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J Dent Res 81(11): 776-783, 2002
© 2002 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Expression Profiling of Periodontal Ligament Cells Stimulated with Enamel Matrix Proteins in vitro: A Model for Tissue Regeneration

P.M. Brett, M. Parkar, I. Olsen*, and M. Tonetti

Department of Periodontology, Eastman Dental Institute, University College London, 256 Gray’s Inn Road, London WC1X 8LD, UK;

* corresponding author, i.olsen{at}eastman.ucl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several studies have examined the role of enamel matrix proteins in root formation and periodontal regeneration, although most of these have focused on a few specific genes which had previously been implicated. However, recent advances in expressional profiling have made it possible to examine the range of genetic responses involved in these processes. In the present experiments, we have therefore utilized this technique to determine the effects of enamel matrix proteins on the gene activities of periodontal ligament cells in vitro. Such cells were found to have an elevated level of RNA synthesis compared with control cells. Moreover, hybridization of the cDNA prepared from this RNA to gene array filters showed that there was differential expression of 121 genes, most of which had not previously been associated with periodontal regeneration. Some of these selective changes in gene activity might thus reflect the fundamental events that underlie periodontal development.

KEY WORDS: periodontium • regeneration • gene arrays • enamel matrix • in vitro


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regeneration of periodontal ligament (PDL) and alveolar bone lost as a result of periodontal disease has been a major objective of periodontal therapy. A variety of treatment modalities has been applied with considerable success, with bone grafting and guided tissue regeneration being widely used procedures. However, both the extent and predictability of outcomes have been limited, and there is still widespread interest in the development of new therapeutic strategies for promoting more effective periodontal regeneration.

Biological mediators have recently received considerable attention, with a variety of growth and differentiation factors being locally applied to periodontal wounds to promote regeneration of periodontal defects. Among these, much attention has been devoted to specific morphogens that are thought to be critical mediators of tooth and periodontal development. Enamel matrix proteins (EM) consist mainly of amelogenins, a group of proline-rich hydrophobic peptides (Eastoe, 1964) formed from a single gene by alternative splicing and post-translational modifications (Fincham et al., 1994). There is increasing evidence that Hertwig’s root sheath cells secrete EM proteins during root formation, and that these are involved in the formation of acellular cementum during tooth development (Slavkin, 1976; Hammarström et al., 1997). Experimental models and human clinical trials have also indicated that amelogenins are effective in inducing regeneration of the PDL, root cementum, and alveolar bone (Hammarström et al., 1997; Heijl et al., 1997).

In an effort to optimize clinical efficacy and to improve understanding of the molecular mechanisms underlying both development and regeneration, several investigators, by in vitro studies, have attempted to clarify the mode of action of EM. Initial investigations have focused on the study of specific cell functions associated with the regenerative response: cell recruitment, proliferation, and differentiation into mature PDL cells and osteoblasts (Tokiyasu et al., 2000; Van der Pauw et al., 2000; Haase and Bartold, 2001; Jiang et al., 2001; Lyngstadaas, 2001). Such studies have evaluated a limited number of known "markers" based on previous investigations, providing confirmation that some genes known to be involved in wound-healing processes are activated as a result of PDL exposure to EM—for example, the PDL response mediated through an increase in intracellular cAMP (Lyngstadaas et al., 2001).

