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Gene expression analysis in cells of the dentine–pulp complex in healthy and carious teeth
Archives of Oral Biology (2003) 48, 273—283
Gene expression analysis in cells of the dentine—pulp complex in healthy and carious teeth Julia L. McLachlan, Anthony J. Smith, Alastair J. Sloan, Paul R. Cooper* Oral Biology, School Of Dentistry, University of Birmingham, St. Chads Queensway, Birmingham B4 6NN, UK Accepted 13 December 2002
KEYWORDS Gene expression; Odontoblast; Pulp; Human; Rodent
Summary Knowledge of the molecular events that occur in carious disease has so far been constrained due to difficulties in obtaining sufficient quantities of the dental tissues and cells involved. Our histological findings indicate that a pulp—odontoblast cellular complex can be obtained from carious and healthy human teeth when exposed to lowtemperatures prior to pulpal extirpation and from rodent teeth processed at roomtemperature. In contrast, pulpal tissue extracted from room-temperature processed human teeth and low-temperature processed rodent teeth resulted in the odontoblast layer remaining attached to the pulp chamber. Semi-quantitative RT-PCR (sq-RT-PCR) analysis confirmed that markers previously shown to be preferentially expressed in odontoblasts, namely dentin sialophosphoprotein (DSPP) and Nestin, amplified more readily from the extracted pulp—odontoblast complex, as compared to pulpal tissue alone, in both human and rodent samples. Subsequent gene expression analysis of collagen-1a and collagen-3a indicated levels were significantly higher in carious pulpal tissue. In addition, analysis characterising the expression of members of the transforming growth factor and bone morphogenic protein families and their receptors indicated in general, that these genes were expressed by healthy odontoblasts and up-regulated in both pulpal cells and odontoblasts in response to carious injury. Use of this temperaturesensitive dental tissue preparation procedure allows detection of differential gene expression in odontoblasts and other pulpal cells in healthy and carious tissue. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction Characterisation of the molecular and cellular events important in dental tissue injury and repair is critical to our understanding of these processes. Abbreviations: TGF, transforming growth factor; BMP, bone morphogenic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcriptase polymerase chain reaction; DSPP, dentin sialophosphoprotein Corresponding author. Tel.: þ44-121-237-2785; fax: þ44-121-625-8815. E-mail address: [email protected] (P.R. Cooper).
Odontoblasts are highly specialised post-mitotic cells aligned in a homogenous monolayer at the periphery of the dental pulp and are solely responsible for both the developmental and reparative formation of dentine.1,2 During mild tissue injury, odontoblasts localised beneath the damaged region can up-regulate their dentine secretory activity.3 However, injury of greater intensity can cause localised odontoblast necrosis. These cells are subsequently replaced by an odontoblast-like cell population, which differentiate from pulpal progenitor cells, and secrete a reparative dentine matrix.4
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(03)00003-7
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At present, our understanding of the transcriptional changes important in these repair processes has been limited due to the nature and location of the odontoblast cells. Attempts to overcome these obstacles has resulted in utilisation of in vitro culturing of immortalised odontoblast-like cells derived from a variety of species.5—7 However, gene expression changes identified using this approach most likely do not accurately or comprehensively reflect events that occur in vivo in man. Transcript profiling utilising clinically derived tissue has also been performed although, reports have indicated that large numbers of teeth are required to provide sufficient quantities of RNA for this analysis.8 Recently, the utility of laser capture microdissection (LCM) has also been demonstrated for the study of gene expression in developing murine dental tissue.9 Whilst this approach offers promise for the study of human dental tissue gene expression, its general application is limited by the requirement for expensive specialist equipment and its ability to provide only relatively small amounts of RNA, limiting the further downstream applications possible. The role of growth factors in dentinogenic processes is well documented.10 In particular, members of the transforming growth factor-b (TGF-b) and bone morphogenic protein (BMP) families, have been implicated in controlling aspects of tooth development and tissue repair.11—14 Experiments have shown that cells of the dentine—pulp complex are capable of synthesising these growth factors, which are subsequently sequestered into the dentine matrix and released during tissue injury, potentially stimulating repair mechanisms.15—18 Whilst the presence of these growth factors and their receptors has previously been reported in dental tissue,19,20 their gene expression profile during health and disease has yet to be determined. The aim of this study was to use semi-quantitative reverse transcriptase polymerase chain reaction (sq-RT-PCR) to examine gene expression levels of molecules important in dental tissue mineralization and growth factors and their receptors within the cells of the dentine—pulp complex in carious and non-carious tissue. This knowledge will contribute to our understanding of the molecular events important in dental tissue injury and repair.
Materials and methods Dental tissue extraction from human and rodent teeth Carious and sound mature human teeth with fully formed roots extracted for orthodontic purposes
J.L. McLachlan et al.
from patients generally in the age range of 20—30 years, were obtained immediately after extraction from clinics at the Birmingham Dental Hospital following informed patient consent. Carious teeth exhibited disease ranging from enamel involvement only to deep dentinal lesions. Rodent maxillary and mandibular incisor teeth were also dissected, using sterilised scalpels and tweezers, from 28-day-old male Wistar rats. Following extraction, human and rodent teeth were immediately frozen in liquid nitrogen for low-temperature processing or submerged in the RNA stabilising solution, RNA Later (Sigma, UK), for subsequent room-temperature processing. Teeth were then longitudinally sliced, using a segmented, diamond-edged rotary saw (TAAB Laboratories, UK) cooled with PBS, and the pulpal tissue carefully removed intact using a sterile dental probe and forceps. Extracted dental tissues were either processed for histological analysis or were used in RNA isolation.
Histological analysis Both dentine and pulp samples (human and rodent) were fixed for 24 h in 10% (w/v) neutral buffered formalin (Surgipath, UK). Dentine samples were subsequently demineralised in 10% formic acid (Sigma, UK) until fully decalcified (approximately 10 days for human samples and 6 days for rodent specimens), as assessed by X-ray examination. Both pulp and dentine samples were routinely processed through a series of graded alcohols and xylene, and finally embedded in paraffin wax. Serial sections of 5 mm thickness were cut and stained in Mayer’s haematoxylin and eosin (H & E).
Histomorphometric analysis Quantitation of cells in the dentine—pulp complex of human and rodent teeth was performed on 5 mm H & E stained longitudinal sections. Histomorphometry was undertaken on healthy and diseased sections from human teeth and rodent intact incisors processed at both low- and room-temperature. One representative section from six teeth was counted for each of the groups described. The sections chosen for counting were all derived from teeth of similar morphology, i.e. molars from humans and incisors from rodents. Histological sections chosen had also been cut to similar levels in all teeth. Counting was performed using an ocular graticule, at 500 magnification. Following Hunting curve analysis, to identify the number of counts needed to produce reproducible results unaffected by further counts, odontoblast cell numbers were
Gene expression in mature dental tissue
counted in 20 random graticule squares along the interface between the dentine and pulp tissue. To estimate the total number of odontoblasts in one 5 mm histological section, the perimeter of the pulp chamber was estimated by accurately counting the number of squares around the pulp chamber perimeter at 50 magnification. The value obtained was multiplied by the average number of odontoblasts found in one square, allowing for the degree of magnification. Similarly, following Hunting curve analysis fibroblast cell numbers were counted in 20 random graticule squares in the pulp tissue. To estimate the total number of fibroblasts within one histological section, the number of squares that overlaid the pulpal area were counted at 50 magnification. The value obtained was multiplied by the average number of fibroblasts found in one square. In diseased tissue, cells not displaying a fibroblast morphology were excluded from cell counts.
