Effect of Enamel Matrix Derivatives on MC3T3 Osteoblasts Gita Safaian

University of Connecticut OpenCommons@UConn

SoDM Masters Theses School of Dental Medicine

June 2004 Effect of Enamel Matrix Derivatives on MC3T3 Osteoblasts Gita Safaian

Follow this and additional works at: https://opencommons.uconn.edu/sodm_masters

Recommended Citation Safaian, Gita, "Effect of Enamel Matrix Derivatives on MC3T3 Osteoblasts" (2004). SoDM Masters Theses. 118. https://opencommons.uconn.edu/sodm_masters/118 EFFECT OF ENAMEL MATRIX DERIVATIVES ON MC3T3 OSTEOBLASTS

Gita Safaian, D.M.D.

B.S., Queen's University, 1995

D.M.D., University of Connecticut, 1999

A Thesis

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Dental Science

At the

University of Connecticut

2004 APPROVAL PAGE

Master of Dental Science Thesis

EFFECT OF ENAMEL MATRIX DERIVATIVES ON MC3T3 OSTEOBLASTS

Presented by

Gita Safaian, B.S., D.M.D.

Qu:~ Major Advisor _ Qiang Zhu, D.D.S., Ph.D.

Associate Advisor ~~~,s\>b_- Lar~:Jg7~~;.~.s.,Ph.D.

Associate Advisor------Kamran E. Safavi, D.M.D., M.Ed.

Associate Advisor -----+-+------r----=---.;~-~----- S., Ph.D.

University of Connecticut 2004

11 ACKNOWLEDGEMENTS

I would like to acknowledge the dedication and guidance of Dr. Qiang Zhu. rlis approach to research is admirable and stress-free. I hope many future endodontic residents take the opportunity to learn from his impressive ability to turn a concept into a meaningful research project.

Dr. Larz Spangberg has been fundamental in helping me combine the technical and the intellectual aspects of endodontics. It was a great honor and privilege to learn from him. I thank him for all his advice.

I am forever grateful to Dr. Kamran Safavi for teaching me the history of endodontology. I thank him for encouraging me to try different techniques while emphasizing proper patient care. I am grateful for his guidance and his emphasis on the ethics of being a Doctor.

I would like to thank Dr. Wang for his assistance with my thesis and Dr.

Ashraf Fouad for my undergraduate training.

Doctor Lisa Sanchez was invaluable in helping me prepare my thesis and oral defense.

Finally, I thank my husband Dr. Tarik Kardestuncer for all his help and support.

iii TABLE OF CONTENTS

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IV LIST OF TABLES

Table 1. Number of genes in response to EMD or TGF-J31---38

Table 2. Full DNA array results------39

v LIST OF FIGURES

Figure1. Adhesion and spreading of MC3T3 cells on Fibronectin, EMDand BSA------42

Figure 2. EMD promotes cell attachment of MC3T3 cells------43

Figure 3. EMD promotes proliferation of MC3T3 cells------44

Figure 4. Increase in alkaline phosphotase activity in cultured MC3T3 cells by EMD------45

Figure 5. Northern blot analysis of EMD on collagena1 mRNA------46

Figure 6. Northern blot analysis of EMD on bone sialoprotein (BSP) mRNA------47

Figure 7. Northern blot analysis of EMD on osteocalcin «()C) mRNA------48

Figure 8. Northern blot analysis of EMD on insulin-like growth factor (IGF-1) mRNA------49

Figure 9. DNA micro array images in control, TGF-B1 and EMD treated groups------50

vi ABSTRACT

During tooth development, cells of Hertwig's epithelial root sheath secrete enamel matrix proteins. A purified enamel matrix product (Enamel Matrix Derivative, EMD) from developing porcine teeth became commercially available and has been successfully employed to restore fully functional periodontal ligament, cementum and alveolar bone. The regenerative capacity of EMD has been demonstrated in clinical and animal studies. However the biological mechanisms are not known. We hypothesize that EMD stimulates growth and differentiation, and regulates certain gene expression of osteoblasts. In this project we will determine the effect of EMD on cell adhesion, proliferation and differentiation of preosteoblastic cell line MC3T3-E1 cells. A DNA array will also be used to identify the cell cycle gene changes regulated by EMD and compare these genes to those regulated by TGF-~1. This study will identify the role of EMD on osteoblasts to understand the mechanisms of EMD induced bone regeneration. By comparing EMD regulated genes to that regulated by TGF-~1, it could be determined whether EMD has a similar function to TGF-~1, which has been shown to induce bone regeneration.

VII INTRODUCTION

During tooth development, cells of Hertwig's epithelial root sheath (HERS) secrete an extracellular matrix similar to enamel matrix proteins secreted by mature ameloblasts (Lindskog S 1982; Schendel et al. 1997; Slavkin et al.

1989b). Immunologic investigation suggest that cellular cementum contains proteins similar to enamel matrix proteins (Slavkin et al. 1989a). In fact, enamel matrix proteins have been recently detected at the apical portion of the forming

root by immunoblot analysis (Fukae 1996) . Hence j new advances j resulting in the development of more sensitive methodology, have shown that many of the so-called 'enamel proteins' have other functions besides that of enamel formation

(Fincham et al. 2000). To fully comprehend the function of these proteins it is essential to review them.

What are enamel proteins?

During the development of the crown of the tooth, two mineralizing matrixes are formed next to each other by two different layers of cells, odontoblasts and ameloblasts. Dentin is a collagen-based matrix. Enamel on the other hand contains no collagen and is mainly composed of hydrophobic proteins known as amelogenins and non-amelogenin proteins (anionic enamel proteins; ameloblastin; enamelin; tuftelin; tuft proteins; sulfated proteins and enamel proteases such as enamelysin. The ameloblasts control the synthesis and secretion of the organic extracellular matrix which is deposited along the dentino-

1 2 enamel junction (DEJ) which in turn controls enamel biomineralization. It is s~ggested that enamel proteins function as nucleators and modulators for calcium hydroxyl apatite crystal formation (Fincham 2000; Robinson et al. 1998;

Simmer et al. 1995; Zeichner-David et al. 1995).

Amelogenins are the most abundant enamel proteins comprising approximately 900/0 of all proteins secreted by ameloblast cells. They are hydrophobic proteins. Multiple amelogenins present in the enamel extracellular matrix are the product of alternative splicing of the amelogenin gene and processing of parent molecules (Gibson 1991). Recent studies shows that hydroxyapatite crystals grown in the presence of amelogenins develop into characterically long crystals associated with enamel formation (Wen 2000).

Tuftelin is a non-amelogenin, anionic enamel protein. This protein is expressed prior to the onset of mineralization. The function of tuftelin in tooth development remains unknown although recent studies suggest that it might function at the level of ameloblast differentiation and/or extracellular matrix secretion (Paine et al. 2000).

Ameloblastin represents 50/0 of the non-amelogenin mRNAs. This molecule is localized to chromosome 4q21, the same region where a family with autosomal dominant Amelogenesis Imperfecta has been linked, suggesting that this protein is important in enamel formation. This protein is present in the secretory stage of enamel formation and plays a role in enamel biomineralization.

