Matti Laaksonen Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes Academic Dissertation To be presented with the permission of the Faculty of Medicine of the University of Helsinki for public examination in the Lecture hall 3 of Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, on 14 th May 2010, at 13 pm. Department of Cell Biology of Oral Diseases, Biomedicum Helsinki, Institute of Dentistry, Faculty of Medicine, University of Helsinki Helsinki 2009
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Dissertation - Associations Among Emdogain, Human Oral Carcinoma Cells and Oral Proteolytic Enzymes
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Matti Laaksonen
Associations among Emdogain®, human oral carcinoma cells and
oral proteolytic enzymes
Academic Dissertation
To be presented with the permission of the Faculty of Medicine of the University of Helsinki for public examination in the Lecture hall 3 of Biomedicum Helsinki, Haartmaninkatu 8, Helsinki, on 14th May 2010, at
13 pm.
Department of Cell Biology of Oral Diseases, Biomedicum Helsinki, Institute of Dentistry, Faculty of Medicine,
University of Helsinki
Helsinki 2009
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Supervised by Professor Tuula Salo, DDS, PhD Department of Diagnostics and Oral Medicine Oulu University Central Hospital Institute of Dentistry University of Oulu Oulu, Finland Professor Timo Sorsa, DDS, PhD, Dipl Perio Department of Cell Biology of Oral Diseases, Biomedicum Helsinki Institute of Dentistry University of Helsinki Helsinki, Finland Rewieved by: Associate Professor Lari Häkkinen, DDS, PhD Department of Oral Biological and Medical Sciences Faculty of Dentistry University of British Columbia Vancouver, BC, Canada Marilena Vered, D.M.D Department of Oral Pathology and Oral Medicine School of Dental Medicine Tel Aviv University Tel Aviv, Israel Opponent: Professor Timo Närhi, DDS, PhD Department of Prosthetic Dentistry and Biomaterials Science Institute of Dentistry University of Turku Turku, Finland ISBN 978-952-10-6198-1 (paperback) ISBN 978-952-10-6199-8 (PDF) Yliopistopaino Helsinki 2010
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CONTENTS: LIST OF ORIGINAL PUBLICATIONS 4 ABBREVIATIONS 5 ABSTRACT 6 1 INTRODUCTION 7 2 REVIEW OF THE LITERATURE 9
2.1 Healthy and diseased periodontium 9 2.2 Gingival crevicular fluid 10 2.3 Matrix metalloproteinases 11 2.4 Carcinogenesis 14 2.5 Emdogain 18 2.6 Effects of Emdogain on mesenchymal cells 19 2.7 Effects of Emdogain on epithelial cells 22 2.8 Effects of Emdogain on neoplastic cells 22
3 A CASE REPORT 24 4 HYPOTHESES AND AIMS OF THE STUDY 28 5 MATERIALS AND METHODS 29
5.1 Patients and sample collection (I) 29 5.2 Study reagents (I,II,III) 29 5.3 EMD degradation assays (I) 30 5.4 Cell lines and cell cultures (I,II,III) 31 5.5 In vitro cell proliferation, migration, wound closure and adhesion assays (I,II) 31 5.6 In vitro MMP production assay (II, III, unpublished data) 33 5.7 Affymetrix cDNA microarrays (III) 34 5.8 Mouse experiments (II) 35 5.9 Data collection for systematic review (IV) 35 5.10 Statistical analysis (I,II,III) 36
6 RESULTS 38
6.1 Degradation of EMD by GCF and MMP (I) 38 6.2 In vitro cell proliferation, viability and behaviour assays (I, II) 38 6.3 Effects of EMD on the in vitro production of matrix metalloproteinases (II, III, unpublished data) 40 6.4 Effects of EMD and TGF-β1 on the gene profile of carcinoma cells (III) 41 6.5 Effects of EMD on metastasis formation in vivo (II) 42
6.6 Results of the systematic review (IV) 42 7 DISCUSSION 44
7.1 Summary of the main results 44 7.2 EMD in periodontal regeneration 44
I Laaksonen M, Salo T, Vardar-Sengul S, Atilla G, Han Saygan B, Simmer JP, Baylas H, Sorsa
T. Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain induced
proliferation of periodontal ligament fibroblasts. Journal of Periodontal Research 2009, Nov 9
Epub ahead of print (DOI: 10.1111/j.1600-0765.2009.01244.x.)
II Laaksonen M, Suojanen J, Nurmenniemi S, Läärä E, Sorsa T, Salo T. The enamel matrix
derivative (Emdogain) enhances human tongue carcinoma cells gelatinase production,
migration and metastasis formation. Oral Oncology 2008;44:733-742
III Laaksonen M, Sorsa T, Vilen ST, Salo T. Emdogain and TGF-β1 target genes in human
tongue carcinoma cells in vitro. Submitted
IV Laaksonen M, Sorsa T, Salo T. Emdogain in carcinogenesis: a systematic review of in vitro
studies. Journal of Oral Science 2010;52:1-11
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ABBREVIATIONS
ALP alkaline phosphatase BM basement membrane BMP bone morphogenetic protein BMSC bone marrow stromal cells BrdU 5-bromo-2’-deoxyuridine BSA bovine serum albumin BSP bone sialoprotein cAMP cyclic adenosine monophosphate CI confidence interval COX cyclo oxygenase CTGF connective tissue growth factor CTT-2 gelatinase inhibitor, (GRENYHGCTTHWGFTLC) DMEM Dulbeccos Modified Eagles’s Medium DNA deoxyribonucleic acid ECL enhanced chemiluminescence ECM extra cellular matrix EDTA ethylendiamine tetraacetic acid EGF epidermal growth factor ELISA enzyme linked immunosorbent assay EMD Emdogain® EMP enamel matrix protein EMT epithelial to mesenchymal transition ERK extracellular signal-regulated kinase ERM epithelial cell rests of Malassez FC fold change FGF fibroblast growth factor GCF gingival crevicular fluid GO gene ontology HNSCC head and neck squamous cell carcinoma IGF insulin-like growth factor IL interleukin MAP mitogen-activated protein MMP matrix metalloproteinase OPG osteoprotegerin PBS phosphate buffered saline PDGF platelet-derived growth factor PGE2 prostaglandin E2 PLF periodontal ligament fibroblast RANKL receptor activator of NF-kB ligand RNA ribonucleic-acid RT room temperature RTK receptor tyrosine kinase SCC squamous cell carcinoma SD standard deviation SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis TCA trichloroacetic acid TGF-β transforming growth factor-β TIMP tissue inhibitor of matrix metalloproteinase TNC Tris-NaCl-CaCl2-buffer TNF-α tumour necrosis factor-α VEGF vascular endothelial growth factor
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ABSTRACT
The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.
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1 INTRODUCTION
Repair and regeneration of periodontal tissues are major goals in periodontal treatment. Periodontal
regeneration is a complex multifactorial process in which molecular interactions in and between
mesenchymal and epithelial cell lines are needed (Nyman et al 1982, Pitaru et al 1994). The
essential stages in successful regeneration are cementogenesis, osteogenesis and assembly of
periodontal ligaments onto the cementum. At the same time, the apical proliferation of epithelial
cells must be arrested (Gottlow et al 1984, Ripamonti & Reddi 1997, Zeichner-David 2006). In
1997, extracted porcine enamel matrix proteins were observed to be capable of inducing new
cementum and bone formation in periodontal defects in monkeys (Hammarström 1997). Since then,
this tissue extract preparation, consisting of hydrophobic enamel matrix proteins from developing
porcine enamel, has been marketed under the commercial name of Emdogain® (EMD). Several in
vivo studies since then have shown EMD to be clinically useful in promoting periodontal
regeneration, including the restoration of alveolar bone, cementum and collagenous ligaments
(Esposito et al 2004).
