The Enamel Matrix Proteins And Regeneration Biology Essay

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Following treatment to periodontitis, the gingival margins may be restored, however, the periodontal attachments are usually not regenerated readily (Bowers et al 1989a). Hence, gingival margins may fall apart more rapidly following another bacterial attack. Therefore, the periodontal ligament needs to be regenerated to ensure a complete recovery from the causative agent. (Esposito et al 2009)

Over the years, there have been many techniques that have been proven to help regenerate the periodontal ligament. These techniques include: Guided Tissue Regeneration (GTR); Bone Grafting (BG) (Bowers et al 1989b) and Enamel Matrix Protein Derivative (EMD). Both GTR and BG techniques are basically comprised of keeping epithelium away from the tooth surface while giving space for the periodontal tissues to regenerate naturally from the surrounding biological structures. (Esposito et al 2009)

EMD, on the other hand, mimics the development of tooth supporting tissues during tooth formation (Venezia et al 2004). It depends on using derivatives obtained from the enamel matrix proteins, such as amelogenins, which are predominately seen in the enamel formation process called amelogenesis (Lyngstadaas et al 2009). These proteins are hard to extract in vivo, as most of these are naturally degraded from the established enamel, during the maturation phase of amelogenesis, as they have little function after enamel has been laid down. Some of these proteins are also tightly bound together by other derivative polypeptides. (Hu et al 2007)

However, amelogenesis is not the only time these specific proteins are witnessed. Oddly, they are also laid down as part of the basement membrane that is deposited by the Hertwig's epithelial root sheathe (HERS) during root development, and help with the formation and proliferation of acellular cementum (Esposito et al 2009). It is, thus, believed that if EMD were to be extracted from other mammals, such as pigs (Hammarström et al 1997), and applied in humans, they would be non-antigenic and would still theoretically mimic proteins in the basement membrane. The exact mechanisms of how EMD induces these changes are still ultimately unknown.

Enamel Matrix Proteins And Their Relation To EMD

Around 90% of enamel matrix proteins are amelogenins. They are secreted in a variety of different isoforms, the main form having the weight of around 25kDa. They are expressed by ameloblasts (Brookes et al 1995). Their function, during the secretory stage of amelogenesis, is to moderate crystal spacing. It does this by passing through the mineralization front, and assembling into nanospheres (Finchem and Simmer 1997). It is also believed that amelogenins can attach ameloblasts to the hydroxyapatite crystals in the enamel, and that they may also aid other cells and molecules to attach to the mineral of teeth, for example, periodontal related cell attachment (Hoang et al 2002). This may be one of the reasons why amelogenins are the most abundant protein in EMD.

However, the reason why EMD has more than just processed amelogenins is that these proteins are more active with a cocktail of other proteins, rather than just the native form of amelogenin. It may also be a possibility that processed amelogenin are more active than the ones found naturally. It is also important to note that the amelogenin protein not only promotes attachment, but it also affects cell spreading in a positive way. (Hoang et al 2002)

Other enamel matrix proteins are mainly called non-amelogenins, this includes a type of protein called enamelin. They are the largest enamel proteins that weigh around 200 kDa, and are also the least abundant proteins (3-5%) of all the major enamel proteins. These proteins are rapidly cleaved following its secretion and are only witnessed on the mineralization front of the enamel (Hu et al 2007). This shows that enamelin may have a function on hydroxyapatite crystal elongation.

Another main non-amelogenin protein is called ameloblastin. These comprise about 5% of the total matrix proteins and occur at the weight of around 70 kDa. Ameloblastins cleave material at the n-terminal sites, and these products accumulate in the sheath space separating the hydroxyapatite prisms and inter-prism enamel (Hu et al 2007). Hence this may indicate function in maintaining prism boundaries.

Both of these non-amelogenins are also secreted by ameloblasts during the secretion stage of amelogenesis (Smith et al 2009). Interestingly, even though both these proteins do not play an active role in the process of laying down mineral, knockout and truncated studies in mice show that no enamel can be formed with the absence of both these proteins (Hu et al 2008, Wazen et al 2009). This may mean that these proteins may activate amelogenins and hence are justified in the inclusion of EMD used the treat periodontal defects.

