University of Connecticut OpenCommons@UConn
SoDM Masters Theses School of Dental Medicine
June 2005 Enamel Matrix Derivative : Mechanisms of Effect on Osteoblasts Amy Z. Lee
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Recommended Citation Lee, Amy Z., "Enamel Matrix Derivative : Mechanisms of Effect on Osteoblasts" (2005). SoDM Masters Theses. 76. https://opencommons.uconn.edu/sodm_masters/76 ENAMEL MATRIX DERIVATIVE" MECHANISMS OF EFFECT ON OSTEOBLASTS
Amy Z. Lee, D.M.D., Ph.D.
D.M.D., University of Connecticut, 2002
Ph.D., Albert Einstein College of Medicine, 1999
A Thesis
Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Dental Science At the
University of Connecticut 2005 APPROVAL PAGE
Master of Dental Science Thesis
ENAMEL MATRIX DERIVATIVE: MECHANISMS OF EFFECT ON OSTEOBLASTS
Presented by
Amy Z. Lee, D.M.D., Ph.D.
Major Advisor Qiang Zhu, D.D.S., Ph.D.
Associate Advisor Larz S t'w. Spftngberg, D:D.S., Ph.D.
Associate Advisor Kamran E.Sa M.Ed.
Associate Advisor yu_Wang, D.I.S., Ph.D.
University of Connecticut
2005
ii Acknowledgements
would like to acknowledge all of the faculty members in the Division of
Endodontology. Their dedication to education has created such a great environment for me to learn endodontology.
It has been a privilege and honor to study Endodontology from Dr. Larz Spangberg. Dr.Spangberg impressed me not only with his renowned comprehension and profound knowledge in all aspects of Endodontology, but also his continued evaluation of those newly developed techniques and concepts.
am also fortunate to be a student of Dr.Kamran Safavi. Dr.Safavi's affection in teaching and his sophisticated knowledge and experience in education have created a wonderful atmosphere to motivate me for study. His intelligence and efforts have made my experience, here at UCONN, joyful, fruitful, and unforgefful.
am especially gratefully to Dr.Qiang Zhu for his guidance and help on my research project. Dr.Zhu has encouraged me to study Endodontology since dental school. believe all of the dental students have benefited greatly from his kindness and encouragement.
would like to take this opportunity to thank Dr.Jin Jiang. She is the person to first introduce me the Endodontology specialty. She had been encouraging me to pursue the career in Endodontology while was applying the program.
iii would like to thank Dr.Yu-Hsiung Wang for his assistance with my thesis. Finally, would to thank for the help of my husband, my parents, and my three daughters. Without their spiritual support, it would have been impossible for me to accomplish my study. TABLE OF CONTENTS
Page TITLE PAGE APPROVAL PAGE ii ACKNOWLEGMENT iii TABLE OF CONTENTS v LIST OF FIGURES vi INTRODUCTION 1 AIM OF STUDY 26 MATERILA AND METHODS 27 RESULTS 31 DISCUSSION 33 CONCLUSION 40 FIGURES 41 REFERENCES 51 LIST OF FIGURES
Figure 1. Millipore culture plate insert placed into a culture well of the 6-well cell culture plate 41
Figure 2. EMD forms precipitated aggregates on the membrane of the culture plate inserts 42
Figure 3. Cell morphology of MC3T3-E1 cells after 3-day incubation 43
Figure 4. The effect of EMD on the proliferation of MC3T3-E1 44
Figure 5. The effect of EMD on secretion of TGFI-I from human osteoblasts 45
Figure 6. The effect of EMD on secretion of TGFI-I from mouse osteoblasts 46
Figure 7. The effect of EMD on secretion of IL-6 from human osteoblasts 47
Figure 8. The effect of EMD on secretion of IL-6 from mouse osteoblasts 48
Figure 9. The effect of EMD on secretion of osteoprotegerin from mouse osteoblasts 49
Figure 10. Model of the effect of enamel matrix derivative on osteoblasts and osteoclasts 50 Introduction
Enamel matrix protein (EMP) is a group of proteins synthesized by ameloblast cells during tooth formation. They are secreted into the enamel extracellular matrix to nucleate and regulate the growth of hydroxyl crystals which form the enamel of teeth (Fincham et al. 1992). A recent study shows EMP is also produced by the epithelial root sheathes of Hertwig (HERS) and is involved in the periodontium formation during root development (Hammarstrom 1997; Schonfeld & Slavkin 1977). Furthermore, new studies have suggested that EMP has osteogenic and chondrogenci properties. Lastly, they have been used to regenerate periodontal tissues in clinical application.
Enamel Matrix Proteins for Crown Formation
During the development of the tooth crown, the deposition of enamel crystallites is highly regulated. The ameloblast in the inner enamel epithelium fabricates and secretes EMP into the extracellular space. As the ameloblast migrates away from the dentine-enamel junction, it leaves behind a tail of secreted mixture of proteins, EMP. This EMP itself assembles to form an enamel extracellular organic matrix to control the initiation, the rate of growth, and the habit of the inorganic crystallites. During the maturation stage, crystallites grow rapidly with the concomitant degradation and eventual removal of enamel matrix components, resulting in stiff and brittle enamel (Eastoe 1979). The major protein of enamel matrix is amelogenin. It constitutes approximately 90% of the matrix. The remaining 10% includes tuftelin, ameloblastin, enamelin and enamel proteases.
Amelogenin is a hydrophobic protein with hydrophilic amino and carboxyl terminals. The amino acid sequences of theN and C terminals of the protein are highly conserved across species, implying a great functional importance (Toyasawa et al. 1998). A study with recombinant amelogenin has shown that the C-terminal facilitates initial orientation of crystallites (Aoba 1989). In vitro experiments with purified (Doi et al. 1984) and recombinant (Wen et al. 2000) proteins have demonstrated that amelogenin directs the growth of crystals.
Amelogenin gene was mapped to human sex chromosomes X and Y (Lau et al. 1989). Individuals with Amelogenesis Imperfecta phenotype have exhibited the mutations in amelogenin gene which reduces the protein expression (Lagerstrom et al. 1991; Aldred & Crawford 1997). A single amino acid change in highly conserved N-terminal of the protein was identified in two unrelated human pedigrees with Amelogenesis Imperfecta (Lench & Winter 1995; Collier et al. 1997). An animal model with amelogenin knock out, in which no amelogenin is expressed, resulted in teeth with enamel not having normal structural organization (Gibson et al. 2001). Ameloblastin represents 5% of non amelogenin mRNAs (Cerny et al.,
1996). Ameloblastin gene is localized to human chromosome 4q21, the same region where Amelogenesis Imperfecta has been linked (Foreman et al., 1994).
Immunostaining of the protein indicates that ameloblastin is present at secretory stage of enamel formation and localized in the Tomes' processes of secretory ameloblasts, suggesting a role in enamel biomineralization of the protein.
Ameloblastin is also detected in HERS, suggesting a role in acellular cementum formation and development of periodontal attachment apparatus (Fong, Cerny et al, 1998; Fong and Hammarstrom 2000).