Although EMD is at present widely used in periodontal therapy, the mechanisms of action and the
exact composition of the product are not completely clear. The predominant compound of EMD is
amelogenin (>90%) (Zeichner-David 2006). In spite of amelogenin not being a growth factor, EMD
possesses several growth factor-like effects. Therefore, many attempts have been made to identify
growth factors in EMD, without satisfying results. However, there is abundant evidence regarding
the effects of EMD on periodontal tissues. EMD can stimulate the migration and proliferation of
periodontal fibroblasts and osteoblasts and induce the production of several cytokines in these cells.
In contrast to periodontal fibroblasts and osteoblasts, other cell lines of the oral cavity, particularly
malignant cell lines, have not been adequately studied. Despite the lack of studies and the fact that
EMD is known to have growth factor-like effects on different cell lines, EMD is said to be
clinically safe (Zetterström et al 1997, Heard et al 2000, Oringer 2002). In studies, however, the
clinical safety has often entailed only the capability of porcine enamel proteins to induce
immunological reactions or local post-operative symptoms such as pain or sensitivity in the teeth
involved. Nonetheless, EMD is applied in high doses directly in periodontal pockets where it
interacts with proteolytic enzymes, e.g. MMPs, of inflamed tissue. Thus, this combination of
proteolytic enzymes together with unknown cytokines of EMD might also be capable of inducing
unwanted effects and alterations in vivo.
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These observations led to two distinct hypothesis of the study. The first hypothesis was that the
proteases present in oral tissues and oral fluids can degrade EMD and the degradation alters the
effects of EMD in vitro. The Second hypothesis was that EMD could be capable of inducing
carcinogenesis. Thus, the purpose of this study was to examine whether gingival crevicular fluid,
containing several different proteolytic enzymes, could degrade EMD and alter the proliferation of
periodontal fibroblasts. In addition, the study aimed to determine if EMD could induce
carcinogenesis-related factors, in particular MMPs, in vitro and in vivo. Furthermore, this study
aimed to contribute to the understanding of the composition of EMD.
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2 REVIEW OF THE LITERATURE
2.1 Healthy and diseased periodontium
Periodontal tissue surrounding the tooth consists of four different developmental, biological and
functional units: gingiva, root cementum, alveolar bone and periodontal ligament (Mueller 2005).
These tissues can support and nourish the related tooth and protect underlying tissues, and thus are
responsible for the main functions of the periodontium.
Figure 1. The structure of the periodontium. A. corresponds to a healthy state and B. to periodontitis. The vertical arrows show the flow direction of the gingival crevicular fluid to the gingival crevice.
The transition from a healthy state or gingivitis to periodontitis is characterised by proteolytic
destruction of periodontal ligament (type I collagen fibers) and alveolar bone accompanied by
apical migration of sulcular epithelial cells. This phenomenon leads to periodontal pocket formation
and eventually to loss of the tooth involved. Periodontal diseases can be grouped according to the
degree, severity and activity of the tissue destruction. In 1999, an International Workshop for the
Classification of Periodontal Disease and Conditions was organised to revise the classification
system of periodontal diseases (Armitage 1999). The major disease categories are: I gingival
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disease, II chronic periodontitis, III aggressive periodontitis, IV periodontitis as a manifestation of
systemic diseases, V necrotising periodontal disease, VI abscesses of the periodontium, VII
periodontitis associated with endodontic lesions, and VIII development of acquired deformities and
conditions.
All the forms of periodontitis are first and foremost infectious diseases and initiated by bacteria. All
of the periodontopathogens, particularly Porphyromonas gingivalis, Aggregatibacter
actinomycetemcomitans and Treponema denticola, can produce proteolytic enzymes directly
capable of degrading host tissues and activating host pro-MMPs (Ding et al 1995, Potempa et al
2000, Eley & Cox 2003). However, host-derived proteolytic enzymes cause most of the tissue
destruction in periodontitis. Lipolysaccarides produced by gram-negative bacteria stimulate
polymorphonuclear leukocytes and macrophages to secrete inflammatory products such as
prostaglandin E2 (PGE2), interleukin-1 (IL-1), -6 and -8, tumour necrosis factor alpha (TNFα), and
matrix metalloproteinases (MMPs). These products in turn activate vascular smooth muscle cells,
MA) or BSA (10 µg/ml, Sigma) and left overnight at RT. HSC-3 and HMK cells were then plated
at 10 000 cells/well and incubated for 2 h. The non-adherent cells were rinsed off and the remaining
cells were fixed with 10 % TCA, stained and quantified using an ELISA reader at 540 nm. The
adhesion study was repeated using the same coating components as described above, but prior to
the experiments the cells were pre-incubated 2 h with EMD (100 µg/ml).
5.6 In vitro MMP production assays (II, III, unpublished data)
Gelatin zymography (II)
In order to determine the effects of EMD on gelatinolytic enzymes produced by HSC-3 carcinoma
cells and periodontal fibroblasts, a gelatin zymography of the culture medium was conducted. HSC-
3 cells at a density of 28 000/well and PLFs at 30 000/well were allowed to grow to sub-confluence
in 24-well plates. The HSC-3 medium was replaced with serum-free medium and 0, 100 or 200
µg/ml EMD was added to both assays. Cells were incubated for 48 h and medium collected, briefly
centrifuged and stored at –20°C. The medium was added to 10 % SDS-PAGE containing 1 mg/ml
of fluorescently labeled gelatin (Sutinen et al 1998). After electrophoresis, the gels were washed,
incubated overnight and stained as described above. The intensities of separate bands were
measured quantitatively using optical densitometry and Quantity one software (Bio Rad Model GS-
700 Imaging Densitometer, Bio-Rad, Richmond, CA, USA).
ELISA immunosorbent assay (II)
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To confirm the effects of EMD on gelatinolytic MMPs observed by zymography, an
immunosorbent assay was done on HSC-3 culture medium. After 48 h of EMD incubation (0, 100
and 200 µg/ml) the MMP-2 and MMP-9 concentrations were analyzed by a commercial ELISA
assay according to the manufacturer's instructions (Biotrak, Amersham Pharmacia Biotech,
Buckinghamshire, UK).
Western immunoblotting (III)
HSC-3 cells at 28 000/well were cultured in serum-free media with EMD (0, 100 or 200 µg/ml) for
24 h. Western blotting for MMP-10 from culture media was carried out as previously described
(Nyberg et al 2003). After SDS-PAGE gel electrophoresis the membrane was incubated with
human monoclonal MMP-10 antibody (1:1000, R&D systems, Minneapolis, MN).
5.7 Affymetrix cDNA microarrays (III)
Microarray sample preparation and hybridization
To assure an adequate amount of RNA, 1x106 HSC-3 cells/flask were seeded and allowed to grow
in their normal media overnight. The study reagents TGF-β1 (10 ng/ml) and EMD (200 µg/ml)
were dissolved from the PBS-stock solutions to serum containing culture media, and the media was
changed. Equal volume of PBS was also added to control cells when the culture media of the cells
was changed. After 6 h and 24 h of incubation the total cellular RNA was extracted using the
Qiagen RNeasy Mini Kit according to the manufacturer’s instructions (Quiagen, Oslo, Norway).
