COMPARISON OF PULPAL RESPONSE FOLLOWING PULPOTOMY PROCEDURE USING ENAMEL MATRIX DERIVATIVE VERSUS FORMOCRESOL IN PRIMARY DENTITION
Ahmed A. Mohamed*, Maha M. F. Mounir**, Nadia A. Wahba***, Omar A. S. El-Meligy****, Jumana M. S. Sabbarini***** ABSTRACT
The aim of the present study was to compare the clinical, radiographical and histological effect of enamel matrix derivative (Emdogain®) versus formocresol on pulpotomized human primary teeth.
Clinical follow-up of formocresol treated teeth at 2 months revealed (93.3%) clinical success rate. Only one tooth suffered from pain and was sensitive to percussion in formocresol group. This dropped to 86.7% at 4 months. At 6 months five teeth showed pain and pain on percussion clinically lowering the clinical success rate to 66.7%. Emdogain® showed an overall clinical success rate of 100% at 2 & 4 months. Only one tooth was reported with pain on percussion at 6 months reducing the clinical success rate to 93.3%. All teeth (100%) were free from mobility, abscess formation or draining sinus at 2, 4 & 6 months among both Formocresol and Enamel Matrix Derivative (Emdogain®) tested groups. Radiographically in Formocresol group, eleven teeth (73.3%) showed no pathological signs at 2 months recall. The radiographic success rate dropped to four teeth (26.7%) at 4 months recall. Two teeth only (13. 3%) were still free at 6 months recall. Emdogain® group showed radiographic success rate of 86.7% representing thirteen free teeth. This succes rate dropped to ten teeth (66.7%) at 4 months follow-up. Nine teeth (60%) were still frees of pathological signs at 6 months. Histological evaluation seemed far more promising for Emdogain® than formocresol. When the pulp wound was exposed to EMD, a substantial amount of reparative dentin-like tissue was formed in a process much resembling classic wound healing with moderate inflammatory infiltrate beneath the injury with subsequent increase in angiogenesis of normal pulp tissue.
*Professor of Pediatric Dentistry and Dental Public Health, Faculty of Dentistry, Alexandria University.
**Professor of Oral Biology, Faculy of Dentistry, Alexandria University.
***Associate Professor of Pediatric Dentistry, Faculty of Dentistry, Alexandria University.
****Lecturer in Pediatric Dentistry, Faculty of Dentistry, Alexandria University.
*****Pediatric Dentist, Ministry of Health, Irbid, Jordan.
At later stages, a fine web of odontoblast-like cells was also observed growing from the central parts of the pulp towards the pulp chamber walls forming a dentin bridge.
The EMD induced hard tissue closely resembled osteodentin early in the process, but later on the hard tissue became more like the secondary dentin. On the contrary, the severe chronic inflammation of pulp tissue acccompained with formocresol eventually produced pulp necrosis with or without fibrosis and incomplete dentin bridging at terminal stages in some cases.
Based on the present findings, it may be concluded that Emdogain® is a bio-inductive material that is compatible with vital human tissues. It offers a good healing potential and is capable of inducing dentin formation, leaving the remaining pulp tissue heathy and functioning.
INTRODUCTION Pulpotomy is removal of the coronal portion of the pulp. It is an accepted procedure for treating primary teeth with carious pulp exposure. The abnormal tissue can be removed, and the healing can be allowed to take place at the entrance of the pulp canal in an area of essentially normal pulp(1).
Historically, pulpotomy therapy for primary dentition has developed along three lines: devitalization, preservation and regeneration. Devitalization, where the intent to destroy vital tisue is typified by formocresol and electrocautery. Preservation, the retention of maximum vital tissue with no induction of reparative dentin bridge is exemplified by glutaraldehyde and ferric sulfate treatment. Regeneration, the stimulation of dentin bridge, as long been associated with calcium hydroxide. Of the three categories, regeneration is expected to develop the most rapidly in coming years(2).
