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J Dent Res 85(5):432-435, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Responses of Rat Pulp Cells to Heat Stress in vitro

T. Amano1, T. Muramatsu1,3,*, K. Amemiya2, K. Kubo2, and M. Shimono1,3

1 Department of Pathology,
2 Department of Dental Anesthesiology, and
3 Oral Health Science Center, Tokyo Dental College, 1-2-2, Masago, Mihama-ku, Chiba, 261-8502, Japan

* corresponding author, tmuramat{at}tdc.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although heat stress can cause irritation in the dentin/pulp complex, little is known about the thermotolerance of pulp cells and their response to heat stress. We investigated cultured rat pulp cell responses to heat stress. Cells were subjected to a temperature of 42°C for 30 minutes, and HSPs, alkaline phosphatase activity, and gap-junctional communication were determined at various time points. Although only low levels of HSP70 expression were detected before heat treatment, heat shock markedly induced HSP70 expression, with it gradually increasing at 1 hour after being heated. HSP25, however, showed no dramatic change. Gap junction protein connexin43 rapidly degraded after heat treatment, recovering to normal levels within the following 6 hours. Alkaline phosphatase activity decreased immediately after heat stress, recovering after 1 hour. These results indicate that dental pulp possesses protective factors, including HSPs, and that it can recover viability of intercellular communication and alkaline phosphatase activity after heat stress.

KEY WORDS: heat • HSP • dental pulp • connexin43 • alkaline phosphatase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Various environmental stimuli—including dental caries, mechanical, physical, and chemical injuries—can cause irritation in the dentin/pulp complex. Heat produced during cavity preparation or laser irradiation is particularly common in a clinical setting. Cavity preparation induces destructive changes, as well as acute inflammation, in the dental pulp at the affected site. The temperature of the pulp increases during cavity preparation as a result of heat stress, so drilling without water spray may result in substantial injury to the pulp. However, little information is available on the responses of dental pulp cells after direct heat stimulation.

The heat-shock response is the protective reaction of cells to a variety of environmental and pathological stimuli (Lindquist and Craig, 1988). The most obvious feature of the heat-shock response is the accompanying increase in the synthesis of heat-shock proteins (HSPs) (Schlesinger, 1990; Sorger, 1991). HSPs act as chaperones (Jakob et al., 1993) and play a role in anti-apoptotic processes (Schlesinger, 1990; Mehlen et al., 1997); they promote cell survival (Lavoie et al., 1993) and are thought to function in the protection and recovery of cells from environmental and pathological stress (Schlesinger, 1990; Sorger, 1991). However, little information is available on the expression of HSPs in dental pulp tissue (Sens et al., 1997; Ohshima et al., 2001; Kitamura et al., 2003). We hypothesized that heat stimulation would induce loss of viability in dental pulp cells, and that HSPs would recover that loss.

To test this hypothesis, we investigated the expression of HSPs and markers of cell viability—including gap junction protein (connexin43; CX43) (Murakami et al., 2001; Muramatsu et al., 2004), intercellular communication, and alkaline phosphatase (ALP) activity (Inoue et al., 1992)—in rat dental pulp cells after heat stimulation in vitro.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture
All experiments were carried out according to the Guidelines for the Treatment of Animals established by Tokyo Dental College. Dental pulp cells were obtained from the incisors of young Sprague-Dawley rats (n = 20), each weighing between 100 and 150 g. Incisors were extracted with the animals under general anesthesia, and pulp tissue was cultured according to the method of Inoue et al. (1992), who used the middle portion of the pulp to avoid epithelial contamination. For 4 wks prior to the experiment, the cells were cultured in minimum essential medium (MEM, Invitrogen, Carlsbad, CA, USA) containing 15% fetal bovine serum (FBS) at 37°C in a humidified incubator containing 5% CO2.

Primary Antibodies
Anti-CX43 monoclonal antibody was purchased from Chemicon International (Temecula, CA, USA). Anti-HSP25 and 70 monoclonal antibodies were obtained from StressGen Biotechnologies (Victoria, BC, Canada). Anti-actin antibody was supplied by Sigma-Aldrich (St. Louis, MO, USA).

