Background
Dentine hypersensitivity is defined as a short, sharp pain caused by exposition of dentine tubules to thermal, tactile, chemical or osmotic stimuli, and that is not referable to any other pathology [
1]. According to the hydrodynamic pain theory liquid fluctuations caused either by rapid changes in temperature or osmolarity activate nerve endings in the pulpal tissue, what is – after centric conduction of the stimulus - experienced as pain [
2].
Epidemiologic data for the prevalence of dentine hypersensitivity generally show a broad range between 3 and 98%. The range is a result of different inclusion criteria, assessment protocols and different populations of the regarding studies [
3]. In patients that have received periodontal treatment, several predisposing factors result in an increased prevalence: In the first place, gingival recessions due to periodontal attachment loss are a typical finding in patients with periodontitis. They result after attachment loss due to inflammation of the periodontal tissues, when therapy results in detumescence of previously swollen soft tissues. The loss of the gingival collar results in exposure of the cervical tooth area, that previously had been covered by well perfused and therefore thermically isolating tissues. Secondly, repeated iatrogenic instrumentation and intense daily brushing of these areas during the periodontal maintenance therapy causes a progressive loss of dental hard tissue, since the regarding tooth area is not protected by hard-wearing enamel but by rather soft and easy abrading dental cementum and dentine. The combination of these impairments is – of course - aggravating the pain symptomatology [
4‐
6]. In particular, erratic tooth brushing techniques and usage of abrasive toothpaste, repeated exposure to erosive nutrition and lactic acid due to the bacteria-induced decomposition process of short-chain carbohydrates are additionally aggravating the affliction. As long as dentine hypersensitivity comes along without or with only minor dental hard tissue defects, the pain sensations can often be successfully treated by topical application of so-called desensitizers: Usually, varnishes cover the exposed dentine tubules and might also superficially penetrate the porous dentine surface [
7]. Then, preparations with a high wetting potential are able to penetrate the dentine tubules. Penetration is facilitated by diffusion of highly concentrated solvents like HEMA [
8] or PENTA [
9]. In the tubules, the compounds precipitate as solid particles that are supposed to bind to the inner tubule surface. Hence, tubules occlude and the evocation of a pain sensation is hampered due to a disabled liquid fluctuation. Even if modern desensitizers clinically work well directly after application, for most of the products the effect begins to fade already after several days [
10,
11]. Furthermore, the effect of products like Gluma® (Haereus Kulzer, Hanau, Germany) or ShieldForce® (Tokuyama Dental, Tokyo, Japan) relies on the precipitation of particles like hydroxyethyl methacrylate (HEMA) or triethylene glycol dimethacrylate (TEGDMA) on one hand and Bisphenol-A-glycidyl-dimethacrylate (BisGMA) on the other hand. These compounds remain embedded in the host tissues. They are, however, considered to be highly biologically effective, what might lead to serious undesired effects: In the case of BisGMA localized cytotoxic effects have been shown in in-vitro models [
12]. Even products with comparatively low toxicity like Seal&Protect® (Dentsply GmbH, Konstanz, Germany) bear the risk of allergic reactions to ingredients like methacrylates or photoinitiators like camphorquinone [
13]. Furthermore, estrogene-like activity of BisGMA [
14] and mutagene activity of TEGDMA [
15] have been suspected based on results from in-vitro studies.
For both reasons, the temporary desensitizing effect and the disadvantage of incalculable side effects, more biocompatible and longer lasting mechanisms for desensitizers are needed.
Accordingly new approaches were designed in order to regenerate dentine-like material in the tubules instead of filling them with potentially harmful substances. DentinoCer contains soluble calcium phophate bioglass diluted in a slightly alkaline gel. Due to its basic character, the saturation concentration for calcium and phosphate are kept up. According to the developper’s conception this might render new formation of apatite on the exposed root surface and in the tubules possible.
It was therefore the aim of our study to assess occlusion of dentine tubules treated with DentinoCer in a simple in-vitro-model, that simulates acid exposure as due to bacterial decomposition of short carbohydrates.
Discussion
The present study is the first to assess the effect of a topical application of a novel dentine desensitizer based of a regenerative approach using a bioglass matrix. Seal&Protect® was taken as comparing solution because of its resistance and its resistivity [
18,
19]. Products were applied on bovine dentine samples that, connected to a pulp fluid pressure simulator, were periodically exposed to lactic acid. Results show, that a homogenous layer covered the openings of previously free tubules after one application only. The tubules remained closed after repeated exposure to lactic acid over a study period of 12 d, corresponding to a total exposition time of 6 h. The assessment of specimens that had been cut in vertical direction, i.e. parallel to the direction of the tubules, showed the apposition of a superficial matrix layer of 2–3 μm on the dentine surface. Furthermore a 20–100 μm thick layer emerges within the dentine, where tubules were not optically detectable, thus suggesting their obturation with a radio-opacity similar to that of dentine. Thereby, the assessment of the effect of the treatment on the outer dentine surface was combined by an assessment of deeper dentine layers to a depth of around 50–100 μm. Though the finding, that dentine seems to show superficially obturated tubules, optical analysis may not answer the question whether obturating material is the matrix of DentinoCer or if newly formed calcium phosphate compounds have precipitated.