These three proteins are not the only ingredients of the EMD solution. There are also two enzymes present, such as MMP-20 and EMSP-1 (Fukae et al 1998, Simmer et al 1998). Recent immunoassay studies have also indicated a presence of growth factors in EMD, namely extracts of TGF-1, BMP-2 and BMP-4 (Iwata et al 2002), which may help in inducing proliferation. However, these growth factors have been isolated developing pig teeth, not the EMD mixture, and hence are not definite components of the mixture (Venezia et al 2004). The reason why they were isolated from pigs' teeth is that that is where the most clinically available EMD is derived from.

Emdogain And The Clinical Safety Of EMD

When considering on how to extract EMD, an avoidance of an immune reaction was at an utmost importance (Venezia et al 2004). As all mammals have chemically and biologically similar enamel proteins, there would be fewer problems for the proteins to be rejected by the human host (Brookes et al 1995). In vitro studies show that EMD does not significantly affect the cellular and humoral immune responses, unless it is applied in an excessive amount. Even then, only the CD4+ lymphocytes have seen minor increase, and B cells have seen a decrease, with all else remaining at nominal levels (Peteinaki et al 1998). This is why piglets may be used, with success and clinical safety, as an EMD bank.

A Swedish company called Biora AB produces the most available EMD, called Emdogain. It is an extract of developing embryonic enamel derived from six-month-old piglets. The mixture acts as a tissue healing modulator that helps to stimulate periodontal regeneration. All three of the proteins described above are included into this mixture. (Venezia et al 2004)

However, as the amelogenins are hydrophobic, they tend to aggregate and hence are inaccessible to the regenerating periodontal tissues at physiological pH and temperature (Hatakeyama et al 2009). This would render the EMD mixture useless at normal physiological conditions. Hence the EMD mixture is acidic to avoid the aggregation. Furthermore, EMD has to be delivered via a medium that allows EMD to not only dissolve during application, but also allows gradual release, or re-precipitation, of the essential proteins needed to aid in regeneration. (Hammarström et al 1997)

Two main mediums, or vehicles, became apparent after an experiment based on a buccal dehiscence model in monkeys. The action of propylene glycol alginate (PGA) was compared to the action of hydroxyethyl cellulose (HEC). The experiment was an analysis of the improvement in periodontal attachment after 8 weeks of application. The results showed that PGA was a better suitor to carry the EMD (Hammarström et al 1997). A major advantage of PGA is that is has a changing viscosity that depends on the application. For example, if PGA was put through a shear force, such as a nozzle through a syringe, the viscosity would decrease, allowing easier clinical handling. It lost viscosity at physiological conditions too, exposing EMD to the tooth root surface (Venezia et al 2004). In addition, using a radiolabeling technique, the PGA was found to leave the surgical area shortly after its application, hence not interfering with EMD and the periodontal treatment. (Gestrelius et al 1997a)

Emdogain comes in two varieties. The first one being PGA supplied in a separate container that has to be mixed with main EMD mixture just before application. This unfortunately took time and so Biora AB produced Emdogain Gel. The gel is a pre-mixed solution of EMD and PGA that was ready and just needed to be applied in the oral cavity (Venezia et al 2004). A study involving 88 patients showed no difference in regenerative ability between both products (Bratthall et al 2001).

How Matrix Proteins Affect The Periodontal Tissues During Development

Before discussing the effects of Emdogain on the periodontal tissues, the link between enamel proteins and periodontal tissues will be briefly discussed. As mentioned earlier, during the root development stages of the teeth, enamel matrix proteins have been witnessed involved in the initial generation of the periodontal tissues (Zeichner 2006). Namely, it has been seen that the proteins have an influencing affect on producing acellular cementum and also promotes the stimulation of periodontal ligament cells. (Zeichner 2006)

Hertwig's epithelial root sheathe (HERS) is classified as the down growth of the enamel sheathe, and hence is similar in contents to the enamel being formed at the coronal aspect of the tooth. As the contents are similar, it also shows that even the HERS has a short secretion phase, just like the secretion phase in amelogenesis, where they

deposit enamel matrix proteins on the root surface prior to cementum formation. There has been some evidence that apically deposited matrix proteins may be the initiating factor in the formation of cementum. (Zeichner 2006)

Cementum is divided into two major classes, acellular and cellular. It is believed that cellular cementum originates from osteoblasts, whereas acellular cementum is believed to originate from precursors of cementoblasts that originate from the dental follicle (Cho and Garant 2000). This is further proved by an experiment conducted on rats, as it showed that cells that formed the cementum like structure that originated from the dental follicle mainly laid down acellular cementum. It was also noticed that these cementum precursors came into contact with enamel matrix proteins, and hence the link between EMD and acellular cementum was established (Yamamoto et al 1996).