Ameloblastin is also detected in pulpal mesenchymal cells and Hertwig's Root 3 sheath (Fong et al. 1998; Fong et al. 2000). The function of ameloblastin in these tissues is unknown.

Enamelin is the largest enamel protein. Based on its presence in forming enamel and the fact that enamelin gene is localized to human chromosome

4q21, the region localized to Amelogenesis Imperfecta, it is believed that it has a

role in enamel biomineralization (Dong et al. 2000).

Enamel proteases are important for processing the secreted amelogenins,

ameloblastin and enamelin in the extracellular matrix and also for their

degradation and removal from mineralizing matrix during the maturation of

amelogenin. A matrix metalloproteinase, enamelysin was recently cloned from tissues and was later localized within the most recently formed enamel. Another

class of proteins present in enamel extracellular matrix are the 'sulfated enamel

proteins' (Smith 1995). The role of these proteins is unknown however.

Role of enamel proteins in Root formation

The involvement of enamel proteins in root formation was first suggested in 1974 (Slavkin 1974). Several different hypothesis have been postulated to explain the function of enamel proteins in root formation. First, they serve in attachment of cementum to root dentine (Ten Cate 1996). Second, they help initiate cementogenesis (Heritier 1982). Third, they serve as an inducer of dental follicle cells to differentiate into cementoblasts (Hammarstrom 1997b;

Hammarstrom 1996). 4

Function of EMD in periodontal regeneration

Recently, with the avent of tissue bioengineering, it has been desirable to

take advantage of biological principles in organ development to induce tissue

regeneration. In 1997 a series of studies were published in the Journal of

Clinical Periodontology showing that when pig enamel matrix was placed in

experimental cavities it could initiate the formation of a tissue identical to

acellular, extrinsic fiber cementum (Hammarstrom 1997a). Extracted enamel

proteins or purified enamel matrix derivative (EMO) resulted in 60-800/0 formation

of new cementum and bone in experimental periodontal pockets in monkeys

(Hammarstrom et al. 1997). There were other studies showing that EMO

suspended in propylene glycol alginate (PGA) adsorbs to Hydroxyapatite and

collagen forming an insoluble complex which can remain on the root surface for

two weeks, sufficient time to promote re-colonization by periodontal ligament

cells (Gestrelius et al. 1997b).

In human studies, an experimental buccal dehiscence defect received

EMD and after four months the tissue was examined histologically. New

periodontal ligaments with functionally oriented collagen fibers and associated alveolar bone were present (Heijl et al. 1997). All these studies suggested that enamel proteins have the ability to promote periodontal regeneration by inducing cementum, periodontal ligament (POL) and bone formation. This product was marketed as EMDOGAIN by Siora, Inc. The active material in this product is

hydrophobic enamel matrix proteins extracted from porcine embryonic enamel.

The possibility of inducing immunologic reaction has been analyzed in vivo 5

(Heard et al. 2000; Heijl et al. 1997; Zetterstrom et al. 1997) and in vitro (Petinaki et al. 1998). It has been shown that EMD exposed to blood lymphocytes caused a slight increase of the proliferation of lymphocytes restricted to CD25, fraction of

DC4 positive cells and decrease of DC19 positive B lymphocytes (Petinaki et al.

1998). There have been several clinical trials where it has been shown that individuals treated with EMDOGAIN have increased periodontium attachment and increased levels of bone formation (Froum et al. 2001; Heijl et al. 1997;

Mellonig 1999; Okuda et al. 2000; Pontoriero et al. 1999; Rasperini et al. 1999;

Sculean et al. 2000; Sculean et al. 1999b). However in many of these cases the results were not significantly different than other treatment modalities such as barriers. Moreover some studies suggested that EMDOGAIN results were unpredictable and other barrier techniques actually have better results (Heden et al. 1999; Parashis et al. 2000; Pontoriero et al. 1999; Sculean et al. 1999b;

Silvestri et al. 2000).

It has been suggested that although the use of EMD for the treatment of periodontal defects appears to be promising, the call for larger and controlled clinical trials is considered necessary (Greenstein 2000; Heard et al. 2000;

Mellonig 1999). Furthermore most of the EMD studies are based on clinical evaluation; such as pocket reduction, periodontium attachment and radiographic images. Few of these studies have a small number of samples and some of their observations are subjective and most importantly there is not a specific marker for cementum, so it is difficult to determine it is cementum being formed or some type of bone-like tissue. 6

Regenerative capabilities of EMD

In order to investigate if enamel proteins really have regenerative properties and how they induce regeneration, in vitro models have been used in several studies. These studies analyze the ability of EMO to influence migration, attachment, proliferation, biosynthetic activity and mineral nodule formation in

POL cells in culture (Gestrelius et al. 1997b). Results indicate that EMO had no effect on migration, and attachment but it enhanced POL cell proliferation. Also

EMO increased total protein synthesis and promoted nodule formation by POL cells. To determine if the effect of EMO could be due to growth factors, immunoassays were used to screen for molecules such as: Calbindin D, GM

CSF, fibronectin, b-FGF, gamma-Interferon, IL-1 beta, 2,3,6; IGF-1,2, NGF,

PDGF, TNF and TGF-beta. None of these molecules were detected but contamination with growth factors can not be ruled out since growth factors produced by ameloblasts such as bone morphogenic proteins (BMPs) were not analyzed.

Several other studies have examined the effect of EMD on different cells.

Hoang (2000) investigated the effect of EMO on wound healing using an in vitro wound healing model; measuring the wound -fill rates of POL, gingival and MG63 osteoblast-like cells in culture where a "wound" was created on a surface of the cell (Hoang et al. 2000). EMD enhanced the wound-fill rate of all cells tested with the POL cells being significantly higher at the early days of treatment. Also the POL cells showed a greater response to EMD than to plasminogen derived growth factor. These studies support the hypothesis that EMO increases cell 7 proliferation. In an experiment on human POL cells, (Lyngstadaas et al. 2001) found that EMD significantly increased the attachment rate, growth and metabolism of these cells. Moreover cells exposed to EMO showed increased intracellular c-AMP signaling and production of TGF-beta1, interleukin 6 (IL-6) and platelet derived growth factor (PDGF-AB).

Conversely with epithelial cells ( HeLa cells ), EMD had no effect on the rate of attachment. Proliferation and growth were inhibited even though CAMP,

PDGF-AB had increased due to EMD. These studies indicate that EMD favors

mesenchymal cell growth while it inhibits epithelium growth. Moreover the autocrine growth factors released by the PDL cells exposed to EMD might contribute to periodontal healing and regeneration mimicking natural root development.

In a study by Schwartz (2000) the effect of EMD on osteoblast cells were analyzed and it was found that EMD regulates proliferation and differentiation

(Schwartz et al. 2000). This regulation is dependent on the 'stage' of maturation of cells. In early stages (Pre-osteoblasts), it stimulates proliferation while in the later stages in enhances differentiation, in dose dependent manner, as measured by alkaline phosphatase activity and osteocalcin production. These studies suggest that EMO might work as a growth factor (in these studies EMD stimulated expression of TGF-~1 and MG63 osteoblast-like cells). On the other hand EMD did not influence the production of collagen and proteoglycans sulfation, while they both increased in the presence of other growth factors such as BMP of TGF-~. It has been shown that EMD prolongs the growth of primary 8 cultures of mouse calvaria osteoblasts (Jiang et al. 2001). In this study, EMD enhanced collagen I, Interleukin-6 and Prostaglandin G/H sythase2 (PGHS-2) mRNA expression, but it did not stimulate the expression of osteocalcin and IGF

I (Jiang et al. 2001). Differences between the results of Jiang et al. and Schwartz et al could be due to differences in the type of cells and\or the developmental stage of cells used. To further understand the effect of EMD in bone formation, ability of EMD to induce de novo bone formation in mouse calf muscle was studied (Boyan et al. 2000). The results indicated that EMD was osteopromotive and not osteoinductive.

