in vivo studies have shown pulp repair and dentin bridging when adhesive resins were applied on micro-mechanical pulp exposures in non-human primate teeth, (Akimoto et al., 1998; Kitasako et al., 1999; Onoe et al., 1995); (Costa et al., 1999). On the contrary, the histological evaluation of human dental pulps treated directly with bonding agents has demonstrated inflammatory responses and lack of dentin bridge development (Costa et al., 2000; Hebling et al., 1999,· Pereira et al., 2000). Here we report that an adhesive resin can interrupt dentin repair by causing apoptosis of pulp cells that are involved in the process of induction of dentin regeneration (i.e. odontoblasts), apoptosis of cells that might replace dead odontoblasts (i.e. undifferentiated pulp cells), and apoptosis of key orchestrators of wound healing responses (i.e. macrophages). Furthermore, we report significant changes in cell cycle profiles upon exposure to unpolymerized or partially polymerized adhesive resin that results in cells that are either quiescent or dying.
The cytotoxic effects of resin monomers have been reported earlier in the literature (Hanks et al., 1991; Ratanasathien et al., 1995). The concentration of substances diffusing across the dentin into the pulp depends in part on the rate of removal of the toxins via pulpal circulation (Pashley, 1979). An adhesive resin releases initially greater amounts of its components, but this release tends to decrease over time (Bouillaguet et al., 1996, 1998; Ferracane 1990, 1994; Hanks et al., 1992; Gerzina and Burne, 1994). This observation suggested that the higher risk of toxicity would happen immediately after placement of the composite material. The presence of unpolymerized monomers might enhance cytotoxicity by diffusion through dentinal tubules due to the small molecular size of them. Rathburn et al., 1991, showed that the toxicity was decreased by 90% following removal of unpolymerized monomers from resin composites with organic solvents. Hanks et al., 1992, reported that when monolayer cultures of Balb/c3T3 cells were exposed to HEMA, it caused irreversible inhibitory metabolic changes for all four parameters tested: DNA synthesis, protein synthesis, succinyl dehydrogenase activity, and total protein content. Gwinnett and Tay (1998) reported an irreversible injury to odontoblast cells when bonding agents were applied closest to the site of exposed cavity preparation.
We observed that soon after exposure to unpolymerized, or partially polymerized Single Bond, all cell types evaluated showed a process of nuclear condensation, rounding and detachment from the culture surface. This phenomenon happened through the process of apoptosis, in which cells commit suicide by activating caspases and downstream DNAses (Thompson, 1995). In agreement with Ferracane (1994) our results also indicated that the degree of polymerization of the adhesive resin is an important factor in the toxicity of the material. An inverse relationship exists between the degree of lightcuring and adhesive resin cytotoxicity. Polymerized adhesive resin discs showed no toxic responses in our study, perhaps because less toxic elements are leached from polymerized resins. However, Gerzina and Hume (1995, 1996) detected the presence ofTEGMA and HEMA released from resin composite material across dentin as late as 100 days following polymerization. This might be explained by degradation with time and leaching from tags, and diffusion towards the pulp. Ferracane et al., 1994, showed that even in fully set restorative materials substantial amount of short-chain polymers remain unbound, with the result that there is possible elution of leachable toxic components toward the pulp. Interestingly, Quinlan eta!., (2002), reported apoptosis of transformed human endothelial cell (ECV -304) when exposed to discs of the composite resin Spectrum or compomer Dyract light-cured for 40 s. This might be explained by increased sensitivity of endothelial cells to composite resin-induced cytotoxicity, as well as differences in study methodologies. In agreement with our study, partially polymerized composite resin (1 s and 4 s) samples caused stronger induction of apoptosis than polymerized specimens. (Quinlan, 2002)
Previous studies have shown that a wet environment interferes with the polymerization of resinous materials (Spahl et al., 1999 Geurtsen et al., 1998). Since humidity inhibits complete polymerization of adhesive resins, an increase in the diffusion of hydrophilic monomers towards the pulp might be observed (Rueggeberg and Margeson, 1990). In sites of pulp exposure, increased oxygen tension caused by the presence of hemorrhagic red blood cells might further inhibit the polymerization of adhesive resins. Therefore, we used unpolished adhesive resin discs to simulate an oxygen-inhibited layer in all our specimens.