"Development of the major collagen bundles, the principle fibres of the periodontal ligament, is closely correlated to root cementum formation" (Cho and Garant 2000). Fibre bundles originate at the surface of the newly formed root, closely related to the active fibroblasts that are also present there. The fibres that make up these bundles are packed together by the action of acellular cementum formation (Cho and Garant 2000). This means that acellular cementum, initiated by the action of EMDs, then promote major periodontal ligament fibres to form (i.e. acts as a cascade of events).

Alveolar bone also depends on the formation of the tooth first. The size, shape, location and function of the tooth greatly influence the morphology of the bone (Schroeder 1989). Alveolar bone starts to form just about the same time as the root formation, however it is not in the shape that is seen in the developed oral cavity (Cho and Garant 2000). Morphological studies and experimental surgical interventions have shown evidence of post-secretory enamel organ and the dental follicle connective tissue work together in reshaping the alveolar bone to erupt the tooth into occlusion. (Cahill and Marks 1980, Wise 1998) This shows again that the enamel proteins may affect the genesis of the alveolar bone and hence shows how the use of EMD can be justified.

How EMD Aids Periodontal Regeneration

Effects of EMDs on the epithelia

Two studies by Gestrelius et al (1997b) and Kawase et al. (2000) examined the proliferation of oral epithelial cells using in vitro rat tongue epithelial cells and in vivo tests respectively. Both concluded that EMDs did not enhance proliferation of epithelial cells; rather they arrested the growth of the cells, by keeping them locked on the G1 phase of the mitotic cycle. There has been evidence that the TGF- may be the factor that causes this suppression. (Bosshardt 2008)

However, it has been witnessed by Rincon et al. (2005), that EMD significantly increases DNA synthesis from a specialised epithelial cell type called the Rests of Malassez. These are the remnant cells of what originally used to be part of the HERS during development that have since migrated into the periodontal space. Their function remains largely unknown, however it has been proposed that their function may be

linked to regeneration, as they react to inflammatory mediators by proliferating and may be one of the factors that may repair damaged tissue (Zeichner 2006).

Although EMD is shown to negatively affect epithelial proliferation, Kawase et al. (2001) proved using 50g EMD/ml culture medium that adhesion of epithelial cells were improved and also showed that it stimulated cytoskeletal actin polymerization. Rincoln et al. (2005) showed that there was increased attachment to EMD coated tissue culture wells, when compared to negative control. However, this is yet to be proven in vivo.

Epithelial cells also showed a rapid and strong secretion of Platelet Derived Growth Factor (PDGF)-AB in the presence of EMD (Lyngstadaas et al 2001). PDGF-AB plays an in important role in cell proliferation, cell migration and angiogenesis (the development of new blood vessels). In addition, they are a required element in fibroblast cellular division (Barres et al 1992). Hence, it can be seen why this is advantageous for regenerating the periodontium.

Effects of EMDs on the gingiva

Van der Pauw et al. (2000) and Rincon et al (2003) showed a significant increase in incorporation of thymidine into the gingival fibroblast DNA compared with controls. Further tests on piglets also showed the strong correlation mentioned (Rincon et al 2005). The thymidine may act as cell proliferation mediators. This was further proven when experiments showed that EMD increased rat gingival fibroblasts by up to two-folds when compared to negative controls (Keila et al. 2004).

EMD greatly increased cell attachment of porcine gingival fibroblast, and this was much more significant than perceived on the porcine periodontal ligament fibroblasts (Rincon et al 2005). Compared with human periodontal ligament fibroblasts, however, the gingival fibroblasts are slower and generally sluggish to respond to EMD (Van der Pauw et al 2000). A further study by the same group showed that intergrins played a major role in the interactions between gingival fibroblasts and EMD (Van der Pauw et al 2002, Bosshardt 2008).