Enamelin is the largest enamel protein. Immunohistochemistry studies show thaht the protein concentrates at the growing tip of enamel crystal. Its precursor, an 186KD glycoprotein, is localized at Tome's process of ameloblast.
The Enamelin gene localized on human chromosome 4q21 is linked to
Amelogenesis Imperfecta (Dong et al. 2000). These evidences suggest
Enamelin has a role in enamel biomineralization.
Tuftelin is an acidic enamel protein. The expression of tuftelin occurs at a very early stage of tooth development, prior to amelogenin expression, indicating this protein might serve as a nucleator for hydroxyl apatite crystal formation (Zeichner-David et al. 1997). However, more recent findings show that Tuftelin is expressed in a wide range of tissues, such as kidney, lung, liver, and testis suggesting that the mineral nucleator is not the primary function of the protein (MacDougall et al. 1998). Enamel proteases are required to degrade and remove enamel matrix proteins during the maturation stage to allow for the rapid growth of crystallite. A recombinant enamel protease, enamelysin, was demonstrated to process the recombinant amelogenin in vitro (Ryu et al. 1999). Enamel Matrix Proteins for Root Formation
The root surface is covered by a layer of calcified structure called cementum. Cementum is classified into three types based on the existing cells and origin of collagenous fiber. Acellular extrinsic fiber cementum contains
Sharpey's fiber embeded in a non cellular ground substance. It covers the cervical 1/3 to 2/3 of the roots in human teeth. Acellular afibrillar cementum has neither cells nor fibers and is found in the coronal cementum. Cellular mixed stratified cementum is found mostly at the apical third of the root. it is composed of extrinsic and intrinsic fibers and cells (Jones 1981). Following crown formation, the inner and outer enamel epithelia fuse below the crown enamel to form a bilayered epithelial sheath, named Hertwig's Epithelial Root Sheath (HERS). As HERS cells migrate apically with proliferation, dental mesenchymal tissues are divided into dental papilla and dental follicle. Mesenchyme cells in dental follicle will be induced to undergo cementogenesis and periodontium generation (Johns 1981).
Studies indicate that enamel matrix protein is involved in the formation of acellular cementum. The idea was first proposed by Slavkin and Boyd (1974).
The inner layer of the root sheath represents the extension of the ameloblast layer in the crown. Early in 1978, Owens found that HERS cells of developing rat molars had organelles, suggesting a secretory activity of these cells (Owen 1978). Later on, scanning electron microscopy and autoradiographic studies found the inner layer of the epithelial root sheath had a secretory stage and a thin layer of extracellular matrix, similar to enamel matrix formed on root surfaces prior to cementogenesis (Lindskog 1982; Lindsdog & Hammarstrom 1982). An immulogical study found that acellular cementum contains proteins that cross- react with the antibodies against amelogenin and enamelin, indicating the presence of these proteins during root formation (Slavkin et al. 1989) In 1996, Fong et al. proved with in situ hybridization that ameloblastin is expressed by HERS cells in rat molars during root formation. Another study with rat molars showed amelogenin formation can also be produced by HERS cells (Hamamoto et al. 1996). Studies by Hammarstrom provide further evidences of the involvement of enamel matrix protein on cementum formation (Hammarstrom 1997). An immunnohistochemistry study was performed with anti-amelogenin antibody applied on the human premolars with less than 2/3 root formation and on developing rat molars. The staining of amelogenin was found at the peripheral surface of the advancing root and on part of the non-mineralized mantle dentin, the area where cementogenesis is initiated. Another experiment was to expose the enamel matrix to the mesenchymal dental follicle cells. The molars of five day old rats were taken out from the mandible and the enamel organs removed to expose the enamel matrix before they were placed back into their position in the jaw. Ten days later, rats were killed and histological exams on the root were performed. The results showed that an acellular collagenous layer was formed on the surface of the exposed enamel matrix. In another study, incisors from monkeys were extracted and the cementum and adjacent layer of the surface dentin were removed. Teeth were reimplanted with or without the application of enamel matrix extract from porcine. Histological staining showed, after eight weeks, root surfaces with treatment of enamel matrix protein healed with a thick layer of acellular cementum. This layer was well attached to the dentin with collagenous fibers extending out from it. The new alveolar bone was found to be formed around the roots. In contrary, the roots without enamel matrix showed a layer of cellular hard tissue poorly attached to the dentin. These results indicated that enamel matrix proteins are not only involved in the formation of acellular cementum during the root development, but also have the potential to induce the regeneration of periodontium and alveolar bone (Hammarstrom 1997). The ability of osteogenic induction by enamel matrix was documented as early as 1971 (Urist 1971). In an animal model, enamel matrix was transplanted into muscle. After several weeks enamel matrix was enveloped by a thin layer of densely calcified irregular eosinophilic matrix containing random scattered osteocytes (Urist 1971). The results indicate that mesenchymal cells from the surrounding tissues were attracted from surrounding tissue and induced for osteogenesis. In recent years, a series of in vitro studies have shown that enamel matrix protein can induce osteoblastogenesis.
Enamel Matrix Derivatives (EMD) and Emdogain
Since the establishment of the regenerative capacity on bone and periodontium, enamel matrix protein has been extracted and purified from the tooth germs of fetal porcine as Enamel Matrix Derivative (EMD). EMD has been used in clinical application and in vitro study.
A commercial enamel matrix derivative, Emdogain, (Biora AB, Malmo,
Sweden) received FDA approval and is available for clinical application. It works as a tissue healing modulator that mimics the events that occur during root development to stimulate bone and periodontium regeneration (Hammarstrom 1997; Heijil et al. 1997). Emdogain is a purified acidic extract of the developing embryonal enamel derived from six month-old piglets. The major component, ameiogenin, is a hydrophobic protein. It can be dissolved in an acidic or alkaline PH environment and at low temperature. At neutral PH and body temperature it is insoluble and aggregates to spherical complexes.
Propylene glycol alginate (PGA) has been used as a vehicle for EMD in
Emdogain product. PGA is commonly used in food and pharmaceuticals as a thickening agent. EMD can be dissolved in PGA at room temperature, resulting in a highly viscous solution. Under physiological conditions the viscosity decreases and EMD is released to precipitate on the exposed root surface to interact with PDL cells. Studies with rats and pigs show that EMD from Emdogain gel can absorb to hydroxyapatite, collagen and roots, for at least two weeks (Gestrlius et al. 1997). A large multicenter randomized control trial showed no differences between EMD and Emdogain gel in treatment of periodontitis (Bratthall et al. 2001). The EMD in Emdogain is heat treated to prevent bacterial and viral infection. An in vitro study on human periodontal ligament (HPDL) cells shows
Emdogain to have greater bioactivities than EMD in aspects of stimulating the biomineralization and osteoblast differentiation (Nagano et al. 2004).