Pulp therapy with adhesive resins allows for continuous release of unreacted resin components, which might be responsible for the cell death in many of the in vivo studies (Hebling et al., 1999; Costa et al., 2000; Pereira et al., 2000; Schmaltz et al., 2002).
Previous studies have shown that in clinical situations where there is still more than 0.5 mm of remaining dentin thickness the cytotoxicity of the resin monomers is decreased by up to 50%, (Hanks et al., 1988, 1989; Meryon & Brook, 1989). Furthermore when more than 0.5 rnrn dentin thickness is left, dentin adsorbs substances in the tubules and further limits the diffusion of these substances (Hanks et al., 1994 ). In the present in vitro study we simulated the direct pulp capping procedure with adhesive resins at the pulp exposure site. Based on our results, we speculate that the adhesive resin causes intense pulp damage under these circumstances in vivo. We have shown that the cytotoxic effect manifests at least in part, through induction of apoptosis of odontoblasts, undifferentiated pulp cells, and macrophages present at the site of exposure.
Many in vitro studies have reported that adhesive resin components are cytotoxic to metabolic activity when applied to fibroblasts (Hanks 1992; Costa et al., 1999). Costa et al., in 1999 evaluated the cytotoxic effect of Single Bond among other one bottle adhesive resins, showing that it was highly cytopathic to MDPC-23. Also cytotoxic effects of resinous materials and their components, such as HEMA, have been reported (Luster, 1989). Since unpolymerized and partially polymerized experimental conditions induced significant cell death which was predominantly caused by the adhesive resin, we decided to study the mode of cell death, t: e programmed cell death (“apoptosis”) or by “accidental” cell death, (“necrosis”). We also evaluated if there were any changes in cell cycle of experimental cells as compared to untreated controls. We observed that unpolymerized and partially polymerized adhesive resin induces a quick and proapoptotic activity. We also observed a change in cell cycle of cultures of odontoblast-like cells (MDPC-23), undifferentiated pulp cells (OD-21) and Raw macrophages that are compatible with cytotoxic cellular responses. In contrast, when cells were exposed to fully cured adhesive resin, cytotoxicity was reduced. We did not observe significant changes in the viability of treated MDPC-23 and OD-21 as compared to controls, but it did affect cell viability of macrophages. This emphasizes the relevance of the degree of polymerization to the cytotoxicity of adhesive resins.
Several authors have reported that direct pulp capping procedures with adhesive resins do not induce dentin bridge formation at the site of pulp exposure (Gwinnett and Tay, 1998; Hebling et al., 1999). /n vitro we observed apoptosis between 80- 100% of odontoblasts, undifferentiated stromal cells and macrophages as a result of exposure of adhesive resin in its unpolymerized (0s) and partially polymerized condition (10s).
Odontoblasts are terminally differentiated cells that do not proliferate in vivo, and undifferentiated pulp cells are believed to be responsible for the replacement of dead odontoblasts. If odontoblast and undifferentiated stromal cells become apoptotic the process of dentin-bridge formation will be hindered. Macrophages, which are important mediators in tissue organization and wound healing, were also found apoptotic upon exposure to Single Bond. Therefore the pulp tissue might be even further compromised in its capacity to organize a healing process.
Requirement of minimum number of cell cycles prior to withdrawal from cell cycle before inductive signaling for differentiation implies that a certain level of competence is required before the cell can respond. Such cell competence would be expected to be especially important in reparative situations in mature tissues and must be more fully understood in terms of recruitment of new odontoblast-like cells (Tziafas, 2000). In addition to the direct induction of apoptosis, we also observed changes in cell cycle pattern that can be translated into reduction in the cell proliferation rate. This means additional evidence to explain the 1observed lack of dentin bridge formation in vivo studies, since cell proliferation is a requirement for any healing process throughout the organism. We also speculate that cell cycle alterations may be responsible for not having pulp cell proliferation, and dentin regeneration when adhesive resins are applied directly over a pulp exposure site.