RNA isolation and cDNA synthesis Following either low- or room-temperature preparation of pulpal tissue from sound or carious human teeth and from rodent teeth, total RNA was extracted using the RNeasy mini kit (Qiagen, UK) as recommended by the manufacturer. Subsequently, 1—5 mg of DNase digested total RNA was used for oligo-dT reverse transcription to generate single stranded cDNA using the Omniscript kit (Qiagen, UK). Both RNA and cDNA concentrations were determined using a BioPhotometer (Eppendorf, UK).
Semi-quantitative reverse transcriptase polymerase chain reaction (sq-RT-PCR analysis) sq-RT-PCR analysis was performed for the human genes DSPP, Nestin, collagen-1a, collagen-3a, TGFb1, TGF-b2, TGF-b3, TGF-b receptor 1 (TGF-bR1), TGF-b receptor 2 (TGF-bR2), TGF-b receptor 3 (TGF-bR3), BMP-7, BMP-receptor 1A (BMP-R1A), and BMP-receptor 1B (BMP-R1B) and rodent genes DSPP and Nestin using the primer sequences provided for the respective species of tissue analysed (Table 1). The housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as a control. In summary, 50 ng of single stranded cDNA was used to seed a 50 ml PCR, which was subjected to between 28 and 42 cycles (as indicated in Table 1). A typical amplification cycle of, 95 8C for 20 s, 51—60 8C for 20 s, and 72 8C for 20 s, was performed using an Eppendorf Mastercycler thermal cycler (Eppendorf, UK). Following the designated
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number of cycles, 8 ml of the reaction was removed and the product separated and visualised on a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide. Scanned gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalised against the GAPDH housekeeping gene control values.
Results Histological examination of human dental tissue following low- or room-temperature processing Following pulpal extirpation from low- or roomtemperature processed human teeth, histological analysis was performed to identify the subsequent localisation of the odontoblasts. Data indicated that their localisation was dependent on the tooth preparation procedure used. Following pulpal preparation from human teeth collected and processed at room-temperature (i.e. in RNA later), the odontoblasts remained lining the pulp chamber attached to the dentine matrix. However, pulp removed from teeth collected and processed at low-temperature (i.e. in liquid nitrogen) had the odontoblast cell layer attached, providing a pulp—odontoblast cellular complex. Tissue separation was unaffected by the extent of the carious process in the diseased teeth. Histological studies were performed on five teeth from each of the four categories of diseased and healthy teeth collected and processed in RNA Later and liquid nitrogen. One representative image from each of the four groups described is presented in Fig. 1.
Histological examination of rodent dental tissue following low- or room-temperature processing The localisation of the odontoblasts following pulpal extirpation from rodent teeth was similarly examined histologically. Again, the data indicated that odontoblast localisation was dependent on the tooth processing procedure used, however, the results were opposite to those obtained for human dental tissue. Following pulpal extirpation from teeth processed at room-temperature, the odontoblast cell layer remained attached to the pulpal tissue, thereby providing a pulp—odontoblast complex. When pulp was removed from teeth processed at low-temperature, the odontoblasts were found to remain attached to the dentine in the pulp chamber. Two representative images
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Table 1
J.L. McLachlan et al.
Primer sequences and cycling conditions used for human and rodent () gene expression analyses.
Gene
Primer sequence (50 ! 30 )
Annealing Cycles temperature (8C)
Product size (bp)
Source
BMP-7
Forward CGC TTC GAC AAT GAG ACG TTC Reverse TGG CGT TCA TGT AGG AGT TCA G
60
35
571
19
BMP-R1A
Forward GAA GAA GAT GAC CAG GGA GGA Reverse CCG TCA TGA AAC CAA GTA TGT
54
32
622
19
BMP-R1B
Forward GTG GCG TGG CGA AAA GGT AGC Reverse TTG GGA ATG AGG GGC GTA AGT
58
30
698
19
TGF-b1
Forward CGC CTT AGC GCC CAC TGC TCC TGT Reverse GGG GCG GGA CCT CAG CTG CAC
60
34
532
Growth factors
TGF-b2
Forward GCT CTG TGG GTA CCT TGA TGC CAT CC Reverse TTC TTC CGC CGG TTG GTC TGT G
60
38
573
Growth factors
TGF-b3
Forward ACT GCC GAG TGG CTG TCC TTT GAT G Reverse AGG CAG ATG CTT CAG GGT TCA GAG TG
60
36
538
Growth factors
TGF-bR1
Forward CGT TAC AGT GTT TCT GCC ACC T Reverse AGA CGA AGC ACA CTG GTC CAG C
53
42
315
AJS personal communication
TGF-bR2
Forward CCC CAA GCT CCC CTA CCA TGA C Reverse AAC TCC GTC TTC CGC TCC TCA G
53
34
642
AJS personal communication
TGF-bR3
Forward TGT CAC CTG GCA CAT TCA TT Reverse CTC AGC ACT GTC TTG GTG GA
60
32
261
Primer 3
Collagen-1a
Forward TGG GAG TGC AAG GAT ACT CTA TAT CG Reverse CCC ATC CCA TCT TCG ACG TAC
60
28
320
32
Collagen-3a
Forward CTG GAC CAA AAG GTG ATG CT Reverse CAG GGT TTC CAT CTC TTC CA
60
28
310
Primer 3
Nestin
Forward GGC AGC GTT GGA ACA GAG GTT GGA Reverse CTC TAA ACT GGA GTG GTC AGG GCT
55
30
718
GenBank accession numbers AJ270321 and AJ270322
DSPP
Forward CCT AAA GAA AAT GAA GAT AAT T Reverse TAG AAA AAC TCT TCC CTC CTA C
51
36
293
35
GAPDH
Forward CCA CCC ATG GCA AAT TCC ATG GCA Reverse TCT AGA CGG CAG GTC AGG TCC ACC
60
28
550
36
DSPP
Forward TGC ATT TTG AAG TGT CTC GC Reverse CCT CCT GTC TTG GTG TGG TT
60
25
277
Primer 3
Nestin
Forward CAT TTA GAT GCT CCC CAG GA Reverse AAT CCC CAT CTA CCC CAC TC
60
30
285
Primer 3
GAPDH
Forward GAT CCC GCT AAC ATC AAA T Reverse GGA TGC AGG GAT GAT GTT CT
60
28
389
Primer 3
Sources of the primer sequences are provided. For primer design the primer 3 program was used at http.//wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.
from studies performed on 10 teeth, 5 teeth collected and processed in RNA later and 5 teeth collected and processed in liquid nitrogen, are presented in Fig. 2.
Histomorphometric analysis of human and rodent dental tissue Table 2 provides data obtained on odontoblast and fibroblast cell numbers in diseased and healthy human teeth. The data indicate that following human pulpal tissue extirpation at low-temperature
approximately 88.6 and 79.3% of the odontoblasts are attached to the pulpal tissue in healthy and diseased teeth, respectively. Whereas following room-temperature processing, approximately 96.0% for healthy teeth and 95.5% for diseased teeth of the odontoblasts cells remained lining the dentine surface. Table 3 provides data obtained on odontoblast and fibroblast cell numbers in rodent incisors. The data indicates that approximately 87% of the odontoblast cells remain attached to the dentine layer following pulpal extirpation at low-temperature.
