Evaluation for the efficacy of haemocoagulase agent (botroclot) as a pulpotomy material as compared to ferric sulfate- an animal study.

Pulpotomy

A pulpotomy is a dental procedure that treats grossly carious primary teeth in children. This type of procedure is carried out when tooth decay reaches the pulp or there is mechanical exposure while cavity cutting. During a pulpotomy, the coronal pulp is removed and the pulp in the root canal is left intact.^1–3,6

All the functions of the radicular pulp are expected to be maintained by the pulpotomy procedure. The main objectives are managing reversible pulpal inflammation and keeping pulp in vital condition as well as maintaining the function of the radicular pulp in deciduous teeth. Many situations such as blood supply, amount of inflammation, hemorrhage control, exposure site disinfection, antibacterial efficacy and tissue compatibility of pulp covering materials and final restoration may affect the success of the procedure.⁴ The most important factor in success is maintaining the vital radicular pulp. Several materials have been used in deciduous teeth, Ferric sulphate being one hemocoagulant material tried and compared with other materials.⁴ Choosing the appropriate Pulpotomy agent is crucial.

Ideal properties of pulpotomy material are as follows⁵

Ferric sulfate and haemocoagulase (botroclot)

Ferric sulphate is extensively used in dentistry as a haemostatic agent (Astringent). It was primarily used in pulpotomy as an aid to haemostasis prior to the placement of calcium hydroxide. However, as an independent therapeutic agent, ferric sulphate pulpotomy has a success rate of 74–99%. Ferric sulphate is thought to react with the pulp tissue, forming a superficial protective layer of iron–protein complex.^2,3

Ferric Sulfate (FS) has agglutination effects due to a reaction between the blood in the pulp.¹ This reaction results in plugs that occlude the capillaries, thus control the bleeding. However, a common side effect of Ferric sulfate is chronic pulpal degradation, which can result in premature exfoliation of the pulpotomized tooth. Some studies have documented internal root resorption after FS pulpotomy.²

Botroclot is a marketed preparation containing hemocoagulase, which is an enzyme having coagulant activity, isolated from the snake botrops atrox. This formulation is used in dental surgeries and other minor surgical wounds. A commercially available hemocoagulant “botroclot” is used widely in Medicine for the stoppage of hemorrhage at wound sites. In dentistry it has been tested at extraction sites of third molars for its efficacy and compared histopathologically for its effects.³ A very small part of snake venom acquired from the snake found in Brazil “Bothrops-jararaca” or atrox which is a nontoxic systemic hemocoagulant (Botroclot). Available everywhere by various names such as Botroxobin which is a WHO accepted outcome and Botropase is a parenteral procoagulant. The formulation of “hemocoagulase agent (botroclot)” as a local application solution and its composition is (a) an aqueous mix of hemocoagulase extracted from “Bothrops atrox” or “Bothrops jararaca” 0.2 Cu/ml. (b) as a preservative “Chlorhexidine” 0.1% v/v and (c) for injection IPq.s water, as instructed in the composition booklet of the available solution bottle.

The blood clotting properties of hemocoagulase are multifaceted. The creation of fibrin monomers is hastened, then it accelerates fibrin clot creation. It triggers Xa factor and thrombin is formed at the hemorrhage site. Its action on Factor XIIIa brings about stabilization of fibrin. Articles have proposed that this parenteral formulation performs as a prohealer, epithelization is enhanced, escalates lesion tensile strength and brings collagen in the curing of the lesion too.⁶

Pulpotomy success is dependent on many factors, and the ability of the pulpotomy medicament being hemostatic is one of the most vital factors to improve success. Considering the limitations of commonly used pulpotomy medicaments for primary teeth i.e. Formocresol and the disadvantages associated with the hemostatic Ferric sulfate, a present study has thought of using hemocoagulase (“botroclot”) which has shown improved hemocoagulant properties in medical field and for healing of extraction socket wounds.⁶

Rationale

Internal resorption and furcation radiolucency was seen in pulpotomies performed with Ferric sulfate.⁷ Also the exact amount of the medicament to be placed in the pulp chamber cannot be standardized with presently available Ferric sulfate as it is available in solution form and it can be used with a cotton pellet dipped in it, where one can not quantify it. To overcome the short falls of these hemostatic pulpotomy agent, Hemocoagulase agent (“botroclot”) needs to be tested and verified as better hemostatic pulpotomy material in gel form for primary teeth.

Hemocoagulase agent (“botroclot”) has not been tried as a pulp therapy material either in primary or permanent teeth and its effect on the pulpal tissue has not been verified. It is a readily available, economic medicament that has been proved as a Heamocoagulant in other surgical fields needs to be tested in the concentration that is readily available or if it needs to be modified to make it suitable for Dental use as Pulpotomy agent.