Haase and Bartold (2001) showed that EMD significantly stimulated the release of matrix glycoproteins, namely versican, biglycan and decorin. Versican works closely with hyaluronin, which is also synthesised in higher levels at the presence of EMD, to maintain the integrity of the extracellular matrix of the gingiva (Bosshardt 2008). It is believed, using knockout experiments in mice, that biglycan is necessary for BMP to show its effects on osteoblasts. BMPs are chemical signallers responsible for developing, or regenerating in this case, bone tissue (Xu et al 1998). Finally, decorin is a proteoglycan closely associated to fibrillogenesis (Ständer et al 1999). In addition, "EMD was found to increase the amount of extracellular matrix and protein content in a dose dependant manner when compared with controls" (Bosshardt 2008).

EMD induced increases in the expression of osteopontin (OPN), which plays an important role in bone remodelling (Rincon et al. 2005). One of its functions includes giving the ruffled border that osteoclasts develop during bone resorption. This would induce bone remodelling and growth. Its other functions are closely associated with cell attachment and wound healing (Choi et al 2008).

EMD also stimulated alkaline phosphatase (ALP) in the gingival fibroblasts. ALP is closely associated with mineralization. Again, it is witnessed that gingival fibroblasts have lower levels of ALP induced when compared to periodontal ligament fibroblasts (Van der Pauw et al 2000). However, this was contrasted by another study that showed no mineralization when dealing with in vitro rat gingival fibroblasts cultured with EMD (Keila et al. 2004).

Effects of EMDs on periodontal ligament cells

A significant increase in cell proliferation was witnessed in human periodontal ligament (PDL) fibroblasts that were exposed to EMD (Gestrelius et al 1997b). EMD was shown to substantially stimulate the proliferation in a dose dependant manner over three days by Kawase et al (2000). A trend was also shown between the time of the application of EMD and the subsequent cell proliferation of the human PDL fibroblasts, showing that the time of application is also an important factor along with dose levels (Okubo et al 2003). Interestingly, short-term exposure to EMD reduced cell numbers compared to the control, however, long-term exposure led to a significant increase in cell numbers (Lossdorfer et al. 2007).

It is also been shown in vitro, that PDL cell density and DNA synthesis were increased in the presence of EMD in prepared cultures (Rincon et al. 2005). This was further proved by another study conducted by Ashkenazi and Shaked (2006), where they cultured human PDL fibroblast with and without the presence of EMD. "The presence of EMD decreased the percentage of cells with the ability of giving rise to colonies with 75%-100% confluence" (Bosshardt 2008). The authors proposed this was due to the increase in cell differentiation effects of EMD.

Another study also showed an uptake of bromodeoxyuridine (BrdU) by PDL fibroblasts in the presence of EMD (Pischon et al 2006). BrdU is a form of experimental assay that shows the presence of newly synthesised DNA in replicating cells (http://www.wellesley.edu/Biology/Concepts/Html/brdu.html). This provides further proof that PDL fibroblasts do replicate under the influence of EMD.

As mentioned earlier, PDL fibroblasts generally respond faster than gingival fibroblasts. PDL cell attachment rate was significantly increased in the presence of EMD in vitro culture. Van der Pauw et al (2002) showed that the EMD achieved this via intergrins that improved attachment between fibroblasts and EMD. However there are many studies that also show that EMD may actually have an inhibitory effect in cell adhesion (Palioto et al 2004, Rodridues et al 2007). To test this, Zeichner et al. (2006) used the core build up of EMD, namely the proteins amelogenin and ameloblastin, from mice on immortomouse derived fibroblasts. They confirmed that EMD did have a positive effect on cell adhesion.

The synthesis of total protein has been shown to increase when the fibroblasts were treated with EMD (Gestrelius et al 1997b). Cell metabolism was also increased when EMD was present in culture. It stimulated increased levels of PDGF-AB to be released when compared with controls (Lyngstadaas et al 2001). Levels of glycoprotiens, such as versican and biglycan, also increased with the increase in dose of EMD (Haase and Bartold 2001). Higher levels of hyaluronan were also witnessed (Hakki et al 2001). Parkar and Tonetti (2004) showed, using a gene array study that in human PDL cells the expression of genes involved early inflammatory events of wound healing were down regulated, whereas genes coding for repair and growth were up regulated. This was further proved by Barkana et al (2007), who showed that EMD up regulated expression of the genes involved in all general metabolism effects in the PDL cells that form mineralised tissue.

Takayangani et al. (2006) examined the effects of EMD on bone related mRNA in human PDL cells in vitro. Their results showed an increase in the expression of cyclooxygenase 2 (COX2) mRNA levels in cells that were exposed to EMD.

