Clearing a tooth to expose internal anatomy

Dr. Craig Barrington demonstrates the steps behind a new technique that clears a freshly extracted tooth to reveal its internal anatomy

Clinical-BarringtonquoteA clear understanding of the root canal anatomy is a prerequisite for conventional endodontic procedures. A consistent level of success in treatment depends on a significant understanding of the root canal anatomy and its morphology.


Current techniques

One way to gain an understanding of the internal anatomy of human teeth is through clearing or diaphonization. Clearing is a histologic term where a specimen or portion of a specimen is rendered transparent. Diaphonization is a term made familiar with clearing of entire or whole specimens or even entire animals. Teeth fall somewhere between both terms; they are small and an entity of their own, but they are also part of the entire human body. With that, the terms are interchangeable depending on the culture or location. With teeth, in order to expose the internal anatomy, using a conventional clearing/diaphonzation method, dyes, or stains must be injected into the former pulp canal space to reveal or expose the internal structures.

To reveal the internal structure of a tooth using the dye or stain injection method, the pulp canal space is accessed creating an injection site wherein the dye or stain can be inserted to reveal the internal structure. This penetration may damage or alter the internal structure, and can create artifacts associated with what would have been the internal structure. The amount of internal structure revealed is based on the pressure used during the injection process and using a stain or dye, for this cannot only be inaccurate, it can be messy and time consuming.
Craig BarringtonUS Pat. Pub. No. US1021952 to Spalteholtz teaches a method that makes it possible to inspect the internal structure or composition of bodies, whether organic or inorganic, by filling the body with a material having a refractive index that corresponds exactly as possible to the body or object being cleared. His method advocates the use of alcohol as a dehydrating agent in the preparation of the body or organ to be cleared. Of course, following this step, the specimen is then immersed in an oil of appropriate refractive index. This method has been applied and modified to human teeth in order to understand the pulpal anatomy.

One prominent method was used by Frank Vertucci, where he cleared the tooth in a process described hereinafter, and developed classifications based on the canal structure. This method for clearing a tooth involves the following steps:

  • Creating an access cavity
  • Washing the tooth with a cleaning solution
  • Decalcifying the tooth with nitric acid
  • Dehydrating using alcohol
  • Finally, immersing the tooth in methyl salicylate

Spalteholtz, Vertucci, Castellucci, and all other documented tooth-clearing methods use alcohol as the dehydrate. Vertucci injects a dye to reveal the structure. The illustration of the canal structure depends on the cleanliness of the canal and the pressure at which the dye is injected. If too much or too little pressure is used when injecting the dye, then the illustration may falsely represent the pulpal anatomy. Additionally, the tooth has to be compromised by creating an injection site, thereby possibly creating other weaknesses and distortions in the pulpal anatomy.

New technique

Clinical-Barringtonquote1However, a new process for clearing a tooth and illustrating the internal structure has now been developed. The goal of the technique was to simply have the internal anatomy of the teeth show up without the injection of any dyes or contrast medium. The internal structures of a tooth are definitively unlike the dentin containing them, so it seemed possible that these internal structures could stay visible in some capacity, yet ensure the dentin goes clear. With that, steps from current methods would have to be altered. This took years of trial and error to discover.

The first major change to current processes is to utilize freshly extracted teeth that have not been stored or cleaned in any fashion once they have been removed from the mouth. The teeth are simply extracted from the socket and taken immediately to a container to dry for 24 hours. The teeth are not treated or washed in any fashion with NaOCl or other chemicals as recommended in other current methods. So, this is the first major alteration from all the current techniques. It is imperative that any periodontal ligament or blood remnants be left to dry on the outside of these samples.

Once the tooth has been dried in its untreated state, it is then decalcified with an acid. After having experimented with many different acids and acid concentrations, it is currently believed that 5%-7% HCl has provided the best final results. Improvements still need to be made with the acid choices and concentrations, but the best results have been achieved with this percentage of HCl.Clinical-Barringtonquote2Clinical-Barringtonquote3The size of the tooth can and will drive the amount of time the sample spends in the acid. This can range from 12 to 24 hours. Larger teeth need to go for longer times; smaller teeth can be decalcified in a shorter period of time. There is also a bit of art to this step that has not quite been settled on yet. By X-raying the tooth, one can see if the tooth is completely demineralized. It has been found that although doing this can help, it is certainly not a definitive or absolute measure to go by.

Once the decalcification step has been completed, it is time to move to the dehydrating step. Water does not have a refractive index that matches human tooth dentin, nor does it match the refractive index of the methyl salicylate oil. Methyl salicylate is the immersion oil of choice due to its shared refractive index with dentin. With that, the water has to be removed or at least reduced in the sample for the final result to be clear. In past methods, this dehydration has been done with gradations of alcohol or simply alcohol.

