A comparative histological analysis of human pulp following

direct pulp capping with Propolis, mineral trioxide aggregate

and Dycal.A Parolia,* M Kundabala,* NN Rao, SR Acharya.

Dr Fatema Yusuf.

1st MDS.

IntroductionThe maintainance of healthy and functional pulp-dentine complex is the principle of restorative dentistry. Healing is a potential characteristic of pulp tissue.

Bacterial penetration Cytotoxicity of dental materials Sensitivity of operative procedureAcc to Ingle:Direct pulp capping is defined as treatment of an exposed vital pulp by sealing the pulpal wound with a dental material placed directly on a mechanical or traumatic exposure to facilitate the formation of reparative dentin and maintenance of the vital pulp.

Compromise pulp healing

• Wide range of materials have been suggested for the dressing of the exposed pulp.Calcium hydroxide is the gold standard for pulp capping in human teeth.

Pereira JC , Segala AD Am J Dent 2000

• Some limitations with calcium hydroxide are :• presence of tunnels in the dentine barrier• extensive dentine formation obliterating the pulp chamber• high solubility in oral fluids• lack of adhesion and degradation after acid etching

Cox CF, Subay RK Oper Dent 1996.

Mineral trioxide aggregate has also shown superior performance as pulp capping agent.

MTA is of two types white and grey MTA. GMTA WMTADicalcium silicate Tricalcium silicate Tricalcium silicate Bismuth oxideBismuth oxide. Masoud P, Torabinajad. JOE Jan 2010.

Evaluation of pulp capping with MTA in monkey teeth, and illustrated superior results of MTA in comparison to calcium hydroxide. Pitt Ford et al. J Am Dent Assoc 1996.

Recently a natural product Propolis has shown to possess potent antimicrobial and anti-inflammatory properties.

Propolis is a resinous substance collected by worker honey bees from

the growing parts of trees. Eg leaf buds,trunk wounds.

Composition: • Resins - flavonoids, phenolic acids and esters- 45-55%• Waxes and Fatty Acids - beeswax and plant origin -25-35%• Essential Oils- 10%• Pollen proteins –arginine and proline • Other Organics and Minerals- 14 trace minerals, iron & zinc.• Ketones, lactones, quinones, steroids, benzoic acid, vitamins.

Materials And Methods• Thirty-six healthy human premolar teeth scheduled to be

extracted for orthodontic reasons were selected from patients ranging from 15 to 25 years old.

• All teeth were examined clinically and radiographically to assure the absence of caries, trauma, periapical and periodontal lesions.

• The procedure commenced with the administration of local anaesthetic. Teeth were cleaned using 0.2% chlorhexidine solution.

• The prepared occlusal cavities were 3.0 to 3.5 mm in occlusal depth, 4.0 to 4.5 mm in mesiodistal width and 3.0 to 3.5 mm in faciolingual width.

• Dimensions of the cavity were checked using a digital caliper.

• Pulp exposure was performed in the centre of the pulp floor by means of a sterilized round diamond bur .

• Complete haemostasis was achieved by applying gentle pressure on the exposed site with cotton pellet moistened with sterile saline. The exposure site was dried with sterile dry cotton pellet.

• The teeth were then divided into six experimental groups (n = 6). After haemostasis, the exposure site was dressed with pulp capping materials directly in contact with the pulp tissue.

• In Groups I & II, 100% Propolis powder was mixed with 70% ethyl alcohol to a thick consistency on a paper pad with the aid of plastic spatula. The mixture was carried to the exposure site in a metal carrier.

• In Groups III & V, ProRoot MTA powder was mixed according to the manufacturer’s instructions. The mixture was carried to the exposure site in a metal carrier.

• In Groups V & VI, Dycal base and catalyst were mixed on a paper pad with plastic spatulas according and the mixture was carried to the exposure site using a Dycal carrier.

• After placement of the pulp capping materials, teeth were restored with composite resin using resin modified glass ionomer cement as a liner.

• Patients belonging to Groups I, III and V were asked about the presence of postoperative sensitivity or pain over 15 days, while patients belonging to Groups II, IV and VI were asked the same over 45 days.

• Teeth from Groups I, III and V were extracted after 15 days, whereas teeth from Groups II, IV and VI were extracted after 45 days. After extraction, the apical third of all teeth were sectioned to facilitate fixation in 10% formalin.

• After this teeth were demineralized formal formic acid (90%formic acid 100ml, formalin 50 ml, distilled water 850 ml)and later washed in distilled water and then dehydrated in ascending grades of N-butyl alcohol and embedded paraffin.

• Serial sections of 6µm were cut using soft tissue microtome in a bucco-lingual plane. It was then placed on gelatin-coated slides and stained with haematoxylin and eosin.

The sections were blindly evaluated by experienced pathologists and calibrated according to the criteria described as follows:

Inflammatory cell response:Grade 1. Absent or very few inflammatory cellsGrade 2. Mild or average number less than 10 inflammatory cells.Grade 3. Severe inflammatory lesion appearing as an abscess or dense infiltrate involving one-third or more of the coronal pulp.Grade 4. Completely necrotic pulp.