While evaluating the response of human follicle cells in vitro to EMD, Kemoun et al (2007) noted increased expression of BMP-2, BMP-7, BSP, cementum attachment protein (CAP) and cementum protein-23 (CP-23). CAP and CP-23 are both thought to increase attachment of cementum.

As mentioned above, PDL fibroblasts have been shown to respond to EMD by also greatly increasing the ALP activity (Van der Pauw et al 2000). This was supported in another study, which showed that mRNA expression was dose-dependently increased in the fibroblasts exposed to PDL cells. Bio-mineralization was also seen to be enhanced (Nagano et al. 2004). Many other test results have all agreed with the above findings, such as Lossdorfer et al (2007), Rodrigues et al (2007) and Kemoun et al (2007).

Effects of EMDs on cementum deposition

There have not been many experiments and papers on the ability of EMD on cementogenic cells, and most that are found show negative results. A quick review will now follow.

Murine cementoblasts exposed to EMD showed significantly enhanced proliferation (Tokiyasu et al 2000). However, other experiments containing the full-length amelogenin or the cleaved N terminal amelogenin peptide showed no effects, or negative (anti-productive) effects at high doses (Viswanathan et al 2003, Swanson et al 2006).

EMD is shown to down-regulate osteocalcin (OC) expression and up-regulate OPN expression. It is believed that osteocalcin may function as a negative regulator of bone modelling, whereas OPN, as mentioned above, has positive effects (Tokiyasu et al 2000). Again, as above, exposure to the full-length amelogenin and the cleaved N terminal peptide showed that there was an increase in OC (Viswanathan et al 2003). Although it was shown that in high concentrations the OC was down regulated, there was an easy recovery with increasing time. A newer study, however, agreed with the original statement, claiming that gene expression of OC was indeed down regulated and OPT was up regulated (Swanson et al 2006).

EMD has been shown to negatively correlate with the formation of mineralization nodules in all of the studies mentioned above (Tokiyasu et al 2000, Viswanathan et al 2003, Swanson et al 2006).

Effects of EMDs on alveolar bone or bone cells

It was shown, using young adult male rats, that EMD failed to stimulate the proliferation of stromal osteoblastic cells derived from the bone marrow (Gurpinar et al 2003). However, many other studies contradict these findings in both rats and human test subjects, even though only one human patient was ever tested (Bosshardt 2008). Another patient test has also concluded that EMD induced the increase in growth of mandibular osteoblasts (Galli et al 2006). However, both human tests were only conducted using one patient each, and hence cannot be reliable source of confirmed information. Although to support these statements, there have been recent in vivo and in vitro experiments that also claim that there was an increased cell proliferation level in a dose-dependant manner to EMD (Pischon et al 2006, Heng et al 2007).

Although EMD has been shown not to be osteoinductive, they are proven to be osteopromotive in vitro (Boyan et al 2000). EMD affects early proliferation and as the cells mature also affects differentiation. The same experimenters also confirmed that EMD increased the number of normal human osteoblasts (Schwartz et al 2000).

More experiments show that EMD in primary osteoblasts enhances gene expression of type 1 collagen and also showed the down regulation or no change to the expression of OC (Tokiyasu et al 2000, Jiang et al 2001). There has been clear evidence shown that EMD may direct the preosteogenic cells C1C12 to differentiate into osteocytes or other cells of the chondroblast lineage (Ohyama et al 2002). Comparisons between two cell lines of osteoblastic cells show that EMD may affect different cell lines in a cell specific manner. For example, for ST2 cells, it was noted that EMD had little, or no effect in stimulating cell growth, whereas the KUSA/A1 cell line was profoundly affected by the application of EMD, as cell proliferation was seen to be greatly enhanced (Bosshardt 2008).

EMD greatly improved ALP activity in rat bone marrow stromal cells at concentrations of 25 g/ml (Bosshardt 2008, Keila et al 2004). It has also been noted that MC3T3-E1 cells, a mouse pre-osteoblastic cell line, responded to EMD with increased ALP activity, this was also linked with an increase in mRNA expression of type 1 collagen (He et al 2004). In another organoid culture system, EMD was shown to enhance "ALP activity, calcium accumulation, and in vitro mineralized nodule formation of osteoblasts isolated from mouse calvaria" (Bosshardt 2008). Furthermore, ALP activity was seen to rise after EMD was applied on commercially available human osteoblasts in vitro. A stimulatory effect was also seen on osteoclasts, which can be an inductive factor for bone remodelling, and EMD was noted to be taken up by osteoblastic cells (Reseland et al 2006, Bosshardt 2008).