Gene expression in mature dental tissue
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Figure 1 Histological analysis of H & E stained human healthy (a, b, e and f) and diseased (c, d, g and h) dental tissue collected and processed at room-temperature (a, b, c and d) and low-temperature (e, f, g and h). Paired images each taken from the same tooth, a and b, c and d, e and f and g and h, following pulpal extirpation are shown. Roomtemperature processing resulted in the odontoblast cells (O) remaining attached to the dentine (D) following pulpal extirpation. Whilst low-temperature processing resulted in the isolation of the pulp (P) and odontoblasts as a complex. Images are at 40 magnification.
Figure 2 Histological analysis of H & E stained rodent dental tissue processed at room-temperature (a, b, c and d) and low-temperature (e, f, g and h). Paired images each taken from the same tooth, a and b, c and d, e and f and g and h, following pulpal extirpation are shown. Processing of rodent tissue at room-temperature resulted in the isolation of the pulp (P) and odontoblasts (O) as a complex, whilst pulpal extirpation of rodent teeth at low-temperature resulted in the odontoblast cells remaining attached to the dentine (D) surface. Images shown are at 40 magnification.
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Table 2
J.L. McLachlan et al.
Average number of odontoblasts and fibroblasts present in 5 mm human tooth sections. Number of odontoblasts
Number of fibroblasts
On dentine
On pulp
Healthy teeth Intact Low-temperature processed Room-temperature processed
11727.7 148 1084.8 50 8930.3 71
11727.7 148 8430.8 122 370.0 42
1707.1 49 1775.9 96 1626.8 15
Carious teeth Intact Low-temperature processed Room-temperature processed
11692.8 345 2086.8 56 8904.8 213
11692.8 345 8011.7 197 413.2 47
1787.6 48 1571.4 22 1548.9 55
Cell numbers are presented for healthy and carious sections in intact teeth and following processing at both lowand room-temperature. Standard error of the mean values are also shown (n ¼ 6).
In contrast, teeth processed at room-temperature have on average 87.5% of the odontoblasts attached to the pulp tissue following removal. The histomorphometric data confirmed the effectiveness of our temperature-sensitive extraction approach in both human sound and carious teeth and rodent incisors.
Human dental tissue gene expression analysis To determine the expression profile of genes in diseased and healthy dental tissue obtained by either low- or room-temperature processing, sqRT-PCR analysis was performed. The genes analysed included; DSPP, Nestin, collagen-1a, collagen-3a, TGF-b1, TGF-b2, TGF-b3, TGF-bR1, TGF-bR2, TGFbR3, BMP-7, BMP-R1A, and BMP-R1B. The data obtained indicated that markers previously shown to be preferentially expressed in odontoblasts, such as DSPP and Nestin, were amplified more readily from low-temperature as compared to room-temperature processed human tissue (see Fig. 3a and b). However, no obvious differences in expression levels for DSPP and Nestin in diseased and healthy
Table 3
dental tissue were detected. Collagen-1a and collagen-3a were found to be more abundantly expressed in diseased pulpal tissue and members of the TGF-b and BMP families, and their receptors, were shown to be basally expressed by odontoblasts and, in general, more abundantly expressed in diseased tissue. Data also indicate that TGF-b1, and the BMP-receptor 1A and BMP-receptor 1B were more abundant in diseased tissue, which comprised of both pulpal and odontoblasts cells (Fig. 3).
Rodent dental tissue gene expression analysis To determine whether genes, known to be selectively expressed in odontoblasts, could be detected preferentially in RNA extracted from the pulp— odontoblast complex as compared to RNA from pulpal tissue alone, sq-RT-PCR analysis was performed. Data indicated that markers previously shown to be preferentially expressed in odontoblasts, such as DSPP and Nestin, were amplified more readily from room-temperature as compared to low-temperature processed tissue (see Fig. 4a and b).
Average number of odontoblasts and fibroblasts in 5 mm rodent incisor tooth sections. Number of odontoblasts
Intact Low-temperature processed Room-temperature processed
On dentine
On pulp
744.1 16 630.2 15 102.3 12
744.1 16 92.8 4 718.7 23
Number of fibroblasts
253.6 11 325.2 24 295.4 15
Cell numbers are presented for intact teeth and following low- and room-temperature processing. Standard error of the mean values are shown for each group.
Gene expression in mature dental tissue
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Figure 3 (a) Semi-quantitative RT-PCR analysis showing differential gene expression analysis in human dental tissue samples. Carious or healthy samples were processed at low- and room-temperature. (b) Densitometric analysis of gel images shown. Gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalised against the GAPDH housekeeping gene control values. Averaged values for each group were calculated and expressed as a percentage of the highest normalised individual volume density obtained and plotted.
Discussion Dental tissue extraction and histological analysis Our histological data from human teeth is consistent with previous reports which indicate that following room-temperature processing and pulpal extirpation the odontoblasts remain lining the pulp chamber (see Fig. 1).21—25 In contrast, however, our study provides the first report that low-temperature processing of human dental tissue results in isolation of a pulp—odontoblast complex (see Fig. 1). Conver-
sely our study has also shown that in rodent incisors following room-temperature processing, pulpal extirpation resulted in a pulp—odontoblast complex being obtained, an outcome consistent with previous reports.26 However low-temperature processing resulted in the novel observation that a relatively pure pulpal tissue was isolated. Whilst it would be valuable to examine an odontoblast only population, isolated from the dentine following pulpal extirpation, other studies have indicated that large amounts of teeth are required to produce detectable amounts of RNA for downstream analysis.8 Our own studies (data not presented) have
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J.L. McLachlan et al.
Figure 4 (a) sq-RT-PCR analysis showing differential gene expression of DSPP and Nestin in rodent dental tissue obtained by low- and room-temperature processing. (b) Densitometric analysis of DSPP and Nestin gel images. Gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalised against the GAPDH housekeeping gene control values. Averaged values for each group were calculated and expressed as a percentage of the highest normalised individual volume density obtained and plotted.
indicated that detectable amounts of RNA cannot be isolated from individual teeth using this approach. The data presented here indicates that our novel dental tissue isolation procedure can be used to analyse the gene expression profile of odontoblasts using a subtraction approach. Currently, we are only able to hypothesise as to why low-temperature processing of human dental tissue results in the isolation of an intact pulp—odontoblast complex, whilst the same approach when used on rodent incisors results in the isolation of a relatively pure pulpal tissue. A possible explanation may arise from the different sizes of the odontoblast process in rodents and humans. In humans, the odontoblast process extends for several millimetres into the dentinal tubule, whilst in rodents this extension is approximately 1 mm.26 In addition there may also be more elaborate lateral branching of the odontoblast processes in human dentine. It is therefore possible that in human tissue, the odontoblasts are more firmly attached to the dentine and therefore, are not removed along with the pulp at room-temperature, unlike in rodent teeth. The snap-freezing of the human teeth may result in a strengthening of
the bond between the pulpal tissue and the odontoblast cells by solidifying the matrix in the area of the cell-free zone. The completion of primary dentinogenesis in these teeth may also have led to some regressive changes in the odontoblasts resulting in dimensional reductions.1 These factors may enable the tissue complex to be removed as one. In contrast, the odontoblasts and their processes in rodent teeth would be at a stage of high secretory activity and unlikely to have shown any regressive dimensional changes.2 The snap-freezing of rodent teeth may cause increased anchoring of the odontoblast due to expansion of the process within the dentine.