The effect of EMD on epithelial cells in vitro was studied using oral epithelial (SCC25) cells (Kawase et al. 2001). After three days of treatment,

EMD inhibited cell division and in tandem arrested cell cycle at the G1 phase.

Prior to this inhibition EMD up regulated p21WAF1/Cip1, a cyclin dependent kinase inhibitor, induced G1- arrest, and inhibited DNA synthesis. In addition,

EMD down regulated expression of cytokeratin-18 (CK18) protein. These studies suggest that EMD acts as a cytostatic agent on epithelial cells and its anti proliferative action is probably due to cyclin-dependent kinase inhibitor,

P21WAF1/Cip1-mediated G1 arrest. This data supports Lyngstadaas et al.

(2000) data and can explain the observation that EMD suppresses the down growth of junctional epithelium onto dental root surfaces, a process that interferes with formation of new connective tissue attachment (Hammarstrom

1997b). 9

EMO promotes periodontal proliferation, enhancement of mineralization in the POL cells and stimulation of collagen synthesis (Gestrelius et al. 1997a;

Gestrelius et al. 1997b). However, EMO inhibits mineralization in vitro when

immortalized cementoblast cells are used (Tokiyasu et al. 2000). All these studies demonstrate that EMD promotes proliferation of cementoblast cells while decreasing osteocalcin gene expression, though the expression of osteopathic is

slightly increased. It also suggests that increasing concentrations of EMD inhibit

mineralization in vitro but do not interfere with mineral formation in vivo when

using EMO-treated cementoblast soaked sponges implanted in

immunocompromised mice. These implants were kept in place for six weeks and

it is unknown how long after EMD treatment of cells they are still under the

influence of its activity (Zeichner-David 2001). Therefore, considering all these experiments, both in vivo and in vitro suggests that EMD can act as a

multipurpose growth factor that stimulates proliferation of mesenchymal cells while it inhibits cell division in epithelial cells or, it can stimulate cell differentiation

in some cells while inhibiting matrix production in others.

Given all these studies, it has been important to identify the actual protein responsible for its function. EMD (EMDOGAIN), is a mixture of acidic extracted enamel proteins. Despite HPLC purification, one cannot assume that it contains only amelogenin. Ameloblast and enamelin are processed into smaller fragments, some with hydrophobic properties similar to amelogenins.

Furthermore the possibility of contamination with growth factors has not been completely ruled out. 10

Are enamel proteins growth factors?

The question arises as to whether or not enamel proteins are indeed growth factors. The only way to determine if enamel proteins (and which enamel proteins) can function as growth factors is to use recombinant proteins. The advantage is due to the fact that enamel recombinant proteins are pure and don't have the problem of contamination with growth factors and each protein can be tested separately or in different combinations. In preliminary studies done by

Zeichner-David et al. (2000), the effect of amelogenin and recombinant ameloblastin was analyzed on cell proliferation of different types of

Immortomouse-derived cell lines. Periodontal ligament and HERS cells were grown in vitro in the presence of different concentrations of recombinant amelogenin and ameloblastin. The rate of proliferation of these cells was determined. The result of this study showed that recombinant ameloblastin had no apparent effect on POL proliferation. However recombinant amelogenin appeared to increase the rate of PDL proliferation. In the case of HERS cells ameloblastin increased the rate of proliferation while amelogenin had a slight effect in higher concentrations. These studies suggest that amelogenin has an effect on PDL proliferation. These results are surprising since amelogenin is not present during root formation. Studies using specific probes for amelogenin mRNA and in situ hybridization shows that HERS cells do not synthesize amelogenins (Luo et al. 1991). In studies using amelogenin and ameloblastin antibodies, epitopes of these proteins were detected in the cervical portion of rat molars but none was evident in the HERS cells or cementum layer (Bosshardt et 11 al. 1998; Nanci et al. 1998). Fong & Hammarstrom reported the presence of epitopes cross-reactive with an amelogenin antibody in the area where cementogenisis is inhibited in human teeth, but not in HERS (Fong &

Hammarstrom 2000). Hamamoto et al. reported the capacity of the epithelial rest cells of Malassez, assumed to be derived from HERS cells, to express amelogenin in response to pulp inflammation (Hamamoto et al. 1996). The amelogenin production only occurred under pathologic conditions in the rest of

Malassez. The mechanism by which amelogenin may act like a "growth factor" and influence PDL proliferation and differentiation remains to be determined.

The expression of ameloblastin by HERS cells has been demonstrated in developing mouse root using in situ hybridization (Fong et al. 1998; Fong &

Hammarstrom 2000; Fong 1996). Ameloblastin is synthesized at the early stages of HERS formation when HERS are actively proliferating to form the roots

(Fong 1996). Recently in vitro studies using the HERS cells, RT-PCR and

Westernblot has confirmed that these cells do not synthesize amelogenin and enamelin, but do synthesize tuftelin and ameloblastin (Ohishi 2003). There has been a suggestion that ameloblastin might have something to do with HERS proliferation during apical migration of these cells to form the roots during tooth development. The mechanisms of amelogenin and ameloblastin actions on proliferation, and whether they have any effect on cell differentiation, remains to be determined.

The 'growth factor'-like activity of amelogenin has been demonstrated

(Veis et al. 2000). They have used a 73 amino acid protein termed A+4 12

(containing exons 2, 3, 4, 5, 6d and 7) representing splice-products of the rat amelogenin. Both peptides were able to increase corporation of 35[S]-804 into proteoglycan in embryonic rat muscle fibroblasts in vitro and in vivo. These proteins also induced the expression of transcription factors SOX9 and Cbfa 1 as well as collagen type II and bone associated protein BAG-75 and BSP followed by mineralization. A+4 appears to induce the osteoblast phenotype. These experiments support previous data by Wang which suggested that amelogenins and soluble dentin proteins could induce the differentiation of muscle mesenchymal cells into chondrocytes and osteocytes (Wang 1993). Also in studies by Nebgen they isolated and characterized a dentin matrix protein exhibiting chondrogenic activity similar to amelogenin (Nebgen et al. 1999). This protein, originally believed to be ameloblast contaminant, represents an actual translation product of odontoblast cells and not the degradation product of enamel matrix proteins.