Using a bone wound healing model on rats, Kawana et al (2001) and Sawae et al (2002) noted a higher bone volume fraction of newly formed bone in seven days when treated with EMD as opposed to the control, which was treated solely with PGA. Another study conducted on rat skull defects revealed an increase in bone formation two weeks after the initial injury (Yoneda et al 2003, Bosshardt 2008).

This is useful for implants, and EMD and implants may go together as EMD may stimulate bone to form around the implant placed in the oral cavity. For example, placing a titanium post in the corticotrabecular area of the bone in rats, it was observed that EMD generated an increased amount of trabecular bone area around the implants when compared with a PGA-only containing control at thirty days after the time when the implant was first placed (Shimizu-Ishiura et al 2002). Furthermore, an experiment that used porcine PDL cells with EMD showed a good bone-to-implant contact. Surprisingly, omitting EMD from this mixture, still lead to a decent bone-to-implant contact with strands of epithelial cells in the implant-connective tissue interface (Craig et al. 2006, Bosshardt 2008).

The studies and information given above give an in depth review of the experiments conducted on EMD thus far, as the effects are still very unknown and in such disagreement, there will be many studies to follow in the future. Hence, at the moment, it is not possible to give the direct mechanisms of EMD. However, a grand overview is provided at the end of this report.

The Use Of EMD Related To Other Cell Regeneration Promotion Techniques

EMD And GTR

Guided tissue regeneration (GTR), as mentioned above, is a physical non-resorbable or resorbable barrier to achieve good clinical results based on histological assessments (Esposito et al 2009). However, the outcome of GTR depends heavily on many factors, some of them being surgical technique, the clinicians experience, tooth morphology and defect morphology. One of the most important factors that effect any regenerative treatment is the being bacterial load. If bacteria heavily colonize this barrier, it would result in negative effects (Venezia et al 2004).

This is where EMD may be used. It is shown that EMD has an inhibitory action on Gram-negative bacteria, thus fighting any colonies that have developed into the aforementioned bacteria in the periodontal region (Spahr et al 2002). This reduces the risk of one of the most common reason for a failure in regenerative treatment. Hence it is thought that EMD or EMD coupled with GTR would be a better treatment option when compared with sole GTR.

However, there are many studies that show no significant difference in the pocket probing depth reduction between EMD and GTR was noted (Minabe et al 2002, Windisch et al 2002). Furthermore, no differences were also noted when considering the clinical attachment gain. In fact, GTR seemed to provide better results than EMD in terms of percentage clinical attachment gain with patients with a baseline clinical loss of 9 mm and above. EMD was shown to provide better results of loss lower than 9 mm (Silvestri et al 2000).

Histologically, GTR again seems to surpass EMD. Almost all GTR treated defects show some sign of true periodontal regeneration, however EMD, although showing signs of new attachments and cell differentiation, is not always followed by proper bone regeneration (Windisch et al 2002).

EMD And BG

The success of any regenerative technique is highly dependant on the available space and stability of the wound under the surgical mucoperiosteal flap (Venezia et al 2004). A bone graft (BG) is used to physically increase the area by acting like natural bone. This would cause a regeneration of the surrounding tissues around the graft. This is useful for implants, as a bone graft can be used to restore previously resorbed tissue to fit the shape of the implant (Esposito et al 2009).

As EMD is supplied in a liquid form, it lacks the physical strength to hold the surrounding tissues in shape (Venezia et al 2004). It would pose huge inaccuracies if EMD were solely used, especially for bone regeneration, as EMD may just flow into different areas. If precipitation has not taken place when the fluid flows away, then the EMD may cause an unwanted effect on other tissues. Hence, it may be used in conjunction with a BG, which adds stability to the effects of EMD.

A study was conducted using naked mouse models to confirm the effectiveness of the combination of EMD with demineralised freeze-dried bone allograft (DFDBA), a specific type of BG. It was concluded that the combination demonstrated osteoinductive activity above a specific threshold dose of 4 mg. Enhanced bone formation, including the production of new bone marrow, was witnessed when this combination was tested against DFDBA with no EMD, or when the combination was applied below the threshold dose (Boyan et al 2000).