EMD in Treatment of Periodontal Defect
Animal Studies
In 1997 two studies on monkey model showed positive effects of EMD on regeneration of cementum, periodontal ligament and alveolar bone (Hammarstrom 1997; Hammarstrom et al. 1997). In the first study, the incisors from monkey were extracted. The experimental cavities on the root surfaces were made by removing cementum and a layer of dentin. The test cavities were treated with EMD and the teeth re-implanted. The histological studies found, after eight weeks, a layer of acellular extrinsic fiber cementum formed on the root surfaces (Hammarstrom 1997). In another study, EMD was applies to the bucal dehiscence defects on the maxilla. After eight weeks, the histological studies demonstrated about 60-80% cementum and bone regeneration. In contrast, almost no new bone and cementum were formed on the control sites where EMD was not applied (Hammarstrom et al. 1997). Recently a histological study was done using a dog model (Sakallioglu et al. 2004). Experimental peridontitis was induced on the premolars of dog. EMD was applied surgically on the defects in the test group. The results showed a significant higher amount of connective tissue and new bone formation in the EMD group than in the control group. Also the rate of bone maturation (number of osteons) was higher in the test group. A firmly attached acellular cementum with collagen fiber insertion was only found in the EMD group. In control the group, a cellular cementum was formed. The amount of cementum formed was significantly higher in the EMD group (Sakallioglu et al. 2004). These in vivo animal studies revealed that EMD has the capacity to stimulate connective tissue proliferation, acellular cementum regeneration and alveolar bone formation.
Several animal studies focused on the characteristics of EMD on bone simulation. Demineralized freeze dried bone allograft (DFDBA) was known to have limited osteoinduction ability. When EMD is implanted together with
EFDBA into the muscle of nude mouse, an enhanced bone induction was observed compared to DFDBA alone (Boyan 2000). In another study bony defects were created by drilling on a rat femur and EMD (test group) or PGA carrier (control group) put into the hole. The volume of newly formed bone trabeculae on day 7 was significantly higher in the EMD group than in the control group (Kawana et al. 2001). It is concluded that EMD is an osteogenic agent.
Clinical Studies Since 1997 multiple randomized controlled clinical trials, cohort studies, case control studies, and case reports have been published for the effectiveness of EMD on improving periodontal health. The comparison were usually based on the assessment of clinical attachment level (CAL) gain, probing pocket depth (PPD) reduction, and restoration of bone radiographically. A multicenter randomized split mouth trial was undertaken to compare
EMD treatment as an adjunct to modified Widman flap (MWF) surgery and MWF plus placebo (PGA). Thirty four paired treatments were included in the study.
The patients were followed for 36 months. EMD group showed better results on the PPD reduction, CAL gain, and bone level improvement (Heijl et al. 1997).
Another prospective, randomized, controlled clinical trial with split mouth design was done by Wachtel et al. (2003). Open flap debriment (OFD) with EMD application or OFD alone was performed on 26 pairs of periodontal defects. At 6 and 12 month evaluations, the CAL gain and PPD reduction were statistically higher in OFD and EMD combination group. Other studies compared EMD with placebo or OFD/MWF alone obtained similar results in terms of clinical and radiographic findings (Pontoriero et al. 1999; Okuda et al. 2000; Silvestri et al. 2000; Sculean et al. 2001; Tonetti et al. 2002). A series of case reports also showed significant improvement in clinical and radiographic parameters following the use of EMD in the treatment of intrabony defects (Heden et al. 1999; Sculean et al. 1999; Heden 2000;
Paradshis & Tsiklakis 2000; Cardaropoli & Leonhardt 2002; Trombelli et al. 2002; Donos et al. 2004).
10 The long term efficacy of EMD was reported in two studies. In Raspernis' report (2005), 3 cases with infrabony defects were treated with EMD in 1999. Significant PAL gain, PPD reduction, and bone fill were evident after the treatment. Surgical re-entry after 7 years in two cases and 5 years in one case all demonstrated the stability of previous findings (Rasperini et al. 2005). In another clinical report, 46 intrabony defects were treated with EMD. PPD and CAL were evaluated clinically before treatment and one year and four years after treatment. There were no statistically significant differences detected at one year and four years after treatment. The conclusion was the clinical improvements obtained following EMD treatment can be maintained over a 4 year period (Sculean et al. 2003). A meta-analysis study on the improvement of PPD and CAL upon EMD application included 28 clinical studies or case reports with 955 intrabony defects through May 2003. When EMD treatments were compared with OFD alone, the EMD group gained significantly more PPD reduction (4.82+0.002mm vs 2.59+0.06mm P=.000) and CAL gain (4.07+.03mm vs 2.55+.04mm P=.000) (Venezia 2004).
Histological Assessment of Effect of EMD on Periodontal Regeneration
The first histological study used Human mandibular incisor scheduled for extraction due to orthodontic reasons. A buccal dehiscence was made and EMD was applied. Teeth with surrounding tissues were removed 4 months later.
]] Histological examination demonstrated regeneration of alveolar bone and new cementum with inserting and functionally oriented collagen fibers at the defect
(Heijl et al. 1997). Other histological studies all revealed similar results (Mellonig 1999; Sculean et al. 2000, 2003; Windisch et al. 2002).
EMD vs Guided Tissue Regeneration (GTR)
GTR is a well-established therapeutic method for clinical periodontal regeneration. However, this is a very technique sensitive procedure. The clinical outcome is highly variable depending on the clinician's experience and surgical skill, tooth morphology and defect morphology (Lu 1992; Tonetti et al 1993; Cortellin et al. 1995).
Researchers have compared EMD with GTR in their studies. Although both regimens revealed significant statistical improvement than their controls within groups, no statistical significant difference was detected in terms of PPD reduction and CAL gain between EMD and GTR groups (Sculean et al. 1999,
2001; Pontoriero et ai. 1999; Minabe et al. 2002; Windisch et al. 2002; Parashis et al. 2004; Donos et al. 2004; Meyle 2004). In Silvestri's study (2000), when intrabony defects with CAL > 9mm, GTR provided better results. However, EMD showed better results for defects with CAL<9mm.
In a randomized clinical trial, 45 patients with 90 comparable buccal class
II furcation defects in mandibular molar were treated with EMD or GTR. Patients were followed for 14 months. The results showed a significantly greater
]2 reduction in horizontal furcation depth in EMD group. The incidence of post- operative pain/swelling is also lower in this group (Jesen et al. 2004). In Venezia's meta-analysis study, the mean PPD reduction is higher in GTR group. Whereas the CAL gain is higher in the EMD group (Venezia 2004).
Combination of EMD with Bone Grafts
Space maintenance under mucoperiosteal flaps is very important for good outcome of any regenerative procedure, particularly when bone formation is one of the treatment objectives (Wikesjo & Selvig 1999). Emdogain is a semi-fluid product and lacks space maintenance ability. Demineralized freeze-dried bone allograft (DFDBA) and alloplast material (Bioactive Glass, BG) have been used with EMD to try to overcome this problem.