It is also claimed that amelogenin splice products are actually transcribed by odontoblasts cells (Veis et al. 2000). The presence of amelogenin epitopes in odontoblast cells has been demonstrated in a study, which postulated that these proteins were translocated from pre-ameloblast to odontoblasts to serve as yet unknown biological function (Nakamura et al. 1994). The expression of amelogenins by odontoblast cells has been reported , however the possibility of contamination of tissue used to obtain mRNA by ameloblast cells has not been ruled out (Dey et al. 2001). Transient expression of specific gene products by other cells also has been shown for dentin sialophosphoproteins using in situ 13 hybridization. However similar studies using amelogenin probes have shown expression exclusively by ameloblast cells. The possibility that amelogenin splice products from root odontoblast cells mediate the signal for POL proliferation and bone formation during root development should be considered.

Clinical evidence of EMD induced regeneration

In 1997, the concept of EMD guided regeneration was introduced to the periodontal community in a series of scientific reports. The clinical efficacy of

EMD in terms of probing measurements and radiographic bone gain was first demonstrated in 2 multicenter studies conducted by a group of Scandinavian investigators (Hejil et al. 1997; Zetterstrom et al. 1997) . Case reports published by several clinicians and researchers confirmed the initial findings and established that EMD application into deep bony lesions promotes significant attachment level gain, probing depth reduction and bone regeneration (Heard et al. 2000; Heden 2000; Heden et al. 1999; Parashis &Tsiklakis 2000; Parodi et al.

2000; Sculean et al. 2001; Sculean et al. 1999a).

In a review by Kalpidis , clinical data from EMD controlled studies were pooled for meta analysis and weighed according to the number of treated defects

(Kalpidis et al. 2002). Clinical attachment gain amounted to 33% of the original attachment level and probing reduction averaged to SOak of the baseline probing depth. Improvements in the clinical parameters achieved with EMO were statistically significant in reference to preoperative measurements. Preliminary histologic investigation with surgically created defects and experimental 14 periodontal lesions demonstrated the ability of EMD guided regeneration to induce formation of acellular cementum and promote significant analysis of the supporting periodontal tissues. The potential of EMD to encourage periodontal regeneration was also confirmed in human infrabony defects. However this review points at the recent human histologic studies where consistency of the histologic outcomes and ability of EMD Guided Regeneration to predict stimulate formation of acellular cementum have been questioned. Moreover, identifying the exact cellular interactions is essential for development of methodologies and enhances predictability of the EMD usage in dentistry. SPECIFIC AIMS

Specific Aim 1:

To study the effect of EMD on adhesion, proliferation, and differentiation of osteoblasts.

Specific Aim 2:

To identify the cell cycle genes regulated by EMD using DNA array, and to compare these genes to those regulated by TGFp1.

15 MATERIALS AND METHODS

Cell culture

Mouse pre-osteoblasts MC3T3 cells were obtained from American Type

Culture Collection (ATCC, Manassas, Va). Cells were cultured in Dulbecco's modified eagle medium (DMEM) (Gibco BRL, Gaithersburg, MD) supplemented with 100/0 fetal bovine serum (FBS, Hyclone Laboratories, Inc, Logan, UT) and

1% of an antibiotic/antimycotic cocktail (300 U/ml penicillin, 300 Jlg/ml streptomycin, and 5 J.lg/ml amphotericin B) under standard cell culture conditions

(37°C, 100% humidity, 95% air and 50/0 CO2).

Cell adhesion

The method for cell adhesion assay was similar as previously described.

Bacterial plastic Petri dishes (100 mm) were coated with 4 ml fibronectin (50 Jlg/ml) or EMD (50 Jlg/ml) in serum free DMEM for 1.5 hrs. Control plates were only incubated with DMEM. The plates were then blocked with 8 ml 0.1 % bovine serum albumin (BSA) in serum free DMEM for 2 hrs. Cells (1x105/dish) were placed into the plate and incubated for 1 hr and 18 hrs. At the end of each incubation period, the total cell number per well was electronically counted, and cell morphology was examined under microscope and photographed.

16 17

Cell proliferation

Cells were seeded onto 6-well or 12-well cell culture plates (Costar,

2 Corning, NY) at an initial density of 5,000/cm , and allowed to attach for 24 hours. Medium was then changed into serum-free medium. 100 J.lg/ml EMD

(BIORA AB, Malmo, Sweden) was added to the treatment groups for 1, 2, 3, 5 and 7 days. Cells cultured without EMD served as negative control. At the end of each incubation period, cells were released with 0.25% trypsin and 1 mM

EDTA in Hanks' balanced salt solution (Gibco BRL, Gaithersburg, MD), and the total cell number per well was electronically counted (Coulter Corporation, Miami,

FL). Data were statistically analyzed using one-way ANOVA by comparing total cell numbers per well.

Cell differentiation

Cell were cultured in DMEM with 2% FBS. Osteoblastic differentiation supplements (50 J.lg/ml ascorbic acid, 8mM ~-glycerophosphotate) were added to the culture media on day 7 when cells reached confluence. Cells were then further incubated for 3 weeks. Media was changed every 2 days. Fresh EMD and supplements were added during each media change. Control group was cultured under the same conditions without EMD. Osteoblastic differentiation was evaluated by ALP activity assay and the expression of bone differentiation markers including collagen a1 (I), SSP, and osteocalcin (OC). 18

Alkaline phosphotase (ALP) activity assay

Cells cultured in differentiating media in the presence or absence of EMD

for 3 weeks were analyzed for ALP activity by ALP staining and ALP activity

assay. For ALP staining, cells were rinsed twice with phosphate-buffered saline

(PBS), fixed with 20/0 paraformaldehyde, ALP substrate mixture (ALP staining kit,

Sigma Diagnostic) was then added and incubated for 15 minutes for color

development. For ALP activity assay, cell layers were scraped off culture plates

in scrapping buffer. Cell pellets were collected after a quick spin. Cells were

then lysed in ALP lysis buffer, and subjected to 3 freeze-thaw cycles. After

centrifuge at 14,000 rpm for 5 minutes, supernatant was collected. Twenty JlI

supernatant from each sample was added to each well in duplicates in a 96-well

plate (Costar, Corning, NY), and incubated with an assay mixture of p-nitrophenyl

phosphate. Plates were then scanned for spectrophometric analysis using a

plate reader (Quant plate reader, Bio-Tek, Winooski, VT). Absorbance was

measured at 405 nm every 5 minutes for 30 minutes. Activity was calculated

using KC junior software (Bio-Tek, Winooski, VT). Protein content in each

sample was determined by BeA assay. ALP activity is expressed as

nMol/min/mg. Group difference of ALP activity was determined using student's t test. 19

Northern blot analysis

Total mRNA from control and EMD-treated cells was extracted using

TRlzol reagent and further purified with phenol-chloroform method after each incubation period. After quantification, 20 J.lg of mRNA from each group was loaded onto 1% agarose gel and separated by electrophoresis. RNA was then transferred onto Gene-Screen Plus Hybridization Transfer membrane by capillary pressure. RNA was cross-linked to each membrane by UV irradiation. The membrane was hybridized with 32P-labeled cDNA probes at 42°C for 20h with 5-6 million counts per minute (cpm) per ml for each probe. The following mouse cDNA probes were labeled with 32p-dGTP using random primer oligonucleotides: collagen a1 (I), SSP, ac. p-actin probe served as loading control. After hybridization, membranes were washed and exposed to a phosphoimager, the intensity of radioactive label was quantified using ImageQuant™. A photographic film was then placed over the membrane for exposure, and was developed using a Kodak developer. The intensity of the bands was normalized to p-actin levels. 20

eDNA micro array

Cell treatment

MC3T3-E1 cells were seeded to the cell culture dishes (Sigma, St Louis

MO) at an initial density of 5000 cells/cm2. Cells were cultured in Dulbecco'a

Modified Eagle Medium (DMEM) (gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Hyclone Laboratories Inc., Logan, UT) under

standard cell culture conditions (37 degrees Celicius, 1000/0 humidity, 95% air

and 5% carbon dioxide). After cells grew to 80% confluence, the medium was

divided into three different groups: Group 1 DMEM (control group) only, Group 2

DMEM plus TGF-J31 (10 ng/ml), and Group 3 DMEM plus EMD (100 IJg/ml)

(Emdogain, BIORA AB, and Malmo, Sweden). Cells were incubated for 24hrs.