As for devitalization, the use of formocresol as pulpal medicament was first introduced in the early twentieth century(3), and has since been a popular choice for use in pulpotomy procedures, mainly because of its ease of use and clinical success. Even though the use of this pulpotomy medicament is common and produces very successful results, concerns regarding its use have led investigators to search for a safe and effective alternative(4). These concerns were represented by many studies. One is a study by Magnusson (1978)(5), in which therapeutic pulpotomies in primary molars with formocresol technique were studied by systematic follow-up. The results showed that the radiographic follow-up revealed periradicular osteitis in 10% of treated teeth. Internal root resorption was seen in 37% of teeth. The histological examination revealed a very capricious diffusion of the medication throughout the pulp tissue. Vital pulp remnants in the apical part of treated roots showed no signs of healing. Pulps presented a varying number of inflammatory cells in the border zone adjacent to the formocresol-fixed region. In 80% of roots histological sections revealed signs of internal resorption. Another study by Ranly & Gracia-Gody (2000)(6), concluded that although rationale for the use of formocresol is not clear, it presumably fixes affected and infected radicular tissue, so that a chronic inflammation replaces an acute inflammation. Other concerns have been raised regarding the toxicity and potential carcinogenicity of formocresol in humans(7).
As for regeneration, the stimulation of dentin bridge, has long been associated with calcium hydroxide which is considerably less harsh on pulp tissue than formocresol(6). It is believed that high pH of calcium hydroxide based materials causes frequent side effects such as internal resorption and necrosis, which prevented their acceptance as universal indicators for reparative dentin formation in primary dentition(8).
Recently, a number of studies have reported that various odontogenic proteins can induce reparative dentin formation. Biologically active molecules, such as the bone morphogenic protiens (BMPs)(9), the osteogenic protein (OP-1)(10), or biometrics, such as the demineralized dentin(11), have all been proposed as specific bioinducers of dentin formation. None of the biomaterials/proteins are commercially available, nor are their safety and toxicity aspects properly assessed for the use in clinical trials(9,10,11).
(Enamel matrix proteins) like amelogenins from the pre-ameloblasts, are translocated during odontogenesis to differentiating odontoblasts in dental papilla, suggesting that amelogenins may be associated with odontoblast changes during development(12). Enamel matrix derivative (EMD), obtained from embryonic enamel of amelogenin, was demonstrated in vitro, using a wound healing model, to be capable of stimulating periodontal ligament cell proliferation at earlier times (i.e., days one to three) compared to gingival fibroblasts and bone cells(13).
Heijl et al (1997)(14), led a clinical trial on enamel extracellular matrix proteins in form of the enamel matrix derivative, EMD commercially presented as EMDOGAIN® which has been successfully employed to incite natural cementogenesis to restore a fully functional periodontal ligament, cementum and alveolar bone in patients with advanced peridontitis.
The ability of EMD to facilitate regenerative processes in mesenchymal tissues is well established. The EMD-induced processes actually mimic parts of normal odontogenesis, and it is believed that the EMD proteins participate in the reciprocal ectodermal-mesenchymal signaling that control and pattern these processes(15). Based on these observations, it has been suggested that amelogenin participates in the differentiation of odontoblasts and the subsequent predentin formation(16).
Nakamura et al (2001)(17), demonstrated that EMD quickly induced a large amount of new dentin-like tissue when applied as direct-capping material onto the exposed pulp tissue of permanent molar teeth in adult miniature swine. The pulp wound showed features of classic wound healing. Subjacent to the healing wound, a bridge of new hard tissue was formed, sealing off the wound from the healthy pulp tissue. The pulp tissue subjacent to this new hard tissue was invariably free of all signs of inflammation. Moreover, a layer of odontoblast-like cells had formed, abutting the newly formed mineralized tissue.
Nakamura et al (2002)(8), in another study designed to examine whether EMD could induce reparative dentin formation without eliciting adverse side effects in pulpotomized teeth in miniature swine. The results demonstrated the potential of EMD as a biologically active pulp dressing agent that specifically induces pulpal wound healing and dentin formation in the pulpotomized teeth without affecting the normal function of the remaining pulp. Furthermore, it was reported that growth of some bacteria including streptococcus mutans, is inibited by the presence of EMD.