Experimental Design
The cultured cells were maintained at 37°C until subconfluence. Heat stress was then carried out at 42°C for 30 min in an incubator (Laing et al., 1998; Shui and Scutt, 2001). After heat stress, the cells were returned to the 37°C humidified incubator (time point 0). Samples were harvested at the specific time points (0, 1 hr, 3 hrs, 6 hrs post-heating). Samples were also obtained at 1 hr prior to heat stress, as a control.

Western Blot
Western blot was carried out according to our previous report (Amemiya et al., 2003). Cells underwent lysis in RIPA buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris [pH = 7.4]) containing inhibitors. A 25-µg quantity of total lysate was subjected to 7.5% SDS-PAGE and then transferred onto a PVDF membrane (BioRad, Melville, NY, USA). The membranes were then incubated with anti-HSP25 antibody (1:10,000), anti-HSP70 antibody (1:1000), anti-CX43 antibody (1:1000), and anti-actin antibody (1:2500) at 4°C overnight. After being washed, the membranes were incubated with horseradish peroxidase (HRP)-conjugated mouse or rabbit Ig (1:1000, Amersham, Rochester, MI, USA) at room temperature for 1 hr. Immunoreactive bands were detected with the use of an ECL Western blot analysis system (Amersham)

Immunohistochemistry
For immunohistochemstry, the cells were seeded on coverslips. They were then fixed in 4% paraformaldehyde-0.1 M phosphate buffer (PB) at room temperature for 2 hrs. Specimens were washed in PBS and blocked with 10% goat serum, and were incubated with anti-CX43 antibody at 4°C overnight. After being washed with PBS, the samples were incubated with Alexa Fluor488-conjugated anti-mouse IgG at 4°C overnight. Specimens were observed under a fluorescence microscope (Axiophot 2, Carl Zeiss, Oberkochen, Germany). As a negative control, immunohistochemistry was carried out without primary antibody.

Dye Transfer
Analysis of gap-junctional intercellular communication was assayed by the scrape-loading/dye transfer technique (el-Fouly et al., 1987). For dye transfer, pulp cells were cultured on coverslips until subconfluence. Cells were cut with a scalpel and incubated with 0.1% Lucifer yellow CH (Sigma-Aldrich) for 2 min at room temperature. After incubation, the cells were washed in PBS and fixed with 4% paraformaldehyde-0.1 M PB. Cells positive for dye transfer were observed under a fluorescence microscope. The number of dye-transferred cells per number of scraped-edge cells was calculated as a ’dye-transferred cell index’.

Alkaline Phosphatase (ALP) Activity
ALP activity was measured with the use of a colorimetric assay kit (ALPopt; Roche Diagnostics, Tokyo, Japan) according to the manufacturer’s protocol. Briefly, cells were washed with PBS and homogenized in distilled water with a homogenizer. The homogenate was centrifuged at 10,000 rpm for 5 min, and the supernatants were assayed. A 1-mL quantity of pre-mixed solution containing 10 mM p-nitrophenol substrate was added to 10 µL of the supernatant. Absorbance at 405 nm was measured at 1, 2, and 3 min, in a spectrophotometer, and average absorbance for each time point was calculated.

Statistical Analysis
Statistical significance of multiple comparisons was evaluated by the Friedman test. Statistical difference was determined as p < 0.01.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of HSPs
Western blot analysis revealed that both HSP25 and 70 were expressed in the pulp cells at all time points. Heat shock induced production of HSP70 at 1 hr following heat stress, with it continuing to rise up to 6 hrs (3.2- to 4.8-fold), after which it fell slightly (Fig. 1Go). In contrast, expression of HSP25 showed no dramatic change compared with HSP70 (Fig. 1Go).


Figure 1
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  		Figure 1. HSP expression over time course by Western blot analysis. HSP70 was expressed in pulp cells at all time points. Heat shock induced production of HSP70 at 1 hr, although HSP70 expression showed no dramatic change between pre-heating and time point 0. HSP70 expression gradually increased between 1 hr and 6 hrs. In contrast, expression of HSP25 was clearly observed before heat treatment, changing slightly, but not dramatically, over time compared with HSP70. Fold change relative to the control (Fold {Delta}) was determined from densitometric data after normalization to actin for HSP25 and 70. Pre-heat value was arbitrarily set at 1.0.