It needs to be understood at this point that if you take an extracted tooth that has no contrast medium or dye and take it through these steps, the final result will be a sample that is clear, and very little to no internal anatomy of any kind will be visible. This is why dyes, inks, and other materials are injected in to the canal systems — it is so that the internal structures will be visible in some capacity.Clinical-Barringtonquote4In the attempt to completely understand the recipes and formulas and what was actually happening, it became apparent that after demineralizing the teeth, internal anatomy was visible (Figures 1-4), but after placing specimens in alcohol, the internal anatomy would disappear. It was a simple deductive step to consider removing the alcohol as the dehydrating agent to see what would happen. After a short search, it was discovered that by simply placing the tooth in items such as cat litter, silica sand, silica gel, or even calcium carbonate, dehydration would occur but not necessarily eliminate the internal structures of the tooth.Clinical-Barringtonquote5Clinical-Barringtonquote6Clinical-Barringtonquote7This has become the second critical, key, and important deviation from all other methods. Without this change, the internal anatomy available will be ruined and/or altered by the typical alcohol step of other methods.
Drying the specimen via a non-alcohol-drying agent accomplishes two things. First, alcohol is not present to break down and destroy any internal blood or pulpal remnants. Second, it provides the opportunity to allow air to replace any voids. Air injection into canal systems exposes the internal anatomy quite well and provides a nice contrast medium. As with all clearing and Clinical-Barrington8diaphonization methods, some shrinkage of the specimen will occur, having the effect of enlarging the internal areas of the tooth where the pulpal complex resided. If alcohol is used, it will most likely destroy the pulpal remnants. It also replaces any air with fluid and therefore obliterates any chance of seeing the internal anatomy via air entrapment. Exposing the anatomy via air entrapment in the former pulp canal space is not only quite accurate, it is a convenience offered by using the non-alcohol based drying agents

Methods that use alcohol as the drying agent use multiple concentrations of the alcohol over a time frame that can involve a couple of weeks. With the non-alcohol drying method, most of the dehydration takes place in about a 2-hour time frame, so non-alcohol based drying agents accomplish the task of dehydrating a sample much more rapidly and efficiently than alcohol-based drying methods.

Clinical-Barrington9Clinical-Barrington10 The larger the volume of the specimen, the more dehydration the sample will require; and therefore, some samples need to take longer than 2 hours. Four hours seems to be the maximum a tooth needs to spend in the dehydrating agent at any one time for this step to be accomplished. Again, there is some art to the method, and what is ideal is not yet fully understood.

The main thing, and the additional benefit to this step, is that the dehydration occurs rapidly with relation to the other established methods. The key is to leave the sample as long as necessary to adequately dry it based on its overall existing volume, but at the same time, leave it as short as possible to minimize overall distortion of the sample from its original state. Like all methods, after dehydration has occurred, the specimen is placed in the methyl salicylate oil where it can become fully clear to allow the internal anatomy to be observed.

This process overcomes the shortcomings of other histologic and diaphonization methods in that it provides a full look at the internal anatomy of a human tooth from pulp chamber to apical region. It does this in three-dimensional fashion for the observer and in high detail. It has been said, and demonstrated repeatedly, that the “microanatomy” is exposed, which cannot be exposed or seen in micro CT-type scan methods due to lack of detail in the resolution of that particular method. Additionally, the thoroughness of the anatomy exposed surpasses the dye/medium injection methods, which can create artifacts through overinjection, or can leave anatomy unseen through under injection.

Instead, the claimed method uses the dried blood and/or pulpal remnants internal to the tooth to illustrate the canal structure after the clearing has been completed. It provides a very accurate depiction of the internal structure and a cleaner method for processing the tooth. The present innovation fulfils a need for illustrating the pulpal anatomy by providing a method that is efficient, with a highly detailed final result.

The process

The following six steps describe the preferred process for clearing a tooth:

  1. Tooth collection
  2. Drying
  3. Decalcification
  4. Dehydration
  5. Clearing
  6. Storage

Tooth collection

Collecting a tooth
Collecting a tooth may be the initial step in the process. The tooth can be freshly extracted from a donor, or it can be a tooth that has been previously extracted but stored in such a manner so as not to destroy the internal anatomy through the method of storage. One such method of storage is freezing, which maintains the freshly extracted tooth in its original state. Once a clinician is ready to complete the process, he/she would remove the tooth from frozen storage and allow it to reach room temperature, at which time both the preferred freshly extracted tooth or a stored tooth would be subjected to the next step in the process: the drying step.