Ishizaki et al (2003)(18), examined the histopathological response of dental pulp tissue to EMD used in pulpotomized teeth of mongrel dogs. The treated teeth histologically demonstrated an increase in tertiary dentin, suggesting that EMD exerts a considerable influence on odontoblasts and endothelial cells of capillaries in dental pulp tissue. These results imply that EMD used as pulp treatment material plays a role in the calcification of dental pulp tissue.
Olsson et al (2003)(19), led a study on the effect of EMD gel on experimentally exposed human pulps and registered postoperative symptoms. After twelve weeks, EMD gel demonstrated extensive amounts of hard tissue that was formed along side the exposed dentin surfaces and in patches in adjacent pulp tissue. Moreover, postoperative symptoms were less frequent.
Based on these experiments, Emdogain® gel has several potential clinical applications and shows promising results as a valuable material for use in pulpotomy procedures especially in primary dentition. However, more experimental data and further human research is needed, before emdogain® gel can be developed as a material for predictable induction of dentin formation, which seems a reasonable challenge that is worthy of investigation.
The aim of this study was to compare the clinical, radiographic and histological effect of Enamel Matrix Derivative (Emdogain®) versus formocresol on pulpotomized human primary teeth.
MATERIALS AND METHODS
This study was carried out on children served by the pediatric dental clinics, Department of Pediatric Dentistry, Faculty of Dentistry, Alexandria University. Fifteen patients with an age range of 4 to 7 years, with bilateral deep carious mandibular primary molars, were selected to meet the criteria recommended for pulpotomy, with no contraindications to pulpotomy in their medical history.
The criteria for tooth selection in this study were:
Primary molars with vital carious pulp exposures that bled upon entering the pulp chambers.
No clinical symptoms or evidence of pulp degeneration, such as pain on percussion, history of swelling, or sinus tracts.
No radiographic signs of internal or external resorption and no periapical or furcation radiolucency.
Teeth would be restorable with posterior stainless steel crowns.
The thirty selected teeth were randomly divided into two treatment groups of fifteen teeth each.
Group I (FC group): In which the pulpotomized teeth were treated with formocresol(Buckley’s, Sultan Chemists Inc., Englewood,NJ,U.S.A.) on one side of the mandible.
Group II (EMD group): In which the pulpotomized teeth were treated with Emdogain® (BIORA AB, Malmo, Sweeden) on the contra-lateral side of the mandible.
Following profound local anaesthesia and rubber dam application, a high sterile high-speed bur was used to remove the roof of the coronal pulp chamber. The coronal pulp tissue was amputated using a sterile sharp spoon excavator. Pulpal hemorrhage was controlled by moist cotton pellet.
In group I (FC group): A sterile cotton pellet lightly moistened with formocresol (1/5 conc.) then blotted, was placed against pulpal stumps for 5 min.
In group II (EMD group): Amputated pulpal stumps were covered with Emdogain® gel.
In all treated teeth, Cavit® (3M ESPE, Germany) base material was placed over treated pulps, followed by a layer of light cured glass ionomer cement. Finally treated teeth were restored with stainless steel crowns.
The children were recalled for clinical and radiographic evaluations after two, four and six months. Clinical success was judged using the following criteria: (1) No pain on percussion, (2) No abscess or fistulation, (3) No pathologic tooth mobility. Radiographic success was judged using the following criteria: (1) Presence of normal periodontal ligament space, (2) Absence of pathologic root resorption, (3) No canal calcification or periradicular radiolucency.
Fourteen carious primary canines indicated for pulpotomy were selected among teeth deemed for serial extraction. These teeth were subjected to the same pulpotomy procedures as previously mentioned in (FC group) and (EMD group).