In summary, we have shown here that adhesive resin induces apoptosis or cell cycle arrest of cells that are major players in the regeneration of dentin. This might explain, at least in part, the lack of dentin bridge formation in teeth treated with direct pulp capping with adhesive resins, and the disorganization observed in the odontoblastic layer of teeth with deep carious lesions that were restored with adhesive resins and composite. Therefore, this study supports the recommendation of the manufacturer (3M) that does not indicate the use of Single Bond for direct pulp capping. We believe that the understanding of the mechanisms of cytotoxic responses induced by restorative materials will provide the clinicians with information that can be used when deciding to restore deeply carious lesions or pulp exposures with composite resins.
Under the conditions of this study:
- Unpolymerized or partially polymerized Single Bond induces apoptosis of odontoblast-like cells, undifferentiated pulp cells, and macrophages.
- Unpolymerized or partially polymerized Single Bond induces escape from cell cycle or cell cycle arrest of odontoblast-like cells, undifferentiated pulp cells, and macrophages.
- The degree of polymerization of the adhesive resin plays a significant role in the intensity of the cytotoxic response of dental pulp cells and macrophages.
- The cytotoxicity of an adhesive resin evaluated in this in vitro study suggests its contra-indication for direct pulp capping procedures.
- Based upon our findings in this study, it is recommended to polymerize more than 10 seconds adhesive resins when those will be placed in deep dentin.
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AN ADHESIVE RESIN AND THE HYDROPHILIC MONOMER HEMA INDUCE VEGF EXPRESSION ON DENTAL PULP CELLS AND MACROPHAGES
Angiogenesis is the fundamental process by which new blood vessels are formed from pre-existing capillaries. Vascular endothelial growth factor (VEGF) plays an important role in orchestrating responses by inducing endothelial cell proliferation, migration and survival. The purpose of this in vitro study was to examine if VEGF is upregulated in dental pulp cells exposed to an adhesive resin or to HEMA (2- hydroxyethyl methacrylate). Mouse odontoblast-like cells (MDPC-23), mouseundifferentiated pulp cells (OD-21 ), fibroblasts and Raw Macrophages (M0), were cultured in triplicate wells with DMEM/10% FBS. After 24 h, cells were exposed to 0, 10, 100 or 1000 nM HEMA. Alternatively, cells were exposed to discs weighing 11 (± 1) mg the adhesive resin Single Bond (3M) at 3 conditions of polymerization: 0, 10 or 40 s light-curing time. The adhesive resin was placed over 0.4 µm pore size polycarbonate transwell inserts. After 24 h, ELISA was performed in conditioned medium for mouse VEGF (Quantikine, R & D systems). T-test or one-way ANOVA were used for statistical analyses. We found that VEGF was upregulated in MDPC-23 exposed to HEMA (p<0.001) and to Single Bond (p<0.018). VEGF was also upregulated in M0 exposed to HEMA (p<0.001) or Single Bond (p=0.001). In contrast, VEGF expression was not upregulated in undifferentiated pulp cells (OD-21) or fibroblasts exposed to either HEMA or Single Bond (p>0.05). These results demonstrate that an adhesive resin or HEMA induces expression of VEGF by macrophages and odontoblasts, but not by undifferentiated pulp cells or fibroblasts.
Vascular endothelial growth factor (VEGF) plays a fundamental role during development, wound healing, and for the maintenance of life. Its importance is underlined by the observation that VEGF knockouts die before birth (Ferrara et al., 1996) VEGF is produced by several cell types, such as keratinocytes, macrophages, mast cells and fibroblasts. VEGF was also shown to increase vascular permeability and to be involved in the occurrence and progression of inflammation (Dvorak, 1995).
During embryogenesis, VEGF is essential for differentiation of “de novo” blood vessels in a process called “vasculogenesis”. VEGF is also involved in the process of “angiogenesis” which is the sprouting of new capillaries from preexisting vessels. The latter term was first used in 1935 to describe the formation of new blood vessels from pre-existing capillaries in the placenta (Hertig, 1935) In adults, angiogenesis is a critical component of wound healing, as well as in inflammatory responses. Disruptions in the regulation of the angiogenic response have been correlated with delayed healing of wounds (Di Pietro et al., 1996). Angiogenesis per se can enhance the severity of the inflammatory process due to the increased transport of inflammatory cell nutrients and oxygen to the site of inflammation (Johnson, 1999).