Dentine bridge formation:

Grade 1. Presence of a dentine bridge directly adjacent to some portion of the medicament interface.Grade 2. Presence of a dentine bridge distant from the medicament interfaceGrade 3. No evidence of any dentine bridge formation in any sections.

The scores attributed for each group were subjected tochi-square test. A p value of <0.05 was established tostate the statistically significantly difference

• RESULTS• Histomorphologic features:• Inflammatory response on the 15th day.• In Group I 33%, • group III 83% • group V 17%. • 67% in Group I, • 17% in Group III and • 83% of group V• Inflammatory response on the 45th day• In Groups II, IV 83% • Group VI 33%

• 17% Groups II ,IV • 67% Group VI

Specimens exihibited very few and scattered inflammatory cells in the pulp

Specimens exihbited moderate infiltrate

Specimens exhibited very few and scattered inflammatory cells in the pulp

Specimens exhibited moderate infiltrate

• Differences in the inflammatory response of three different materials were statistically calculated using chi-square test and were found to be non-significant on the 15th day

(p = 0.054) and on the 45th day (p = 0.105).

Statistical analysis of samples showinginflammatory cell response

Days Material Grade 1 Grade 2 Grade 3 Grade 4 total

15 Propolis 2 4 - - 6

MTA 5 1 - - 6

Dycal 1 5 - - 6

Propolis 5 1 - - 6

45 MTA 5 1 - - 6

Dycal 2 4 - - 6

Statistical analysis of samples showingdentine bridge formation

Days Materials Grade 1 Grade 2 Grade 3 Total15 Propolis 2 - 4 6

MTA 3 1 2 6

Dycal 1 1 4 6

45 Propolis 5 1 - 6

Mta 5 1 - 6

Dycal 3 2 1 6

• Hard tissue bridge on 15th day • Group I 33% , • Group III 67%• Group V 33%

• Hard tissue bridge on 45th day• Groups II ,IV 100%• Group VI 83%

Specimens exhibited hard tissue bridge

Specimens showed dentin bridge formation

Pulp cap with dycal. No bridge formation and presence of moderate inflammation after 15 days.

Pulp cap with propolis. Formation of thin dentine bridge after 15 days.

Pulp cap with MTA. Formation of thin calcified bridge after 15 days.

Pulp cap with propolis.Formation of thick dentine bridge after 45 days.

Pulp cap with MTA.Formation of thick dentine bridge after 45 days.

Pulp cap with dycal.Formation of thick dentine bridge after 45 days.

Discussion• Pulp capping is clinically successful if the tooth functions free of

symptoms, reacts adequately to sensibility tests and has a normal radiographic appearance.

• However, the clinical criterion is inadequate for an evaluation of the long-term prognosis for teeth treated by pulp capping.

• In the present study, specimens capped with Propolis exhibited less inflammation on both the 15th day and 45th day. This could be related to the anti-inflammatory property of Propolis.

• Flavonoids and caffeic acid present in Propolis are known to play an important role in reducing the inflammatory response.

• Flavonoids and caffeic acid also aid the immune system by promoting phagocytic activities and stimulating cellular immunity.

• The stimulation of various enzyme systems, cell metabolism, circulation and collagen formation could contribute to the hard tissue bridge formation by Propolis.

• These effects have been shown to be the result of the presence of arginine, vitamin C, provitamin A, B complex and trace minerals such as copper, iron, zinc as well as bioflavonoids.

• All these factors assist in faster healing of the wound. In addition to its wound healing ability, Propolis is a good antimicrobial agent. It breaks down bacterial cell wall, cytoplasm and prevents bacterial cell division

• In the present study, Dycal was used as a conventional pulp capping material which has been a gold standard. Dycal with its lower pH may avoid major tissue damage and directly stimulate reparative dentine.

• Most of the Dycal specimens in the present study showed incomplete bridge formation with more inflammation of the pulp tissue.

• In one specimen, black particles of the capping material were detected within the phagocytes. This finding may suggest that the material is gradually dissolved. A recent study suggested that Dycal should be substituted with MTA because the latter showed less inflammation and more predictable results after pulp capping

• MTA and calcium hydroxide have a similar mechanism for hard tissue formation.

• MTA such as high alkalinity (that has been related to its bactericidal properties), excellent seal and biocompatibility.

• The results of the present study confirmed the favourable outcome of this material when compared to Dycal in terms of lack of inflammation and better quality of bridge formation.

Conclusion• In the present study, Propolis showed no statistically significant

difference in pulp response when compared toDycal and MTA as a pulp capping agent.

• The effect of various dental materials on the prognosis of pulp exposure has been evaluated in several studies. It is generally accepted that accidental pulp exposure offers more favourable prognosis when compared to carious pulp exposure.

• The therapeutic influence of pulp capping material was observed in the induction of reparative dentinogenesis, free of any supplementary chemicals or products which can mimic such an action.

Critical appraisal• The title is relevant and appropriate and indicates the topic

and focus of the study.• The author’s names are mention and correspondence address

is also mentioned.• The sample size was statistically accurate.• Blinding of subject, practitioner and investigator was carried

out efficiently.• All teeth used in this study were caries free teeth, therefore it

is difficult to compare the results of this study with those using cariously exposed teeth.