DSPP and Nestin levels in extracted dental tissue samples Based on our histological findings we hypothesised that genes such as DSPP and Nestin, which have previously been shown to be preferentially expressed by odontoblasts,27,28 should amplify more readily from RNA isolated from human and rodent pulp—odontoblast complex tissue as compared to pulpal tissue alone. Our results indicated
Gene expression in mature dental tissue
this was indeed the case and thereby supported our histological findings (see Figs. 3 and 4). It was notable, however, that there was a more marked difference in detectable levels of DSPP and Nestin between human samples collected and processed at low- or room-temperature, than in rodent samples (see Figs. 3 and 4). Histomorphometric data (Table 2) indicated that for both healthy and diseased human samples there was an approximately 20-fold increase in the number of odontoblasts attached to the pulp following extirpation at lowtemperature as compared to room-temperature. In contrast, in rodent dental samples the cell count data (Table 3) indicated that there was only a 7—8fold increase in the number of odontoblasts attached to the pulp tissue following extirpation at room-temperature as compared to low-temperature. Whilst this histological data only provides twodimensional information it does however indicate that the ratio of odontoblasts on the pulp—odontoblast complex as compared to pulp enriched tissue is significantly higher in humans. This therefore most probably explains the apparent difference in relative expression levels for DSPP and Nestin between low- and room-temperature processed dental tissue in rodent and human samples.
Analysis of gene expression levels in carious and non-carious teeth Histological analysis and histomorphometric data combined with our DSPP and Nestin expression analysis data lead us to hypothesise that our temperature dependent dental tissue extraction approach could be used to assay gene expression levels specific to odontoblasts or pulpal fibroblasts in carious and non-carious teeth. Whilst previous reports have indicated that protein levels for DSPP and Nestin are higher during dentinogenesis,29,30 we did not note any observable gene expression level difference between healthy and diseased samples (Fig. 3) Our results may indicate that DSPP and Nestin gene expression changes are relatively small due to lack of reparative response in the specimens examined and/or are occurring only in a very localised group of odontoblasts beneath the site of injury. In addition, it is noteworthy that changes in protein levels are not always reflected by similar changes in gene expression. Gene expression data for both collagen-1a and collagen-3a indicated they were more abundant in carious teeth and that levels in the pulp—odontoblast complex from carious teeth were not observably higher than in carious pulpal tissue alone. This data therefore clearly indicated that these genes
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were predominantly expressed by cells within the pulpal tissue (see Fig. 3). Whilst previous studies have indicated that odontoblasts express collagen1a, our data indicates its expression is more abundant in carious pulpal tissue. This result may reflect a generalised inflammatory response by the pulpal fibroblasts at the transcriptional level and may represent the associated fibrotic processes that occur.31 In contrast, the results indicated that TGF-b and BMP family members and their receptors are predominantly expressed by odontoblasts in healthy teeth and in addition, generally showed higher levels of expression in odontoblasts and pulpal tissue from carious teeth. The sq-RT-PCR data (see Fig. 3) also indicated that TGF-b1, BMP-receptor 1A and BMP-receptor 1B were up-regulated in odontoblast cells during disease. Support for these observations is provided by previous immunohistochemical studies, which have shown pulpal and odontoblast expression of the TGF-b isoforms 1—3 and their receptors in both healthy and carious human molar teeth.17,20 Whilst gene expression of BMP’s and their receptors has previously been reported in human dental pulp,19 our sq-RT-PCR analysis is the first to provide transcription profiling data for these molecules in odontoblast and pulp cells in sound and carious human teeth. In addition, we feel it is noteworthy that as previous studies have shown that several members of the TGF-b superfamily and their receptors are expressed by immune and inflammatory cells, it is possible that their increased expression levels detected here are contributed to by these infiltrating cells.
Implications of this study Previous reports8,32 have indicated that following pulpal tissue extirpation, RNA can be isolated from both the extirpated pulpal tissue and the odontoblasts which remain lining the vacated pulp chamber of human teeth. However, these studies have reported that RNA isolated from five pulp samples and 20 odontoblast preparations require pooling to enable single gene analysis by RT-PCR to be performed. Our results now indicate that sufficient amounts of RNA can be obtained from a single human or rodent tooth to enable multiple differential gene expression analyses in pulpal and odontoblast cells. In addition, we anticipate that this approach can also be applied for the study of in vitro cellular specific gene expression changes in human and rodent organ cultures exposed to a variety of stimulants and insults.33,34 Our procedure is tech-
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nically simple, robust, reproducible, efficient and economical and can be applied in any laboratory equipped with standard molecular biology apparatus. Combining this temperature-sensitive dental tissue extraction technique with powerful molecular biological techniques, such as DNA array technology, will facilitate further enhancement of our knowledge of the molecular events associated with dental disease.
J.L. McLachlan et al.
12.
13.
14.
15.
Acknowledgements JLM is supported by a University of Birmingham, Scientific Projects Committee grant number EBX 1193. This study was also supported in part by a University of Birmingham, Scientific Projects Committee grant number EBX 1194. We would like to thank Miss Sally Kerr (Oral Surgery) for providing the teeth for this investigation.
16.
17.
18.
19.
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(BMP-2) on the differentiation of odontoblasts in-vitro. Int J Dev Biol 1992;36:491—503. Tucker AS, Sharpe PT. Molecular genetics of tooth morphogenesis and patterning: the right shape in the right place. J Dent Res 1999;78:826—34. Sloan AJ, Smith AJ. Stimulation of the dentine—pulp complex of rat incisor teeth by transforming growth factor-b isoforms 1—3 in vitro. Arch Oral Biol 1999;44:149—56. Smith AJ, Lesot H. Induction and regulation of crown dentinogenesis embryonic events as a template for dental tissue repair. Crit Rev Oral Biol Med 2001;12:425—37. Finkelman RD, Mohan S, Jennings JC, Taylor AK, Jepsen S, Baylink DJ. Quantitation of growth factors IGF-I, SGFIIGF-II, and TGF-b in human dentin. J Bone Miner Res 1990;5: 717—23. Cassidy N, Fahey M, Prime SS, Smith AJ. Comparative analysis of transforming growth factor-b isoforms 1—3 in human and rabbit dentine matrices. Arch Oral Biol 1997;42:219—23. Sloan AJ, Perry H, Matthews JB, Smith AJ. Transforming growth factor-beta isoform expression in mature human healthy and carious molar teeth. Histochem J 2000;32: 247—52. Zhao S, Sloan AJ, Murray PE, Lumley PJ, Smith AJ. Ultrastructural localisation of TGF-b exposure in dentine by chemical treatment. Histochem J 2000;32:489—94. Gu K, Smoke RH, Rutherford RB. Expression of genes for bone morphogenetic proteins and receptors in human dental pulp. Arch Oral Biol 1996;41:919—23. Sloan AJ, Matthews JB, Smith AJ. TGF-b receptor expression in human odontoblasts and pulpal cells. Histochem J 1999; 31:565—9. Pincus P. Some physiological data on human dental pulp. Br Dent J 1950;89:143—8. Kramer IRH. The isolation and examination of odontoblasts, in the fresh unfixed state. Proc R Soc Med 1956;49:545—6. Flieder DE, Fisher AK. The rate of endogenous oxygen consumption in bovine dental pulp. J Dent Res 1955;34: 921—3. Rockert H. Methods for isolating odontoblasts and determination of intracellular potassium. Acta Odont Scand 1964; 22:373—8. Chada S, Bishop MA. Effect of mechanical removal of the pulp upon the retention of the odontoblasts around the pulp chamber of human third molars. Arch Oral Biol 1996;41: 905—9. Gotjamanos T. A method for isolating an intact dental pulp from rat dentine. Arch Oral Biol 1969;14:729—30. MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4. J Biol Chem 1997;271: 21695—8. Feng JQ, Luan XH, Wallace J, Jing D, et al. Genomic organization, chromosomal mapping, and promoter analysis of the mouse dentin sialophosphoprotein (DSPP) gene, which codes for both dentin sialoprotein and dentin phosphoprotein. J Biol Chem 1998;273:9457—64. About I, Laurent-Maquin D, Lendahl U, Mitsiadis TA. Nestin expression in embryonic and adult human teeth under normal and pathological conditions. Am J Pathol 2000;157: 287—95. Papagerakis P, Berdal A, Mesbah M, et al. Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth. Bone 2002;30:377—85. Varga J, Jimenez SA. Modulation of collagen gene expression: its relation to fibrosis in systemic sclerosis and other disorders. Ann Int Med 1995;122:60—2.