Several recent manuscripts address the potential role of VEGF in the biology of the dentin-pulp complex. VEGF among other angiogenic factors was found to be expressed in dentin matrix. Its expression in dentin suggests a contribution to the overall reparative response of the dentine-pulp complex (Roberts-Clark and Smith, 2000). Artese et al., 2002 reported that VEGF expression in stromal cells in healthy pulps was strongly positive in the inflammatory infiltrate in irreversible pulpitis. In contrast, it was found to be slightly, but significantly decreased in the stromal cells in irreversible pulpitis. VEGF ranged from 20-100% and irreversible pulpitis ranged from 0-100%. The microvessel density in healthy pulps was higher as compared to those with irreversible pulpitis. It is known that adhesive resins or calcium hydroxide induce pulp-vascularization.
Neovascularization is regulated by several molecules, which can either have a positive or a negative regulatory effect (Folkman and Klagsbrun, 1987). The nature and origin of angiogenic stimuli in pulp tissue are still uncertain. The aim of this study is to examine if vascular endothelial growth factor (VEGF) is upregulated in pulp cells exposed to an adhesive resin or HEMA in vitro, and to determine which cells synthesize this factor.
An in vitro system was designed to study VEGF expression levels induced by exposure to 2-hydroxyethyl-methacrylate (HEMA) or an adhesive resin. Mouse odontoblast-like cells (MDPC-23), and undifferentiated mouse pulp cells (OD-21), (gift from C.T. Hanks), as well as mouse fibroblasts or mouse macrophages (Raw, ATCC) were cultured at 37°C in a humidified C02 incubator. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 250 )lg/ml L-glutamine, 125 units/ml Penicillin, and 125 )lg/ml Streptomycin (all from Gibco BRL, Grand Island, NY, USA). Cells were plated in 12-well-plates, in a concentration of 6 x 104 cells/well for MDPC-23 and OD-21, 8 x 104 cells/well forfibroblasts and 5 x 104 cells/well for macrophages. Zero, 10, 100, or 1000nM of HEMA (batch # 868-77-9, Aldrich Chemical Company, St. Louis, MO) was added to the culture medium in triplicate wells per condition. Experiments using the adhesive resin, Single Bond (3M, Minneapolis, ~), were performed by culturing the cells in 12-well plates and placing the specimens over permeable membrane inserts (0.4 )lm pore size, Coming, Costar, Cambridge, MA) to prevent direct physical interaction between adhesive resins and cells.
To measure baseline expression of VEGF of each cell line, 4 x 104 cells/well were seeded in 12-well plates and the medium was changed after 24 hours. VEGF expression was quantified by ELISA from harvested medium.
Single Bond discs measuring 5 mm x 2 mm were prepared under sterile conditions with a prefabricated mold. The specimens were polymerized with an Optilux 401 light-curing unit (Demetron, Kerr, Danbury, CT) calibrated at 800 mW/cm2. Specimens were light-cured for 10s, to obtain the partially polymerized group; and were light-cured for 40s, to obtain the completely polymerized group. All specimens were weighed in a calibrated electric balance and only those weighing 11 ± 1 mg were utilized. Unpolymerized (0s light-curing time) specimens (11 ± 1 mg) were used as positive control and untreated cells were used as negative controls.
Trypan Blue Exclusion Assays
Cell viability was assessed using trypan blue exclusion assay technique, which measures integrity of the cell membrane (Freshney, 1987). After a 0-24 h exposure to either Single Bond or HEMA, MDPC-23, OD-21, fibroblasts or macrophages were retrieved with a solution of 0.25% trypsin supplemented with 1 mM EDTA (Gibco). Aliquots of harvested cells from each well were mixed with 0.4 % trypan blue dye (1: 1 by volume). The number of necrotic cells and the total number of cells was determined from triplicate wells for each condition by means of a hemacytometer with an optical microscope at 100x.