During both development and wound healing, external stimuli lead to rapid changes in the catalogue of genes that are expressed, in the proteins that are produced, and eventually in the cellular phenotype. Describing the profile of early gene expression after exposure to external stimuli is thus fundamental to the understanding and possible modification of these responses (Heller et al., 1997; Khodarev et al., 1999). Until recently, Differential Display (DD) reverse-transcription/polymerase chain-reaction (RT-PCR) (Liang and Pardee, 1992) was used to examine the differences in gene expression profiles between and among different cell types (Liang et al., 1992) and in the same type of cell exposed to different stimuli (Francia et al., 1996; Furumura et al., 1998; Hooper et al., 2001). However, technical difficulties associated with the simultaneous study of multiple gene products and the labor-intensive identification of the expressed gene products have hampered progress in the use of this technique. These difficulties can now be overcome by the use of cDNA arrays that allow for the simultaneous evaluation of hundreds of genes associated with specific cell functions (Granjeaud et al., 1999) and is at present the method of choice for expression profiling (Hooper et al., 2001; Xynos et al., 2001). This technique has the advantage of simplicity, high throughput capacity, and reduction of the down-stream analysis involved in conventional DD. The present study has, therefore, used gridded cDNA arrays to elucidate some of the changes in gene expression in PDL cells exposed to EM, to clarify the molecular basis of periodontal regeneration and root development.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
PDL Tissue and Cell Culture
PDL tissue was obtained from premolar teeth extracted for orthodontic reasons from one healthy individual (Caucasian male, 25 yrs of age), after informed consent was obtained in accordance with a protocol reviewed and approved by the Joint Research and Ethics Committee of the Eastman Dental Institute/Hospital. PDL cells were obtained from the tissue after 2 to 4 wks of culture as previously described (Parkar et al., 1996; Kuru et al., 1998). They were incubated at 37°C in 5% CO2 in air in alpha-Minimum Essential Medium ({alpha}-MEM) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 25 µg/mL fungizone, 2 mM L-glutamine (GIBCO Life Technologies, Paisley, UK), and 10% fetal calf serum (FCS) (PAA Laboratories, Linz, Austria). Trypsin-EDTA (GIBCO) was used to detach the adherent monolayer of cells, which was subcultured and used between the third and fifth passages. To determine the ability of the PDL cultures to form mineralized nodules in vitro, we grew the PDL cells to confluence in six-well plates as described above and supplemented the medium with 50 µg/mL of ascorbic acid and 10 mM ß-glycerophosphate. Von Kossa staining was carried out after 3, 4, 5, and 6 wks of culture. The cells used in all the experiments described below were derived from this same patient.

DNA, RNA, and Protein Synthesis
To measure the kinetics of DNA, RNA, and protein synthesis, we seeded the PDL cells in triplicate into 24-well plates and grew them to log phase, removed the media, and replaced them with media containing EM proteins at 100 µg/mL (Emdogain®; Biora AB, Malmö, Sweden). Control cultures were incubated in the absence of EM. At various time points, 3H-thymidine, 3H-uridine, and 3H-amino acid mixtures (Amersham Pharmacia Biotech, Little Chalfont, UK) were added to the cultures at a final concentration of 1 µCi/mL and incubated for 2 hrs for measurement of the synthesis of DNA, RNA, and protein, respectively. At each time point, as shown, the media were removed and the cells washed with phosphate-buffered saline (PBS), followed by 3 washes with 5% (w/v) trichloroacetic acid to precipitate the DNA, RNA, and protein. A 100-µL quantity of 0.5 M NaOH was added to each well and the macromolecules transferred directly to scintillation vials and counted by means of a Wallac 1409 liquid scintillation counter. The results are shown as the mean of the isotope incorporated per culture + standard deviation (SD). The experiments were repeated three times.

Cell Exposure to EM
EM was dissolved in 0.1% acetic acid at a concentration of 10 mg/mL. The PDL cells were grown to confluence in 175-cm2 flasks (Marathon Laboratory Supplies, London, UK), the medium was removed, and fresh medium was added containing 100 µg/mL of EM, a concentration used previously by other groups (Tokiyasu et al., 2000; Van der Pauw et al., 2000; Haase and Bartold, 2001; Jiang et al., 2001; Lyngstadaas, 2001). After 2 and 24 hrs, the cells were trypsinized and washed with PBS. Control cells were obtained from replicate confluent cultures harvested prior to the addition of EM.