 
Gap Junction Protein and Intercellular Communication
Western blotting of total lysates confirmed CX43 and actin bands in all samples. Degradation of CX43 after heat treatment (42°C, 30 min) was observed at time point 0 (Fig. 2AGo). Western blot analysis showed that the 43- and 45-kDa bands, which correspond to slightly and highly phosphorylated forms of CX43, respectively (Fig. 2BGo), decreased significantly at time point 0 (Fig. 2AGo). Expression of phosphorylated forms of CX43 returned to normal levels within 3 hrs following heat stress, and then increased again up to 6 hrs. The 41-kDa band, which corresponds to the non-phosphorylated form, was unchanged at time 0 (Fig. 2AGo). Expression of non-phosphorylated CX43 increased from 1 hr to 6 hrs following heat stress.


Figure 2
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  		Figure 2. Expression of connexin43 over time course. (A) Western blot analysis showed rapid degradation of CX43 at time point 0. CX43 expression returned to normal levels within 3–6 hrs following heat stress. (B) The 43- and 45-kDa bands, which correspond to phosphorylated forms of CX43, decreased dramatically after heat treatment. However, the 41-kDa band, which corresponds to the non-phosphorylated form, changed only slightly. (C) CX43 was observed between cell bodies or cell processes in pulp cells prior to being heated (Pre). Immunohistochemical findings revealed that heat stress led to dramatic degradation of CX43 at time point 0, which agreed with Western blot findings. Heat-induced CX43 degradation was reversible, since CX43 expression increased after cultures were returned to 37°C for 3 hrs. Bars = 20 µm.

 
Immunohisotochemically, CX43 was observed between cell bodies or cell processes in pulp cells prior to being heated. Immunohistochemical findings revealed that heat treatment led to a dramatic degradation of CX43 at time point 0, which was confirmed by the Western blot findings. Heat-induced CX43 degradation was reversible, with expression increasing again when the cultures were returned to 37°C for 3 hrs (Fig. 2CGo).

We performed intercellular transfer of Lucifer yellow dye to investigate intercellular communication from scraped-loaded cells to contiguous cells. When cells were pre-heated, a definite transfer of Lucifer yellow dye was detectable. Cell stimulation at 42°C for 30 min significantly decreased the extent of dye transfer, thus indicating disruption of gap-junctional communication (Fig. 3AGo). Communication resumed after 1 hr, and the number of coupled cells increased at 3 hrs. Significant differences were confirmed between the pre-heating and time 0 groups, and between the pre-heating and one-hour groups (p < 0.01) (Fig. 3BGo).


Figure 3
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  		Figure 3. Gap-junctional communication. (A) Micrograph of dye transfer. Prior to being heated (Pre), definite transfer of Lucifer yellow dye was detectable. Stimulating cells at 42°C for 30 min significantly decreased the extent of dye transfer, suggesting disruption of gap-junctional communication. Degradation of gap-junctional communication was reversible, since it increased after cultures were returned to 37°C for 3 hrs. Dashed lines = cutting edge; arrows = transferred cell. Bars = 50 µm. (B) Communication resumed after 1 hr (1.6 ± 0.2), and the number of coupled cells increased at 3 hrs (1.9 ± 0.2). Significant differences were confirmed between pre-heating and time point 0 groups, and between pre-heating and one-hour groups (p < 0.01, n = 20).

 
ALP Activity
ALP activity was quantified by a modified colorimetric assay, with p-NPP acting as a substrate. The results showed clear differences in ALP activity between the preheating and zero-, three-, and six-hour groups. At time point 0, values decreased dramatically. ALP activity increased after 1 hr, and the values in coupled cells increased further at 6 hrs. Significant differences were observed between preheating and the time point zero-, three-, and six-hour groups (p < 0.01) (Fig. 4Go).


