Comparison of Pulpal Response Following Pulpotomy Procedure Using Enamel Matrix Derivative

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.

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*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

A- Clinical study:

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:

1- Primary molars with vital carious pulp exposures that bled upon entering the pulp chambers.

2- No clinical symptoms or evidence of pulp degeneration, such as pain on percussion, history of swelling, or sinus tracts.

3- No radiographic signs of internal or external resorption and no periapical or furcation radiolucency.

4- 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.

B- Histological study:

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 .

results

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

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

M= Months.

FC= Formocresol.

EMD= Enamel Matrix Derivative (Emdogain®).

*Significant at 5% level.

3- Histological results

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.