RNA extraction

Total cellular RNA was isolated with guanidine thiocyanate followed by

phenol chloroform extraction and ethanol precipitation (Chomczynski and Sacchi,

1987). The RNA was digested with 10 units of RNase-free DNase for 30 minutes at 37 degrees Celicius, extracted with Sodium acetate and ethanol. The

recovered RNA was quantitated by spectrophotometry. A twenty microgram aliquot of RNA from each group was fractionated by electrophoresis through a

1% agarose/2.2M formaldehyde gel. The gel was stained with ethidium bromide to visualize the 18S and 28S RNA bands and to assess the RNA integrity. The

RNA samples were stored at -700 C for further use. 21

cDNA probe preparation and array hybridization

Labeled probes were prepared by reverse transcriptase of 3 micrograms

of RNA in the presence of Biotin labeled dUTP (Super Array, Bioscience

Corporation). GE arrayTM cDNA expression array membranes (Super Array,

Bioscience Corporation) containing human cell cycle gene array series, which was developed to profile the expression of 96 genes involved in cell cycle

regulation, were used. Cell cycle progression is precisely controlled by cyclin

dependent kinases (CDKs) and proteins that regulate COKs. These CDKs and

CDK-modifying proteins, including cyclins, COK inhibitors, COK phosphatases,

and COK kinases, are included in the human cell cycle GE Array series. Genes

essential for ONA damage and mitotic spindle checkpoints, as well as genes in the SCF and APC ubiquitin-conjugation complexes, are also represented. These

membranes were prehybridized and hybridized with biotinylated cDNA probes

and washed twice with two wash solutions (2X SSC, 1%SOS and 0.1XSSC,

05 % S0S) as specified in the instruction manual (Super Array, Bioscience corporation).

Chemiluminescent signal detection

The hybridized filter membranes were incubated with alkaline phosphatase-conjugated Streptavidin (AP). Filters were washed and 0.1 ml

COP-star chemiluminescent substrate was added. The membranes were then exposed to x-ray film. The image from x-ray film was scanned and analyzed

using GE ArrayTM Analyzer software. GE Array Analyzer matches the raw data 22 table with the gene list for the particular array_ Genes with two fold induction or repression were selected and further analyzed. RESULTS

MC3T3 -E1 cells adhere and spread on EMD

After 1-hr incubation cells attach and spread on fibronectin and EMD (Fig.

1). However cells spread less on EMD than fibronectin (Fig. 1). Cells in the BSA group are rounded and most of the cells can be washed out (Fig. 1). After 18-hr incubation cells spread extensively both on fibronectin and EMD (Fig. 1).

However cell morphology looks different. Cells did not attach on BSA coated plates after 18-hr incubation (Fig. 1).

The cell number in the EMD group was significantly more than that in the

BSA group (P < 0.05) after 1-hr incubation (Fig. 2). The cell number in the fibronectin group is greater (P < 0.05) than that in the BSA and EMD groups (Fig.

2). After 18-hr incubation, there is no cell adhesion in the BSA group, and there is no cell number difference between the EMD group and fibronectin group.

EMD promotes proliferation of MC3T3-E1 cells.

Total cell counts in control and treatment groups after each incubation period are shown in figure 3. Cell morphology in both groups appeared normal.

No significant difference was seen between control and EMD-treated groups on day 1. Starting from day 2, EMD treatment resulted in a marked increase in cell number (p<0.01). This increase was persistent into day 7. Cell numbers in both groups increased with extended incubation time, and reached plateau around day 3.

23 24

EMD promotes differentiation of MC3T3-E1 cells.

ALP staining showed more and bigger ALP-positive nodules in EMD showed no significant difference between control and treatment groups determined by SeA assay (data not shown). ALP activity was up-regulated in cells exposed to EMD after normalized to total protein levels (Fig. 4).

Densitometry of Northern blot with 32P-labeled collagen a 1 (I) probes

revealed that the levels of collagen a1 (I) mRNA expression were increased after

3-week culture in differentiating media treated wells (Fig. 5). SSP and OC were

also enhanced by EMD treatment (Fig. 6, 7). Increased expression of growth factor IGF-I was also observed after 3 weeks in differentiation medium in EMD treated group (Fig. 8).

Both EMD and TGF-p1 upregulate large number of common genes in the cell cycle array

The DNA Array results showed that thirty two genes in the cell cycle are upregulated by both EMD and TGF-~1 (see table 1). There are also fifty six genes which are not affected by either EMD or TGF-~1. There are only two genes which are upregulated by EMD alone and not TGF-~1. Moreover two genes are down regulated only by EMD and not by TGF-~1. Fig. 9 shows the

DNA micro array images. Table 2 shows the complete list of cell cycle genes from the DNA array. DISCUSSION

I. Effect of EMD cell adhesion, proliferation, and differentiation of osteoblasts.

A BACKGROUND ON EFFECTS OF EMD ON OSTEOBLASTS

EMD had been used in promoting periodontal tissue regeneration. Its osteogenic activities have been shown clinically in the repair of periodontal infrabony defects in alveolar bone (Cardaropoli et al. 2002; Hammarstrom 1997a;

Heden et al. 1999; Heijl et al. 1997; Rasperini et al. 1999; Sculean et al. 1999a;

Trombelli et al. 2002). This study investigates the effects of EMD in osteoblast adhesion, proliferation, differentiation and gene expression using DNA array technology. To accomplish this purpose, our study was divided into two parts.

First, a thorough understanding of the adhesion, proliferation and differentiation of MC3T3 mouse pre-osteoblastic cells in the presence of EMD was studied.

With a thorough understanding of the effect of EMD on this pre-osteoblastic cell line we can then focus our attention on the specific aim of this study. The main objective of this project was, therefore, to identify cell cycle genes regulated by

EMD and compare these genes to those regulated by TGFJ3-1 using DNA array technology. By using this powerful molecular tool, DNA Array was applied in the identification of up regulated and down regulated genes in EMD treated cells compared with untreated cells.

25 26

Other studies have shown that during tooth development, cells of

Hertwig's epithelial root sheath secrete enamel matrix proteins (Lindskog S 1982;

Schonfeld et al. 1977; Slavkin et al. 1989b). A purified enamel matrix protein called EMD, is a protein mixture with no detectable growth factors (BIORA AB,

Malmo, Sweden). EMD is 90°A, amelogenin plus other proteins including enamlein, ameloblastin, tuftelin and dentine sialophosphoprotein (Brookes et al.