Research gap and scarce existing literature

Ferric sulfate is tested as a pulpotomy material as it shows hemostatic qualities. It produces a metal protein mass at the pulp surface that acts as a barricade to irritating constituents of the sub-base. Although Ferric sulfate pulpotomies exhibited superior clinical success rates, it may show moderate to severe inflammatory changes histologically as shown in studies.⁸ Ferric sulfate and laser have been tested for conservative pulp therapy to assess and histological changes were compared in the pulp.⁹ It was found that the materials produced significant extraneous body and cell reactions that are inflammatory in the pulpal tissue. At 45 days follow up of all the samples, two models of laser group showed severe necrosis. Lasers are not cost effective and there is no control on penetration of the laser, which may lead to destruction of normal tissue along with the affected tissue.⁹

Modern developments in dentistry are searching for more biocompatible and effective materials as the best medicament for primary teeth pulpotomies. There is limited scientific evidence which can illustrate the comparative efficacy of botroclot versus Ferric sulfate as an effective pulpotomy material. The present proposed research will be implemented to assess the efficacy of these two agents as pulpotomy materials. The available solution of hemocoagulase agent (“botroclot”) needs to be tested for its tissue compatibility and hemocoagulant efficacy when used as pulpotomy material specially for deciduous teeth in a more suitable form to be used as chairside.

Study materials

Suitable consistency material (gel) from readily available haemocoagulase agent (botroclot) solution will be formulated. The preparation will be carried out in the research laboratory using Hydroxypropyl methylcellulose (HPMC) as excipient to change the consistency of the solution to gel.

Formulation of gel – Phase I

The weight quantity of HPMC will be dispersed accurately in purified water at 50° C. After that, 50 ml of haemocoagulase “botroclot” will be added and the mixture will be stirred continuously at a constant rate to form a uniform gel.¹⁰

In vitro tests of the prepared gel will be performed as follows.¹¹

Dose calculation

1 ml haemocoagulase solution will be equivalent to 0.2 CU (Clotting Unit)

About 5 drops used for hemcoagulase action.

1 drop = 0.06 ml, 5 drops = 0.3 ml

0.3 ml = 0.06 CU. and 0.6 ml = 0.12 CU

The prepared gel formulation will have 50 ml of hemocoagulase solution. Upon adding HPMC, the weight of the gel would be around 55 gm (55 gm = 10 CU). To have effective hemocoagulase action, 0.5 mg to 1gm of gel will be used in the study.

Comparison with other material Phase II

Animal studies will be carried out with the newly developed material and it will be compared with ferric sulfate. The animals will be anesthetized and the pulpotomy procedure will be carried out in two molars of each rat under aseptic conditions. High speed handpiece will be used to access pulp chamber in maxillary and mandibular first molar. Upon pulp extirpation, haemocoagulase agent botroclot and ferric sulfate will be placed in teeth of the rats as per groups. Tissue reaction will be checked by histopathological evaluation at 24 hours, 7 days, 15 days and 30 days.

Sources of the data

The Albino Wistar rats to be studied will be selected from the “Animal House, Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra”. For animal study, total 32 albino Wistar rats having weight 200 – 300 grams will be chosen randomly. All the animals will be divided in 2 groups and each group will have 16 animals.

Methods of collection of data

Grouping of animals and methodology

There will be two groups for animal trials which are as follows:

Group A – Hemocoagulase agent “(botroclot)” preparation group (Test group).

Group B – Ferric sulfate group.

The animals will be cared as per CPCSA guidelines of ‘Animal Ethical Committee’.

Pulp tissue reaction to the pulpotomy materials will be assessed by in vivo and in vitro methods. The follow up period will be 24 hours, 7 days, 15 days and 30 days. The teeth will be extracted at these intervals and histopathological assessment of the pulp tissue will be done.

Statistical analysis

The data will be subjected to a statistical analysis by using “Student t test” by using SPSS version 21.0 “(SPSS. Inc., Chicago, IL, USA)” with a p value set at 0.05. The statistical analysis of preclinical study results will be done using Two way “ANOVA”, Student’s paired “t-test”, Unpaired t-test, Multiple Comparison: Tukey test and Kruksal-Wallis test. A proprietary free software alternative is R or PSPP.

Dissemination

Hemocoagulase agent (botroclot) in a newer form which may exhibit good coagulant efficacy and can be utilized as a “pulpotomy” material for primary teeth. Based on clinical experiences, topical hemocoagulase may be an effective haemostatic agent after tooth extraction procedures including pulpotomy, which also reduces pain, swelling and improves wound healing. However, more clinical trials are required to explore the benefits of topical hemocoagulase solution in various dental procedures.

The hemocoagulase may be equally beneficial as a local haemostatic agent compared to other chemical haemostatic agents like ferric sulfate. The other effective agent that is ferric sulfate is a commonly used astringent solution that has multiple uses in dentistry. It is widely used in dentistry as haemostatic agent. However, the evidence about its comparative effectiveness with heamogoacgulase as haemostatic agent in pulpotomy procedure has not been widely illustrated in the existing literature.

Study status

Animal study on going in vitro component completed.

Ethical considerations

The research Protocol is approved by institutional animal ethics committee of the respective institute.

The approval number is DMIMS/IAEC/2020-21/41.