• Radiographic evaluation of the treatment should have also been included.

Cross references• The radiographic outcomes of direct pulp-capping procedures

performed by dental students: a retrospective study. Al-Hiyasat AS, Barrieshi-Nusair KM,

J Am Dent Assoc. 2006 Dec;137(12):1699-705

• The aim of the authors' study was to evaluate the outcome of direct pulp-capping procedures performed by dental students.

• The authors followed the treatment outcomes of 193 patients with 204 pulp exposures with direct pulp capping. They determined the outcome of pulp capping radiographically using periapical radiographs taken at least three years after pulp exposure.

• The outcome was considered as successful if the tooth was present and not associated with periapical radiolucency or root canal treatment; otherwise, the outcome was considered as being a failure.

• RESULTS: Overall, the success rate of pulp capping was 59.3%.The success rate of direct pulp capping was 92.2% with mechanical exposure & 33.3 % with carious exposure.

Cross reference 2• Mineral trioxide aggregate or calcium hydroxide direct pulp capping:

an analysis of the clinical treatment outcome Mente J, Geletneky B ,Ohle M. JOE 2010 May;36(5):806-13

• One hundred forty-nine patients treated between 2001 -2006 who received direct pulp capping treatment in 167 teeth. Treatment was performed by supervised undergraduate students (72%) and dentists (28%). Assessment of clinical and radiographic outcomes was performed by examiners 12-80 months after treatment.

• RESULTS: One hundred eight patients (122 treated teeth) were available for follow-up (72.5% recall rate). A successful outcome was recorded for 78% of teeth in the MTA group & for 60% of teeth in the the calcium hydroxide group.

• MTA appears to be more effective than calcium hydroxide for maintaining long-term pulp vitality after direct pulp capping.

Cross reference 3• Evaluation of Mineral Trioxide Aggregate and Calcium Hydroxide Cement as Pulp-capping Agents in Human Teeth. Maria R, Roberto H JOE 2008;34(1):1-6

• This study evaluated the histomorphologic response of human dental pulps capped with mineral trioxide aggregate (MTA) and Ca(OH)2. Pulp exposures were performed on the occlusal floor of 40 human premolars.

• After that, the pulp was capped either with Ca(OH) 2 or MTA and restored with composite resin. After 30 and 60 days, teeth were extracted and processed for histologic exam and categorized in a histologic score system.

• A lower response of CH30 was observed for the dentin bridge formation, when compared with MTA30 and MTA60 groups. Although the pulp healing with calcium hydroxide was slower than that of MTA, both materials were successful for pulp capping in human teeth.

Cross reference 4• Comparative study of in vitro methods used to analyse the activity of propolis extracts with different compositions against species of Candida A.C.H.F. Sawaya1, A.M. Palma1

Applied Microbiology 2002:35 (3) 203-207• Brazilian propolis was extracted by maceration using different concentrations of

ethanol and water. The resultant extracts were analysed by chromatographic methods. Several microbiological methods were compared to determine which one best evaluated the activity of the propolis extracts against species of Candida, with average minimal inhibitory concentration values between 6 and 12 mg/ ml.

• Agar dilution in plates showed the clearest results. These were in agreement with the chromatographic analyses, which also identified the active substances.

Although the active substances identified in this sample are typical of Brazilian propolis, their activity against Candida had not been recognized previously, demonstrating the importance of standardizing the correct combination of microbiological and chromatographic analyses.

Cross reference 5• Study of the effectiveness of propolis extract as a storage

medium for avulsed teeth Casaroto AR, Hidalgo MM

Dental Traumatology 2010;1(4) 323-331

• The purpose of the present study was to evaluate the efficacy of propolis extract in maintaining the viability of human periodontal ligament (PDL) cells, and to radiographically analyze tooth replantation and the adjacent periodontium in dogs.

• After storage in this extract Human PDL cells were incubated with the experimental media propolis, milk, saliva, Hank’s balanced salt solution (HBSS), and Dulbecco’s modified Eagles medium, and distilled water. Cell viability was determined 0, 1, 3, 6, 12, & 24 h later by colorimetric MTT assay.

• Thirty incisors from dogs were divided into two storage time blocks (1 & 3 h) and were maintained in the experimental media. HBSS served as a positive control, and dry teeth (on gauze) as a negative control.

• The replanted teeth were radiographed once per month for 6 months. The radiographic images were standardized by the shortening/lengthening factor.

• The in vitro results showed that the efficacy of propolis in maintaining functional viability of PDL cells was similar to that of milk. Propolis and milk were significantly better than controls from the 6-h time period.

• The in vivo results showed that teeth maintained in propolis medium exhibited replacement resorption with significant reduction in tooth length, similar to teeth maintained in saliva and dried teeth. This resorption was less intense with the 3-h storage time than the 1-h storage time. Conditions close to normal were found in teeth maintained in milk, similar to the HBSS control.

• Therefore, although propolis was effective in maintaining the viability of human PDL cells, resorption of the tooth replantation in dogs occurred under these experimental conditions.

Thank you.