The preferred next step in this process can be to dry the tooth by exposing it to ambient air. The speed at which the tooth dries depends on various environmental conditions such as temperature, humidity, pressure, and the internal structure and contents of the pulp chamber. Typically, a tooth will take 24 hours to dry under normal room conditions. The speed at which drying occurs can be changed by increasing the room temperature to reduce the drying time required or decreasing the drying speed by lowering the room temperature.
Additionally, a tooth may be dried by other methods, including (but not limited to) mechanical drying devices, such as an oven or blow dryer. After the tooth is dried, it may proceed to the decalcification step.

To decalcify the tooth, it should be exposed to a decalcifying solution to remove the calcification that may prevent clearing. A clinician may select a decalcifying solution from a family of strong acids, weak acids, and chelating agents. Examples of these types of decalcifying solutions include nitric acid, hydrochloric acid, formic acid, and the decalcifying solution known as Decalcifier Solution II.

Clinical-Barringtonquote11Additionally, the type of solution that may be used will depend on the speed and depth of the decalcification of the tooth desired by the clinician. The strength of the decalcifying solution determines the speed at which the solution will decalcify the tooth. To decalcify the tooth quickly, a strong solution may be used; however, there are risks associated with the strong solution wherein it may decalcify so quickly that it exceeds the depth of the decalcification desired, and inevitably, it can damage the tooth. In such a case, the strong solution may be diluted to control the speed or rate of decalcification.

Furthermore, if the solution is too weak, it may never achieve the desired level of decalcification for this step and may hinder the clearing of the tooth, thus preventing the internal tooth’s anatomy from being sufficiently exposed. A preferred method of decalcifying a tooth for this process is to use the Decalcifier Solution II mentioned previously and leave the tooth in the solution from somewhere between four to 36 hours. The tooth should be checked frequently in order to determine the level of decalcification and may be removed once the tooth has reached the preferred level of decalcification. Once the decalcification is complete, the tooth may proceed to the next step in the clearing process, which is the dehydration step used to remove any excess water.

Dehydration occurs by exposing a de-calcified tooth to a non-alcohol dehydrant, thereby producing a dehydrated tooth. This step is designed to remove all or most of the water from the tooth. The clinician may use one of the many types of dehydrants available with the limitation being that the dehydrant may not contain alcohol.
Some of the types of dehydrants available are magnesium sulfate, diatomaceous earth, calcium chloride, and silica gel. The preferred dehydrant is silica sand. Typically, this dehydrant is commonly known as cat litter.
The decalcified tooth is exposed to the dehydrant for approximately 2 to 8 hours, wherein a clinician would constantly monitor the condition of the dehydration process. Once the tooth has obtained the desired level, the tooth is removed from the de-hydrant. Any dehydrant remaining should be cleaned from the sample before proceeding to the following step.

The next step in the preferred process is the clearing of the tooth. Clearing occurs when a dehydrated tooth is exposed to a clearing agent in a clear container until the desired amount of clearing occurs. The clearing agent should have a refractive index that is compatible with human tooth dentin.

Typically, the clinician may choose a clearing agent with a refractive index ranging from 1.4 to 1.7 in order to be compatible with the decalcified/dehydrated tooth structure. The preferred refractive index for clearing a tooth is 1.535. The tooth may be exposed to the clearing agent from 5 minutes to 48 hours, and the tooth will be monitored during this time and photographed at intervals selected by the clinician to capture the tooth’s internal anatomy as the clearing progresses. Under this step, the preferred time for clearing is 24 hours. However, once the maximum amount of clearing has occurred, exceeding the time of the range will not damage the tooth, unlike the dehydration and decalcification steps.

Once the tooth has completed the clearing process to the satisfaction of the clinician, it must be stored in such a way as to maintain its final condition (i.e., the level of clearing). The tooth should remain in the clearing agent indefinitely in order to maintain its translucence. If the tooth is removed from the clearing agent, it will start reverting to its former state of opaqueness.
The steps described in this article illustrate the preferred process for clearing a tooth and capturing its internal anatomy.

Author’s words

My main focus from the start was simply my interest in the actual chemical process itself. I first sought to understand what was happening, and from there I just let my imagination take over. The recent results that I have achieved have also been simply to make the work process shorter and easier, with better outcomes.
Nobody wants to inject dyes into canals — it takes a lot of work and time. So, my goal, once I better understood the chemical process, was to see if I could get the internal anatomy to stay without having to inject a dye of some kind. With that, the results delivered greater detail with less work, so there were benefits all the way.

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