In Venezia's meta-analysis study, EMD is superior to EMD combined with bone graft based on PPD and CAL assesments (Venezia 2004). Some studies showed no statistical differences between these two groups (Gurinsky et al. 2004; Sculean et al. 2005).
A recent histological study revealed the superiority of EMD with BG over BG alone (Sculean 2005). Six patients' teeth with intrabony defects were included in this study. Six months later, teeth were extracted with surrounding tissues for histological study. All three teeth treated with EMD plus BG showed new PDL, new cementum with inserting collagen fibers and signs of ongoing mineralization. All BG treated teeth revealed downgrowth of epithelia. Only one
]3 of the BG treated teeth showed cementum formation on the most apical part of the defect (Sculean 2005).
EMD in Endodontic Application
Direct Pulp Capping
During odontogenesis, amelogenin is translocated from ameloblasts to differentiating odontoblasts, suggesting its role in odontoblast induction (Inai et al. 1991). Several studies have proved the capacity of EMD on the induction of reparative dentin formation.
In a dog model study, all 32 teeth showed reparative dentin in histological examination 4 weeks after EMD treatment (Ishizaki et al. 2003).
When EMD was compared with its carrier PGA, EMD promoted earlier and higher amounts of reparative dentin and dentin bridge formation in rat molars (Igarashi et al. 2003). When EMD and Calcium hydroxide were compared, EMD treated teeth induced higher amounts of reparative dentin formation than Calcium hydroxide (Hammarstrom et al. 2001; Nakanura 2004). A recent human study claims opposite result. When EMD was used for pulp capping, patients claimed less post operative symptoms compared to
Calcium hydroxide. However, EMD was not effective for the formation of a hard tissue barrier (Olsson 2005).
14 Replantation and Auto Transplantation
The most serious complication following replantation of avulsed teeth and autotrasplantation is root resorption (Andreasen 1995). Inflammatory root resorption can be prevented by endodontic therapy. Replacement resorption is the result of PDL and cementum damage. Wound healing around roots results from the competition between PDL fibroblasts and osteoblasts in sockets. If PDL cells are damaged, PDL will be replaced by repair from osseous tissue, resulting in ankylosis and replacement resorption. To prevent replacement resorption, the therapeutic goal will be to stimulate periodontal regeneration. The important step of which is the early induction of cementogenesis and assembly of newly formed
PDL fibers into the acellular cementum on root surfaces (Ripamenti 1997).
Since EMD was demonstrated to enhance the regeneration of acellular extrinsic fiber cementum in monkeys after replantation (Hammarstrom 1997),
Emdogain has been used clinically and in animal studies for PDL regeneration after replantation and transplantation.
In an animal study, beagle dog incisors were extracted and re-implanted after 15, 30, or 60 minutes of dry storage. Teeth and surrounding tissues were removed after 12 weeks for histological exam. The restults showed higher incidence of PDL healing in Emdogain group. The control group (without Emdogain application on root sudaces) showed higher incidence of ankylosis (Iqbal & Bamaas 2001). In a subcutaneous transplantation study, rat molars were extracted and air dried 30 minutes before they were transplanted into a subcutaneous position in the abdominal wall. Histological study revealed one of the 14 teeth treated with
EMD had root resorption. However, in the control group, all 12 teeth showed resorption (Hamamoto 2002).
Two case reports also indicated good results using EMD in transplantation and replantation (Ninomiya 2002; Caglar et al. 2005). In Caglar's report an avulsed tooth from a 9 year old patient was dried and stored for 5 hours before replantation. The tooth was followed for 12 months. No sign of ankyiosis and resorption was detected (Caglar et al. 2005).
A recent retrospective clinical study indicated that EMD can enhance the healing of compromised PDL. The PDL was categorized as hopeless when the teeth were stored under non-physiological conditions for extended periods of time before replantation. In this group, EMD treatment did not prevent occurrence of anylosis and replacement resorption (Pohl et al. 2005).
Two other studies indicated that EMD is not effective in preventing resorption. In an animal study, monkey teeth were extracted and air dried for
60min before transplantation. EMD treatment on root surfaces did not appear to significantly reduce the chance of replacement resorption (Karen 2004). A human clinical study also indicated EMD was not able to prevent or cure ankylosis (Schjott & Andreasen 2005). Mechanisms of EMD Actions
Antimicrobial Properties of EMD
Bacterial load is one of the factors adversely affecting the outcome of every regenerative procedure in periodontal treatment. For example, bacteria may heavily colonize barrier membrane in the GTR procedure (Demolon et al. 1993). There is a negative relationship between attachment gain and bacterial colonization of the membrane (Demolon et al. 1993).
In vitro studies showed Emdogain gel has a marked inhibitory effect on the growth of the gram-negative periodontal pathogens, such as A. actinomycetemcomitans, P. gingivalis, and P. intermedia, without significant inhibition in gram positive bacteria (Spahr et al. 2002; Van der Pauw et al. 2001). In addition, two studies demonstrated Emdogain to significantly reduce the vitality of biofilm derived from patients with peridontitis (Sculean 2001; Arweiler et al. 2002).
An in vitro study suggested the anti-P, gingivalis activity of EMD should be attributed to the PGA vehicle (Newman et al. 2003).
Cellular Mechanism of EMD Action
The effects of EMD on PDL cells have been explored by in vitro studies.
In cell culture studies, EMD enhanced proliferation of PDL cells, increased total
]7 protein production, and promoted mineralized nodule formation of PDL cells (Gestrlius et al. 1997). Later on, another study found EMD to not only significantly increased the proliferation and metabolism of PDL cells, but also increased the attachment rate of the cells (Lynstadaas et al. 2001).
The proliferation enhancement on PDL cells was further proved by an in vitro wound-healing model (Hoang et al. 2000). Wounds were created by incisions on a mono layer of cells cultured on plates. When cells were exposed to EMD, wound-fill was significantly enhanced. The rate of wound filling was even higher than the positive control in which the cells were exposed to platelet derived growth factor (PDGF). EMD was also proved to promote the proliferation of PDL fibroblasts by cell culture and wound healing (Rincon et al. 2003). In contrary, EMD appears to inhibit the proliferation and growth of epithelial cells in cell culture studies (Gestrelius et al. 1997, Kawase et al. 2001). This may explain the biological guided tissue regeneration effect of EMD observed in vivo.
The direct effects of EMD on osteoblasts were demonstrated by in vitro studies. EMD was shown to enhance the proliferation, differentiation, and metabolism of both primary and immortalized ostoblasts (Schwarz et al. 2000; Yoneda 2002; Yoneda et al. 2003). In a study of pluripotential mesenchymal cells (C2C12), EMD promoted differentiation of C2C12 into osteoblast lineage, but strongly inhibited the development of cells into myoblast (Ohyama et al. 2002).
18 Results from these studies indicate that EMD could regulate multiple cell types in healing sites.
Molecular Mechanism of EMD Action
All of the cellular activities that EMD regulated, including cell proliferation, differentiation, and attachment, are known to be controlled by cytokines and growth factors. In PDL cells, the mitogenic response to EMD was found to be associated with the activation of an intraceilular signaling molecule, extraceilular signal-regulated kinase (ERK) (Matsuda et al. 2002). This signaling pathway induced by EMD is similar to that activated by epidermal growth factor (EGF).