RNA quality and quantity were determined using the Agilent 2100 Bioanalyzer (Agilent
Technologies, DE, USA). Micro-array experiments were carried out using the Affymetrix Human
Genome U133 Plus 2.0 chip, for analysis of over 47,000 transcripts. Total RNA (8 µg) from each
sample was used for target cDNA synthesis according to the Affymetrix protocol
(www.affymetrix.com). Three biological replicates were produced at each time point (Lee &
Whitmore 2002)
Gene Ontology enrichment analysis
For the differentially expressed genes, Gene Ontology (GO) enrichment analysis was performed
using the DAVID annotation tool (http://david.abcc.ncifcrf.gov/home.jsp) according to Huang et al
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(2009). The analysis was conducted using the list of all statistically significantly up- and down-
regulated genes separately. The whole genome list of the Human Genome U133 Plus 2.0 chip was
used as a background. An EASE score (E-score), which is a modified Fisher Exact P-Value, was
used to identify enriched categories. An E-score of <0.05 was considered statistically significant.
Furthermore, the enriched terms were grouped into functional clusters according to terms having
similar biological meaning due to sharing similar gene members.
5.8 Mouse experiments (II)
The animal experiment was approved by the Ethics Committee for Animal Experimentation in the
State Provincial Office of Southern Finland. For inoculation, the 1x107 detached HSC-3 cells
diluted and suspended in 200 µl serum-free culture medium were injected subcutaneously into the
right and left back of twenty 6- to 8-week old Harlan-Spraque Dawley athymic nude mice. On days
4-8 after the inoculation, ten mice were daily injected subcutaneously with 100 µg/ml EMD in 200
µl saline, whereas ten control mice were daily injected with the same volume (200 µl) of saline.
Three times a week the mice were weighted, the tumour growth measured and the survival
observed. The criteria for euthanasia were tumour size over 10 mm in diameter, weight loss of more
than 15 %, or symptoms of suffering. After euthanasia, the mice were dissected and enlarged
axillary and peritoneal lymph nodes (n=6) were collected for metastasis analysis. The presence of
metastasis in the lymph nodes was confirmed histologically.
5.9 Data collection for the systematic review (IV)
An information specialist-assisted search was made from PubMed and EMBASE databases. The
search terms were Emdogain and/or ‘enamel matrix derivative’ and language definition English.
The publication date was restricted to the period of January 1997 to December 2008. Altogether
403 publications were found. No clinical reports concerning association of Emdogain with oral
carcinoma were found. Thus, the review was restricted to in vitro studies and the criteria for a
systematic review was applied after the in vivo exclusion.
Thus, exclusion criteria were:
1) Article not written in English
2) Clinical studies, e.g. studies dealing with radiology or treatment techniques, outcomes and
surveys.
3) In vivo and in vitro studies concerning histology, immunohistocemistry, etc.
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4) In vitro studies designed to study bacteria or cells other than oral cells or malignant cells.
5) Review articles and conference reports.
Altogether 76 in vitro studies were included in this study, designed to obtain all available biological
data relevant for carcinoma-related factors and the behaviour of oral cells. Thus, inclusion criteria
were:
1) Articles published in English between 1. January 1997 and 31. December 2008.
2) Assays designed to study human cells present in oral tissues or malignant cells.
3) Assays designed to study cytokines in Emdogain or regulated by Emdogain.
5.10 Statistical analysis (I,II,III)
Analysis of variance models was used to estimate the effects of the experimental substrates on the
various in vitro outcomes. The results are presented as relative comparisons of means between
relevant treatment groups, expressed in terms of fold change or percentage from a given baseline,
together with the 95% confidence intervals (CI). In study I, the CI was calculated with the web-
based Texas University Confidence Interval Calculator
(http://www.stat.tamu.edu/~jhardin/applets/index.html). In study II, the R environment for
statistical computing and graphics was used in computations, especially the function lm() for fitting
linear models (URL:www.R-project.org.).
Reporting statistical significance using P-values has deliberately been avoided where possible.
Instead, statistical certainty/uncertainty has been expressed using 95% confidence intervals, which
are known to be more informative than P-values. When reporting the 95% confidence intervals
(CI), it means that the data is well consistent within these limits. At the same time, the CIs point out
the effect size and error margins. In addition, the ’statistical significance’ can also be judged when
CIs are present. In contrast, P-value is generally uninformative, fails to convey important
information about effect size and is often misleading (lack or over emphasis of the biological
relevance). This statistical method follows also the explicit recommendations given by leading
medical journals (Brennan et al 1994, Sterne et al 2001), and these recommendations are also
adopted in the Uniform Requirements for Manuscripts from 1988 onwards (International
Committee of Medical Journal Editors. Uniform Requirements for Manuscripts Submitted to
Biomedical Journals, URL:www.icmje.org). However, the P-values are presented for the
microarray data and a P-value of <0.05 is considered statistically significant.
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Microarray data analysis
The microarray data analysis was performed using open source Chipster software
(http://chipster.csc.fi). For normalization, the Robust Multi-Array method was used. The regulated
genes by EMD and TGF-β1 were compared with the gene list of the control cells by using the
empirical Bayes t-test to estimate the statistical significance of individual genes with a cut-off
threshold of P<0.05. The fold change (FC) of the statistically significantly regulated genes was also
calculated.
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6 RESULTS
6.1 Degradation of EMD by GCF and MMP (I)
GCF was observed to be capable of degrading EMD-embedded zymography gels. The degradation
depended on the state of periodontal disease, with statistically significantly more degradation
observed in all states of periodontal disease (gingivitis, chronic periodontitis and aggressive
periodontitis) compared to healthy control GCF.
To further determine the EMD-degrading proteases observed in periodontic GCF, the effects of
recombinant human MMPs -1, -2, -8, -9, -13 and -14 on EMD- embedded gels were studied. Only
MMPs -2 and -8 caused barely detectable lysis of the gel and in a restricted range compared to GCF
samples.
6.2 In vitro cell proliferation, viability and behaviour assays (I, II)
Effects of EMD, GCF and amelogenin on the proliferation of periodontal ligament fibroblasts
The proliferation of PLFs was markedly induced by EMD (100 µg/ml and 200 µg/ml) after 24 and
48 h of incubations compared with the untreated controls. When the cells were treated with EMD
together with GCF (10 µg/ml; dissolved in TNC-buffer), the induction of the proliferation of PLFs
by EMD was decreased compared to treatment with EMD alone. Thus, EMD at the lower
concentration of 100 µg/ml together with GCF could not induce the proliferation of PLFs. The GCF
alone as well as TNC-buffer alone had no effect on the proliferation of PLFs (data regarding TNC-
buffer not shown). Amelogenin, the most abundant protein in EMD, had a drastic inhibitory effect
on the proliferation of PLFs. On average, 200 µg/ml amelogenin caused a 54% decrease in the
proliferation of PLFs with respect to control.