1995 & Robinson et al. 1998). It is well known that these enamel proteins play an important role in the formation of hydroxyapetite crystals in mature enamel

(Robinson et al. 1998). Recently. EMD was also shown to be associated with cementogenesis, periodontal tissue development and regeneration

(Hammarstrom 1997b; Lindskog S 1982; Slavkin et al. 1989a). Now, purified

EMD from porcine teeth is commercially available and has been used for its regenerative capacity in preclinical studies and in clinical treatment. However, the biological mechanisms of EMD in periodontal regeneration and in alveolar bone are not clearly elucidated.

What is known, however, is the effect of EMD on bone regeneration in vivo. EMD adsorbs to denuded root dentin surfaces and induces the formation of a new layer of acellular cementum and alveolar bone in humans and monkeys

(Gestrelius et al. 1997a; Hammarstrom 1997a; Heijl et al. 1997; Safavi et al.

1999). Formation of new bone, cementum and alveolar bone was also demonstrated histologically in cases with periodontal defects. Clinical studies in the treatment of intrabony periodontal defects with EMD found bone regeneration and a significant gain in clinical attachment (Heden et al. 1999; Heijl et al. 1997; 27

Rasperini et al. 1999; Sculean et al. 1999b). Controlled clinical trials comparing the treatment of infrasonic defects with EMD and guided tissue regeneration fund that there was no difference between the two treatment modalities. Both resulted in significant improvement of probing depth, clinical attachment level and other investigated clinical parameters (Pontoriero et al. 1999; Sculean et al. 1999a) .

In tissue implanted with EMD, there is enhanced new bone formation induced by active demineralized freeze-dried bone allograft which may indicate that EMD is an osteopromotive agent (Boyan et al. 2000).

Previous studies have shown that EMD enhances cell attachment spreading the proliferation of cultured periodontal ligament cells (Gestrelius et al.

1997b; Lyngstadaas et al. 2001; Van der Pauw et al. 2000). It also stimulates

TGF p1, platelet derived growth factor and a variety of other growth factors in

PDL cells (Lyngstadaas et al. 2001; Van der Pauw et al. 2000). Alkaline phosphatase activity has also been enhanced by the presence of EMD.

The role of EMD on cell specific osteoblastic proliferation and differentiation is less well understood. Others have shown that it stimulates proliferation but not differentiation of preosteoblastic 2T9 cells and it inhibits proliferation and stimulates differentiation of osteoblastic like MG63 cells and increases proliferation and differentiation of normal human osteoblasts

(Schwartz et al. 2000). Yet the effect of EMD on the well understood preosteoblastic MC3T3 cell line was part of the focus of this study. 28

EMD PRMOTES ADHESION OF MC3T3-E1 CELLS

Our adhesion data is quite interesting. We have shown that after one hour

of incubation of MC3T3 cells on either fibronectin, BSA or EMD shows that cells

spread less on EMD than on fibronectin. Cells in the BSA group were rounded

and were washed out. However, after 18 hours of incubation cells spread

extensively both on fibronectin and EMD. However the cell morphology looked

quite different. Cells did not attach on the BSA coated plates after 18 hours. A

statistical significant increase in cell number was found after only 1 hour of

incubation between the cell numbers in the EMD group compared with the BSA

group. In the one hour incubation period the cell number in the fibronectin group was greater that in either of the two other groups. After 18 hours there was no

cell adhesion in the BSA group and there was no difference in cell number

between EMD and fibronectin. These results clearly show that adhesion of these

MC3T3 is enhanced in the presence of EMD.

EMD PROMOTES PROLIFERATION OF MC3T3-E1 CELLS

With regards to proliferation of these cells, EMD also showed interesting

results. First, cell morphology in both groups appeared normal. On day one there was no difference between the control group and the EMD group. On day two, EMD treatments resulted in a statistically significant increase in cell number.

This increase was persistent into day seven with a peak at day three. Both groups reached a plateau by day three and then slowly declined. Therefore EMD treatment in MC3T3-E1 cells resulted in increased cell numbers compared to 29 control which suggest cell proliferation. This effect is consistent with results obtained in some studies (He et al. 2004) but inconsistent with other reports using the same culture system (Tokiyasu et al. 2000). This robust proliferation of MC3T3 cells in the presence of EMD compared with control leads one to conclude that part of the molecular action of EMD perhaps results from this significant proliferation of these pre-osteoblastic cell line.

EMD PROMOTES DIFFERENTIATION OF MC3T3-E1 CELLS

With regards to differentiation, EMD also showed a stunning effect on the

MC3T3 cell line. Alkaline phosphatase staining showed much larger and greater

number of positive wells after three weeks in culture. However, total soluble protein levels showed no difference between control and EMD groups as determined by PCA assay. Alkaline phosphatase activity was up regulated in cells exposed to EMD after being normalized to total protein levels.

Densitometry of northern blot with collagen alpha-1 probes revealed that the amount of collagen mRNA expression was increased after three weeks of culture in EMD treated groups. SSP and OC were also enhanced by EMD treatment.

Type I collagen, SSP and OC are important extracellular matrix proteins and contribute to bone matrix formation and mineralization (Stein et al. 1996).

The expression levels of theses genes represents different stages of cell cycle. 30

Levels of collagen alpha 1 m RNA was increased under both the short and long term differentiating conditions. SSP and OC are relatively later markers for

osteoblast differentiation and are not expressed in early culture period (Stein et al. 1996). However the expression levels of both SSP and DC were significantly

increased in cells treated with EMD when sells were cultured under differentiating

conditions. Up-regulation of these genes indicates that EMD enhance

osteoblastic differentiation at both early and late stages.

In summary, this portion of this study shows that, in the presence of EMD, the MC3T3 cell line shows a significant increase in the amount of adhesion,

proliferation and differentiation. 31

II. Both EMD and TGF-131 upregulate large number of common genes in the cell cycle

The objective of the second part of the study is to identify cell cycle genes regulated by EMD and compare these genes to those regulated by TGF:-~1. The

results from the DNA Array shows a remarkable similarity between the genes

regulated by EMD compared to TGF-~1. Table 1 summarizes the number of overlapping genes between EMD and TGF-~1. There are 32 cell cycle genes that are unregulated both by TGF-~1 and EMD. EMD unregulated two cell cycle genes which are not affected by TGF-~1 and, TGF-~1 up regulates another two genes which are not influenced by EMD. There are 56 genes in the cell cycle which are not affected either EMD or TGF-~.1. Also it is important to mention that

EMD and TGF- ~.1 down regulate multiple genes but these genes are not common to both EMD and TGF-~.1. These results require further analysis.

What is TGF:- 131?

Transforming growth factor-~1 (TGF-~.1) is a 25 kD homodimetric polypeptide, belonging to a super family of multifunctional cytokines, which participates in a broad array of biologic activities such as normal development and wound repair, as well as pathologic processes (Attisano et al. 1996; Choi et al. 1997). TGF:- ~1 regulates multiple cellular functions including inhibition and stimulation of cell growth, cell death or apoptosis, and cellular differentiation.

TGF:-~1 is also a potent inducer of extra cellular matrix (ECM) protein synthesis 32 and has been implicated as the key mediator of fibro genesis in various tissues

(Border et al. 1994).

