Evaluation of different therapeutic Carnoy's formulations on hard human tissues: A Raman microspectroscopy, microhardness, and scanning electron microscopy study

Abstract

Purpose

To evaluate different therapeutic Carnoy's solution formulations on hard human tissues.

Materials and methods

An in vitro study was performed with human teeth (n = 36) and bone fragments (n = 18), randomly divided into two experimental groups (Group I = Carnoy solution with chloroform; Group II = Carnoy solution without chloroform) and a control group (saline solution). The groups were subdivided into pre-conditioning, post-conditioning, and post-conditioning with saline washing. Raman microspectroscopy, Knoop microhardness test, and scanning electron microscopy with energy dispersive X-ray spectroscopy were used.

Results

There was demineralization of dental structures regarding mineral/matrix and carbonate/phosphate ratios (GI versus GII, p < 0.05). The presence of chloroform resulted in a statistically significant reduction of the teeth surface microhardness (p = 0.036), but not exceeding 0.01 μm. Both GI and GII showed significant structural changes by using scanning electron microscopy with energy dispersive X-ray spectroscopy.

Conclusion

Carnoy's solution altered the organic and inorganic matrix of the human calcified specimens analyzed in vitro, and its effect was more pronounced when chloroform was present.

Introduction

Benign lesions (odontogenic and non-odontogenic), but locally aggressive and with a high percentage of recurrence, may occur within the maxillofacial region. (Abou Neel et al., 2016) Conservative treatment of these jaw lesions can lead to high recurrence rates; however, a radical approach usually is followed by severe functional and aesthetic impairments (Costa et al., 2010, Pogrel, 2015). Adjunct therapies allied to surgical treatments, such as the use of Carnoy's solution or liquid nitrogen as a cryosurgical agent, have been widely used since the 1980s, aiming to improve clinical outcomes (Costa et al., 2011).

Voorsmit, Stoelinga and van Haelst in 1981 (Voorsmit et al., 1981) were the first investigators that used Carnoy's solution in intraosseous-related jaw lesions, recommending the use of this cauterizing agent in the treatment of odontogenic keratocysts, as this lesion shows important infiltration patterns and high rates of recurrence (Voorsmit et al., 1981). Since its first use in the field of surgical interventions, Carnoy's solution has also been reported in the adjunct treatment of other pathologies such as glandular odontogenic cyst (Cano et al., 2012), ossifying fibroma of the jaws (Gurol et al., 2001, Rajeshkumar et al., 2013), and ameloblastomas (Lee et al., 2004).

Based on previously published studies (Costa et al., 2010, Voorsmit et al., 1981, Hellstein et al., 2007, Cutler and Zollinger, 1933), Voorsmit established a clinical protocol for applying Carnoy's solution over a period of 5 min on the bony defect, which promoted necrosis of approximately 1.5 mm in depth (Voorsmit, 1985). Initially, the Carnoy's solution consisted of 9 ml of 95% ethanol, 3 ml of glacial acetic acid, and 1 g of ferric chloride (Hellstein et al., 2007). Subsequently, there were two important changes in its formulation: (1) presence of 6 ml of absolute alcohol instead of ethanol; and (2) addition of chloroform (Cutler and Zollinger, 1933). However, the addition of chloroform has been discussed in the literature (Hellstein et al., 2007, Dashow et al., 2013). Since 2000, the US Food and Drug Administration has prohibited its use in cosmetic products because of its carcinogenicity as observed in animal studies and a probable risk to human health (US Food and Drug Administration, 1992).

The evidence of bony penetrability and therapeutic effect have been demonstrated in experimental (Voorsmit et al., 1981) and clinical studies (Dashow et al., 2013), respectively. In some clinical situations, there is a possibility of this chemical agent covering teeth root surfaces after its application in surgical cavities closely adjacent to vital teeth. However, there is a lack of knowledge regarding the in vitro effects of Carnoy's solution, with or without chloroform, on the tooth root surface. In comparison with cryotherapy, which has yielded important data in the study by Pollan et al. (1974), which described the effects of liquid nitrogen on root teeth obtained from dogs, there is no in vitro or in vivo similar investigations involving Carnoy's solution to date.

In addition to uncertain effects of Carnoy's solution when in contact with dentin from root surfaces, possible mechanisms explaining the clinical outcomes after its use in human bone tissue remain unclear. In the field of Carnoy's solution applied in oral and maxillofacial surgery, it could be beneficial if chemical analysis was performed. For this purpose, there are specific methodologies such as Raman microspectroscopy.

In 1928, the Indian physicist Sir Chandrasekhara Venkata Raman observed and analyzed the phenomenon of light inelastic scattering through matter using a microspectroscopy technique called the Raman effect (Kann et al., 2015). Raman microspectroscopy is recognized as a valuable analytical technique for measuring the chemical composition of complex biological samples, such as biofluids, cells, and tissues. In addition, it has been considered as a modern molecular fingerprint of different substrates, providing quantitative information regarding its chemical composition (Kong et al., 2015). This methodology is considered to be a noninvasive, chemically selective modality that has been reported in oral surgery−related scientific publications (Zizzari et al., 2016, Carvalho et al., 2017; Owosho et al., 2014).

We have recently conducted physicochemical and rheological characterizations of different Carnoy's solutions (Carvalho et al., 2017); however, in vitro effects on mineralized oral tissues were not reported. Thus, the objective of the present study was to perform a novel investigation of possible physico-chemical alterations of different calcified human substrates (teeth and bone tissue) that have undergone different Carnoy's solution protocols through Raman microspectroscopy, the Knoop microhardness test, and scanning electron microscopy approaches. To date, there are no published studies that have conducted a similar investigation with distinct Carnoy's solution formulations.