Effects of EMD on the proliferation and apoptosis of carcinoma cells
In HSC-3 cell proliferation, no differences were found between the control and the EMD-treated
(100 and 200 µg/ml) cells after 12, 24, 48, 72, and 96 h of incubation. Furthermore, EMD (100 and
200 µg/ml) had no effect on apoptosis of HSC-3 or HMK cells.
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Effects of EMD on in vitro migration and wound closure of carcinoma cells
EMD stimulated the random migration of HSC-3 cells in Transwell-assays during overnight culture.
EMD at 100 µg/ml increased the migration of HSC-3 cells by 43% (95% CI: 21% to 69%) and at
200 µg/ml by 57% (95% CI: 33% to 86%) in relation to control. EMD had no effect on the
migration of benign HMK cells.
CTT-2 (50 µg/ml), a selective inhibitor of MMP-2 and -9, decreased the migration of HSC-3
carcinoma cells by 89% (95% CI: 65% to 97%) compared to the control. In combination with EMD
at 100 µg/ml, CTT-2 decreased the migration by 97 % (95% CI: 91% to 99%), but with EMD at
200 µg/ml the observed migration was decreased to only 57% of the control level (95 % CI: -87%
to +41%). CTT-2 (50 µg/ml) also decreased the migration of benign HMK cells.
EMD was able to induce in vitro wound closure of carcinoma cells, which requires both cell
proliferation and migration. In the beginning of the assay the mean of the wounded areas of HSC-3
cells was 46 mm2 (SD 7.1 mm2). After 24 h incubation the mean areas of control wounds were
reduced to 35.2 mm2 (SD 13.8 mm2). EMD at 10 µg/ml concentration decreased the wound areas
by 51% (95% CI: 10% to 74%), and at 100 µg/ml by about a similar amount: 57% (95% CI: 20% to
77%) compared to the control wounds.
Effects of EMD on carcinoma cell adhesion
EMD was able to alter the attachment of HSC-3 cells to various extracellular matrix components.
The mean attachment of HSC-3 cells was similar for both EMD and BSA coated surfaces. Matrigel
and fibronectin coating increased the attachment of HSC-3 cells relative to BSA by 20% (95% CI:
14% to 26%) and by 8% (95% CI: 4% to 13%), respectively. Pre-incubation with EMD-containing
medium (100 µg/ml) followed by the adhesion experiment increased the HSC-3 adhesion to BSA
by 10% (95% CI: 5% to 16%), to fibronectin by 11% (95% CI: 5% to 16%), but decreased the
adhesion to Matrigel by 7% (95% CI: 2% to 11%), whereas the effect on the adhesion to EMD was
unclear, the observed increase being 3% (95% CI: -2% to +9%)
6.3 Effects of EMD on the in-vitro production of matrix metalloproteinases (II, III, un-
published data)
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Emdogain-induced production of MMP-2 by periodontal ligament fibroblasts
The production of 72 kDa pro-MMP-2 was observed to be induced by EMD in the conditioned
medium of periodontal ligament fibroblasts when analysed with gelatin zymography. The induced
pro-MMP-2 production was on average 1.7-fold (95% CI: 1.5 to 1.8) with EMD at 100 µg/ml and
1.8-fold (CI 1.6 to 2.0) with EMD at 200 µg/ml with respect to control.
Figure 5. An example of a gelatin zymography gel from the culture medium of periodontal ligament fibroblasts. EMD dose-dependently induced pro-MMP-2 production.
Emdogain-induced production of MMP-2, MMP-9 and MMP-10 by carcinoma cells
The production of 72 kDa pro-MMP-2 and 92 kDa pro-MMP-9 was markedly induced by EMD in
the conditioned medium of HSC-3 carcinoma cells when analyzed with gelatin zymography. The
induction caused by EMD was also stronger in carcinoma cells than in periodontal ligament
fibroblasts. The induced pro-MMP-2 production was on average 2.3-fold (95% CI: 1.6 to 3.2) with
EMD at 100 µg/ml and 2.6-fold (95% CI: 1.8 to 3.7) with EMD at 200 µg/ml with respect to
control. The induced pro-MMP-9 ratios were 2.4-fold (95% CI: 2.0 to 2.8) with EMD at 100 µg/ml
and 2.3-fold (95% CI: 1.9 to 2.7) with EMD at 200 µg/ml with respect to control. The production of
MMP-2 and MMP-9 was similarly induced by EMD in the conditioned medium of HSC-3 cells
analyzed with ELISA.
Western immunoblots showed that the untreated HSC-3 cells did not produce MMP-10 but the
production was clearly induced at both EMD concentrations (100 and 200 µg/ml) after 24 h of
incubation.
6.4 Effects of EMD and TGF-β1 on the gene profile of carcinoma cells (III)
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Comparison of the genes regulated by TGF-β1 and EMD treatment
The Affymetrix microarray revealed a total of 32 genes to be statistically significantly (P<0.05) up-
regulated and 16 genes down-regulated in HSC-3 carcinoma cells after 6 h of EMD treatment
compared to untreated control cells. After 24 h of EMD incubation, this effect on the regulated
genes (P<0.05) was attenuated, showing only four up-regulated and eight down-regulated genes.
TGF-β1 altered regulation of markedly more genes than EMD after 6 h and 24 h of treatment. A
total of 239 genes were statistically significantly (P<0.05) up-regulated and 154 genes down-
regulated in HSC-3 cells after 6 h of TGF-β1 treatment compared to untreated control cells. After
24 h of TGF-β1 incubation, a total of 242 genes were up-regulated and 104 genes down-regulated
compared to untreated carcinoma cells.
About half (53%) of the genes (n=17) up-regulated by the 6 h EMD treatment were the same as
those up-regulated by the 6 h TGF-β1 treatment. Five (31%) of the genes down-regulated by EMD
were the same as those by TGF-β1. Of the four up- and eight down-regulated genes at 24 h of EMD
treatment, two up- and two down-regulated genes were the same as those affected by the TGF-β1
treatment.
Effects of EMD and TGF-β1 on MMP family genes
MMP-9 was the most significantly (P<0.05) up-regulated of all the genes after 24 h of EMD
treatment as well as after 6 and 24 h of TGF-β1 treatments. MMP-10 was the most up-regulated
gene after 6 h of EMD treatment, but was then the most down-regulated gene after 24 h. In TGF-β1
treated carcinoma cells, MMP-10 was first among the most up-regulated genes after 6 h of
treatment but was then absent at 24 h. Furthermore, MMP-1 was also statistically significantly up-
regulated after 6 and 24 h of TGF-β1 treatments.
Effects of EMD and TGF-β1 on Gene Ontology (GO) groups
GO enrichment analysis revealed several enriched terms after 6 h EMD treatment (Appendix 1).
After grouping the enriched terms into functional clusters, the analysis revealed keratinocyte
differentiation, inflammatory response and proteolysis to be the most up-regulated. Among the
down-regulated genes, the most enriched clusters were cell growth and morphogenesis, circulatory
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system and cellular developmental processes. Because of the relatively few genes regulated by
EMD after 24 h, the GO enrichment analysis was not performed for that treatment.
GO analysis of 6 h TGF-β1 treatment revealed several enriched terms (Appendix 1), which were
grouped into functional clusters. The most enriched clusters among up-regulated genes were system
development, chemokine activity/inflammatory response, hemopoiesis and angiogenesis. Those
among down-regulated clusters were cell cycle processes, mitosis/M-phase and cell growth. After
24 h TGF-β1 treatment, the most enriched clusters among up-regulated genes were developmental
processes, inflammatory/defence response and angiogenesis. Down-regulated clusters were
regulation of cellular processes, intracellular organelle and calcium homeostasis.