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32. Levin LG, Rudd A, Bletsa A, Reisner H. Expression of IL-8 by cells of the odontoblast layer in vitro. Eur J Oral Sci 1999; 107:131—7. 33. Sloan AJ, Shelton RM, Hann AC, Moxham BJ, Smith AJ. An in vitro approach for the study of dentinogenesis by organ culture of the dentine—pulp complex from rat incisor teeth. Arch Oral Biol 1998;43:421—30. 34. Dobie K, Smith G, Sloan AJ, Smith AJ. Effects of alginate hydrogels and TGF-b1 on human dental pulp repair in vitro. Conn Tiss Res 2002;43:387—90.
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35. Buchaille R, Couble ML, Magloire H, Bleicher F. A substractive PCR-based cDNA library from human odontoblast cells: identification of novel genes expressed in tooth forming cells. Matrix Biol 2000;19:421—30. 36. Yousefi S, Cooper PR, Mueck B, Potter SL, Jarai G. cDNA representational difference analysis of human neutrophils stimulated by GM-CSF. Biochem Biophys Res Commun 2000;277:401—9.
Gene expression analysis in cells of the dentine—pulp complex in healthy and carious teeth Julia L. McLachlan, Anthony J. Smith, Alastair J. Sloan, Paul R. Cooper* Oral Biology, School Of Dentistry, University of Birmingham, St. Chads Queensway, Birmingham B4 6NN, UK Accepted 13 December 2002
KEYWORDS Gene expression; Odontoblast; Pulp; Human; Rodent
Summary Knowledge of the molecular events that occur in carious disease has so far been constrained due to difficulties in obtaining sufficient quantities of the dental tissues and cells involved. Our histological findings indicate that a pulp—odontoblast cellular complex can be obtained from carious and healthy human teeth when exposed to lowtemperatures prior to pulpal extirpation and from rodent teeth processed at roomtemperature. In contrast, pulpal tissue extracted from room-temperature processed human teeth and low-temperature processed rodent teeth resulted in the odontoblast layer remaining attached to the pulp chamber. Semi-quantitative RT-PCR (sq-RT-PCR) analysis confirmed that markers previously shown to be preferentially expressed in odontoblasts, namely dentin sialophosphoprotein (DSPP) and Nestin, amplified more readily from the extracted pulp—odontoblast complex, as compared to pulpal tissue alone, in both human and rodent samples. Subsequent gene expression analysis of collagen-1a and collagen-3a indicated levels were significantly higher in carious pulpal tissue. In addition, analysis characterising the expression of members of the transforming growth factor and bone morphogenic protein families and their receptors indicated in general, that these genes were expressed by healthy odontoblasts and up-regulated in both pulpal cells and odontoblasts in response to carious injury. Use of this temperaturesensitive dental tissue preparation procedure allows detection of differential gene expression in odontoblasts and other pulpal cells in healthy and carious tissue. ß 2003 Elsevier Science Ltd. All rights reserved.
Introduction Characterisation of the molecular and cellular events important in dental tissue injury and repair is critical to our understanding of these processes. Abbreviations: TGF, transforming growth factor; BMP, bone morphogenic protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcriptase polymerase chain reaction; DSPP, dentin sialophosphoprotein Corresponding author. Tel.: þ44-121-237-2785; fax: þ44-121-625-8815. E-mail address: [email protected] (P.R. Cooper).
Odontoblasts are highly specialised post-mitotic cells aligned in a homogenous monolayer at the periphery of the dental pulp and are solely responsible for both the developmental and reparative formation of dentine.1,2 During mild tissue injury, odontoblasts localised beneath the damaged region can up-regulate their dentine secretory activity.3 However, injury of greater intensity can cause localised odontoblast necrosis. These cells are subsequently replaced by an odontoblast-like cell population, which differentiate from pulpal progenitor cells, and secrete a reparative dentine matrix.4
0003–9969/03/$ — see front matter ß 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0003-9969(03)00003-7
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At present, our understanding of the transcriptional changes important in these repair processes has been limited due to the nature and location of the odontoblast cells. Attempts to overcome these obstacles has resulted in utilisation of in vitro culturing of immortalised odontoblast-like cells derived from a variety of species.5—7 However, gene expression changes identified using this approach most likely do not accurately or comprehensively reflect events that occur in vivo in man. Transcript profiling utilising clinically derived tissue has also been performed although, reports have indicated that large numbers of teeth are required to provide sufficient quantities of RNA for this analysis.8 Recently, the utility of laser capture microdissection (LCM) has also been demonstrated for the study of gene expression in developing murine dental tissue.9 Whilst this approach offers promise for the study of human dental tissue gene expression, its general application is limited by the requirement for expensive specialist equipment and its ability to provide only relatively small amounts of RNA, limiting the further downstream applications possible. The role of growth factors in dentinogenic processes is well documented.10 In particular, members of the transforming growth factor-b (TGF-b) and bone morphogenic protein (BMP) families, have been implicated in controlling aspects of tooth development and tissue repair.11—14 Experiments have shown that cells of the dentine—pulp complex are capable of synthesising these growth factors, which are subsequently sequestered into the dentine matrix and released during tissue injury, potentially stimulating repair mechanisms.15—18 Whilst the presence of these growth factors and their receptors has previously been reported in dental tissue,19,20 their gene expression profile during health and disease has yet to be determined. The aim of this study was to use semi-quantitative reverse transcriptase polymerase chain reaction (sq-RT-PCR) to examine gene expression levels of molecules important in dental tissue mineralization and growth factors and their receptors within the cells of the dentine—pulp complex in carious and non-carious tissue. This knowledge will contribute to our understanding of the molecular events important in dental tissue injury and repair.
Materials and methods Dental tissue extraction from human and rodent teeth Carious and sound mature human teeth with fully formed roots extracted for orthodontic purposes
J.L. McLachlan et al.
from patients generally in the age range of 20—30 years, were obtained immediately after extraction from clinics at the Birmingham Dental Hospital following informed patient consent. Carious teeth exhibited disease ranging from enamel involvement only to deep dentinal lesions. Rodent maxillary and mandibular incisor teeth were also dissected, using sterilised scalpels and tweezers, from 28-day-old male Wistar rats. Following extraction, human and rodent teeth were immediately frozen in liquid nitrogen for low-temperature processing or submerged in the RNA stabilising solution, RNA Later (Sigma, UK), for subsequent room-temperature processing. Teeth were then longitudinally sliced, using a segmented, diamond-edged rotary saw (TAAB Laboratories, UK) cooled with PBS, and the pulpal tissue carefully removed intact using a sterile dental probe and forceps. Extracted dental tissues were either processed for histological analysis or were used in RNA isolation.