EMD was also tested with other BG materials and derivatives. Major examples of specific types of BG include bovine-derived bone xenograft (BDX) and alloplastic synthetic bone graft (ASBG). It is shown that BDX enhances the action of EMD when combined compared with the application of sole EMD in intrabony periodontal defects. Furthermore, it was also revealed that adding a membrane to the combined mixture of EMD and BDX improved clinical attachment, reduced probing depths, and promoted defect fill levels even further (Camargo et al 2001). However, surprisingly, when EMD was tested together with ASBG, there was no significant change in the defects between the combination and ASBG alone (Sculean et al 2002).

The effectiveness of EMD in combination with DFDBA has yet to be properly compared to the effectiveness of the BDX combination (Venezia et al 2004). However, it is stated that by comparing the studies performed on each type of combination, the DFDBA combination has better clinical outcome than that of BDX (Rosen and Renolds 2002). Hence, at the moment, the best type of graft to be used in conjunction with EMD is DFDBA.

The Advantages Of The Usage Of EMD

Even though the mechanism of EMD action has not been truly understood yet, EMD are under severe research due to its many advantages over the other periodontal regenerative techniques. One of the main advantages includes the fact that EMD treatments fall under the Minimally Invasive Surgery (MIS) category (Cortellini et al 2008). This means that the patient feels less pain during treatment, as the surgery is not as deep and penetrating as other forms of regeneration treatment can be. However, it should be noted that the patient still needs a form of anaesthetics. Post-operatively, the patient would also comparatively take less or weaker painkillers to subside the pain.

Another advantage of being in the MIS category is that the patient would have to go through less surgical trauma. The access insertion does not need to be as large as other techniques, such as BG. The surgical flap may be reduced which would cause less iatrogenic damage to the tissues. It adds stability to the open flap. This also means that patients have less post-operative difficulties to deal with, such as accumulation of a lot of granulation tissue. Therefore, this surgery would leave a smaller scar tissue, which is formed in response to the treatment.

As it requires little surgery, the clinical in-chair time for patients is also relatively shorter compared to other techniques. A study showed that average time for a patient is in the range of 45 minutes to 89 minutes (Cortellini et al 2008). This means that not only is this technique less traumatising, it is also a relatively quick procedure. This allows the patient to return to their social lives, and allows the health profession to deal with more patients.

Conclusion

As a result, it can be noted that EMD may be used in periodontal regeneration. They are a relatively new advancement in periodontology. The main mixture of EMD without the vehicle is mainly consisted of amelogenins, with a cocktail of other drugs also witnessed in amelogenesis, such as enamelins and ameloblastins. A few lysis enzymes are also included in the mixtures that are also witnessed during the formation of enamel.

The natural tendencies of amelogenins are to coagulate and form nanospheres. This is combated by two factors. The first being the fact that the EMD mixture is slightly acidic, this alters conditions and stops the aggregation. EMD may also be dissolved into a vehicle, such PGA, which may slowly release the proteins on to the target site.

There are not many companies that manufacture EMD, the first and main one being Biora AB. The company started commercial distribution after the positive results from the Hammerstrom et al. study. They produced Emdogain and Emdogain gel, with the only difference being that the latter came pre-mixed with the PGA vehicle.

The exact mechanisms of EMD are still unknown, however, as described above, EMDs have been shown to improve cell attachment in the epithelial, gingival and PDL cells. It is also revealed that generally human PDL fibroblasts proliferate faster than the gingival fibroblasts. On the other hand, no proliferation of the cells in the epithelia was witnessed. It has been noted that EMDs tend to have an inductive effect on general up-regulation of protein synthesis, and also an effect on the release of promotive molecules. An example of this can be the increased activity of ALP, which helps at the bone mineralization front. As mentioned earlier, there are still studies running to assess the exact mechanisms of EMD, and these are only a few of the properties that EMD exhibit on the periodontal tissues.

EMD can be used with other regeneration techniques, such as BG and GTR. It seems that EMDs antibacterial and inductive effects coupled with these physical barriers is proven to show an improvement in regenerative ability. The fact that EMD fall in the MIS category of treatments mean that it is a better option for both patients and dentists.

EMDs have only started to be studied in the past 13 years, starting from 1997. This shows that the understanding of EMD is still a very hot topic for researchers and the application in the clinic is just starting to gain popularity amongst dentists. Hence, an increase of understanding and clinical use of EMD in regeneration may be an outcome in the future years to come.

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