6.5 Effects of EMD on metastasis formation in vivo (II)
The mean number of enlarged lymph nodes containing metastases was 3.1 (range 1 to 5, SD 1.6) in
the EMD group, and 1.5 (SD 0.9) in the NaCl group, the difference being 1.6 (CI 0.2 to 2.8). The
evidence was insufficient to determine whether there were differences in mortality between the two
groups.
6.6 Results of the systematic review (IV)
Altogether 76 studies were found to meet the inclusion criteria, e.g. in vitro studies concerning
cytokines in EMD or regulated by EMD and assays designed to study the effects of EMD on human
oral cells or malignant cells. EMD was found to enhance the in vitro production of several factors
related to carcinogenesis in malignant and benign cell lines. TGF-β1 and MMPs are crucial
regulators in carcinogenesis and their production is enhanced by EMD in many different cell lines
including osteoblasts, fibroblasts and malignant osteosarcoma cells (Schwartz et al 2000,
Mirastschijski 2004, Goda et al 2008). In addition, EMD promotes the production of several other
carcinogenesis-related cytokines by benign cells. These include CTGF, FGF-2, ALP and
osteopontin produced by osteoblasts; IGF-I, PDGF, VEGF, IL-6, ALP and OPG produced by PDL
fibroblasts; and PDGF, VEGF and osteopontin produced by epithelial-derived cells (Hakki et al
2001, Rincon et al 2005). Together with the effects of EMD on production of MMP- and TGF-β1
by malignant cells, EMD also stimulates collagenolysis by osteosarcoma cells (Goda et al 2008).
Furthermore, EMD increases cAMP levels and PDGF-AB production of HeLa cells and stimulates
43
phosphorylation of several MAP family kinases in SCC-25 cells (Lyngstadaas et al 2001, Kawase et
al 2001, 2002). Despite these findings, EMD has been shown to possess negligible or inhibitory
effects on the proliferation of malignant cells and to inhibit breast cancer cell attachment to bone
matrix (Gestrelius et al 1997, Schwartz 2000, Lyngstadaas 2001, Kawase et al 2001, Takayama
2005).
Table 1. In vitro effects of Emdogain on carcinogenesis-related cytokines and cellular processes in malignant and benign cell lines. [+] corresponds to stimulatory, [−] to inhibitory, [0] to unchanged and [X] to non-relevant effects of EMD on the corresponding carcinogenesis-related variable. Each symbol [+], [−] and [0] corresponds to one study, or to one cell line when several cell lines were studied.
44
7 DISCUSSION
7.1 Summary of the main results
The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains
several different proteolytic enzymes, could degrade EMD and alter its biological functions. In
addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in
particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the
understanding of the composition of EMD.
GCF was observed to degrade EMD depending on the level and severity of the periodontal disease.
The GCF-degraded EMD was less effective in enhancing PLF proliferation than non-degraded
EMD. EMD was observed to enhance the proliferation of PLFs whereas amelogenin inhibited the
proliferation. In contrast, TGF-β1 was more effective and regulated more genes in HSC-3 cells than
EMD. EMD was capable of enhancing in vitro migration and wound closure of HSC-3 cells and to
promote metastasis formation in vivo. In addition, EMD was capable of inducing MMP-2, MMP-9
and MMP-10 production at the protein level and MMP- 9 and MMP-10 expression at the gene
level.
7.2 EMD in periodontal regeneration
The mechanisms of action and the exact composition of Emdogain are not completely clear. In
addition, a systematic Cochrane review suggests that the overall treatment effect of EMD might be
overestimated and that the actual clinical advantages of using EMD are unknown (Esposito et al
2005). Despite the lack of knowledge, EMD is extensively used in regenerative periodontal therapy
in all kinds of patients including medically compromised subjects. Periodontal inflammation is
characterised by increased apical proliferation of gingival sulcular epithelial cells together with
increased activity of proteolytic enzymes in gingival tissue and GCF. Matrix metalloproteinases
(MMPs) and matrix-degrading serine proteinases have been shown to be the main mediators of
pathologic tissue destruction in periodontitis (Sorsa et al 2004, 2006). These proteinases, in
particular MMPs, can degrade most, if not all, extracellular matrix and basement membrane
components of periodontium (Birkedal-Hansen et al 1993, Sorsa et al 2004, 2006).
45
In periodontal treatment, a full-thickness flap procedure is used to apply the EMD in the deepest
periodontal pockets and in contact with periodontally-exposed root surfaces. Detectable amounts of
EMD remain at the site of application on the root surface for two to four weeks after which EMD is
degraded (Gestrelius et al 1997, www.straumann.com). This degradation is presumably due to
proteolytic enzymes present post-operatively and during the healing process in connective tissue
and thus secreted also in GCF.
The tissue breakdown and healing after the periodontal therapy and after the surgical trauma
involve collagen destruction via an MMP-mediated pathway (Birkedal-Hansen 1993, Kinane et al
2003). It has been shown that non-surgical periodontal therapy decreases the MMP levels of GCF
weeks to months after periodontal therapy (Haerian et al 1996, Kinane et al 2003, Marcaccini et al
2010). However, the MMP levels of GCF are not statistically significantly reduced during the
healing period of the first weeks (Kinane et al 2003,) or in sites that do not respond to the treatment
(Mäntylä et al 2003). Furthermore, it has been shown that periodontitis-affected sites continue to
display high levels of MMP-8 and collagenolytic activity also after effective periodontal treatment,
and these levels and activity are in the range of those found in gingivitis (Figueredo et al 2004).
In this study it was observed that GCF can, depending on the periodontal status, degrade EMD.
Clinically EMD is not directly interacting with GCF but with the connective tissue fluids instead.
Furthermore, EMD should only be used after infection control and proper scaling and root planing.
In this assay, GCF was mainly used to mimic the circumstances and proteases present in connective
tissue after periodontal therapy. Thus, the strongest EMD degrading effect caused by GCF from
chronic periodontitis states may be clinically overestimated. However, also healthy GCF was
capable of degrading EMD, thus implicating that connective tissue fluids are capable of degrading
EMD also after the periodontal therapy.
In addition, this study suggest, that MMPs are not the main factors contributing to the observed
degradation, since only MMPs-2 and -8 were capable of degrading EMD, and only to a relatively
low extent. The results suggest that the role of other proteases in GCF are most likely more
important for the degradation of the EMD in vivo. Interestingly, both metalloendoproteinase and
serine protease activities have also been observed in EMD itself (Maycock et al 2002).
The effects of EMD on the proliferation of periodontal ligament fibroblasts have been extensively
studied. Also, the observations of the present study confirmed that EMD can enhance the
46
proliferation of PLFs in vitro. Surprisingly, the recombinant amelogenin protein was observed to
depress the proliferation of PLFs significantly. This observation suggests, as Chong et al (2006)
have previously postulated, that amelogenin itself has very little to do with the proliferation of
periodontal ligament fibroblasts, and that the stimulatory effect of EMD on the proliferation of
PLFs is caused by other factors in EMD. Another novel observation was that EMD together with
GCF was less effective in promoting the proliferation than EMD alone, and with the lower
concentration of EMD (100 µg/ml) the proliferation was not different from that of the control cells.