Histological analysis Both dentine and pulp samples (human and rodent) were fixed for 24 h in 10% (w/v) neutral buffered formalin (Surgipath, UK). Dentine samples were subsequently demineralised in 10% formic acid (Sigma, UK) until fully decalcified (approximately 10 days for human samples and 6 days for rodent specimens), as assessed by X-ray examination. Both pulp and dentine samples were routinely processed through a series of graded alcohols and xylene, and finally embedded in paraffin wax. Serial sections of 5 mm thickness were cut and stained in Mayer’s haematoxylin and eosin (H & E).
Histomorphometric analysis Quantitation of cells in the dentine—pulp complex of human and rodent teeth was performed on 5 mm H & E stained longitudinal sections. Histomorphometry was undertaken on healthy and diseased sections from human teeth and rodent intact incisors processed at both low- and room-temperature. One representative section from six teeth was counted for each of the groups described. The sections chosen for counting were all derived from teeth of similar morphology, i.e. molars from humans and incisors from rodents. Histological sections chosen had also been cut to similar levels in all teeth. Counting was performed using an ocular graticule, at 500 magnification. Following Hunting curve analysis, to identify the number of counts needed to produce reproducible results unaffected by further counts, odontoblast cell numbers were
Gene expression in mature dental tissue
counted in 20 random graticule squares along the interface between the dentine and pulp tissue. To estimate the total number of odontoblasts in one 5 mm histological section, the perimeter of the pulp chamber was estimated by accurately counting the number of squares around the pulp chamber perimeter at 50 magnification. The value obtained was multiplied by the average number of odontoblasts found in one square, allowing for the degree of magnification. Similarly, following Hunting curve analysis fibroblast cell numbers were counted in 20 random graticule squares in the pulp tissue. To estimate the total number of fibroblasts within one histological section, the number of squares that overlaid the pulpal area were counted at 50 magnification. The value obtained was multiplied by the average number of fibroblasts found in one square. In diseased tissue, cells not displaying a fibroblast morphology were excluded from cell counts.
RNA isolation and cDNA synthesis Following either low- or room-temperature preparation of pulpal tissue from sound or carious human teeth and from rodent teeth, total RNA was extracted using the RNeasy mini kit (Qiagen, UK) as recommended by the manufacturer. Subsequently, 1—5 mg of DNase digested total RNA was used for oligo-dT reverse transcription to generate single stranded cDNA using the Omniscript kit (Qiagen, UK). Both RNA and cDNA concentrations were determined using a BioPhotometer (Eppendorf, UK).
Semi-quantitative reverse transcriptase polymerase chain reaction (sq-RT-PCR analysis) sq-RT-PCR analysis was performed for the human genes DSPP, Nestin, collagen-1a, collagen-3a, TGFb1, TGF-b2, TGF-b3, TGF-b receptor 1 (TGF-bR1), TGF-b receptor 2 (TGF-bR2), TGF-b receptor 3 (TGF-bR3), BMP-7, BMP-receptor 1A (BMP-R1A), and BMP-receptor 1B (BMP-R1B) and rodent genes DSPP and Nestin using the primer sequences provided for the respective species of tissue analysed (Table 1). The housekeeping gene glyceraldehyde3-phosphate dehydrogenase (GAPDH) was used as a control. In summary, 50 ng of single stranded cDNA was used to seed a 50 ml PCR, which was subjected to between 28 and 42 cycles (as indicated in Table 1). A typical amplification cycle of, 95 8C for 20 s, 51—60 8C for 20 s, and 72 8C for 20 s, was performed using an Eppendorf Mastercycler thermal cycler (Eppendorf, UK). Following the designated
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number of cycles, 8 ml of the reaction was removed and the product separated and visualised on a 1.5% agarose gel containing 0.5 mg/ml ethidium bromide. Scanned gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalised against the GAPDH housekeeping gene control values.
Results Histological examination of human dental tissue following low- or room-temperature processing Following pulpal extirpation from low- or roomtemperature processed human teeth, histological analysis was performed to identify the subsequent localisation of the odontoblasts. Data indicated that their localisation was dependent on the tooth preparation procedure used. Following pulpal preparation from human teeth collected and processed at room-temperature (i.e. in RNA later), the odontoblasts remained lining the pulp chamber attached to the dentine matrix. However, pulp removed from teeth collected and processed at low-temperature (i.e. in liquid nitrogen) had the odontoblast cell layer attached, providing a pulp—odontoblast cellular complex. Tissue separation was unaffected by the extent of the carious process in the diseased teeth. Histological studies were performed on five teeth from each of the four categories of diseased and healthy teeth collected and processed in RNA Later and liquid nitrogen. One representative image from each of the four groups described is presented in Fig. 1.
Histological examination of rodent dental tissue following low- or room-temperature processing The localisation of the odontoblasts following pulpal extirpation from rodent teeth was similarly examined histologically. Again, the data indicated that odontoblast localisation was dependent on the tooth processing procedure used, however, the results were opposite to those obtained for human dental tissue. Following pulpal extirpation from teeth processed at room-temperature, the odontoblast cell layer remained attached to the pulpal tissue, thereby providing a pulp—odontoblast complex. When pulp was removed from teeth processed at low-temperature, the odontoblasts were found to remain attached to the dentine in the pulp chamber. Two representative images
276
Table 1
J.L. McLachlan et al.
Primer sequences and cycling conditions used for human and rodent () gene expression analyses.
Gene
Primer sequence (50 ! 30 )
Annealing Cycles temperature (8C)
Product size (bp)
Source
BMP-7
Forward CGC TTC GAC AAT GAG ACG TTC Reverse TGG CGT TCA TGT AGG AGT TCA G
60
35
571
19
BMP-R1A
Forward GAA GAA GAT GAC CAG GGA GGA Reverse CCG TCA TGA AAC CAA GTA TGT
54
32
622
19
BMP-R1B
Forward GTG GCG TGG CGA AAA GGT AGC Reverse TTG GGA ATG AGG GGC GTA AGT
58
30
698
19
TGF-b1
Forward CGC CTT AGC GCC CAC TGC TCC TGT Reverse GGG GCG GGA CCT CAG CTG CAC
60
34
532
Growth factors
TGF-b2
Forward GCT CTG TGG GTA CCT TGA TGC CAT CC Reverse TTC TTC CGC CGG TTG GTC TGT G
60
38
573
Growth factors
TGF-b3
Forward ACT GCC GAG TGG CTG TCC TTT GAT G Reverse AGG CAG ATG CTT CAG GGT TCA GAG TG
60
36
538
Growth factors
TGF-bR1
Forward CGT TAC AGT GTT TCT GCC ACC T Reverse AGA CGA AGC ACA CTG GTC CAG C
53
42
315
AJS personal communication
TGF-bR2
Forward CCC CAA GCT CCC CTA CCA TGA C Reverse AAC TCC GTC TTC CGC TCC TCA G
53
34
642
AJS personal communication
TGF-bR3
Forward TGT CAC CTG GCA CAT TCA TT Reverse CTC AGC ACT GTC TTG GTG GA
60
32
261
Primer 3
Collagen-1a
Forward TGG GAG TGC AAG GAT ACT CTA TAT CG Reverse CCC ATC CCA TCT TCG ACG TAC
60
28
320
32
Collagen-3a
Forward CTG GAC CAA AAG GTG ATG CT Reverse CAG GGT TTC CAT CTC TTC CA
60
28
310
Primer 3
Nestin
Forward GGC AGC GTT GGA ACA GAG GTT GGA Reverse CTC TAA ACT GGA GTG GTC AGG GCT
55
30
718
GenBank accession numbers AJ270321 and AJ270322
DSPP
Forward CCT AAA GAA AAT GAA GAT AAT T Reverse TAG AAA AAC TCT TCC CTC CTA C
51
36
293
35
GAPDH
Forward CCA CCC ATG GCA AAT TCC ATG GCA Reverse TCT AGA CGG CAG GTC AGG TCC ACC
60
28
550
36
DSPP
Forward TGC ATT TTG AAG TGT CTC GC Reverse CCT CCT GTC TTG GTG TGG TT
60
25
277
Primer 3
Nestin
Forward CAT TTA GAT GCT CCC CAG GA Reverse AAT CCC CAT CTA CCC CAC TC
60
30
285
Primer 3
GAPDH
Forward GAT CCC GCT AAC ATC AAA T Reverse GGA TGC AGG GAT GAT GTT CT
60
28
389
Primer 3
Sources of the primer sequences are provided. For primer design the primer 3 program was used at http.//wwwgenome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi.