Enzyme-linked lmmunosorbent Assay (ELISA)
The Quantikine Cytokine ELISA Kit (R&D Systems, Inc. Minneapolis, MN) was used to quantify mouse VEGF expression in the supernatants of MDPC-23, OD-21 , fibroblasts, and macrophages induced by exposure to either Single Bond, or HEMA. Cells were cultured for 24 hours as previously described and conditioned medium was collected and centrifuged for 5 min, at 4°C at 13,000 rpm. Conditioned medium (1 00 µl) was placed in each well of a 96-well plate, which was previously coated with monoclonal antibody for VEGF (Quantikine, RD Systems), and incubated for 2 hours at room temperature. Subsequent to aspirating and washing away any unbound substances, a secondary antibody was added to each well, and incubated for 2 additional hours. After aspirating and washing the wells, incubation with a chromogenic substrate solution was performed and after 30 minutes, the reaction was stopped by stop solution. Readings for optical densities were recorded with Spectrophotometer calibrated to 450 nM wavelength (DU-20, Beckman, USA).
The statistical analyses of the data were performed by Student’s t-test and one-way ANOVA with SigmaStat 2.0 software (SPSS, Chicago, IL). Statistical significance was determined at p 0.05.
Effect of Single Bond on VEGF Expression
To examine how this angiogenic process is regulated by dental pulp cells, fibroblasts and macrophages, we first measured the baseline expression of VEGF by ELISA’s from 100µl of harvested medium at 24 h. We observed that odontoblast-like cells and undifferentiated pulp cells express higher baseline levels of VEGF as compared to fibroblasts and macrophages. For example, MDPC-23 expressed 256 (±4.8) pg/ml and OD-21 expressed 495 (± 46.5) pg/ml, whereas macrophages expressed 52.03 (±16.68) pg/ml and fibroblast expressed 175.5 (±49.3) pg/ml (Figure 3.1). Then, we evaluated how the four cell types respond to stimulation by an adhesive resin (Single Bond). We observed that the expression of VEGF was upregulated in odontoblast-like cells (p=0.004) and macrophages (p<0.001), but not in undifferentiated pulp cells and fibroblasts (p>0.05) (Figure 3.2)
In light of the results described above and taking into consideration the fact that HEMA is one of the most common components of adhesive resins, we exposed cells to 0-1000 nM HEMA and evaluated VEGF expression levels (Figure 3.3). We observed that the monomer HEMA is sufficient to upregulate VEGF expression in both odontoblastlike cells (p<0.001) and macrophages (p<0.001), but not in undifferentiated pulp cells (p>0.05) and fibroblasts (p>0.05) (Figure 3.3).
Effect of Polymerized Single Bond or HEMA on Cell Morphology
We also observed that the induction of VEGF expression mediated by Single Bond or HEMA was not accompanied by changes in cell morphology that could be detected at the light-microscopy level (Figure 3.4 and 3.5).
The direct application of an adhesive resin to sites of pulp exposure was shown to induce an increase in pulp vascularization (Costa et al., 2000). The release of inflammatory mediators and cytokines can contribute to the wound healing process in the pulp or lead to generalized inflammatory reactions and pulp necrosis through increased vascular permeability and tissue pressure in the low-compliancy pulpo-dentinal environment (Heyeraas eta!, 1999). VEGF is an angiogenic growth factor that binds to specific surface receptors in endothelial cells and enhances tissue vascularization and vascular permeability.
In the present study, we observed high expression levels of VEGF in pulp cells. This finding suggests that this growth factor plays an important role in dental pulp vascularization. We observed an upregulated expression of VEGF in odontoblast-like cells (MDCP-23) and Raw macrophages when cells were exposed to Single Bond or HEMA. However VEGF was not upregulated in a precursor odontoblastic cell line (OD-21), or in fibroblasts. This suggests that the odontoblasts play an important role in the angiogenic response observed in healthy pulps. And macrophages may intensify angiogenesis when inflammation is present in the pulp tissue. Artese et al., 2002, demonstrated that VEGF is expressed in healthy human dental pulp tissues. These authors analyzed pulp microvessel density and VEGF expression by immunostaining.