These results suggest that GCF can degrade or inactivate the growth factor-like molecules in EMD
responsible for proliferation. It should also be noted that due to the high consumption of GCF in
these assays, the GCF samples were pooled, and thus the assay also contained less effective EMD-
degrading GCF from healthy subjects. Thus, this approach is also closer to clinical circumstances
where EMD is not in contact with chronically infected tissues, but healing tissues instead. Taking
these observations together, it can be concluded that the inductive effects of EMD on the
proliferation of PLFs are not caused by amelogenin, and thus the presence of cytokines in EMD is
presumable. In addition, the stimulatory effect of EMD on the proliferation of PLFs, one of the
most crucial steps in periodontal regeneration, may be inhibited if the product comes in contact with
proteolytic enzymes in periodontal tissues also in vivo.
7.3 EMD and carcinogenesis
As stated above, the overall knowledge of the effects of EMD on periodontal tissues is relatively
good. However, many relevant issues, including the cytokine content of EMD, still remain
unresolved. There is evidence suggesting that the epithelial-mesenchymal interactions are important
during periodontal tissue development and regeneration, and that EMD can mimic these processes
(Gestrelius et al 2000). In carcinogenesis, epithelial-mesenchymal interactions act in an
uncontrolled manner and one of the first steps towards malignancy is the epithelial to mesenchymal
transition. After the initial formation of the epithelial malignancy, tumour growth depends on the
proteolytic enzymes and cytokines produced by stromal cells surrounding the tumour. EMD has
been observed to stimulate the production of several cytokines and cellular processes normally
related to periodontal regeneration but also to carcinogenesis (See Table 1).
One of the major cytokines in carcinogenesis is TGF-β1. TGF-β1 stimulates cell growth, MMP
production, angiogenesis, invasion and metastasis in head and neck squamous cell carcinomas
47
(HNSCC) (Derynck et al 2001, Akhurst et al 2001). In addition, TGF-β1 together with other
cytokines, for example those in epidermal growth factor families, can enhance morphological and
invasive phases of the EMT phenotype (Wilkins-Port and Higgins 2007). The present study
examined the effects of TGF-β1 on gene expression of carcinoma cells using Affymetrix
microarrays. TGF-β1 was observed to induce angiogenesis-related genes but to down-regulate
proliferation-related genes in carcinoma cells after 6 h of incubation. However, the inhibitory effect
of TGF-β1 on the proliferation of carcinoma cells was only temporary and was absent after 24 h,
whereas the stimulatory effect of TGF-β1 on angiogenesis remained constant over the observation
period.
EMD has been postulated to contain TGF-β1 (Kawase et al 2001, Suzuki et al 2005) and to enhance
the production of TGF-β1 by several cell lines (Schwartz et al 2000). In the present study, when the
effects of EMD and TGF-β1 on carcinoma cell gene expression were compared, TGF-β1 was
observed to regulate markedly more genes than EMD. This result suggests that EMD might not
contain TGF-β1, or that the cytokine is present in other concentrations as studied here. The latter
assumption is supported by the result showing, that the expression of TGF-β family genes was
down-regulated by EMD. Thus, EMD may contain TGF-β1 or have TGF-β1-like activity. In
addition, several regulatory functions for both of the study reagents were similar, for example both
EMD and TGF-β1 up-regulated the expression of MMP-9 and MMP-10 in carcinoma cells. Thus,
this study cannot confirm or reject the hypothesis that EMD contains TGF-β1.
Together with TGF-β1, MMPs are considered to be crucial in tumorigenesis (Lόpez-Otίn &
Matrisian 2007). Typically, several MMPs are expressed in human malignant tumours, and elevated
MMP levels correlate with aggressive and invasive tumours and poor prognosis (Stetler-Stevenson
& Yu 2001). MMPs have an important role at all stages of tumorigenesis: they enhance tumour-
induced angiogenesis, regulate and activate growth factors, and break down ECM and BM to allow
tumour cell invasion and metastatic spread (McCawley & Matrisian 2000; Liotta and Kohn 2001;
Stetler-Stevenson & Yu 2001, Nyberg et al 2008). In this study EMD was observed to enhance the
production of MMP-2 and MMP-9 by carcinoma cells as well as by non-malignant keratinocytes.
The MMPs from the culture media of different assays were analysed both by gelatin zymography
and ELISA immunosorbent assay. The present study confirmed EMD-induced up-regulation of
MMPs at the gene level and the production of MMP-10 in protein level. In addition, the GO
enrichment analysis revealed the term proteolysis being one of the most up-regulated by carcinoma
48
cells after EMD incubation. Also, EMD-induced migration of carcinoma cells was observed to
depend on MMP-2 and -9, since an inhibitor of these MMPs (CTT-2) arrested the effects of EMD
on the migration. Thus, these studies provide extensive results showing EMD to be capable of
regulating MMPs produced by oral carcinoma cells. In addition, other studies have confirmed that
EMD can enhance the production of MMPs in different cell lines. Goda et al (2008) observed EMD
to enhance the production of MMP-1 in malignant MG-63 osteosarcoma cells and to promote the in
vitro degradation of type I collagen. In benign cells, it has been shown that EMD increases more
that threefold the release of MMP-2 from cultured rat skin fibroblasts as well as from endothelial
cells (Mirastschijski et al 2004). This effect of EMD on MMPs is highly interesting and may
potentially cause carcinogenic effects. However, the effects of EMD on MMP production can also
vary between cell lines, MMPs studied and according to other factors and cytokines present in vivo.
As with carcinoma cells, EMD was also observed to enhance the MMP-2 production by periodontal
ligament fibroblasts in vitro. However, EMD decreases the levels of MMP-1 and MMP-8 in GCF
after flap surgery in vivo. In that study, the decreased MMP levels in GCF may just be due to the
decreased inflammation in periodontal tissues after periodontal treatment. Even then the result
highlights the limits of in vitro studies and the variability in the response to different cytokines by
different cell lines and tissues.
As with the production of MMPs, the effects of EMD on other cellular functions also vary between
the cell lines investigated. In this study, notably increased in vitro migration and wound closure of
carcinoma cells were observed after EMD treatment. The effect of EMD on the migration and
wound closure of benign keratinocytes (HMK cells) was less obvious. These findings with a shorter
culture period are similar to previous findings in which enhanced wound-fill of osteosarcoma (MG-
63) cells was observed after a longer culture period (6 to 9 days) (Hoang et al 2000). According to
the effects of EMD on migration and wound closure of carcinoma cells, it would be logical to find
EMD to enhance the proliferation of carcinoma cells as well. However, it has been demonstrated
that EMD has no effect on highly malignant MCF-7 breast carcinoma cell proliferation during short
180 min incubations (Takayama et al 2005). The findings of the present study also demonstrated
that EMD exhibits no effect on the proliferation of oral carcinoma cells after 12h to four days of
treatment. In contrast, EMD inhibited HMK cell proliferation after 12 and 24 hours incubation.
