Clearing Protocol by dr. Augusto Malentacca

TRANSPARENT SECTION A METHOD FOR TEACHING AND RESEARCH

   Dr. Augusto Malentacca

 

MATERIALS AND METHODS

The main condition to get good transparent sections is a perfect hydration of the tooth, The dentine should not get dehydrated in any steps of the process as microscopic air bubbles get into the dentinal tubules ,make dentine opaque and thus prevent us from getting a thoroughly transparent section. The best thing is to work on freshly extracted and, if possible, vital teeth, which should be preserved in water immediately after extraction,  then  washed with a mild peroxide solution, scaled  and cleaned under running water and finally preserved in 2% glycerol-formaline solution. The teeth are then placed on a large Petri dish and dried with absorbent paper  to remove the excess  glycerol. They are then brushed with a 30% alcohol solution and under running water the pulp cavity is opened removing the whole pulp. (fig.3) Once this step is completed, the teeth will be soaked into a solution of 0;5%  blue anilin   and 8% citric acid for about 10 min.

If you have accurately cleaned the root surface, the staining will penetrate into the canal system and highlight pulp tissue foramens (fig.2) .At this point, paying great attention not to allow the teeth to dry out, we will study the anatomy of the root surface with the microscope and decide how to cut it to better highlight the structures we are mostly interested in.

The next step consists in wearing the roots (fig 12,15) following the direction of the canal, taking great care to include it well in the section. The canals often have a double curvature on two different planes and therefore in the end our sections will prove to be curved too. This is certainly the most delicate phase of the whole process. In fact, if the cut happens to touch the canal in any way, the section cannot be used, furthermore the amount of dentine that will be worn within the canal by the instruments during preparation must be calculated in order to prevent the section from perforating in an ulterior phase. One must also take into consideration that the section will undergo a further reduction at the end of the staining phase.

After this first rough-shaping of both the roots and crown, the teeth are soaked in distilled water and then dyed with one of the many  coloring methods for pulp tissue and root cement(e.g. Mallory mix 1) for 2 or 3 days according to the thickness of the section.

  The section   is dyed and then washed under running water and finished first with a fine-grain drill for turbine up to a thickness of 2-3 mm, and then polished with abrasive disks mounted on a contrangle always under running water. The grain progressively becomes finer and finer.

The section is then washed with distilled water. Water is then replaced by alcohol and the section is soaked in alcohol solutions at progressively increasing concentrations (30%,50%,80% and 90%) then it is left in pure alcohol until the staining in the dentinal tubules is totally removed leaving dentinal tissue finally transparent and pulp tissues a reddish-brown color.

At this point, the sections are transferred to Petri capsules and soaked in pure alcohol to better analyse their anatomy with a stereomicroscope and then place their root in an appropriate position.

The sections are then transferred   to methylbenzoate for some days until the dentine becomes totally transparent and then to xilene working under the hood to avoid highly toxic vapours.

The xilene has a very fast evaporation and it is very easy for the section to dry in this phase

So you have to remove this solution  very quickly  with absorbent  paper and immediately fix with resin for inclusion  or attack glue  on the slide for histology  covering with it all the section

Before inclosing in the  resin, it is necessary to scout the canal with 0.8 or 10   file and leave it there with the tip  1 or 2 mm out of the  apex. Both pulp cavity and apex are covered with Vaseline to prevent any resin penetration into the canal and thus a difficult if not impossible  utilization of the section.

The section is now ready and can be used both for educational and experimental purposes, we can then observe how instruments work in actual conditions.

These sections are  extraordinary educational tools as they reproduce  the best conditions to watch canal preparation, check the filling phases, note any  mistakes and possible complications which may occur in human teeth. (fig.11)

In retreated cases these sections allow you to observe high risk conditions for instrument separation (fig. 12) and test the most effective strategies to remove broken instruments.

In endodontic surgery they offer the possibility to see how the different instruments work in retrogade preparation and how materials behave in the different filling phases.

 

 

fig 1  artificial plastic canals

 fig 2  apex with foramens picked out by coloring

 Fig 3

In this section it is clear  why one must open the pulp cavity as the staining only penetrates into the canal by a few mm if we do not create this opening

Fig 4  

A root in which we were not able to cut on a single plane therefore the section follows the natural curvature of the canal

Fig 5

The section after coloring with Mallory 1° solution 

Fig 6

Observation of cutting  planes in the sections for  better positioning on slides after the last phase of the transparency process  

Fig 7 

The section after cutting is left with a thickness of at least 2-3 mm to include the whole  canal

Fig 8 efficiency of the different canal preparation techniques in a canal with an apical foramen

the size of a 45 file

a) b)  inconsistency between the rotary instrument (point taper 20) and the apical portion of the canal.

c)      apical deformation due to preparation; the rotary instrument was pushed beyond the apex

d) e)  manual preparation of the apical portion

f)       the filling shows the two different types of canal taper: 0.12 in the mid-portion prepared with  a rotary instrument and 0.05 in the apical portion negotiated manually.

Fig 9

a)b)c) action of the rotary instruments in ellipsoid-shaped canals; the instruments negotiates a preferential path in the canal and follows it till the end of preparation leaving large areas untouched; they will then have to be finished manually.

d)e)f) the same problems in these next sections. The unremoved staining shows an unclean area very close to the apex and in coronal third of the canal.

Fig 10 the combined effect of instruments and irrigant in cleaning the canal

A) b)  the section was dyed with a coloring which changes from red to yellow on contact with sodium hypoclorite. In these first two sections, canals were prepared with rotary instruments. The areas of the two main canals which have changed colour are clearly apparent but a large portion between the two has not been negotiated either by instruments or by hypoclorite.

C) d)  manual instrumentation with a stainless steel 10-taper file. The pre-curved instrument reaches even the most inaccessible areas of this canal and thus brings the irrigant into contact with the staining and changes its colour

E)      filling phase

F)       the x-ray shows that the irrigant has reached the areas negotiated by instruments during the cleaning phase. The filling material has penetrated, too, sealing all the endodontic space.

Fig 11 checking efficiency of ni.ti rotary instruments in particularly stressing situations

a)    An apex with a very small bending radius which can be easily probed with a precurved hand instrument

b)    The rotary instrument undergoes  excessive pressure and perforates the root.

c)    Post-filling view of both apex and perforation

d)    Canal with  nearly a 90° angle of curvature and a wider bending radius apical preparation is carried out manually

e)    During the preparation with ni-ti rotary instruments. Here the instruments fail to negotiate beyond the curvature

f)     Continuation with rotary instruments creates a ledge.  The path can be found only with precurved steel file

       

G)      Curvature at the opening of the canal. Preparation of the opening orifice with gates.

H) i)   Canal preparation with rotary instruments. At the end of preparation the curvature has been rectified because the largest and least flexible part of the instrument works in the coronal third and does not follow the natural path of the canal

Fig 12 typical bifurcation risks for ni-ti instrumentation

A) b)  scouting bifurcation with a 10 file

C) d)  after having swiftly and appropriately prepared the straightest  canal, in the final phase  the instrument engaged the untreated  part of the bifurcation and inevitably the point breaks.