This study demonstrated higher microvessel density in healthy pulps when compared to irreversible pulpitis. The downregulation in the irreversibly inflamed cells could be due to their presence in a low compliance space such as it is in the dental pulp. Matsushita and collaborators (1999) found that human pulp cells expressed VEGF spontaneously and in response to lypopolysacharide (LPS) and pro-inflammatory stimulants in vitro. The same authors conducted a study that suggests that VEGF promotes chemotaxis and proliferation of human pulp cells (Matsushita, 2000).
VEGF upregulation will result in enhanced angiogenesis. Thus VEGF might be beneficial in pulp healing processes that require increased blood supply. However, this will only be possible if cells are available for this response. We have previously shown that adhesive resins induce apoptosis of both odontoblasts, and undifferentiated pulp cells. Furthermore, VEGF expression will make existing blood vessels more permeable. The net effect of VEGF expressions is an increase in intra-pulpal pressure and pulp tissue might not capable be capable of surviving the consequences of this pressure. This process may contribute to advancement to pulp necrosis.
Reactive dentinogenesis is mediated by upregulation of odontoblast synthetic and secretory activities from a relative dormant state during physiological dentinogenesis (Linde et al., 1993). Recently, Roberts-Clark and Smith showed that dentine matrix is not an inert dental hard-tissue, but rather a potential tissue store house of a cocktail of bioactive molecules. Previously have been reported that a number of growth factors are sequestered within the dentine matrix (Finkelman eta! 1990; Cassidy et al., 1997; Pearce et al., 1996). Release of growth factors may account for the increased local angiogenesis at sites of dental tissue repair (Baume eta!, 1980, Schroder et al., 1985)
We conclude that VEGF is upregulated in some cell populations of the dental pulp, noticeably the odontoblasts in response to an adhesive resin. In a fashion similar to the brain tissue, the dental pulp is restricted within rigid walls. If VEGF plays a role in dental pulp tissue responses , the use of VEGF-blocking antibodies in situations of uncontrolled vascularization may help to prevent over-exuberant angiogenesis in the confined space of the pulp chamber. It might be expected that anti-angiogenic agents would aid for the decrease blood flow leading to transient normalization of blood vessel permeability and density.
- Exposure to HEMA resulted in up-regulation of VEGF expresswn by odontoblast-like cells (MDPC-23) and mouse Macrophages but not in undifferentiated pulp cells (OD-21) or mouse fibroblasts.
- Exposure to Single Bond resulted in up-regulation of VEGF expression by odontoblast-like cells (MDPC-23) and mouse Macrophages but not undifferentiated pulp cells (OD-21) or mouse fibroblasts.
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Adhesive resins have been used to reduce microleakage, postoperative sensitivity and the incidence of recurrent caries. Vital pulp therapy aims the treatment of reversible pulpal injuries by the application of a protective liner or base. Previous studios have questioned the biocompability of bonding agents applied directly to dental pulp cells. The results described in Chapter II are in agreement with Costa et al (2000). They demonstrate that single bond induced apoptosis of pulp cells and should not be recommended for vital pulp therapy.
Dental pulp cells, as any other cell m the body, depend on homeostasis of extracellular matrix to remain viable and functional. If this equilibrium is lost, even a modest increase of fluid volume may raise tissue pressure, compress blood vessels, and lead to ischemia and pulp necrosis. If VEGF is upregulated when bonding agents are applied to dental pulp cells, it may increase vascularization of dental pulp tissues, increase vascular permeability and lead to edema and necrosis, because dental pulp has a relative low-compliant environment due to its enclosure between rigid dentin walls, where there is minimum chances of expansion. Therefore the upregulation of VEGF expression might result in additional damage to dental pulp cells.
Our results suggest that an adhesive resin can cause pulp tissue damage through, at least, two mechanisms:
- Direct cytotoxicity: Adhesive resin causes apoptosis of dental pulp cells; therefore it impairs pulp healing and dentin regulation.
- Indirect effect: Adhesive rosin upregulates VEGF expression of dental pulp cells. Upregulated VEGF has been correlated with increased tissue microvessel density and permeability, when is translated into enhanced interstitial pressure. Due to the confinement of the pulp tissue within nonexpanding dentin walls, the increase in pressure may result in disruption of pulp homeostasis and additional death of both cells in vivo.