Kawase et al (2001) have shown that EMD inhibits the proliferation of human squamous cell
carcinoma SCC-25 cells after three days of treatment. In the same study, it was also shown that
EMD dose-dependently arrested the cell cycle of SCC-25 cells at G1 and did not discernibly
increase SCC-25 apoptosis over 8 days of treatment. These results indicate that the effects of EMD
49
on cell proliferation are likely to depend on the cell type investigated. The discrepancy between the
results with SCC-25 cells of Kawase et al (2001) and those with HSC-3 cells used in this study may
reflect the fact that HSC-3 cells are highly invasive whereas the SCC-25 cells are moderately
differentiated, less invasive and often, like in the study by Kawase et al (2001), used as a substitute
for normal epithelial cells (Matsumoto et al 1989). Eventually, the less invasive carcinoma cells are
likely to be more sensitive to the regulatory cell proliferation signals of EMD relative to more
invasive and aggressive counterparts.
After having observed carcinogenesis-related factors to be regulated in several in vitro assays, this
study aimed to examine the effects of EMD on carcinogenesis in vivo. Despite the small number of
study animals, ten subjects in the study group and ten in the control group, the inductive effect of
EMD on metastasis was clear, and also statistically significant. Due to the small number of subjects
no differences were observed in survival or tumour growth between the two study groups. Thus, a
more extensive in vivo study would be needed to confirm the observed effects.
In addition to the present study, only a few studies concerning EMD and malignant cells have been
conducted. None of these have focused on carcinogenesis as such, but malignant cells have rather
been used as an “easy growing” substitute for their benign precursors. Also, many of these assays
have been carried out with cells very different from those in oral tissues. Thus, these studies are not
of major relevance in the context of EMD and oral cancer. No matter their original scientific goals,
the present study reviewed all in vitro studies in which malignant cells were treated with EMD.
According to the reviewed studies, EMD has been shown to stimulate the production of several
cytokines known to be up-regulated in carcinomas, including MMP-1, -2, -9, IL-6, TGF-β1 and
PDGF. In addition, EMD can enhance the expression of these and several other cytokines by benign
cells. In carcinogenesis, the cytokines produced by stromal cells surrounding the malignant tissue
are crucial in regulating tumour behaviour and growth. Thus, in a situation in which EMD were
present in direct contact with either malignant tissue or nearby normal tissue, EMD could be
capable of promoting carcinogenesis by inducing cytokine production and transformation of the
tissues.
The initial hypothesis for the present study arose from a clinical observation in which a dysplastic
lesion was converted into an invasive carcinoma shortly after EMD treatment. The presented
clinical situation is rare, but it should be noted that according to the manufacturer of EMD
50
(Straumann, Basel, Switzerland), mucosal dysplasia is not a contraindication for EMD treatment.
However, the treatment choices should not be based solely on the statements made by
manufacturers, and performing a periodontal surgery to an area with an erosive and dysplastic
lesions is not in accordance with the standard of care. In this case, the treatment should have
included the management of the periodontal pocket by conventional scaling and root planing,
together with the treatment of the mucous dysplastic lesions by local medication or by surgical
means. In addition, when considering the possible causal role of Emdogain in the development of
carcinoma, it should be noted that preceding factors existed, as the patient already had widespread
dysplastic pre-malignant non-homogenous leukoplakia lesions. Before the EMD-therapy, several
biopsies were taken around the mucous membranes, and also near the EMD-treated sites, showing
moderate dysplasia. However, it is well known that the degree of dysplasia can vary from site to site
and thus it may be possible that the patient already had a carcinoma in situ. Nonetheless, the
location of the initial cancer within the bone, close to the apex of d.12, and the close chronological
and spatial relationship to the EMD-treatment were unusual. Based on the follow-up history, it is
highly possible that this patient had genetic susceptibility to dysplastic transition (“field
cancerization”), since the recurrences of mucous dysplastic and cancer lesions have occurred
several times during the recent past. However, the possibility of EMD influence on the cancer
induction of this patient could not be completely ruled out. Further studies of the effects of EMD on
cytokines produced by benign and malignant oral tissues together with studies focusing on the
composition of EMD are needed before the actual safety of EMD on patient with dysplastic
mucosal lesions can be confirmed.
7.4 Methodological considerations
The first sub-study examined whether the degradation of EMD caused by GCF has effects on the
proliferation of PLFs. The proliferation was determined with a commercial Cell Proliferation
ELISA BrdU kit. BrdU is incorporated into newly synthesised DNA, which is optically measured.
Thus, the assay actually measures the DNA synthesis of the cells assumed to reflect the
proliferation instead of directly measuring the proliferation of the cells. However, the method was
selected because it is simple and sensitive, does not rely on microscopical counting of the cells, and
is therefore objective and currently often used.
The main result of the proliferation assay was that GCF together with EMD cannot stimulate the
proliferation of PLFs, as EMD did alone. In addition, the GCF treatment alone had no effect on the
51
proliferation. During the proliferation assay, the cells were kept in serum containing media to
simulate in vivo circumstances, in which the cells normally have enough nourishment. However, the
high concentrations of serum in the media are likely to override the effects of tiny amounts of
cytokines and other effectors in GCF. Thus, the result concerning the GCF only treated control cells
may be interfered by the serum. However, this problem cannot cause bias to the main result of the
study, as it was shown that GCF can degrade EMD or inactivate its effects despite the presence of
serum.
The general aim of the second and third sub-studies was to study whether EMD could induce
carcinogenesis. This was studied with in vitro cell assays and in vivo mice experiment. With the
selected in vitro assays, several crucial cellular processes related to carcinogenesis could be studied,
including migration, proliferation and MMP production. However, carcinogenesis relies on several
other factors as well which were not studied. These include the production of other MMPs and
growth factors as well as the invasion capabilities of the cells. In addition, it has been shown that
malignant cells with different phenotypes behave differently in various assays (Matsumoto et al
1989). However, the assays in the present study were done using only one highly malignant cell
line, the HSC-3 oral carcinoma cells. Furthermore, the selected in vitro assays were relatively
simple and thus are not comparable with the in vivo circumstances in which the malignant cells are
constantly interacting with the stromal cells and numerous different cytokines. Thus, the results
from these in vitro studies are presumably partly cell type-specific, and cannot be directly
generalised to all dysplastic cell types or to in vivo circumstances.
In the present assays, EMD, amelogenin and TGF-β1 were dissolved in PBS. In previous studies,
EMD has most often been dissolved in acidic acid (Hoang et al 2000), trifluoroacetic acid (Kawase
et al 2000) or propylene glycol alginate (PGA, Yuan et al 2003) instead of PBS (Klein et al 2007).
In the study, PBS was chosen because it is neutral and thus closer to the neutral pH of the oral
cavity compared to the acids. However, enamel matrix proteins, in particular amelogenin, are
hydrophobic and do not completely dissolve at neutral pH. To maximise the dissociation of EMD to
PBS the solution was gently mixed 24 h in RT. Microscopically, EMD was observed to be properly
dissolved in the culture media after incubation at +37°C. However, there is the possibility that some
minority of the insoluble proteins may not have dissolved and were thus not present in the assays.
This could cause slightly higher concentrations of the possible water-soluble growth factors in
EMD in assays compared to studies in which acid vehicles were used.
52
The used EMD and amelogenin concentrations were selected based on preliminary assays and/or
previous studies. In the case of EMD, the selected concentrations are most likely well below the
concentrations used clinically, since gel-like EMD is applied locally, whereas such gel application
and high concentrations are not possible in cell cultures. This discrepancy between clinical and the
study concentration causes bias, and the observed effects can be highly concentration depend.
