Comparative ultrastructural analysis of dentin surfaces ... Comparative... · techniques is to be...

Romanian Biotechnological Letters Vol. 22, No. 4, 2017 Copyright © 2017 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER

Romanian Biotechnological Letters, Vol. 22, No. 4, 2017 12775

Comparative ultrastructural analysis of dentin surfaces mechanically

prepared using carbon steel conventional burs and polymer burs

Received for publication, January 10th, 2016 Accepted, April 4th, 2016

IRINA-MARIA GHEORGHIU1*, VALENTINA MARASCU2,3, DUMITRU STAICU2, DACIANA ZMARANDACHE1 and PAULA PERLEA1 1Faculty of Dental Medicine, University of Medicine and Pharmacy „Carol Davila” Bucharest, Romania. 2Faculty of Physics, University of Bucharest, 405 Atomiștilor Street, 077125, Măgurele, Romania. 3National Institute for Laser, Plasma and Radiation Physics, 409 Atomiștilor Street, 077125, Măgurele, Romania. *corresponding author: [email protected]

Abstract

Adhesion of modern restorative materials to dental tissues occurs via the hybrid layer created at the interface between the filling material and the dental tissue. The physical characteristics of the remaining dentin surface through which dentinal adhesion is realized are influenced by the method of cavity preparation, basically the technique that is used to remove caries-infected tissues.The present study is conducted in vitro on a total of 20 premolar and molar human teeth which have cavities with altered dentine. Teeth were extracted from orthodontic or periodontal reasons. We used two groups of tests: in group A, the excision of the altered dentine was made by means of stainless steel burs, while in the group B polymer burs were employed. Optical microscopy and AFM (Atomic Force Microscopy) techniques were used for analyzing the prepared dentin surfaces. Using a CC (Continuum Current) plasma reactor we studied the possibility of removing the smear layer debris which is present on the dentin surface after using dental burs.The ultrastructural analysis of the prepared dentin surfaces by well-established techniques like optical microscopy and AFM showed significant differences in the morphology of the samples from the two test groups. Each method of dentine excavation determines different characteristics of its surface, thus influencing the bond strength of dental adhesives.

Keywords: carbon steel burs, polymeric burs, dentine surface, AFM, plasma 1. Introduction

The concept of minimal invasive dentistry in combination with the adhesion dentistry techniques have revolutionized the traditional preparation and restoration of carious and non-carious dental lesions. Nowadays, thanks to the use of adhesive materials, cavity preparation is radically different and the final shape is given only by the size and expansion of the dental caries itself. The removal of altered hard dental tissues represents the essential element of cavity tooth preparation and many modern methods of excavation are currently available: mechanical methods, rotating and non-rotating, chemo-mechanical methods, laser ablation (A. de ALMEIDA NEVES & al. [1]). The current perception for the caries excavation techniques is to be atraumatic and conservative and refers to the removal of all infected tissues by the caries process, without unnecessary loss of hard substance, therefore limiting as much as to affect the healthy or only slightly affected hard dental tissues (T.M. ROBERSON [2]). Adhesion of modern materials to dental tissues occurs via the hybrid layer created at the interface between the filling material and the dental tissue (K.W. HSU & al. [3]). Among