RNA Extraction
A lysate of the cells was prepared by treatment with denaturing solution (4 M guanidine isothiocyanate, 25 mM sodium citrate, pH 7.5, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol). Total RNA was extracted from the cell lysate according to the method of Chomczynski and Sacchi (1987). The RNA pellet was washed with 70% ethanol, re-centrifuged, and re-suspended in diethyl-pyrocarbonate-treated water. The total RNA was then treated with DNase I (Gibco) for removal of any DNA contamination. The quantity and purity of the RNA were measured by means of a spectrophotometer.

Expression Profiling
Expression profiling was performed with the use of the AtlasTM Human Cancer 1.2 gene array containing 1176 genes (Clontech Laboratories Ltd., Basingstoke, UK). This particular array was selected because it contains genes involved in cell proliferation and differentiation, some of which are likely to be responsive to EM, including growth factors and their receptors, transcription activators and repressors, and cell-cycle regulating genes. The full list of the genes on this array can be found at: http://atlasinfo.clontech.com/atlasinfo/AtlasInfo2.

A 5-µg quantity of the extracted total cellular RNA was used to produce cDNA probes labeled with 32P-dCTP (Amersham Pharmacia Biotech). The labeled probes were then hybridized to the AtlasTM gene array filters at 68°C (Xynos et al., 2001). The filters were then washed according to the manufacturer’s instructions [once with 2 x standard saline-citrate (SSC), 1% SDS; twice with 0.1 x SSC, 0.5% SDS]. The filters were exposed to autoradiographic film overnight and for 3 days at -70°C. The autoradiographs were scanned with the use of an Alpha Imager 1200 and AlphaEase version 5.5 image capture software (Flowgen, Ashby de la Zouch, UK), and saved for later analysis.

Data Analysis
We assessed and compared the patterns of gene expression generated under the different conditions using the AtlasImageTM 2.0 software for analysis of the images obtained from the autoradiographs. We used the gene-based signal threshold function to determine the specific signals which were above the background level of hybridization. This value was set at 75% above background. The images were normalized by the global normalization function, which allowed for quantitative analysis to be performed between different filters. The detection parameters of the program were arbitrarily set to report greater than two-fold changes in expression levels either up or down. Differences in gene expression greater than two-fold are presented as the ratios of gene expression at 2 hrs compared with the control (0 hr) cells, at 24 hrs compared with the control cells, and at 24 hrs compared with 2 hrs.

To assess the reliability and repeatability of the methods, we performed the expression profiling experiments twice, each time point in duplicate and each set of RNA labeled and hybridized twice. Comparison of the individual replicate filters showed that there were no differences between them which were above the threshold limits set as described above (data not shown). For the final analysis, the replicate images were therefore merged to produce a composite image.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The early-passage cultures of PDL cells used in these experiments all produced mineralized nodules by 3 wks of culture in vitro, as assessed by von Kossa staining (data not shown). The Fig.Go shows the effects of EM on macromolecular synthesis by these cells. In both the control and EM-treated cultures, the incorporation of 3H thymidine increased progressively at a similar rate until approximately 10 hrs, after which DNA synthesis remained constant (Fig., AGo). Protein synthesis also increased to the same extent in both types of cultures, although maximal 3H amino acid incorporation was not observed until after 15 hrs (Fig., CGo). In marked contrast, the results in Fig. BGo show that the incorporation of 3H uridine far more rapidly increased in the EM-treated cultures and by 6 hrs was nearly 150% greater than in the non-treated control cultures, which increased only slowly over a period of 24 hrs. Thus, RNA synthesis in the PDL cells was elevated within the first 6 hrs of incubation in the presence of EM, whereas there was no differential effect of EM on either protein or DNA synthesis, even at 24 hrs.