Teeth were extracted after one week, two weeks and six months to compare and asses the response of the pulp to both formocresol and Emdogain® gel. After extraction, teeth were fixed in 4% neutral buffered formaline, then apical foramina were occluded with wax. Demineralization was performed in 5% trichloro-acetic acid. Longitudinal serial sections of (5μm) were prepared, processed and stained with Haematoxylin & Eosin and Trichrome stain. The specimens were examined under light microscope to assess histological response of the treated pulpal tissue. Each specimen was observed for pulp vitality, pulp inflammation, odontoblastic layer integrity, dentin bridge formation and calcific deposits. Series of sections containing pulp tissue were observed under light microscope equipped with a digital camera (Olympus Micro-Image, Maryland, U.S.A.) and computerized for histometric analysis(8).
Clinical and radiographic data was collected, tabulated and statistically analysed .
The clinical study consisted of fifteen patients with bilateral pulpotomized mandibular primary molars for clinical and radiographic evaluation. They were chosen according to the previously determined criteria. The mean age was 5 years ± 0.73. They were five females and ten male patients. The treated teeth comprised twenty two lower first primary molars and eight lower second molars. Teeth of both groups were checked clinically one week post-operatively. At this time, no pathological signs or symptoms were reported in any of the treated teeth.
1- Clinical results
At 2 months, on clinical examination only one tooth suffered from pain and was sensitive to percussion in the formocresol group. This accounts for (93.3%) clinical success rate. This dropped to 86.7% at 4 months. At 6 months five teeth showed pain and pain on percussion clinically, lowering the clinical success rate to 66.7%.
Emdogain® showed an overall clinical success rate of 100% at 2&4 months. Only one tooth presented with pain on percussion at 6 months reducing the clinical success rate to 93.3% .
All teeth (100%) were free from mobility, abscess formation or draining sinus at 2, 4 & 6 months among both Formocresol and Enamel Matrix Derivative (Emdogain®) tested groups (Table 1).
Table (1): Comparison between Formocresol and Enamel Matrix Derivative (Emdogain®) as regards to clinical success at 2, 4 & 6 months follow-up
P-Value of Mc Nerma
M = Months.
FC = Formocresol.
EMD = Enamel Matrix Derivative (Emdogain®).
2- Radiographic results In the formocresol group eleven teeth (73.3%) showed no pathological signs at 2 months recall. The radiographic success rate dropped to four teeth (26.7%) at 4 months recall. Two teeth only (13.3%) were still free at 6 months recall.
Emdogain® group showed radiographic success rate of 86.7% representing thirteen free teeth at 2 months recall. This success rate dropped to ten teeth (66.7%) at 4 months follow-up. Nine teeth (60%) were still free of pathological signs at 6 months.
The radiographic success rates for FC tested group at four and six months were 26.7% and 13.3%,respectively , and for EMD tested group they were 66.7% and 60% respectively . Statistically significant difference was evident between FC and EMD groups at four months recall (P=0.03). Likewise, a statistical significant difference was evident between the tested materials at 6 months recall (P=0.04) (Table 2).
Figures (1-6) shows periapical radiographs of lower second primary molars of the same patient treated with formocresol ( on the left side ) and Emdogain® ( on the right side ).
Table (2): Comparison between Formocresol and Enamel Matrix Derivative (Emdogain®) as regards to radiographic success at 2, 4 & 6 months follow-up
P-Value of Mc Nerma
M = Months.
FC = Formocresol.
EMD = Enamel Matrix Derivative (Emdogain®).
*Significant at 5% level.
Figure (1): At 2 months no radiographic changes after formocresol pulpotomy
Figure (2): At 2 months no radiographic changes after Emdogain® pulpotomy
Figure (3): At 4 months PDL widening after formocresol pulpotomy
Figure (4): At 4 months no radiographic changes after Emdogain® pulpotomy
Figure (5): At 6 months furcation radiolucency (), periapical radiolucency (), internal resorption () after formocresol pulpotomy
Figure (6): At 6 months no radiographic changes after Emdogain® pulpotomy
At 7 days Emdogain®, the amputated pulpal surface is lined by a thin nearly continuous cellular layer. Generalized congestion accompanied by an increase in angiogenesis is evident in the deeper parts of the pulp tissue below application sites. Moderate inflammatory cell infiltration is seen within the otherwise normal pulp architecture.A nearly continuous layer of odontoblast cells line dentin cells.