TGF-~.1, a basic protein with high hydrophobicity, regulates proliferation and differentiation of fibroblasts, keratinocytes and osteoblasts, extracellular matrix metabolism in the bone or the connective tissue, and immunological system. TGF:-~1 molecules bind to latency associated peptide (LAP), and latent

TGF-~.1 binding protein (LTBP) through non covalent and covalent bonds, respectively. This binding enables in vivo unstable TGF:-~1 molecule to be stored in the extracellular matrix thereby regulating its cellular functions. TGF-~.1 molecules are released in an active form from the extracellular matrix by disulfide bond cleavage.

TGF-~.1 exerts its multiple biologic actions by the interaction with two transmembrane serine/threonine kinase receptors, types I and II, which are co expressed by most cells. Initiation of signaling requires the binding of TGF- ~.1 to

TBR-II, a constitutively active serine! threonine kinase, resulting in the recruitment and phosphorylation of TBR-I to produce a heterometric signaling complex that in turn activates downstream signaling pathways (Vivien et al. 1995;

Wrana et al. 1994). 33

Mechanism of TGF- 131 signaling

To understand the mechanisms involved in TGF- ~1 signaling from the cell membrane to the nucleus, it is crucial to unravel the intracellular events that provide a link between the activated cell surface TGF- ~.1 receptors and the downstream effects of TGF:-r31, such as cell growth inhibition and matrix induction. With the identification of the Smad family of signal-transducing proteins involved in mediating TGF-r3.1 signals downstream of the transmembrane serine/threonine kinase receptors, the focus of many investigators has centered in studies of the Smad proteins. In particular, it is now known that Smad2 and Smad3 both act as signaling proteins and transcription factors and are serine phosphorylated in a TGF-~.1 dependent fashion and associated with Smad4 to translocate to the nucleus where they bind to target sites on specific gene promoters (Massague 1996).

Other researchers have demonstrated the critical involvement of the mitogen-activated protein kinase (MAPK) pathways in TGF:-~1 signaling (Atfi et al. 1997; Chin et al. 1999; Hartsough et al. 1996). MAPK is a major signaling system used by eukaryotic cells to transducer extracellular signals to intracellular responses (Seger et al. 1995; Vojtek et al. 1995). The signal transduction cascades involved in the activation of MAPKs require a well-coordinated cascade of tree protein kinase reactions that transducer signals by sequential phosphorylation and activation of the next kinase in their respective pathways.

The MAPKs require dual phosphorylation at the threonine and tyrosine sites by

MAPK kinases, which are in turn activated by MAPK kinase kinases through 34 phosphorylation (Vojtek & Cooper 1995). The MAPK cascades are considered to play essential roles in the signal transduction of many biological events, such as the regulation of the cell growth, differentiation, and apoptosis and cellular

responses to environmental stresses. TGF- J3.1 has been demonstrated in various cell types to be capable of activating each of the three major MAPK

members.

Potential functions of amelogenins which are the main constituent of EMD

To better understand the exact mechanism of action of EMD and compared it to TGF-J31, it may be worthwhile to investigate the postulated functions of amelogenins since EMD is essentially a mixture of amelogenins

(Gestrelius et al. 1997a; Hammarstrom 1997b). In the studies by Maycock et al

(2002) whereby EMD was compared to porcine enamel matrix derivative, it was shown that EMD was heterogeneous but all the amelogenin components such as degradation products and specific splice products were concentrated in EMD.

In addition to the direct role that amelogenins are postulated to have in the organization and mineralization of the enamel matrix (Moradian-Oldak et al.

1995), they have been proposed to have signaling effects. As Lyngstadaas et al.

(2001) have suggested for DL cells , EMD interactions with mesenchymal cells could initiate signaling pathways leading to secretion of transforming growth factor-B1, interleukin-6 or platelet-derived growth factor or similar growth factors which then permit tissue regeneration, depending on the cells involved. 35

Moreover it has been suggested t different peptide fractions which are produced as a result of gene splicing in amelogenins may be essential in induction of tissue generation (Gibson 1991; Viswanathan et al. 2002). In a study by Veis et al.2000 it has been shown that two peptide fractions GST [A-4] and [A+4] which were produced in the process of amelogenin splicing had activities comparable to recombinant human BMP-2. These peptides incorporated statistically significant levels of sulfate compared to controls. Same group have also shown that [A+4] and [A-4] induce the appearance of type II collagen message. In vivo implants into muscle, using a polylactide-polyglycolide carrier confirmed that [A+4] and [A-4] were active in inducing cellular in growth into the implants, followed by extracellular matrix production, vascularization and mineralization after 4 weeks (Veis et al. 2000).

Therefore it has been suggested that smaller proteins produced by minor

(Fincham et al. 2000) spliced forms may have very specific functions. These functions may relate to epithelial-mesenchymal signaling that is a prominent part of tooth morphogenesis. It has been suggested that [A+4] may promote odontoblast development and [A-4] may inhibit ameloblast maturation until a layer of mineralized dentin is formed (Veis et al. 2000) .

Recently the preliminary studies on the use of [A+4] and [A-4] on tooth repair via dentin bridge formation or pulp capping and differentiation effects on fibroblasts and stem cells , presents a possibility for cartilage and bone tissue regeneration or repair based on initiating factors other than the BMPs. 36

Our data agrees with other investigation whereby it appears that EMD have, besides their activity in regulating hydroxyapatite crystal growth and enamel formation, other functions as growth factors such as TGF- ~1. EMD may act as a signaling molecule such as TGF:- ~1 in exerting its functions or it may simply be involved in induction of growth factors such as TGF:- ~1. It is still not conclusive that EMD can eliminate the damage of periodontal or bone disease by the regeneration of new tissue. Furthermore it is unclear whether they could be some other growth factor in the EMD preparation in addition to enamel proteins.

It remains to be determined if any of the other 'enamel' or 'dentin' proteins also have functions in enamel or dentin mineralization. What are the signaling mechanisms? Are they receptors? It is still so much to understand and all the studies thus far are just the beginning of a new era of discovery. CONCLUSION

1. EMD promotes cell adhesion of MC3T3-E1 cells at levels comparable to fibronectin.

2. Compared to serum free medium, EMD enhances cell proliferation of

MC3T3-E1 cells.

3. EMD increases expression of osteoblastic makers including CoI1-a1, ALP, BSP and OC. 4. EMD and TGF-(31 have similar effects on cell cycle gene expression.

37 38

Table 1* Number of genes in response to EMD or TGF-~1.