However, since clinical concentrations are higher than the used study concentrations, it is likely that
the proteins and possible cytokines of EMD diffuse into the surrounding tissues in vivo, thus
resulting in the study concentrations at some point near the EMD treated site.
Also the selected TGF-β1 concentration (10 ng/ml) in microarrays is somewhat contradictory. The
used concentration was selected to be biologically meaningful according to previous studies
(Pääkkönen et al 2008), while it is unclear whether EMD contains TGF-β1 or not and in what
concentration. However, the response of carcinoma cells to growth factors and to TGF-β1 can be
variable depending on the concentration. Thus, the current microarray analysis can only prove that
EMD cannot contain TGF-β1 at the concentration of 10 ng/ml, but other concentrations of TGF-β1
in EMD are still possible which warrants further studies.
In addition to TGF-β1 concentration, also the data analysis of microarray results had limitations.
Due to the lack of more sophisticated statistical methods available in the used microarray analysis
software, and in microarray analysis software in general, the statistical significance in gene
expression between study and control groups were judged according to P-values instead of CIs,
which were otherwise used in this study. Also the fold change (FC) of the genes between control
and study groups was calculated, but all the statistically significant results were regarded as true
observations with biological relevance. This may cause overstatements in the results since
′statistically significantly′ differing genes can have only relatively small difference in fold change at
which point the biological relevance of the observation may be questionable.
The fourth sub-study aimed to systematically review the existing studies focusing on the effects of
EMD on carcinogenesis-related factors in vitro. There were no previous studies directly focusing on
the associations among oral carcinoma cells and EMD. Therefore, the aim of the study was met by
including all in vitro studies relevant to carcinoma-related factors and behaviour of oral cells. With
the chosen method, the study was able to present and summarise all available biological data
53
concerning the topic. However, at the same time, the inclusion/exclusion criteria had to be relatively
imprecise and it did not thus completely meet the criteria of systematic reviews. First and foremost,
the inclusion/exclusion of the studies was not done according to the methodological diversities of
the reviewed studies. Thus, in the included studies, different experimental set-ups were used
including different cell lines, assay designs, EMD concentrations, dissolvent of EMD, time points,
and controls. However, the underlying biological mechanism, and thus the results of a study, may
depend on the assay design. Therefore, the conclusions of the review must be made taking these
limitations into account.
54
8 CONCLUSIONS
This study aimed to find out the effects of proteolytic enzymes present in GCF on the degradation
and functions of EMD. In addition, the objective was to find out whether EMD could induce
carcinogenesis-related genes, proteins and cellular processes in vitro or in vivo. Furthermore, the
study aimed to contribute to the understanding of the composition of EMD, which has been
observed to contain mainly amelogenin and possibly TGF-β1. The results of this study led to the
following conclusions:
I. GCF containing various proteases degrades EMD. EMD degradation depends on the state of
periodontal disease being stronger in all states of periodontal disease (gingivitis, chronic and
acute periodontal) compared to a healthy state. MMPs are probably not responsible of the
degradation of EMD.
II. GCF can decrease EMD induced proliferation of periodontal ligament fibroblasts. Unlike
EMD, also amelogenin decreases the migration of PLFs. Thus, the effect of EMD on the
proliferation is likely to be mediated by other cytokines in it and not the amelogenin itself.
III. TGF-β1 regulates more and mostly different genes than EMD. This suggests that TGF-β1
cannot be present in EMD at the studied concentration, if present at all.
IV. EMD can induce several factors closely related to carcinogenesis in gene, protein, cell and in
vivo levels. EMD enhance the expression of MMP-10 and MMP-9 in gene level and the
production of gelatinases MMP-2 and MMP-9 and MMP-10 by cultured carcinoma cells.
EMD stimulates the migration and in vitro wound closure of carcinoma cells. Furthermore,
EMD also promotes metastasis formation in athymic mice bearing human tongue carcinoma
xenografts. These results show that EMD can regulate several different factors, not only in
benign cells, but also in highly malignant oral carcinoma cells. These results may have clinical
implications and warrant further studies.
V. Review of the literature confirms that EMD can regulate the production of several cytokines
related to carcinogenesis in benign and malignant cell lines, but the evidence is scarce and in
part contrasting.
55
ACKNOWLEDGEMENTS
This study was carried out at the Department of Cell Biology and Oral Diseases, Biomedicum
Helsinki, Institute of Dentistry, University of Helsinki and at the Department of Diagnostics and
Oral Medicine, Institute of Dentistry, University of Oulu, during the years 2004-2010. Animal
experiments were performed at the Viikki Laboratory Animal Center of the University of Helsinki.
The study was financially supported by the HUCH-EVO and OUCH-EVO grants and by the
Finnish Dental Society Apollonia.
I have been very fortune to have had Professor Tuula Salo and Professor Timo Sorsa as my
supervisors. Their unbeatable team - full of enthusiasm, ideas and passion for science - has inspired,
guided and encouraged me during these years. Thank you.
I wish to express my gratitude to the former and present Deans of the Institute of Dentistry,
University of Helsinki, Professor Jukka H. Meurman and Professor Jarkko Hietanen, for providing
the excellent research facilities at my disposal. I am grateful to Professor Lari Häkkinen and Dr.
Marilena Vered for reviewing my thesis and offering constructive suggestions to improve the work.
I warmly thank all my co-authors for their collaboration: Professor Esa Läärä for his expertise in the
statistics, Dr. Saynur Vardar-Sengul, Professor Gül Atilla, Dr. Buket Han Saygan, Dr. Haluk Baylas
and Professor James P. Simmer for providing the study reagents and samples at my disposal, and
Dr. Juho Suojanen, Sini Nurmenniemi, MSc and Suvi-Tuuli Vilen, DDS, for their participation in
the study assays. I am also thankful to Kirsti Kari, MSc, the Head of the Scientific Laboratory of the
Institute of Dentistry, University of Helsinki, for her organisational skills and kind help with
anything needed in the lab, and to Docent Taina Tervahartiala for her practical support and
guidance. I acknowledge Ritva Keva, Jukka Inkeri, and Marjatta Kivekäs (University of Helsinki)
as well as Maija-Leena Lehtonen (University of Oulu) for their excellent technical assistance. I
thank the entire staff, colleagues and friends at our laboratory for the enjoyable and encouraging
working atmosphere. Outside the lab, I also wish to thank Professor Tuomas Waltimo (University
of Basel), Roope Lehtinen, DDS, and Pirjo Rissa, DDS, for their flexibility, support and
understanding towards my work on the thesis during these years.
My dearest thanks go to my friends and family for their support and for providing me with activities
and thoughts unrelated to science. Finally, words fail me to express my appreciation to my wife
Elina. She has been my third supervisor, the unnamed co-author and the most valuable reviewer of
56
this work at its every phase. She has shared the moments of joy and encouraged me in
disappointments. Thank you for everything.
Basel, April 2010
Matti Laaksonen
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APPENDIX The 15 most enriched GO terms according to P-value by 6 h (A1/A2) of EMD and 6 h (B1/B2) and 24 h (C1/C2) of TGF- β1 treatments of HSC-3 cells. Table shows the term, p-value and number of genes (N. G.) related to various terms. * correspond to terms, which were the same for EMD and TGF-β1 after 6 h of treatment. “ correspond to terms, which were the same for 6 h and 24 h of TGF-β1 treatments.