IRINA-MARIA GHEORGHIU, VALENTINA MARASCU, DUMITRU STAICU, DACIANA ZMARANDACHE, PAULA PERLEA


other factors, the hybrid layer properties are influenced by the cavities preparation technique, practically by the method of altered tissues excavation found inside the carious process S.K.MOURA & al. [4]; I.V. LUQUE-MARTINEZ & al. [5]). Mechanical rotary excavation of dental caries uses stainless steel or tungsten-carbide burs, ceramic burs or polymer burs (A. BANERJEE & al. [6]). The classical drills used for altered dentin excision are made of stainless steel or tungsten carbide. These burs are active on dental tissues, having a higher mechanical resistance to abrasion than enamel or dentin. They basically produce an uncontrolled and unselective removal of both the affected and healthy tissues. The major risk in using this type of instrumentation is over preparation and over contouring cavity preparation, followed by an increased risk of accidental pulp exposure (C.MELLER & al. [7]; M. BELOICA & al. [8]). The carbon steel and the tungsten carbide burs are used in contra-angle handpiece at conventional speed (between 1.000 - 40.000 rpm). Stainless steel burs have good abrasive capacity and give fine tactile sense to the dentist. We normally use round burs for excavation of altered dentine, while the size of the bur is dictated by the size of the caries. By contrast, polymer drills are made of polymers (polyamide) with their own hardness (Knoop 50), higher than decayed dentine (Knoop 10-40), but inferior to the sound dentine (70-90 Knoop). As such, when removing dentin tissue affected by caries process, we obtain only a limited safe removal, according to the modern principles of minimally invasive caries treatment. The polymer bur is inactive in healthy sound hard dentine. Clinical use of polymer burs has therefore significant advantages that highly recommend the technique: low working speed, reduced vibration, thus avoiding overheating of the tooth. This excavation method is painless for the patient, does not require anesthesia and provides an increased pulp protection against of the risk of accidentally pulp exposure (T. DAMMASCHKE & al. [9]; A. PRABHAKAR & N.K. KIRAN [10]). The recent second-generation polymer burs are available in three different sizes (No. 4, No. 6, No. 8), and for excavation we have to choose the appropriate size for the carious lesion. Dentin adhesion depends upon multiple factors, among which we may mention the characteristics of the dentine, the type of the used restorative material, different dental and topographic anatomical features, and the methods of achieving dental adhesion (C. VÂRLAN & al. [11]. Meanwhile, dentin surface resulted from drilling, which is involved and responsible for the occurrence of the hybrid layer, shows different characteristics by employing two types of mechanical preparation (C. VÂRLAN & al. [11]; A. BANERJEE & al. [12]). A smoother and less full of smear layer dentin surface is an advantage for achieving a superior sealing and dentinal adhesion. The dentin surface prepared with rotating mechanical carbon steel burs generally shows a homogeneous appearance, entirely covered by smear layer and also dentinal tubules with dental plugs (S. SHERAWAT & al. [13]). According to some studies, the use of the polymer burs produces an irregular dentine surface, thus a lower value of the dentinal adhesion of the dental restoration is obtained (N.R.F.A. SILVA & al. [14]). Meanwhile, an increasing number of recurrences of caries are presented, mainly by producing a thick layer of smear layer, as a consequence of the preparation of the dentin tissue (C. FERRAZ & al. [15]). 2. Materials and methods

The present study is conducted in vitro on a total number of 20 premolar and molar human teeth which have dental caries with altered dentine. The teeth were extracted from orthodontical or periodontal reasons, after the patient's consent was obtained. All teeth have carious cavities without pulpal involvement. We used two groups for the tests: the teeth included in the study were divided into two equal sets (10 teeth/set) and placed in the

Comparative ultrastructural analysis of dentin surfaces mechanically prepared using carbon steel conventional burs and polymer burs


corresponding numbered bottles group to which they belong (A1-A10 and B1-B10, respectively). After the extraction, the teeth were cleaned of soft tissue debris and brushed with a prophylactic paste for professional cleaning (Cleanic™®, Kerr). After rinsing, the teeth were immersed in a solution of chloramine T (Fischer Chemical, Fair Lawn NJ), which contains 12% chloramine active solution of distilled water at room temperature for proper infection control until the start of the study. Subsequently, altered tissue excision was performed and the teeth were then ready for microscopic investigation. The opening of the carious lesions in both groups was performed with round tungsten carbide cutters active in enamel (Bur Carbide 1558, SS White Burs™®), and by using high rotational speed (200.000 rpm). In the first group (group A), the excision of the altered dentine was made using classic stainless steel burs (Round steel bur RA6, Dendia™®) and low conventional speed (1000 rpm). Altered dentine excision was performed according to the classical principles, from the periphery to the center of the caries process. The removal of the carious dentin was performed until the hard dentin was detected. Its consistency was verified using straight dental probe. In the second group (group B), caries excavation process was performed using the second generation polymer burs (Smart Burs™® II, SS White Burs No.6) at a speed of 800 rpm. The polymer burs show a high enough mechanical hardness to achieve soft altered dentine excision, but are self-limiting, becoming inactive in the sound dentin. Altered dentine excision was performed according to the method of work for this type of instruments, from the center of the caries process to the periphery. Dentin hardness of the deepest area of the cavity was also verified by using a dental probe. 3. Experimental technique