Although it is controversial, a study demonstrated that EMD product itself does not contain cytokines and growth factors, such as macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), nerve growth factor
(NGF), platelet derived growth factor (PDGF), TGFII, and insulin like growth factors (IDFs) (Gestrelius et al. 1997). However it was revealed that EMD could stimulate the autocrine production of TGF I1, IL-6, and PDGF from PDL cells (Lyngatadass et al. 2001).
A cDNA arrays analysis on PDL cells showed that 38 genes out of 268 genes coding for cytokines, growth factors, and growth factor receptors were differentially expressed when PDL cells were cultured in the presence of EMD.
Among the 38 genes, 12 were found to be down-regulated, mostly being inflammatory genes. Whereas 26 genes demonstrated up-regulation, many of those coding for growth factors and growth factor receptors (Parkar & Tonetti 2004). As for the study on osteoblasts, it is proved that EMD enhances the differentiation and biomineralization of the cells. Bone sialoprotein (BSP)is an early phenotypic marker of osteoblast differentiation. BSP is implicated in the nucleation of hydroxyapatite during bone formation. A northern hybridization experiment showed that the expression of BSP gene was increased 2.8 fold when osteoblasts were treated by EMD (Shimizu et al. 2004).
The mechanisms of BSP gene activation was studied by the method of transient transfection of osteoblast cell line (ROS 17/2.8) with chimeric constructs of a rat BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assay (Shimizu et al. 2004). The results indicated that the increased BSP gene expression is related to the activation of the promoter of the gene, which may be mediated by the increased binding of transcription factors to TGF-I activation element (TAE) in the promoter (Shimizu et al. 2004).
Bone Remodeling
The bone remodeling process starts with bone resorption by osteoclasts and follows with bone reconstruction by osteoblasts. This process is highly regulated by hormone and local regulators.
Osteoclasts
2O Osteoclasts are hematopoitic originated cells. The precursors of osteoblasts reside in blood and bone marrow. The differentiation of precursors into mature cells is regulated by a cytokine, RANK ligand which is produced by osteoblasts. When osteoblasts are activated by systemic hormone (such as 1, 25 dihydroxyvitamin D3) or cytokines (such as IL6), RANK ligand is produced. When RANK receptors on osteoclast precursors bind to RANK ligand, precursor cells are activated to transform into mature osteoclasts. Osteoprotegerin (OPG) is a soluble factor. OPG can bind with RANK ligand to interfere with the interaction between RANK ligand and RANK. Therefore, the osteoclastogenesis is inhibited. Osteoclasts resorb bone by secreting acids and enzymes from its ruffled border onto the bone surface. After the bone resorption process is finished, osteoclasts undergo apoptosis, or programmed cell death. Apoptosis is also mediated by growth factors and cytokines.
Osteoblasts
Osteoblasts originated from precursor stem cells in bone marrow. The transformation of stem cells into osteoblasts is regulated by multiple growth factors and cytokines. The mature osteoblasts secrete proteins into the extracellular space, forming an organic matrix, to control the mineralization of the bone. Finally, osteoblasts will transform into osteocytes, bone lining cells, or undergo apoptosis.
Hormones
2] Bone remodeling is a closely regulated procedure. Hormones produced by parathyroid, thyroid, ovaries, testes, adrenal glands, and pituitary gland are all involved in the bone metabolism.
Parathyroid Hormone, Glucocorticoids, Thyroid Hormone, and Vitamin D metabolites increase bone resorption. Calcitonin and Gonadal steroids decrease bone resorption. Bone formation is enhanced by Growth hormone, Vitamin D metabolites, and Gonadal steroids and inhibited by Glucocorticoids.
Growth Factors and Cytokines
Growth factors and cytokines are local regulators. They are secreted to the extracellular space to act on the nearby cells.
Both osteoblast and osteoclast cells can produce and secret certain local regulators to mediate the proliferation, differentiation, and survival of the cells. Bone morphogenetic proteins (BMP) BMP are produced in bone or bone marrow. When BMP binds to BMP receptors on mesenchymal stem cells, the stem cells are activated to produce Cbfal. Cbfa 1 is able to stimulate the gene transcription. The increased expression of certain proteins leads to the differentiation of the stem cells into osteoblasts.
Insulin-like growth factors (IGFs)" When osteoblast cells are activated by parathyroid hormone, estrogens, or BMPs, osteoblasts would produce IGFs.
During bone remodeling, IGF stimulates the osteoblasts proliferation. IGF also could stimulate the differentiation of cells.
2.2 Interleukin-1 (IL-1), interleukin-6(IL-6), and tumor necrosis factor (TNF) are produced by osteoblast cells in response to systemic hormones or other cytokines. IL6 can cause the differentiation of bone marrow stem cells into preosteoclasts; mediate the proliferation and differentiation of osteoblasts; inhibit the apoptosis of osteoblasts.
23 The Molecular triad OPGIRANK/RANKL in Bone Remodeling
During bone remodeling, bone formation and resorption are two tightly coupled processes. Osteoclastogenesis is under direct control of factors expressed by osteoblasts including RANKL and osteoprotegerin (OPG) (Khosla, 2001). RANKL exists in both membrane-bound and secreted forms. RANKL-
RANK interaction triggers a series of intracellular changes in pre-osteoclasts leading to increased osteoclastogeneis and enhanced osteoclast function. OPG is a soluble protein secreted by osteoblasts, it is the equivalent of a decoyed receptor for RANKL. OPG binds to RANKL, occupies the binding site for RANK and blocks its effects. Therefore, OPG acts as a negative regulator for bone resorption (Khosla, 2001). OPG/RANK/RANKL are key molecules for osteoclastic differentiation and bone remodeling.
Despite extensive studies, the mechanism of EMD function is still largely unknown. It was proposed that insoluble matrix proteins are the components which attract and interact with cells to stimulate their biological functions. In this situation, the direct contact between EMD and cells is required. Another hypothesis is certain bioactive molecules released from EMD take the responsibility.
In this study, first-it will be explored whether direct contact between EMD and cells is needed for its bioactive functions. Then osteoblast cells will be used
24 to detect if cytokines and growth factors typical for bone cell growth are activated to release when cells are stimulated by EDM.
25 SPECIFIC AIMS
Specific Aim 1-
To determine whether the effect of enamel matrix derivative (EMD) on osteoblast proliferation is dependent on direct contact between EMD and the cells.