Likewise, it is discussable if not unfavourable orientation of the tubules might render them invisible in the reported greyish zone. Regarding this issue it is important to state, that this greyish area was not observed in deeper dentine areas, eventually leaving space for a belt with visible tubules more marginally in the dentine. Neither was the reported layer observed in one of the samples of group A (control) or B (Seal&Protect®).
In order to further assess the physical permeability, the passage of methylene blue was assessed in the study set-up, simulating pulp fluid pressure based on a previously published model bei Jungbluth et al. [
16].
Even though the study design was adapted as best possible to the clinical situation, several issues depict compromises and have to be discussed: Previous to the application of dentine desensitizers, dentine discs had been pretreated in order to open and clean the dentine tubules. Figure
1 shows that cleansing with EDTA resulted in perfect purification of the tubule lumina. Thus however, samples at baseline depicted a situation that is much worse in terms of osmotic and fluid movements as compared to the clinical situation, where tubules are at least partially filled with organic compounds, toothpaste particles and dentine detritus due to brushing [
20]. Therefore, this first study to assess the application of DentinoCer started from a more difficult baseline situation as compared to the clinical situation. This fact renders the observed complete tubules closure over several days clinically even more promising with regard to the possible desensitizing effect.
Since pre-study data showed substantial problems due to bacterial overgrowth after already few experimental days, thorough care was taken to decontaminate specimens, desensitizers and the whole tube system by which the pulp chamber pressure was simulated: The dentine discs had been exposed to a radiation of 12 kGy, while tubes, instruments and applicators which were used during the experiment had previously been gas-sterilized. Applied liquids, Seal&Protect® and DentinoCer were gamma-sterilized by a total of 23 kGy. The liquid in the tubes, which simulated the pulp fluid pressure, contained sterile water and only ultrafiltrated components. Nevertheless, first bacteria were detected on day 4 and – in higher numbers – on day 8 and 12. Especially group C, which was treated with the experimental liquid DentinoCer, showed bacterial colonization. Though the exact origin of these bacteria is not clear, airborne bacterial contamination or contamination directly by manipulation during exposition to acid is possible. Since the coloured fluid which simulated the pulp chamber pressure is one of the possible sources, different effects on the study set-up are possible: On the one hand, bacterial overgrowth in the tubes might have led to lower or even suspended pulpal fluid. On the other hand it is possible that due to bacterial growth in the surface layers the initial sealing due to application of the test liquids got hampered or that present obturation degraded due to bacterial metabolism, colonization and movement. In any case, the observation that finally only few bacteria were detected suggests a rather small effect of those microorganisms for the present investigation. If the source of bacteria was DentinoCer itself, what might still be possible despite very strong exposure to radiation [
21], a clinical effect in the oral cavity with a plethora of quickly proliferating and highly adapted bacterial species does not seem to constitute a matter of great concern – as long as no specific pathogens are harboured in the liquid [
22].
Like in every study with an optical assessment of individual samples, some inhomogeneity for SEM-imaging was found within each group. Accordingly, in the set of three samples for every group and every time point some areas showed surface morphologies, that seemed “anormal” with regard to the vastly predominating characteristics in the major part of the analyzed surface. Therefore, we found lumina of single tubules in the control group, which still harboured small particles of detritus, or a small area on a sample from group B at day 4 that had not been completely covered by Seal&Protect®. Anyhow, best efforts were taken to generally describe the vastly dominating and therefor “normal” aspects.
Furthermore, cracks were found after 12 d on several samples of the control group. Since these cracks are not filled with Technovit, they must have emerged late in the fixation process, maybe during SEM analysis due to the specific atmospheric conditions in the vacuum chamber of the microscope.
The results from the optical assessment were qualitatively supported by the findings from the spectrometric assessment: While methylene blue penetrated the untreated dentine discs and stained the water in the measure glasses in higher concentrations, staining was low for the test groups and especially for treatment with DentinoCer. Principally, the fact that the application of desensitizing agents allows for residual dentine permeability is in accordance with the respective literature [
23,
24]. The reason for the fact that the penetration maximum is at day 2–4 for the untreated discs and the discs of group B before concentrations decline to zero after day 8–12, is unknown. One reasonable explanation is a possible agglutination of the coloured medium in the tubes or in the tubules of the discs. Since in the electron microscope images no such plugs were detectable only a partial obturation of the proximal tubule areas (i.e. distant from the treated surface) beginning after day 5 is possible. On the other hand this would not explain the rising stain penetration till day 5.
The present study aimed to assess the effects of lactic acid on the treated dentine surface in order to simulate cariogenic activity, but no mechanic force was applied to simulate brushing. Since it seems likely that mechanical ablation completely removes the silicate matrix and also have an impact of obturated tubules, future studies should include repeated brushing in combination with lactic acid attack in order to show whether dentine tubules remain closed or if superficially obturating remnants will be washed out, before clinical trials test applicability and pain-reducing effect of DentinCer. Likewise, future studies should assess whether the tubules are filled by the glass matrix itself, or whether a desirable regeneration in terms of a precipitation of calciumphosphate compounds took place.