At 7 days Formocresol, residual formocresol line the amputated pulpal surface. Generalized congestion accompanied by moderate inflammatory infiltration of pulp tissue is seen. A discontinuous odontoblastic layer line dentin walls.
At 14 days Emdogain® the amputated pulpal surface is lined by a thin, continuous cellular layer. Increase in angiogenesis and congestion are persistent in nearly most of the specimen. Mild to moderate inflammtory cells infiltrate. One specimen few resorption lacunae in the walls of dentin.
Generalized homogenous collagenous deposition is seen within the pulpal matrix. Most of the teeth showed small islands of dentin-like tissue at different stages of mineralization. Some of these islands tend to coalesce together subjacent to the amputation site, at the junction between vital pulp tissue and amputated superficial part .
At 14 days formocresol, increase in congestion is noted in pulp tissue accompanied by mild to severe inflammatory cell infiltrate. Discontinuity of odontoblastic cell layers lining dentin walls and increase in fibrous content of pulp tissue is also noted.
At 6 months/24 weeks Emdogain®, most of the teeth showed coalescing island of dentin-like tissue trying to bridge the full width of the coronal pulp at the interface between the wounded and unharmed pulp tissue below amputation site accompanied by deposition of reparative dentin along site pulpal walls narrowing the pulp canal (Fig. 7). pulp tissue shows inflammatory infiltration.
Few teeth showed the amputated pulpal surfaces covered by a thick infiltrating layer of cellular condensation accompanied by massive deposition of reparative dentin parts of the whole pulpal wall both coronal and apical (Fig. 8a,b). Inflammatory cell infiltration is moderate in coronal pulp and decreases apically.
Only one tooth showed small coalescing dentin islands covering the surface of amputated pulp not completely bridging the site. The pulp tissue is homogenous and shows small areas revealing signs of partial degeneration. Dentin walls shows resorption lacunae covered by predentin (Fig. 9).
At 6 months/24 weeks formocresol, partial bridging of dentin is seen together with coronal pulp necrosis. chronic inflammatory infiltration, increase in fibrous content of pulp and pulpal degeneration is also seen. nearly complete absence of odontoblast cells are recognized.
Quantitative analysis of new hard tissues
When the thickness of dentin bridges formed in EMD-treated teeth after 24 weeks they were assessed by histomorphometry. The mean thickness of dentin-like tissues bridging was measured.
The results were mean thickness of dentin (mm) 0.137 ± 0.105, 0.021 ± 0.004 and 0.276 ± 0.905 in different teeth.
6 months Emdogain®
Fig. (7): Micrograph showing deciduous canines at 24 weeks after treatment with Emodogain. 110x H&E micrograph of amputated pulp showing coalescing islands of dentin-like tissue trying to bridge the full width of the coronal pulp at the interface between the wounded and unharmed pulp tissue below amputation site accompanied by deposition of reparative dentin along site pulpal walls narrowing the pulp canal.
Dentin (D), pulp (P) and dentin bridge (DB).
6 months Emdogain® (Cont.)
Fig. (8): Micrographs showing deciduous canines at 24 weeks after treatment with Emdogain®. H&E micrograph, of amputated pulp showing the amputated pulpal surface covered by a thick infiltration layer of cellular condensation accompanied by massive deposition of reparative dentin along the whole pulpal wall (a) coronally and (b) apically ().
6 months Emdogain® (Cont.)
D P PD
Fig. (9): Micrograph showing deciduous canines at 24 weeks after treatment with Emdogain®. 110x H&E micrograph of amputated pulp showing small coalescing dentin islands covering the surface of amputated pulp (). The pulp tissue is homogenous and shows signs of partial degeneration (*). Dentin walls shows resorption lacunae ( ) covered by predentin (PD). Dentin (D) and pulp (P).