Specific Aim 2:
To evaluate the influence of EMD on the release of transforming growth factor beta1 (TGF-131), interleukin-6 (IL-6), insulin-like growth factor (IGF-I), bone morphogenetic protein 2 (BMP2), Prostaglandin G/H synthase 2 (PGE2), and osteoprotegerin (OPG)in human and mouse osteoblasts. MATERIALS AND METHODS
Cell Culture
Mouse pre-osteoblasts MC3T3-E1 cells and Human MG63 osteoblasts were obtained from American Type Culture Collection. Cells were cultured in
Dulbecco's modified eagle medium (DMEM, Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories, Inc, Logan, UT) and 1% of an antibiotic/antimycotic cocktail (300 U/ml penicillin, 300 ug/ml streptomycin, 5 ug/ml amphotericin B) under standard cell culture conditions (37 C, 100% humidity, 95% air and 5% 002).
For Aim 1 Experiment
MC3T3-E1 cells from Petri dishes were released with 0.25% trypsin and 1 mM EDTA in Hanks' balanced salt solution (Gibco BRL, Gaithersburg, MD). Then cells were seeded onto 6-well culture plates (Costar, Corning, NY) at an initial density of 5,000 cells/cm2 in DMEM with 10% FBS and allowed to attach for 24 hours. Medium was then changed into serum-free DMEM with antibiotics.
Four different treatment groups were included"
Group 1, cells were cultured in serum-free DMEM only and served as negative control. Group 2, 100 pg/ml EMD (BIORA AB, Malmo, Sweden) was added directly to the culture medium.
27 Group 3, cells were cultured in DMEM with a culture plate insert (Millipore Corp. Bedford, MA) only (Fig. 1). The Millipore culture plate insert (30 mm diameter; 0.4 um pore size) was placed into the culture well with the porous membrane 1 mm away from the bottom of the well. Group 4, 100 IJg/ml EMD was added to DMEM on the top surface of the culture plate insert, so that there was no direct contact between EMD and cells
(Fig. i). After 3-day incubation, cell morphology was examined under a phase- contrast microscope (CK40 Culture Microscope, Olympus American Inc. Melville NY) and photographed. The appearance of EMD on the culture plate inserts and in culture media was also examined and photographed.
Cell proliferation was evaluated by total cell number count after treatment.
Cells were detached with 0.25% trypsin and 1 mM EDTA in Hanks' balanced salt solution (Gibco BRL, Gaithersburg, MD). The resuspended total cell number per well was electronically counted using cell coulter (Coulter Electronics, Hialeah, FL).
Six culture wells were included in each group. Experiments were repeated three times. Results from the representative experiments are shown.
Total cell number per well is expressed as mean + SD. The difference between treatment and control groups was analyzed using one-way analysis of variance (ANOVA). A p value less than 0.05 is considered significant.
For Aim 2 Experiment Human MG63 cells and MC3T3-E1 cells are seeded onto cell culture dishes (Costar, Corning, NY) at an initial density of 5,000 cells/cm2 in DMEM with 10% fetal bovine serum and allowed to attach for 24 hours. After cell adhered to the culture well, medium are changed into serum-free medium.
Then cells are divided into two groups for evaluation"
Group 1, cells are cultured in DMEM only.
Group 2, cells were cultured in DMEM with 100 IJg/ml EMD (BIORA AB, Malmo, Sweden). After 1, 3, 7, 10 days incubation, cell morphology was examined under a phase-contrast microscope (CK40 Culture Microscope, Olympus American Inc. Melville NY) and photographed.
Culture medium was collected after 1, 3, 7, 10 days culture. The following bioactive molecules were measured by use of a commercially available sandwich ELISA kit (R&D System, Minneapolis, MN, USA)" Transforming growth factor beta1 (TGF-131) Interleukin-6 (IL-6) Insulin-like growth factor (IGF-I) Bone morphogenetic protein 2 (BMP2) Prostaglandin G/H synthase 2 (PGE2) Osteoprotegerin (OPG) For ELISA assay
After 1, 3, 7, 10 days incubation Then cells were detached with 0.25% trypsin and 1 mM EDTA in Hanks' balanced salt solution (Gibco BRL,
29 Gaithersburg, MD), and the total cell number will be electronically counted (Coulter Electronics, Hialeah, FL).
Statistical analysis of differences in values of bioactive molecules between different groups and the cell number between EMD treated and control groups will be performed by one-way ANOVA.
3O RESULTS
Aim 1
After EMD was added onto the culture insert, precipitated aggregates were formed on the surface of the porous membrane (Fig. 2). The appearance of these aggregates was similar to that observed in the culture dish when EMD was added directly to the medium.
Cells appeared healthy and grew normally after incubation with EMD or culture plate inserts. The culture plate insert itself did not cause any changes in cell morphology (Fig. 3). After 3-day treatment, the total cell number in group 2 (DMEM with EMD added directly to the medium) was significantly greater than that in group 1 (DMEM only) and in group 3 (DMEM with culture insert only) (p < 0.01) (Fig. 4). The total cell number in group 4 (DMEM with EMD added into the culture insert) was significantly greater than that in group 3 (DMEM with culture insert) and group 1 (p < 0.01) (Fig. 4). No significant difference was observed between group 1 and group 3, or between group 2 and 4. The presence of cell culture insert did not have any effect on cell proliferation. The addition of 100 lg/ml EMD significantly increased cell number regardless the presence of the culture plate insert.
Aim 2 EMD treatment significantly increased the release of TGF I1 (p<0.05) from both human and mouse osteoblasts at all tested time points (Fig. 5, 6).
3I The secretion of IL-6 is barely detectable without EMD treatment (Fig. 7,
8). EMD treatment significantly increased IL-6 secretion by 3 to 10 folds in both human and mouse osteoblasts (p<0.05) (Fig. 7, 8). The release of osteoprotegerin was also significantly increased in mouse osteoblasts (Fig. 9). As no human OPG antibody is available for ELISA test, the release of osteoprotegerin from human osteoblasts was unable to be tested.
The levels of bone morphogenetic protein 2, insulin-like growth factor I, and prostaglandin synthase were not changed under the experimental conditions between EMD treated and control groups.
32 DISCUSSION
Emdogain has shown great potential in promoting tissue regeneration in different dental fields including the treatment of periodontal defects, avulsion, and pulp capping. Although the beneficial effects of EMD is well recognized in both clinical and laboratory settings, the mechanisms of its action are still under debate. It has been proposed that EMD supports tissue regeneration by forming an insoluble matrix that favors the attachment and proliferation of regenerative cells such as fibroblasts and osteoblasts (Gestrelius et al. 1997). This insoluble matrix also promotes cells to produce growth factors, including TGF-13, PDGF, etc (Lyngstadaas et al. 2001; Rude et al. 2001). In this theory, direct contact with cells is required for EMD to exert its functions. Another hypothesis is that bioactive molecules released from EMD are responsible for the tissue regenerative activity of EMD. The bioactive molecules could be growth factors absorbed to EMD during its preparation or amelogenin peptides (Veis et al. 2000; Veis. 2003).
In this present study, a Millipore culture plate insert was used to separate the aggregated enamel matrix protein from attached cells in the culture plate well. A 40% increase in cell proliferation was observed when EMD was added to the culture plate insert without any direct contact with the cells. This increase is similar to that observed when EMD was added directly to the culture media.