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  		Figure. Differential effect of EM on RNA synthesis by PDL cells in vitro. A representative of three separate experiments showing the synthesis of DNA (A), RNA (B), and protein (C) by PDL cells cultured in the absence (open squares) and presence (closed circles) of EM. The cells were incubated with the corresponding radioactive substrates for periods of 2 hrs, as indicated, and the average amount of isotope (dpm) incorporated by three replicate cultures measured as described in MATERIALS & METHODS. Error bars are shown in (B) but not in (A) and (C) because of the very close values obtained, the variation of the SD being less than 10% of the mean in all samples in (A) and (C). The same results were also obtained in two other experiments (data not shown).

 
Using the AtlasImageTM software settings as described in MATERIALS & METHODS, we obtained evidence that there were differences in the expression of 121 genes by PDL cells cultured in the presence of EM, of a total of 198 genes detected on the hybridized filters. In Table 1Go, these 121 differentially expressed genes have been assigned to 7 groups based on the functional activity of the corresponding protein. Although there is some overlap in the functions of several of the gene products, each group contains between 14 and 22 different genes, nearly every gene having a unique response to the presence of EM protein in the culture medium. Among individual genes, fibronectin, bone proteoglycan II, MMP 14, and BIGH3 were markedly up-regulated at 2 hrs, while two DNA-associated proteins were particularly strongly down-regulated by EM, even after this short period of exposure. By 24 hrs, the calmodulin 1, integrin beta 8, AXL tyrosine protein kinase receptor, arachidonate 5-lipoxygenase, and cytokeratin 12 genes were additionally up-regulated. Substantial numbers of individual genes were also no longer detected at this later time, generally those expressed at only very low levels at 2 hrs. Three cell-cycle proteins showed a greater than 10-fold difference between 24 and 2 hrs, while one DNA-binding protein increased more than 50-fold in the presence of EM.


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  		Table 1. The 121 Genes Differentially Expressed by PDL Cells in Response to EM in vitroa
 
There were also several genes of interest not included in Table 1Go, since they did not reach either the two-fold level or the intensity limits used in the analysis. Some of these genes, which were positive at lower thresholds, have previously been identified in other studies of single gene changes induced by EM. Thus, transforming growth factor beta was up-regulated at 2 hrs but was not found to be expressed at 24 hrs (Van der Pauw et al., 2000; Lyngstadaas et al., 2001). Several other growth mediators were also found to be elevated by culture in the presence of EM protein, including connective tissue growth factor (CTGF), vascular endothelial growth factor receptor 1, and platelet-derived growth factor receptor alpha (data not shown).

The designation ’ON’ in Table 1Go indicates that the gene was detected in the EM-treated culture compared with no expression in the control culture, while ’OFF’ indicates that the particular gene was not detected in the test culture compared with the zero-hour cells. Thus, for many individual genes, we could not evaluate the extent of quantitative change within the defined confidence limits. A summary of gene expression changes in each functional category was therefore calculated in which all the ON and up-regulated (> 2.0 relative increase) genes are combined (the ’UP’ group), and all the OFF and down-regulated (< 0.5 relative decrease) genes are combined (the ’DOWN’ group). The data in Table 2Go indicate that, at 2 hrs, a high proportion of transcription factor and DNA-associated genes is down-regulated by EM protein. In contrast, at this early time period, the surface receptor-ECM and cell signaling groups are up-regulated, the expression of 68 and 41% of the total genes in each group having increased. By 24 hrs, the proportion in these groups increased further, to 77 and 59%, respectively, and the cell cycle and metabolic gene groups were also strongly induced in the presence of EM (50 and 63% of the genes being up-regulated). Moreover, in each of the functional groups, the majority of the genes showed no change between 2 and 24 hrs in the presence of EM protein (64 to 87% of the total, as shown in Table 2Go).