Pulpotomy of the deciduous teeth is not only the most frequent endodontic treatment performed on children, but also the most controversial. Based on the amputation of the pulp chamber and the conservation of the inflammation-free root canals, the clinical results can be good, depending on the materials used. In this, formocresol remains the reference even if its clinical toxicity is still reported in literature on very controversial basis. Nevertheless, this was sufficient to trigger and stimulate a search for alternatives, and led to the proposition to use several other new materials and even most recent regenerative materials as new bases for the treatment of the pulp stumps after coronal pulp amputation(20).
In the present study, clinical success rate ranged from 66.7% to 93.3%. One tooth only showed pain through the three periodic recalls. However, two teeth showed pain on percussion at 4months follow-up and five teeth at 6 months. One tooth was considered to have failed clinically due to pain on percussion. The same tooth, however, did not present any radiographic changes. Similarly, some of the teeth, which were considered radiographic failures did not present any clinical signs and symptoms through the 6 months follow-up. Due to the small sample size in this study, a difference of one tooth could be significant. However, if the recall periods were continued longer, more cases might have started to show clinical or even more radiographic failures. Furthermore, it was reported that an 81% agreement between clinical and histological diagnosis of chronic coronal pulpitis in carious primary teeth exists. A tooth may appear to be a good candidate for pulpotomy clinically, but pulpal inflammation may not be confined to the coronal portion of the pulp only, jeopardizing the success of the procedure. The fixative properties of formocresol and the possibility of mummifying a broad zone of the remaining pulpal tissue make a tooth treated with this medicament still remain clinically successful(21).
In some clinical studies, the criteria of clinical failure went to the extreme and was determined based on the fistula formation and even buccal swellings only, ignoring moderate pain or even pain on percussion which gives rise to real difference in success results (22,23).
In the present study, radiographic success rate ranged from 13.3% to 73.3% at 2,4 & 6 months follow-up. Only two teeth of fifteen were free of any radiographic changes including periodontal membrane widening which resembles early sign of pulpal pathosis.
Almost all other studies ignored the PDL widening and considered it normal pulpal change or even questionable success but not failure. Moreover, a recent study has categorized internal resorption as radiographic success(24). In their study, Smith et al (2000)(24), claimed that since this radiographic finding does not involve osseous changes it would not affect the permanent successors. For this reason, it was proposed that internal resorption, as long as it is confined to the tooth, should not be defined as failure. Of course, this gives comparatively higher radiographic success rate. In this study, internal resorption amounted to 26.7% of pulpotomized molars which is moderately higher than previous studies that ranged from 11% to 19% of the treated teeth(25,26,27).
Periapical radiolucency was described in many studies as part of the pathological external root resorption(28). Moreover, it was reported that formocresol from the pulpotomized molars leaches into the surrounding periodontal tissue causing the radiolucency and accelerates root resorption due to chronic inflammatory reaction. This accounts for 39% to 45% of pulpotomized molar teeth that show periapical radiolucency with the presence of external resorption(25,29).
In this study, the furcation and perapical radiolucency ranged from 26.7% to 53.3% of the pulpotomized molar teeth. These results coincide with the previous studies.
Pulp calcification was observed in 13% of teeth treated with formocresol after 24 months, which is similar to frequencies reported in many previous studies (25,26,27). In the present study, only 6.7% of teeth treated with formocresol at 6 months follow-up showed calcific degeneration. This low percentage was due to the short period of this study. These results did not coincide with the findings of Fei et al(23) who reported 44% of pulp calcification after application of diluted formocresol in 27 human molars. His explanation seemed logical due to less harmful effect of the diluted formocresol, which might be keeping the pulpotomized tooth more healthy and might as well allow a degree of odontoblastic activity.