Figure 4
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  		Figure 4. ALP activity. At time point 0, values decreased dramatically (303 ± 24), and ALP activity increased after 1 hr (374 ± 24). Values increased further at 6 hrs (485 ± 13). Statistically significant differences were observed between pre-heating and zero-, three-, and six-hour groups (p < 0.01, n = 15).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Heat stress modulates the degradation of numerous proteins (Lindquist and Craig, 1988; Schlesinger, 1990; Sorger, 1991). Although heat stimuli as a result of cavity preparation and laser irradiation are particularly common in a clinical setting, there have been few studies on the expression of HSP in dental tissue (Sens et al., 1996, 1997; Chen et al., 2000; Kitamura et al., 2003; Ohshima et al., 2003). There was a report that HSP70 was not detected in human third molar dental pulp (Sens et al., 1997), while expression of HSP70 was confirmed during reparative dentinogenesis in rat dental pulp tissue (Chen et al., 2000). The results of this study clearly demonstrated the presence of HSP25 and 70 in rat dental pulp, and that HSP25 did not change dramatically after heat stress, while expression of HSP70 was elevated at 1 hr after stimulation. These responses to stimuli are important in repair and regeneration, since HSP plays an anti-apoptotic role in the inhibition of the JNK pathway early in the healing process, and may contribute to the thermotolerance of pulp tissue (Kitamura et al., 2003). These findings suggest that HSP70 plays a role in the rapid recovery of dental pulp after heat stress.

Dental pulp is frequently subject to irritation by environmental stimuli, and particularly by heat stimuli resulting from cavity preparation, which, if done without water spray, can result in substantial pulp injury (Ohshima et al., 2003). Dental pulp is susceptible to heat stimulation. In contrast, some investigators have suggested that pulp cells may survive after such injuries (Chiego, 1992; Amemiya et al., 2003; Ohshima et al., 2003). We investigated the viability of pulp after heat stress by determining ALP activity and CX43. ALP is an enzyme expressed in early-stage mineralization, and is one marker of viability in pulp cells (Inoue et al., 1992). Also, we previously found a decline in CX43 in old odontoblasts in rats and in aged pulp in humans, suggesting that this reflected viability of dental pulp cells (Murakami et al., 2001; Muramatsu et al., 2004). Therefore, we believe that this decrease in ALP activity and CX43 by heat stimulation indicates a loss of viability in dental pulp cells. In the present study, heat-induced degradation of pulp viability recovered within 3 hrs after heat stress. This suggests that dental pulp possesses survival or recovery potential against heat stimulation.

Hyperthermia therapy stimulated bone remodeling and the formation of new bone, thus increasing cortical bone density (Leon et al., 1993a,b). These reports suggest that hyperthermia accelerates local bone formation. There is only one paper showing thermotolerance of the pulp cells following heat stress (Kitamura et al., 2005). However, the effect of heat stimulation on dental pulp is not fully evident in vitro. Mild heat shock induced proliferation, ALP activity, and mineralization in bone marrow stromal cells (Shui and Scutt, 2001). Furthermore, ALP activity in normal and inflamed dental pulp was investigated, and reversible pulpitis tissue exhibited high levels of ALP activity when compared with normal and irreversible pulpitis tissues (Spoto et al., 2001). Our results, taken together with those of previous reports, suggest that heat stress not only facilitates the recovery of cells but also enhances ALP activity in dental pulp cells.


   ACKNOWLEDGMENTS
 
We thank Prof. Takashi Inoue (Dept. of Clinical Pathophysiology, Tokyo Dental College) for his helpful suggestions and Associate Professor Jeremy Williams (Lab. of International Dental Information, Tokyo Dental College) for his editing of this manuscript. This study was supported by Oral Health Science Center Grant 982A01 from Tokyo Dental College. Takahiro Amano and Takashi Muramatsu contributed equally to this work.

Received July 21, 2004; Last revision January 21, 2006; Accepted February 7, 2006


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Amemiya K, Kaneko Y, Muramatsu T, Shimono M, Inoue T (2003). Pulp cell responses during hypoxia and reoxygenation in vitro. Eur J Oral Sci 111:332–338.[ISI][Medline]

Chen Z, Fan M, Bian Z, Zhang Q, Zhu Q, Lu P (2000). Immunolocalization of heat shock protein 70 during reparative dentinogenesis. Chin J Dent Res 3:50–55.[Medline]