These results show that direct contact between EMD and osteoblasts is not required for EMD-induced cell proliferation, and supports the hypothesis that
33 soluble factors contained in EMD are responsible for the proliferative effects of EMD.
The question now is that, what are these soluble factors that promote cell proliferation? Growth factors are attractive candidates, but their presence and participation in EMD-induced tissue regeneration have not been clearly defined.
Previous study using various detection systems could not detect the presence of any common growth factors in the EMD, such as macrophage colony stimulating factor (GM-CSF), epidermal growth factor (EGF), nerve growth factor (NGF), platelet derived growth factor (PDGF), TGF-I, and insulin like growth factors
(IGFs) (Gestrelius et al. 1997). Furthermore, the outcome of periodontal regeneration stimulated by EMD is totally different from that induced by bone morphogenetic protein-2 (BMP-2); suggesting BMP-2 is not responsible for the effects of EMD (Cochran et al. 2003, Sigurdsson et al. 19955). However, a recent study showed that significant levels of TGF-II were present in EMD preparations which led to rapid phosphorylation of the MAP kinase family and translocation of smad2 into the nucleus in both oral epithelial and fibroblastc cells (Kawase et al. 2001). Therefore, growth factors, especially TGF-I 1, remain to be a candidate mediating the effects of EMD.
Amelogenin is the major component of EMD (Gestrelius et al. 1997;
Maycock et al. 2002; Hoang et at. 2002). Recombinant amelogenin stimulates PDL cell proliferation in a concentration-dependent manner (Sigurdsson et al.
1995). A recent study shows that amelogenin regulates the expression of bone sialoprotein (BSP) and osteoclacin (OCN) (Viswanathan et al. 2003). At low
34 doses amelogenin enhances BSP and OCN expression, while at high doses amelogenin decreases their expression with a corresponding decrease in mineral nodule formation (Viswanathan et al. 2003). In amelogenin knockout mouse, there is a dramatic reduction in the expression of BSP mRNA and protein
(Viswanathan et al. 2003). Multiple amelogenin gene products exist as a result of alternative splicing (Veis et al. 2000; Veis. 2003). The larger forms are important for enamel mineralization while small amelogenin peptides may have signal transduction function, and have been shown to enhance the expression of type !! collagen, Sox 9 and Cbfa 1 mRNA in vitro (Veis et al. 2000; Veis. 2003).
These small amelogenin peptides were able to induce bone formation around implants in vivo by enhancing the production of extracellular matrix, matrix vascularization and mineralization (Veis et al. 2000; Veis. 2003, Nebgen et al.
1999). They have comparable osteogenic activities to recombinant human BMP-
2 (Veis et al. 2000; Veis. 2003). The amelogenin peptides also induce the formation of reparative dentin bridge, which are comparable to BMP-7 and calcium hydroxide (Goldberg et al. 2003). Protein analysis of EMD revealed the presence of proteolytic enzymes such as metalloendoproteases and serine protease in this commercial preparation (Maycock et al. 2002). During cell culture, amelogenin could be proteolytically processed into smaller amelogenin peptides, which pass through the Millipore membrane and promote cell proliferation.
This study suggests that direct contact is not required for EMD-induced cell proliferation; soluble factors contained in EMD may be responsible for the stimulating effects of EMD. Growth factors such as TGF-131 and small amelogenin peptides are potential candidates mediating the effects of EMD.
Further study is needed to determine the exact nature of these soluble molecules and their mechanisms of action.
Studies have shown that EMD induced autocrine release of growth factors. EMD significantly increased the attachment rate, growth and metabolism of these cells. Cells exposed to EMD showed increased intracellular cAMP signaling and production of transforming growth factor (TGF-II), interleukin 6 (IL-6) and platelet-derived growth factor (PDGF-AB) (Lyngstadaas et al. 2001). Conversely, when epithelial cells were used (HeLa cells), EMDs showed no effect on the rate of attachment; proliferation and growth were inhibited even though both cAMP and PDGF-AB secretion were induced by the presence of
EMDs in the culture (Lyngstadaas et al. 2001). Transforming growth factor-Il (TGF-I) is a basic 25 kD homodimetric polypeptide with high hydrophobicity that is a member of a super family of multifunctional cytokines. It participates in a broad range of biologic activities such as growth development and wound repair, as well as some pathologic processes (Attisano and Wrana 1996; Choi et al. 1997). TGF-II has been shown to regulate numerous cellular functions including inhibition and stimulation of cell growth, apoptosis, and differentiation
(Attisano and Wrana 1996). TGF-II is also an inducer of extracellular matrix (ECM) protein synthesis and has been implicated as a key mediator of fibro genesis in various tissues (Border and Noble 1994). TGF-II has recently been suggested to regulate proliferation and differentiation of fibroblasts, keratinocytes, oseoblasts, and extracellular matrix metabolism in the bone (Hartsough et al. 1996; Atfi et al. 1997; Chin et al. 1999). IL-6 has effects on skeletal homeostasis by regulating osteoblast and osteoclast development and function. (Manolagas et al. 1996).
The enhanced release of TGF-II by EMP treatment was also substantiated by other investigators. The effects of EMD on the behavior of human periodontal ligament fibroblasts (HPDL) and gingival fibroblasts (HGF) were investigated with special focus on cell attachment properties, the expression of alkaline phosphatase (ALP) activity, the release of transforming growth factor (TGF-II), and cell proliferative rates. It was found that HGF barely attached and spread on EMD-coated substrate, whereas HPDL attached and spread within 24 hours. However, when cultured on purified collagen type I, both cell types showed rapid attachment and spreading. Furthermore, the expression of ALP activity was significantly enhanced under the influence of EMD, especially in HPDL. HPDL and HGF both released significantly higher levels of TGF-II in the presence of EMD (Van der Pauw, et al. 2000). There are increased alkaline phosphatase activity, increased production of osteocalcin and TGF-131 with unaffected prostaglandin 52 in MG63 cells (Schwartz et al. 2000). EMD prolongs mouse primary osteoblasts growth (Jiang et al. 2001a), and upregulates expression of collagen I, interleukin-6, and prostaglandinG/H synthase 2 (Jiang et al. 2001b).
Our results that EMD induced both human and mouse osteoblasts secrete
TGF-II and IL-6 agree with the previous studies by using different cells. The effects of endogenous TGF-II on cell growth are demonstrated by using anti- TGF-II (Okubo et al. 2003). The EMD-stimulated cell growth was suppressed by cotreatment with anti-TGF-bl (Okubo et al. 2003). Anti-TGF-II antibody also blocks EMD-induced upregulation of p21WAF1/cip1 in Oral epithelial cells (Kawase et al. 2001; Kawase et al. 2002). The results that EMD increases osteoblasts secretion of osteoprotegerin (OPG) suggest that EMD indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG. During bone remodeling, bone formation and resorption are two tightly coupled processes. Osteoclastogenesis is under direct control of factors expressed by osteoblasts including RANKL and
OPG (Khosla, 2001). RANKL exists in both membrane-bound and secreted forms. RANKL RANK interaction triggers a serial of intracellular changes in pre- osteoclasts leading to increased osteoclastogeneis and enhanced osteoclast function. OPG is a soluble protein secreted by osteoblasts. It is the equivalent of a decoyed receptor for RANKL. OPG binds to RANKL, occupies the binding site for RANK and blocks its effects. Therefore, OPG acts as a negative regulator for bone resorption (Khosla, 2001). In this study, we examined the effects of EMD on the expression of OPG in MC3T3-E1 cells to understand the indirect influence of EMD on bone resorption. Under our experimental conditions, EMD increased the production of OPG, which in turn suppresses osteoclastogenesis and osteoclast activities, therefore inhibits bone resoption and shifts the balance towards increased bone formation. This study's result agrees with the previous
38 investigation that mRNA expression of OPG was up-regulated in cells treated with EMD (He et al. 2004).
