Histological Evaluation of Hard Tissue Formation After Direct Pulp Capping With RetroMTA

The study was conducted in accordance with the tenets of the World Medical Association Declaration of Helsinki. Seven caries-free, intact, maxillary, and mandibular third molars from humans, aged 30-37 years, which were scheduled for extraction for orthodontic or surgical purposes, were included in the study. The patients received a thorough explanation of the experimental rationale, clinical procedures, and possible complications. All experimental protocols were independently reviewed and approved by the Local Ethics Committee of Pomeranian Medical University, Szczecin, Poland (approval number KB-0012/27/13).

Clinical protocol. For all included teeth, periapical radiographs were taken to exclude the presence of caries or periapical pathology. Tooth sensitivity was assessed by thermal testing (Kältespray; M\&W Dental, Büdingen, Germany) and electric sensitivity testing (Vitality Scanner Pulp Vitality Tester; SybronEndo, Orange, CA, USA). These teeth were subjected to pulp exposure, direct capping with RetroMTA, and restoration with a composite resin. Seven months later, the teeth were clinically and radiographically evaluated. Clinical assessment was performed at 1 week and at 7 months after the procedure. During clinical interviews, the patients were asked about the presence of pain, and its type and duration. Reactions to thermal stimuli were classified into three categories: 1 = normal response (pain lasting up to 10 s); 2 = an extended response (pain > 10 s); 3 = no response. An electrical test was performed using a Vitality Scanner, and was repeated three times. Seven months after the procedure the teeth were extracted and subjected to histological processing and evaluation.

Histological processing. Immediately after extraction, the teeth were immersed in a 10% neutral buffered formalin solution, and gently washed from blood and saliva. After brief prefixation (≤10 minutes), special precautions were taken to facilitate pulp tissue fixation. The roots were separated 5 mm apically to the cemento-enamel junction, using a diamond disk under copious water cooling. Then the teeth were grinded with a high speed cylindrical diamond bur under water spray in a mesio-distal or bucco-lingual plane until exposing one or more pulp horns. Photographs and radiographs were taken of each sample before successive steps. For demineralization, the specimens were immersed for 3 to 4 weeks in an aqueous solution consisting of a mixture of 22.5% (v/v) formic acid and 10% (w/v) sodium citrate, with radiographic determination of the end-point. Then all specimens were washed in running tap water for 24 hours, dehydrated using ascending grades of ethanol, cleared in xylene, infiltrated, and embedded in paraffin (melting point of 56 °C) following standard procedures. The paraffin blocks were trimmed. With the microtome set at 5 μM, serial sections were taken until exhausting the entire pulp tissue in the chamber. Six to eight sections were collected on each slide. Every fifth slide was stained with haematoxylin and eosin for screening and evaluation of mineralized tissue formation. These stained sections were used to locate the areas exhibiting the most severe inflammatory reactions, and those with less favourable bridging. Based on this initial evaluation, all slides adjacent to the location with the less favourable conditions were stained. In addition, the selected slides were subjected to a modified Brown and Brenn technique (Taylor 1966) for staining bacteria. Next, cover slips were placed on the slides, and the sections were examined using a light microscope. For each pulp, the worst histologic condition observed was recorded. If bacteria were observed on the cavity walls or in the area of exposure, the case was excluded from evaluation.

Two intact teeth (one maxillary and one mandibular third molar) extracted for pericoronitis were used as control. To test the reliability of the bacterial stain, two teeth with deep caries were examined, extracted because deemed non-restorable. These teeth were processed using the same protocol as the experimental group.

Slides were observed under a light microscope (Leica DMLB; Leica Microsystems, Wetzlar, Germany) and digital photographs were taken (Leica DFC420; Leica Microsystems, Wetzlar, Germany). Following aspects were observed with particular care: 1) Presence and type of mineralized tissues formed in the area of the surgical exposure; 2) Morphology of cells layering this mineralized tissue; 3) Presence and degree of inflammatory reactions in the pulpal area subjacent to the newly formed tissue; and 4) Presence of stainable bacteria in the experimental cavity or the area of pulp exposure.