Chiego DJ Jr (1992). An ultrastructural and autoradiographic analysis of primary and replacement odontoblasts following cavity preparation and wound healing in the rat molar. Proc Finn Dent Soc 88(Suppl 1):243–256.

el-Fouly MH, Trosko JE, Chang CC (1987). Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res 168:422–430.[ISI][Medline]

Inoue T, Chen SH, Usuda J, Morohoshi Y, Shimono M (1992). Osteogenic activity of cells from dental pulp, periodontal ligament, bone marrow and muscle in vitro: an ultrastructural study and alkaline-phosphatase activity. Bull Tokyo Dent Coll 33:7–12.[Medline]

Jakob U, Gaestel M, Engel K, Buchner J (1993). Small heat shock proteins are molecular chaperones. J Biol Chem 268:1517–1520.[Abstract/Free Full Text]

Kitamura C, Ogawa Y, Nishihara T, Morotomi T, Terashita M (2003). Transient co-localization of c-Jun N-terminal kinase and c-Jun with heat shock protein 70 in pulp cells during apoptosis. J Dent Res 82:91–95.[Abstract/Free Full Text]

Kitamura C, Nishihara T, Ueno Y, Nagayoshi M, Kasugai S, Terashita M (2005). Thermotolerance of pulp cells and phagocytosis of apoptotic pulp cells by surviving pulp cells following heat stress. J Cell Biochem 94:826–834.[ISI][Medline]

Laing JG, Tadros PN, Green K, Saffitz JE, Beyer EC (1998). Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes. Cardiovasc Res 38:711–718.[Abstract/Free Full Text]

Lavoie JN, Hickey E, Weber LA, Landry J (1993). Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J Biol Chem 268:24210–24214.[Abstract/Free Full Text]

Leon SA, Asbell SO, Edelstein G, Arastu HH, Daskal I, Sheehan S, et al. (1993a). Effects of hyperthermia on bone. I. Heating rate patterns induced by microwave irradiation in bone and muscle phantoms. Int J Hyperthermia 9:69–75.[Medline]

Leon SA, Asbell SO, Arastu HH, Edelstein G, Packel AJ, Sheehan S, et al. (1993b). Effects of hyperthermia on bone. II. Heating of bone in vivo and stimulation of bone growth. Int J Hyperthermia 9:77–87.[Medline]

Lindquist S, Craig EA (1988). The heat-shock proteins. Annu Rev Genet 22:631–677.[ISI][Medline]

Mehlen P, Mehlen A, Godet J, Arrigo AP (1997). hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells. J Biol Chem 272:31657–31665.[Abstract/Free Full Text]

Murakami S, Muramatsu T, Shimono M (2001). Expression and localization of connexin 43 in rat incisor odontoblasts. Anat Embryol (Berl) 203:367–374.[Medline]

Muramatsu T, Hamano H, Ogami K, Ohta K, Inoue T, Shimono M (2004). Reduction of connexin 43 expression in aged human dental pulp. Int Endod J 37:814–818.[ISI][Medline]

Ohshima H, Nakakura-Ohshima K, Yamamoto H, Maeda T (2001). Responses of odontoblasts to cavity preparation in rat molars as demonstrated by immunocytochemistry for heat shock protein (Hsp) 25. Arch Histol Cytol 64:493–501.[Medline]

Ohshima H, Nakakura-Ohshima K, Takeuchi K, Hoshino M, Takano Y, Maeda T (2003). Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells. Microsc Res Tech 60:483–490.[Medline]

Schlesinger MJ (1990). Heat shock proteins. J Biol Chem 265:12111–12114.[Free Full Text]

Sens DA, McGuirt JP, Khan W, Todd JH, Howell RM (1996). Expression of heat shock protein 27 in adult human third molar dental pulp. J Oral Pathol Med 25:382–387.[Medline]

Sens DA, McGuirt JP, Khan W, Howell RM, Todd JH (1997). Expression of hsc 70, but not hsp 70, in human third molar dental pulp. Eur J Oral Sci 105:271–277.[Medline]

Shui C, Scutt A (2001). Mild heat shock induces proliferation, alkaline phosphatase activity, and mineralization in human bone marrow stromal cells and Mg-63 cells in vitro. J Bone Miner Res 16:731–741.[ISI][Medline]

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