from studies performed on 10 teeth, 5 teeth collected and processed in RNA later and 5 teeth collected and processed in liquid nitrogen, are presented in Fig. 2.
Histomorphometric analysis of human and rodent dental tissue Table 2 provides data obtained on odontoblast and fibroblast cell numbers in diseased and healthy human teeth. The data indicate that following human pulpal tissue extirpation at low-temperature
approximately 88.6 and 79.3% of the odontoblasts are attached to the pulpal tissue in healthy and diseased teeth, respectively. Whereas following room-temperature processing, approximately 96.0% for healthy teeth and 95.5% for diseased teeth of the odontoblasts cells remained lining the dentine surface. Table 3 provides data obtained on odontoblast and fibroblast cell numbers in rodent incisors. The data indicates that approximately 87% of the odontoblast cells remain attached to the dentine layer following pulpal extirpation at low-temperature.
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277
Figure 1 Histological analysis of H & E stained human healthy (a, b, e and f) and diseased (c, d, g and h) dental tissue collected and processed at room-temperature (a, b, c and d) and low-temperature (e, f, g and h). Paired images each taken from the same tooth, a and b, c and d, e and f and g and h, following pulpal extirpation are shown. Roomtemperature processing resulted in the odontoblast cells (O) remaining attached to the dentine (D) following pulpal extirpation. Whilst low-temperature processing resulted in the isolation of the pulp (P) and odontoblasts as a complex. Images are at 40 magnification.
Figure 2 Histological analysis of H & E stained rodent dental tissue processed at room-temperature (a, b, c and d) and low-temperature (e, f, g and h). Paired images each taken from the same tooth, a and b, c and d, e and f and g and h, following pulpal extirpation are shown. Processing of rodent tissue at room-temperature resulted in the isolation of the pulp (P) and odontoblasts (O) as a complex, whilst pulpal extirpation of rodent teeth at low-temperature resulted in the odontoblast cells remaining attached to the dentine (D) surface. Images shown are at 40 magnification.
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Table 2
J.L. McLachlan et al.
Average number of odontoblasts and fibroblasts present in 5 mm human tooth sections. Number of odontoblasts
Number of fibroblasts
On dentine
On pulp
Healthy teeth Intact Low-temperature processed Room-temperature processed
11727.7 148 1084.8 50 8930.3 71
11727.7 148 8430.8 122 370.0 42
1707.1 49 1775.9 96 1626.8 15
Carious teeth Intact Low-temperature processed Room-temperature processed
11692.8 345 2086.8 56 8904.8 213
11692.8 345 8011.7 197 413.2 47
1787.6 48 1571.4 22 1548.9 55
Cell numbers are presented for healthy and carious sections in intact teeth and following processing at both lowand room-temperature. Standard error of the mean values are also shown (n ¼ 6).
In contrast, teeth processed at room-temperature have on average 87.5% of the odontoblasts attached to the pulp tissue following removal. The histomorphometric data confirmed the effectiveness of our temperature-sensitive extraction approach in both human sound and carious teeth and rodent incisors.
Human dental tissue gene expression analysis To determine the expression profile of genes in diseased and healthy dental tissue obtained by either low- or room-temperature processing, sqRT-PCR analysis was performed. The genes analysed included; DSPP, Nestin, collagen-1a, collagen-3a, TGF-b1, TGF-b2, TGF-b3, TGF-bR1, TGF-bR2, TGFbR3, BMP-7, BMP-R1A, and BMP-R1B. The data obtained indicated that markers previously shown to be preferentially expressed in odontoblasts, such as DSPP and Nestin, were amplified more readily from low-temperature as compared to room-temperature processed human tissue (see Fig. 3a and b). However, no obvious differences in expression levels for DSPP and Nestin in diseased and healthy
Table 3
dental tissue were detected. Collagen-1a and collagen-3a were found to be more abundantly expressed in diseased pulpal tissue and members of the TGF-b and BMP families, and their receptors, were shown to be basally expressed by odontoblasts and, in general, more abundantly expressed in diseased tissue. Data also indicate that TGF-b1, and the BMP-receptor 1A and BMP-receptor 1B were more abundant in diseased tissue, which comprised of both pulpal and odontoblasts cells (Fig. 3).
Rodent dental tissue gene expression analysis To determine whether genes, known to be selectively expressed in odontoblasts, could be detected preferentially in RNA extracted from the pulp— odontoblast complex as compared to RNA from pulpal tissue alone, sq-RT-PCR analysis was performed. Data indicated that markers previously shown to be preferentially expressed in odontoblasts, such as DSPP and Nestin, were amplified more readily from room-temperature as compared to low-temperature processed tissue (see Fig. 4a and b).
Average number of odontoblasts and fibroblasts in 5 mm rodent incisor tooth sections. Number of odontoblasts
Intact Low-temperature processed Room-temperature processed
On dentine
On pulp
744.1 16 630.2 15 102.3 12
744.1 16 92.8 4 718.7 23
Number of fibroblasts
253.6 11 325.2 24 295.4 15
Cell numbers are presented for intact teeth and following low- and room-temperature processing. Standard error of the mean values are shown for each group.
Gene expression in mature dental tissue
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Figure 3 (a) Semi-quantitative RT-PCR analysis showing differential gene expression analysis in human dental tissue samples. Carious or healthy samples were processed at low- and room-temperature. (b) Densitometric analysis of gel images shown. Gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalised against the GAPDH housekeeping gene control values. Averaged values for each group were calculated and expressed as a percentage of the highest normalised individual volume density obtained and plotted.
Discussion Dental tissue extraction and histological analysis Our histological data from human teeth is consistent with previous reports which indicate that following room-temperature processing and pulpal extirpation the odontoblasts remain lining the pulp chamber (see Fig. 1).21—25 In contrast, however, our study provides the first report that low-temperature processing of human dental tissue results in isolation of a pulp—odontoblast complex (see Fig. 1). Conver-
sely our study has also shown that in rodent incisors following room-temperature processing, pulpal extirpation resulted in a pulp—odontoblast complex being obtained, an outcome consistent with previous reports.26 However low-temperature processing resulted in the novel observation that a relatively pure pulpal tissue was isolated. Whilst it would be valuable to examine an odontoblast only population, isolated from the dentine following pulpal extirpation, other studies have indicated that large amounts of teeth are required to produce detectable amounts of RNA for downstream analysis.8 Our own studies (data not presented) have
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J.L. McLachlan et al.