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  		Table 2. Expression of Different Functional Groups of PDL Genes Changed as a Result of Incubation with EM in vitro
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study has shown that the addition of EM protein to PDL cells in vitro elicited a substantial increase in RNA synthesis, without affecting DNA and protein. This differential effect on RNA synthesis was found to occur within the first 6 hrs and persisted at least until 24 hrs, at which time the overall levels of protein and DNA synthesis were still the same in the presence and absence of EM. Although the present study did not examine the effects of more prolonged exposure, previous studies have shown that protein and DNA synthesis were also significantly elevated after 48 hrs of incubation in the presence of EM (Van der Pauw et al., 2000; Lyngstadaas et al., 2001). The majority of the new ’early’ RNA production which we detected is undoubtedly ribosomal and would be expected to lead eventually to a greater capacity of the cells for protein synthesis. Nevertheless, the DD RT-PCR technique with cDNA filter arrays clearly demonstrated that there were also major qualitative changes in mRNA expression which were induced by EM protein. This procedure could thus provide an extensive profile of the complex sequence of changes in gene expression that accompany the development and regeneration of the periodontium and which are, at present, only poorly understood. Moreover, the use of cDNA array technology can also reveal genes which have been identified in other wound-healing responses but have not been implicated specifically in periodontal regeneration as well as gene responses to other forms of periodontal therapy.

The cancer gene array used in the present experiments is comprised of more than 1000 oncogenes, growth factors and their receptors, transcription factors, and cell cycle proteins. It is therefore not unexpected that approximately 20% of these were found to be expressed by early-passage PDL cells, with more than half (121 genes) being differentially affected by EM. Of these, it was notable that only one transcription factor, the fos-related antigen 1(FRA1), which has been shown to have an important role in osteoclast differentiation and can reverse osteopetrosis in Fos knockout mice (Matsuo et al., 2000), was up-regulated at 2 hrs or even after longer incubation. However, a most striking feature of the effects of EM on PDL gene expression was the rapid, persistent, and strong up-regulation of many surface-receptor-ECM genes. Thus, after 2 hrs of exposure to EM protein, 15 of the 22 differentially expressed genes in this group were elevated, including the MMPs 1, 3, 11, and 14, which are involved in wound healing and ECM remodeling processes. The fibronectin, collagens 6 and 16, tenascin, integrin beta-8, and bone proteoglycan genes were also up-regulated, suggesting that the ECM plays a fundamental and perhaps an essential regulatory role in PDL regeneration induced by EM protein, even at an early stage. Notably, the most strongly down-regulated genes were among the DNA-chromatin group, perhaps reflecting an EM-induced program of changes in gene activity directed away from DNA replication/repair and toward more rapid differentiation of bone and other precursor cells present in the PDL.

Independent assays of the expression of specific genes, for example, by Western and Northern blotting, are clearly required to corroborate the array results we have obtained before definitive biological conclusions can be drawn. Thus, while our results do not establish unequivocally that one or a small group of genes is essential in EM-induced PDL regeneration, the technique of expression profiling has nevertheless highlighted a range of multiple gene activities that warrant further analysis. This would help to clarify the fundamental molecular events underlying this process and might also identify previously unsuspected gene targets for clinical therapy.


   ACKNOWLEDGMENTS
 
This work was supported by an educational grant from Biora AB, Malmö, Sweden, and by the Eastman Periodontal Research Fund.

Received January 29, 2002; Last revision August 6, 2002; Accepted September 10, 2002


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chomczynski P, Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 16:156–159.

Eastoe J (1964). The chemical composition of bone and tooth. In: Advances in fluorine research and dental caries prevention. Hardwick JL, Held HR, König KG, editors. Oxford: Pergamon Press, pp. 5-17.

Fincham AG, Moradian-Oldak J, Simmer JP, Sarte P, Lau EC, Diekwisch T, et al. (1994). Self-assembly of a recombinant amelogenin protein generates supramolecular structures. J Struct Biol 112:103–109.[Medline]

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Granjeaud S, Bertucci F, Jordan BR (1999). Expression profiling: DNA arrays in many guises. Bioessays 21:781–790.[Medline]

Haase HR, Bartold PM (2001). Enamel matrix derivative induces matrix synthesis by cultured human periodontal fibroblast cells. J Periodontol 72:341–348.[Medline]

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