EMD only EMD and TGF-~1

Unregulated 2 3 32 Down regulated 2 1 a No Effect 4 4 56

* Please see Table 2 for a complete list of the genes which are summarized above= 39

Table 2: Full DNA array results

Gene expression increased by both EMD and TGF-131:

Abelson murine leukemia oncogene ataxia telangiectasia gene mutated in human beings Bcl2-associated X protein cyclin A2 cyclin B Mus musculus cyclin 82 (Ccnb2) Cyclin C Cyclin D1(Ccnd1) Cyclin E Cyclin E2 cyclin G Mus musculus cyclin H (Ccnh) mRNA CDC16 (cell division cycle 16, S. cerevisiae, homolog) Mus musculus mmCdc20 mRNA Mus musculus cell division cycle 6 homolog (S. cerevisiae) (Cdc6) Mus musculus M015-associated kinase (M015) mRNA cyclin-dependent kinase inhibitor p21Waf1 Mus musculus checkpoint kinase Chk1 (Chk1) mRNA Mus musculus cullin 3 (CuI3) mRNA Cullin 4A, RIKEN cDNA 2810470J21 gene Similar to human E2F2, EST clone Mus musculus transcriptional activator (E2F3) mRNA E2F transcription factor 4, p107/p130-binding E2F transcription factor 5 Mus musculus mitotic checkpoint component Mad2 mRNA M. musculus mRNA for P1 protein (P1.m) Mus musculus mini chromosome maintenance deficient 2 Mouse mRNA for mcdc21 protein meiotic recombination 11 homolog A (S. cerevisiae) Mus musculus nibrin (Nbn) Mus musculus Rad51 homolog (S. cerevisiae) (Rad51) Mus musculus RAD9 homolog (S. pombe) (Rad9)

Gene expression increased by EMD (2):

Mus musculus Cdc25a (cdc25a) mRNA cyclin-dependent kinase 6 40

Gene expression decreased by EMD (2):

Transformed mouse 3T3 cell double minute 2 Mus musculus DNA repair protein RAD50 (RAD50) mRNA

Gene expression increased by TGF-J31 (3):

apoptotic protease activating factor 1 Cyclin-dependent kinase inhibitor p57Kip2 Mouse CDK4 and CDK6 inhibitor p16ink4a

Gene expression decreased by TGF 131 (1):

Retinoblastoma protein (Rb)

Gene expression no changes (56):

B-cell leukemia/lymphoma 2 breast cancer 1 Mus musculus cyclin A1 (Ccna1) Cyclin D2 (Ccnd2) Cyclin D3 (Ccnd3) M.musculus mRNA for cyclin F Mus musculus cyclin G2 mRNA Mus musculus cell division cycle 25 homolog 8 (S. cerevisiae) Cyclin-dependent kinase 1(Cdc2) Mus musculus cdc37 homolog mRNA Mus musculus testes CDC45L (Cdc451) mRNA Mus musculus muCdc7 mRNA, complete cds cyclin dependent kinase 2L cyclin-dependent kinase 4 Mus musculus cyclin-dependent kinase 5 Homolog of Human Cyclin-dependent kinase 8, image clone Mouse cyclin-dependent kinase inhibitor p27Kip1 cyclin-dependent kinase inhibitor p151NK4b Cdk4 and Cdk6 inhibitor p18 cyclin-dependent kinase inhibitor p19 Mus musculus CDC28 protein kinase 1 (Cks1) Mus musculus SCF complex protein cul-1 mRNA Cullin 2,Mus musculus adult male liver cDNA, RIKEN full-length enriched library, clone: 1300003018, f Cullin 48, RIKEN cDNA 2700050M05 gene E2F-related transcription factor (DP-1) 41 protein regulating cell cycle transcription factor DRTF1/E2F E2F1 Mus musculus E2F-like transcriptional repressor protein mRNA, complete cds Mus musculus forkhead box M1 (Foxm1) DNA-damage inducible transcript 1 Mus musculus Hus1 homolog (S. pombe) (Hus1) Mouse mRNA for mCDC46 protein mini chromosome maintenance deficient 6 (S. cerevisiae) Mus musculus mRNA for mCDC47 antigen identified by monoclonal antibody Ki 67 neural precursor cell expressed, developmentally down-regulated gene 8 Mus musculus I-kappa B alpha chain Proliferating cell nuclear antigen protein regulator of cytokinesis 1 Mus musculus RAD17 homolog (S. pombe) (Rad17) Mus musculus protein kinase Chk2 (Rad53-pending) Retinoblastoma-like protein 1 (p107) Retinoblastoma-like protein 2 (p130) Mus musculus ring-box protein 1 (Rbx1) mRNA GTP-binding protein ROC2 ESTs, Highly similar to REPLICATION PROTEIN A 14 KD SUBUNIT [Homo sapiens] SCF complex protein Skp2 (Skp2) SCF complex protein Skp1 (Skp1) Tissue inhibitor of metalloproteinase 3 Transformation related protein 53, Tumor antigene Transformation related protein 63 Ubiquitin C Mus musculus ubiquitin-activating enzyme E1, Chr X (Ube1x) Mus musculus E6-AP ubiquitin protein ligase mRNA Mus musculus ubiquitin-homology domain protein (UbI1) mRNA Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon polypeptide 42

Fibronectin EMD BSA

1 hr incubation before washing

1 hr incubation after washing 3 times with DMEM.

18 hrs incubation and washing 3 times with DMEM

Fig 1. Adhesion and spreading of MC3T3-E1 cells on fibronectin (A),

EMD (B), and BSA (C). 43

BSA .EMD D Fibronectin

Fig 2. EMD promotes cell attachment of MC3T3-E1 cells. 44

750000 ---Control ... - ... - EMD G) * * .Q 500000 * E ::::J C -G) 0 250000

O-+------,..------r--...... ---,...------. 1 2 3 5 7 Time (day)

Fig 3. EMD promotes proliferation of MC3T3-E1 cells. 45

Control EMD

Fig. 4. Increase in alkaline phosphatase (ALP) activity in cultured MC3T3 E1 cells by EMD. 46

~:w= '

~ , if ,'" " '

'N.". ~~,;:&, _,_

c 50 ;i (.) 40 ...... ca '-e- ca 30 ~ 20 -0 0 10 0

Con EMD

Fig. 5. Northern blot analysis of EMD on collagen a1 (I) messenger RNA expression. Results are shown by autoradiography (A) and quantitatively analyzed by ImageQuant (B). 47

'C~ wiffi ' 'g.

(o~ "" ... :,: oX' ~ Yo ... "'" ~ "'-

• x '" '\' - . ,,- , ... ~~ »;;::0;:- ...... ~ ,,'" ~ :-z: :jh

c ; 5 u -!! Q. U) m 2

Q Control EMD

Fig. 6. Northern blot analysis of EMD on bone sialoprotein (BSP) messenger RNA expression. Results are shown by autoradiography (A) and quantitatively analyzed by ImageQuant (B). 48

'"' ~ ;.: :=::::;'"'"'" v '" ... ~ '... ;.: ...;}-::~::; "' ... \: ....

~ , ~" 1

",&. 01h~'l'W:'"""" v'1m" ,

c ~ (,) CV (3 o

Control EMD

Fig. 7. Northern blot analysis of EMD on osteocalcin (OC) messenger RNA expression. Results are shown by autoradiography (A) and quantitatively analyzed by ImageQuant (B). 49

:i- ~ ...,:-; "'..m: ~~ ,

z ~% :-:: .... 7'( ~

> ,

, 1': <

% '

~ mid, "'~~","~ ,

Control EMD

Fig. 8. Northern blot analysis of EMD on insulin-like growth factor I (IGF-I) messenger RNA expression. Results are shown by autoradiography (A) and quantitatively analyzed by ImageQuant (B). . 50

Fig. 9. DNA micro array images in control, TGF-p1, and EMD treated groups. REFERENCES

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