Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

The overall goal of this protocol is to present the direct pulp-capping procedure in mice. This method can help you answer key questions in pulp biology, such as, how pulp heals, or how reparative dentin forms, when you place trapped pulp-capping materials on the exposed pulp. The main advantage of this technique is that the actual direct pulp-capping procedure performed in humans can also be done in mice, in exactly the same manner.

The implications of this technique extends towards improving material properties to save tooths that is otherwise doomed to be more invasive treatments, such as root canal therapy. After properly anesthetizing the mouse for the procedure, position the mouth holder in its mouth, and secure the other end to the table such that the head is facing upward. Using good lighting and a stereoscope, observe the first maxillary molar.

Then, using the quarter-round bur in a high speed handpiece at 200, 000 rpm, remove the central region of the tooth's enamel until the pulp is visible through the transparent dentin. Be careful to not penetrate into the pulp. Next, using a no.

15 endodontic k-file, perforate through the dentin and expose the pulp. Do not push any debris into the pulp;this can be avoided by rotating the k-file quarterly, and then pulling the k-file out. Now, prepare the MTA, and using the explorer, deliver the MTA, then pack it into the exposed region using the back side of the paper point and gentle tapping.

Next, using a syringe loaded with 35%phosphoric acid etchant, cover the tooth with the etchant, avoiding the gingival tissues, and let the etchant work for 15 seconds. After 15 seconds, suck the etchant off the tooth, and use a water-moistened cotton applicator to wipe off any remaining etchant. Suck and wipe until all the etchant is removed.

Follow by applying the dental adhesives using the backside of the paper point. Make the adhesive layer thin by blowing it with compressed air for a few seconds. Then cure the adhesive for 30 seconds with the appropriate light.

Now, place small amounts of composite onto the tooth using the tip of the explorer to flow the composite into the tooth grooves. Once applied, cure the composite for 30 seconds. After euthanizing the mouse and removing the entire maxilla, place the jaw into 4%paraformaldehyde in PBS at pH 7.4.

Keep the tissue at 4 degrees Celsius overnight, and on the following morning, transfer it to 70%ethanol for storage until it can be processed. To take a micro CT scan of the maxilla, first secure it in gauze soaked with 70%ethanol, then pack it into a 15 milliliter cell culture tube. Then mount the tube onto the micro CT scanning stage.

Next, set the x-ray source to deliver a 145 microamp current with 55 peak kilovolts for 200 milliseconds. Using a 0.5 millimeter aluminum filter, acquire images with 20 micron resolution. After the scans are completed, start decalcifying the maxilla with 5%EDTA and 4%sucrose in PBS, at a pH of 7.4.

Let this reaction go for two weeks at 4 degrees Celsius. After embedding the maxilla, make five micron thick slices. The pulp-capping areas usually coincide with the distal palatal root, which can be used as a landmark.

Determine the precise area of interest by examining the histology under the light microscope, and comparing the results to the micro CT scans. For H&E staining, remove the paraffin, and rehydrate the slides with two baths in xylene for a few minutes each. Then pass them through a series of serially diluted ethanol.

Each bath should be applied for just one minute. Follow the rehydration with a rinse. And then apply the hemotoxylin solution for two and a half minutes.

Remove the dye with a rinse, and then put the slides in 95%ethanol for one minute. Next, stain the slides with eosin solution for one minute, and then rinse them off. Reverse the hydration steps to dehydrate the stained tissues, starting with ethanol.

Then, treat them with xylene. The slides can then be mounted and imaged. Using the described procedures, pulp-capping was performed on 8-week-old mice.

Six weeks later, their maxilla were harvested and examined. Curiously, H&E staining revealed dentin formation throughout the pulp chamber and the root canals. Comparatively, the uncapped molar did not show the same dentin formation.

In the capped molar, there were characteristics of both dentin-like and bone-like formation, as both dentinal tubules, and osteocytes were found in the reparative dentin, suggesting that injured pulp may be mineralized by both residing odontoblasts and immigrated osteoblasts. Use of DMP1 immunohistochemical staining further confirmed increased activities of cells forming reparative dentin. Just like real dental clinical practices, having an assistant is extremely helpful.

For example, with an assistant, this technique can be done in just ten minutes, once mastered. Importantly, when you're exposing the pulp, remember to use endofile, not the bur. And when you're handing the MTA, be careful.

Don't forget that working with fixatives like paraformaldehyde can be extremely hazardous. Precautions such as wearing personal protective equipment should always be taken.