Prominent pulpal destruction was the most outstanding histological feature accompanied by fibrosis and incomplete dentin bridging. This observation is consistent with previous studies by Garcia-Godoy et al (1982)(30), and Hill et al (1991)(31), implying that the use of formocresol results in pulpal inflammation and necrosis.
Some authors consider signs of dentin bridging as an indicator of healing process(30). The severe chronic inflammation of pulp tissue accompanying formocresol will eventually proceed to pulp necrosis with or without fibrosis in almost all cases. This is consistent with the present study.
In this study, the percentage of clinical success rate of Emdogain® was 93.3% at 6 months where only one tooth out of fifteen showed pain. However, an outstanding disadvantage was the gel consistency of emdogain®, which made its application rather difficult. Moreover, it was impossible to condense any material over it. Furthermore, the amount of the gel in 0.3 ml syringe was enough for five primary molars. It was to be used within 2 hours or it would lose its effect. This entailed that any remaining quantity was discarded if not used in proper time. This, of course, should be considered when evaluating the cost effectiveness of the material.
Since scarce human clinical and radiographic information exists on the use of emdogain® on pulpotomized teeth, its comparison with other regenerative materials of different mode of action was found necessary.
The high clinical success rate of emdogain® coincides with the results reported on calcium hydroxide as pulpotomy material in deciduous teeth by Brown (1967)(32), Hannah (1972)(33), and Heilig et al (1984)(34) as the reported, clinical success rates ranging from 88.2% to 95.4%.
Massoud (2002)(35), in his study on freeze-dried bone as a regenerative pulpotomy material showed a high clinical success rate of 93.3% coinciding with that of emdogain®.
Radiographic results seemed far more promising for Emdogain® than formocresol. Periodontal membrane widening was significantly decreased in Emdogain® group. The same was true for Periapical and /or furcation radiolucencies but the difference between the two materials was not significant.
Internal resorption was not considered a radiographic failure by some authors as previously mentioned (24). Only 1/5 of the cases showed signs of internal resorption. On the other hand, no pulp calcifications were reported, which might indicate a different pattern of odontoblastic activity.
Histological Evaluation of Emdogain®
According to Nakamura et al (2002)(8), when pulp wound is exposed to EMD, substantial steps occur in a process resembling classic wound healing with subsequent neogenesis of normal pulp tissues and repair of dental pulp, i.e., rapid fibrodendin matrix formation and subsequent reparative dentinogenesis. The present study is in full agreement with Nakamura et al. At 2 weeks period, the pulp matrix itself showed homogenous fibrous deposition together with reparative dentin islands. The formation of new dentin started from within the pulp at some distance from the amputated site. There was also a marked tendency for angiogenesis in the deeper parts of the pulps indicating an increased level of cell growth and/or metabolism. After the initial phase of healing in these teeth, at later stages, a fine web of odontoblast-like cells was also observed growing from the central parts of the pulp towards the pulp chamber walls forming a dentin bridge. The EMD induced hard tissue closely resembled osteodentin early in the process, but later on the hard tissue became more like the secondary dentin. In addition, at the 6 months stage most of the treated pulp tissues revealed characteristic morphological findings of extensive reparative dentin formation along the root canal walls and the apex itself, narrowing the pulp space and extending to the coronal pulp. This is in accordance with Paine & Snead(36) and Hammarstron et al(37).
The current study revealed that the incidence of inflammation did not significantly influence the outcome of hard tissue formation. In agreement with Nakamura et al (2002)(8), non of the teeth treated with emdogain® showed signs of irreversible pulp damage, but rather resembled normal wound healing including the formation of a scab covering the amputation site, moderate inflammatory infiltrate beneath the injury and a local increase in angiogenesis and cell proliferation. All these descriptions also coincide with previous animal studies by Nakamura et al (2001)(17) on miniature swine and Igarashi et al (2003)(38), on Wistar rats.
Ishizaki et al (2003)(18) reported different results in their study on molar teeth of mongrel dogs. Congested capillaries in pulp tissue were observed in early stages. Later on, the pulp tissues revealed characteristic morphological findings of tertiary dentin along the root canal walls accompanied by atrophic pulpal degeneration. However, dentin bridge formation could not be reproduced in this study.