From the results of this study the effect of EMD on osteoblasts could be described in the proposed model (Fig.10). Bioactive factors released from EMD stimulate osteoblasts releasing TGFII, IL-6, and OPG. TGFII has been demonstrated to enhance the proliferation and differentiation of osteoblasts and inhibit apoptosis of osteoblasts (Fig. 10). IL6, in one hand promotes osteoblasts function by activating proliferation and differentiation and inhibiting apoptosis, and in other hand activates the transforming of stem cells into osteoclasts to accelerate bone remoldeling (Fig. 10). OPG block the binding of RANK Ligand to RANK to interfere the activation of osteocalst. Therefore, osteoclastogenesis is inhibited (Fig. 10). Thus the stimulatory effects of EMD on tissue regeneration are mediated by the upregulation of local mediators released by osteoblasts.
The enhanced bone turnover may also explain the beneficial effect of clinical appication of EMD (Fig. 10). CONCLUSION
EMD formed precipitated aggregates on the membrane of the culture insert as when it was added directly to the culture medium.
Direct contact between EMD and osteoblasts may not required tO induce cell proliferation. EMD treatment significantly increased the production of IL-6 and TGFI-I in osteoblasts.
The release of OPG was also increased by EMD in mouse osteoblasts.
The levels of IGF-I, BMP2 and PGE2 were not changed under the experimental conditions.
This study suggests that bioactive factors released from EMD may be responsible for the stimulating effects of EMD, and the stimulatory effects of EMD on tissue regeneration may be mediated by the upregulation of local mediators released by osteoblasts.
4O Culture plate insert Culture well Medium Porous membrane Cells
Figure 1. Millipore culture plate insert placed into a culture well of the 6-well cell culture plate. MC3T3-E1 cells were seeded onto 6-well culture plates and allowed to attach for 24 hours, medium was changed into serum-free medium. The Millipore culture plate insert (30 mm diameter; 0.4 pm pore size) was then placed into the culture well. EMD was placed onto the top surface of the culture plate insert. The porous membrane of the insert was 1 mm away from the bottom of the well, and prevented direct contact between aggregated EMD and cells.
41 Figure 2. EMD forms precipitated aggregates on the membrane of the culture plate inserts. After cell incubation, culture plate inserts were taken out from the culture wells and observed under a phase-contrast microscope, a. The membrane of the culture plate insert without EMD (original magnification X l00). b. EMD forms precipitated aggregates on the membrane of the culture plate insert (original magnification Xl00).
42 Figure 3. Cell morphology of MC3T3-E1 cells after 3-day incubation, a. DMEM only; b. DMEM with 100 Ig/ml EMD directly added to the culture medium; c.
DMEM with a culture plate insert only; d. DMEM with 100 Ig/ml EMD placed onto the top surface of the culture plate insert. After cells were incubated 3 days in the different groups, cell morphology was observed and photographed under a phase-contrast microscope (original magnification X l00).
43 750000
250000
Control EMD Inse EMD+inse Groups
Fig. 4. The effect of EMD on the proliferation of MC3T3-E1 cells. After 3-day incubation under four different culture conditions, cells were released from the culture wells and electronically counted. The total cell number per well is expressed as mean + SD. Results were statistically analyzed using one-way
ANOVA. Control, Cells were cultured with DMEM only; EII/ID, Cells were cultured with DMEM and 100 IJg/ml EMD directly added to the culture medium;
Insert, Cells were cultured with DMEM and culture plate insert only;
ED+in8ert, Cells were cultured with DMEM and 100 lg/ml EMD added onto the top surface of the culture plate insert. * Statistically significant difference from control and insert groups, p < 0.01.
44 500 ntrol
450
400
350
300
20
200
150
100
2 5 9 Days
Fig. 5. The effect of EMD on secretion of.TGFI-I from human osteoblasts.
Control, Cells were cultured with DMEM only; EMD, Cells were cultured with
DMEM and 100 lg/m EMD. * Statistically significant difference from control and insert groups, p < 0.01.
45 45O
400
350
300
250
200
150
100
5O
2 5 9 Days
Fig. 6. The effect of EMD on secretion of TGFI3-1 from mouse osteoblasts. Control, Cells were cultured with DMEM only; EMD, Cells were cultured with
DMEM and 100 #g/ml EMD. * Statistically significant difference from control and insert groups, p < 0.01.
46 35
3O
15
10
2 6 2
Fig. 7. The effect of EMD on secretion of IL-6 from human osteoblasts.
Control, Cells were cultured with DMEM only; EMD, Cells were cultured with
DMEM and 100 #g/ml EMD. * Statistically significant difference from control and insert groups, p < 0.01. 400
350
300
250 200 150
O0
5O
2 5 9
Fig. 8. The effect of EMD on secretion of IL-6 from mouse osteoblasts.
Control, Cells were cultured with DMEM only; ED, Cells were cultured with
DMEM and 100 #g/ml EMD. * Statistically significant difference from control and insert groups, p <-0.01.
48 3500
3000
2500
2000 Control
1500
1000
5OO
2 5 9
Fig. 9. The effect of EMD on secretion of osteoprotegerin from mouse osteoblasts. Control, Cells were cultured with DMEM only; ED, Cells were cultured with DMEM and 100 #g/ml EMD. * Statistically significant difference from control and insert groups, p < 0.01. T Fopl
Proliferation & ifferenti
Fi(::J, 10. Proposed model of the effect of enamel matrix derivative on osteoblasts and osteoclasts. Bioactive factors released from EMD (one possible way of EMD function) stimulate osteoblasts releasing I-_GFII, IL-6, and OPG. TGFII enhances the proliferation and differentiation of osteoblasts and inhibits apoptosis of osteoblasts. L6, in one hand, promotes proliferation and differentiation, and inhibiting apoptosis of oeteoblasts, and, in other hand, activates the transforming of stem cells into osteoclasts to accelerate bone remoldeling. OPG binds RANK Ligand, blocks the binding of RANK Ligand to RANK to interfere the activation of osteocalst. Therefore, osteoclastogenesis is inhibited. Thus the stimulatory effects of EMD on tissue regeneration are mediated by the upregulation of local mediators released by osteoblasts.
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