Figure 4 (a) sq-RT-PCR analysis showing differential gene expression of DSPP and Nestin in rodent dental tissue obtained by low- and room-temperature processing. (b) Densitometric analysis of DSPP and Nestin gel images. Gel images were imported into AIDA image analysis software (Fuji, UK) and the volume density of amplified products calculated and normalised against the GAPDH housekeeping gene control values. Averaged values for each group were calculated and expressed as a percentage of the highest normalised individual volume density obtained and plotted.
indicated that detectable amounts of RNA cannot be isolated from individual teeth using this approach. The data presented here indicates that our novel dental tissue isolation procedure can be used to analyse the gene expression profile of odontoblasts using a subtraction approach. Currently, we are only able to hypothesise as to why low-temperature processing of human dental tissue results in the isolation of an intact pulp—odontoblast complex, whilst the same approach when used on rodent incisors results in the isolation of a relatively pure pulpal tissue. A possible explanation may arise from the different sizes of the odontoblast process in rodents and humans. In humans, the odontoblast process extends for several millimetres into the dentinal tubule, whilst in rodents this extension is approximately 1 mm.26 In addition there may also be more elaborate lateral branching of the odontoblast processes in human dentine. It is therefore possible that in human tissue, the odontoblasts are more firmly attached to the dentine and therefore, are not removed along with the pulp at room-temperature, unlike in rodent teeth. The snap-freezing of the human teeth may result in a strengthening of
the bond between the pulpal tissue and the odontoblast cells by solidifying the matrix in the area of the cell-free zone. The completion of primary dentinogenesis in these teeth may also have led to some regressive changes in the odontoblasts resulting in dimensional reductions.1 These factors may enable the tissue complex to be removed as one. In contrast, the odontoblasts and their processes in rodent teeth would be at a stage of high secretory activity and unlikely to have shown any regressive dimensional changes.2 The snap-freezing of rodent teeth may cause increased anchoring of the odontoblast due to expansion of the process within the dentine.
DSPP and Nestin levels in extracted dental tissue samples Based on our histological findings we hypothesised that genes such as DSPP and Nestin, which have previously been shown to be preferentially expressed by odontoblasts,27,28 should amplify more readily from RNA isolated from human and rodent pulp—odontoblast complex tissue as compared to pulpal tissue alone. Our results indicated
Gene expression in mature dental tissue
this was indeed the case and thereby supported our histological findings (see Figs. 3 and 4). It was notable, however, that there was a more marked difference in detectable levels of DSPP and Nestin between human samples collected and processed at low- or room-temperature, than in rodent samples (see Figs. 3 and 4). Histomorphometric data (Table 2) indicated that for both healthy and diseased human samples there was an approximately 20-fold increase in the number of odontoblasts attached to the pulp following extirpation at lowtemperature as compared to room-temperature. In contrast, in rodent dental samples the cell count data (Table 3) indicated that there was only a 7—8fold increase in the number of odontoblasts attached to the pulp tissue following extirpation at room-temperature as compared to low-temperature. Whilst this histological data only provides twodimensional information it does however indicate that the ratio of odontoblasts on the pulp—odontoblast complex as compared to pulp enriched tissue is significantly higher in humans. This therefore most probably explains the apparent difference in relative expression levels for DSPP and Nestin between low- and room-temperature processed dental tissue in rodent and human samples.
Analysis of gene expression levels in carious and non-carious teeth Histological analysis and histomorphometric data combined with our DSPP and Nestin expression analysis data lead us to hypothesise that our temperature dependent dental tissue extraction approach could be used to assay gene expression levels specific to odontoblasts or pulpal fibroblasts in carious and non-carious teeth. Whilst previous reports have indicated that protein levels for DSPP and Nestin are higher during dentinogenesis,29,30 we did not note any observable gene expression level difference between healthy and diseased samples (Fig. 3) Our results may indicate that DSPP and Nestin gene expression changes are relatively small due to lack of reparative response in the specimens examined and/or are occurring only in a very localised group of odontoblasts beneath the site of injury. In addition, it is noteworthy that changes in protein levels are not always reflected by similar changes in gene expression. Gene expression data for both collagen-1a and collagen-3a indicated they were more abundant in carious teeth and that levels in the pulp—odontoblast complex from carious teeth were not observably higher than in carious pulpal tissue alone. This data therefore clearly indicated that these genes
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were predominantly expressed by cells within the pulpal tissue (see Fig. 3). Whilst previous studies have indicated that odontoblasts express collagen1a, our data indicates its expression is more abundant in carious pulpal tissue. This result may reflect a generalised inflammatory response by the pulpal fibroblasts at the transcriptional level and may represent the associated fibrotic processes that occur.31 In contrast, the results indicated that TGF-b and BMP family members and their receptors are predominantly expressed by odontoblasts in healthy teeth and in addition, generally showed higher levels of expression in odontoblasts and pulpal tissue from carious teeth. The sq-RT-PCR data (see Fig. 3) also indicated that TGF-b1, BMP-receptor 1A and BMP-receptor 1B were up-regulated in odontoblast cells during disease. Support for these observations is provided by previous immunohistochemical studies, which have shown pulpal and odontoblast expression of the TGF-b isoforms 1—3 and their receptors in both healthy and carious human molar teeth.17,20 Whilst gene expression of BMP’s and their receptors has previously been reported in human dental pulp,19 our sq-RT-PCR analysis is the first to provide transcription profiling data for these molecules in odontoblast and pulp cells in sound and carious human teeth. In addition, we feel it is noteworthy that as previous studies have shown that several members of the TGF-b superfamily and their receptors are expressed by immune and inflammatory cells, it is possible that their increased expression levels detected here are contributed to by these infiltrating cells.
Implications of this study Previous reports8,32 have indicated that following pulpal tissue extirpation, RNA can be isolated from both the extirpated pulpal tissue and the odontoblasts which remain lining the vacated pulp chamber of human teeth. However, these studies have reported that RNA isolated from five pulp samples and 20 odontoblast preparations require pooling to enable single gene analysis by RT-PCR to be performed. Our results now indicate that sufficient amounts of RNA can be obtained from a single human or rodent tooth to enable multiple differential gene expression analyses in pulpal and odontoblast cells. In addition, we anticipate that this approach can also be applied for the study of in vitro cellular specific gene expression changes in human and rodent organ cultures exposed to a variety of stimulants and insults.33,34 Our procedure is tech-
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nically simple, robust, reproducible, efficient and economical and can be applied in any laboratory equipped with standard molecular biology apparatus. Combining this temperature-sensitive dental tissue extraction technique with powerful molecular biological techniques, such as DNA array technology, will facilitate further enhancement of our knowledge of the molecular events associated with dental disease.
J.L. McLachlan et al.
12.
13.
14.
15.
Acknowledgements JLM is supported by a University of Birmingham, Scientific Projects Committee grant number EBX 1193. This study was also supported in part by a University of Birmingham, Scientific Projects Committee grant number EBX 1194. We would like to thank Miss Sally Kerr (Oral Surgery) for providing the teeth for this investigation.
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