It is generally known that the ratio of surface area of tissue in contact with capping materials relative to the remaining tissue volume is considerably higher in pulpotomy, thus if a material can elicit unwanted side effects, it is more likely to do so in narrow pulp canals than in the wider coronal pulp(39) eliciting necrosis and / or internal root resorption. Few specimens showed internal root resorption in the present study and non showed necrosis. Few degenerative changes accompanied by root resorption were only seen in one specimen in the 24 weeks period while the rest of the pulp showed signs of total pulp regeneration.
Furthermore, a recent Immunohistochemical study by Nakamura
et al (2004)(40), demonstrated that enamel matrix proteins were present as an insoluble protein matrix in detectable amounts at the application site for about 4 weeks. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix nanospheres at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome. This can explain the changes of the whole enamel matrix as early as 2 weeks towards a favourable milieu for starting mineralization and actual formation of dentin islands. The formation of dentine islands and dentine bridge below amputation site and along dentine walls suggests that EMD treatment not only stimulates pre-existing odontoblasts, but also recruits new odontoblasts of unknown origin from the central part of the pulp through mimicking biological mediators normally active during early dentinogenesis.
Concerning the mechanism of EMD biological action, it has been reported that EMD increases the autocrine release of TGF-β and PDGF-BB, and enhances alkaline phosphatase expression of periodontal ligament fibroblasts(41). Both TGF-β and PDGF-BB are known to be associated with the onset of odontoblast differentiation. The TGF- β expressed by odontoblasts has been reported to be embedded within the dentin matrix and involved in tissue homeostasis and reparative dentinogenesis after pulp amputation(42,43). The PDGF-BB secreted by macrophages at the wound site is also reported to stimulate reparative dentinogenesis after surgical pulp exposure and direct pulp capping in rat incisor model(41).
The mechanism underlying the induction of hard tissue formation is still not known in detail. However, it has recently been reported that EMD induces an intracellular cyclic-AMP signal in mesenchymal cells. This intracellular signal is followed by secretion of autocrine growth factors and other transcription factors that subsequently increase proliferation and maturation of extracellular matrix secreting cells(44). Furthermore, EMD has been shown to induce expression of integrins in cultured fibroblast(45). Integrins are known to play important roles in mesenchymal cell development and function. The effect of EMD on mesenchymal cells appears to be general since different connective tissue cells react similarly to these stimuli(44,45). These mechanisms may thus be at play also in the EMD-induced pulpal healing.
It was also reported that growth of some bacteria including Streptococcus mutans, is inhibited by the presence of EMD(46). This restriction of bacterial growth is a clinically beneficial side-effect because most pulpotomies are performed under non-sterile conditions, i.e. in decayed or traumatized teeth. Thus EMD components may not only act as signal for induction of mesenchymal cell differentiation and maturation, but also form a stable extracelluar matrix that provide a beneficial and protective pulp environment.
The results reported in this study strongly supported the hypothesis that certain enamel matrix proteins, like amelogenins, participate in differentiation and maturation of odontoblastic cells and that these enamel matrix molecules play important role(s) during early dentin formation. When the pulp wound is exposed to EMD, a substantial amount of reparative dentin- like tissue is formed in a process much resembling classic wound healing with subsequent neogenesis of normal pulp tissue(8).
Thus EMD components may not only act as a signal for induction of mesenchymal cell differentiation, maturation and biomineralization but also forms a stable extracellular matrix that provides a beneficial and protective pulp environment.
CONCLUSION Based on the present findings, it may be concluded that Emdogain® is a bio-inductive material that is compatible with vital human tissues. It offers a good healing potential and is capable of inducing dentin formation, leaving the remaining pulp tissue healthy and functioning.
Further investigations are recommended to validate the long term human pulp tissue response to Emdogain® as pulpotomy agent in both primary and young permanent teeth, using larger samples and longer intervals.
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