Our millimeter size samples were obtained by cropping, by means of a superflexible diamond disk (Dental Future Systems™®, Germany). Afterwards, they were stabilized onto glass microscope slides (22 × 22 × 0.2 mm) by employing an UV polymerized acrylic resin. Regarding the experimental investigations, these were focused on determining the morphology of the surfaces of the samples after removing the carries (in the size range from a few nm up to tens of microns) (V. MARASCU & al. [16]). Therefore, for the optical microscopy we have used an Olympus™® BX51 instrument, equipped with a single-CCD color camera. The images were acquired by using reflection mode and 5x or 10x magnification values. Atomic Force Microscopy can solve surface structure down to the nanometer scale, but the great advantage is that it is not limited to conductive and semiconductive samples (as the Scanning Electron Microscopy technique). The instrument used for our measurements was a NTegra Prima™® model. The AFM images were evaluated by means of multiple surface scans (sizes 150x150 µm, 60x60 µm, 50x50 µm, 10x10 µm). We obtained the individual topographical images as well as averages for the various samples. The values of the Root Mean Square (RMS) for roughness were obtained by analyzing various sections, making use of the algorithms of the Nova AFM software. The plasma treatment of the samples was performed in a continuous current (CC) plasma reactor chamber installation experimental laboratory device. The working discharge current was 0.1A and the chamber pressure 0.1 torr. The incident angle of the plasma jet may be tuned in the range 0 - 70 degrees. Figure 1 is a Schematic representation of the cold plasma reactor chamber. Nowadays, there is an increasing attention in using plasma polymerization techniques for obtaining polymers, biopolymers and biomaterials used to the medical and pharmaceutical fields (E. GÂTIN & al. [17]; C. MIRON & D. NEDELCU [18]; C. BERLIC & al. [19]; E.ST. BARNA & al. [20]; C. MIRON & F. SIMION [21]; V. BARNA & al. [22]). During the time,



a similar installation was used in laboratory to obtain polymeric films used as substrates for liquid crystal cells that were experimentally and theoretically investigated (C. MIRON [23]; L. GEORGESCU & al. [24]; C. BERLIC & al. [25]; C. BERLIC & V. BARNA [26]).

Figure 1. Schematic representation of the cold plasma reactor chamber.

4. Results and Discussion

Samples were selected both from the sidewall and also from the bottom of the prepared cavity. After securing the samples on the microscopic glass, they were washed with saline solution and dried in oven at 39°C. The first round of investigations were made at micrometric scale by means of an optical microscope used in reflection mode. Different aspects of dentin surface were highlighted, depending on the type of the employed dental bur.

Figure 2. Optical microscopy investigations of the dentin layer. (Left) Group A sample. Optical microscope

images of the dentin surface show an irregular morphology with numerous fissures and grooves. (Right) Group B sample. Dentin surface presents a relatively smooth appearance with only small irregularities and a few cracks.

Inset represents the size scale (200 microns).

These different aspects of the dentin surface morphology are a direct result of the specific

properties of the two types of instruments we used. For samples in group A, it should be mentioned that classic stainless steel burs have a good cutting efficiency and the mechanical strength is greater than sound dentin. By rotation action during the excavation of dentine chips are broken, causing irregular and rough appearance, with multiple grooves and superficial scratches (Figure 2 Left). It is a more aggressive procedure than the dentin excavation by means



of polymer burs. The polymer burs used to perform altered dentin excision in group B have a different mechanism of action: they compress carious dentin which has a reduced hardness, to the dentin interface with normal resistance, followed by its removal. The polymer burs, being softer than healthy dentine, become inactive in contact with it. Subsequently, the samples (Figure 2 Right) show a relatively smooth appearance with only small irregularities and a few cracks compared to the ones obtained by using conventional carbon steel burs. The dentin substrate plays an essential role in the dentin adhesion mechanism, following its intrinsic characteristics: the structure itself (various structural aspects for peritubular dentin, intertubular dentin, sound dentin, sclerotic dentin etc.), surface energy, surface roughness, the presence on its surface of the "smear layer" (as a result of the dentin excavation process). The smear layer is an amorphous film composed of hydroxyapatite and altered collagen. The existence and, most importantly, the thickness of the smear layer influence the dentin surface characteristics and also the receptivity to the adhesive systems. The differences in topographic dentin characteristics were also highly visible when we investigated the samples with the Atomic Force Microscope (Figures 3, 4, 5, 6). We may notice from the AFM experiments that for sample group A (where excavation was made with the carbon steel burs) the dentinal surface presents a rough appearance, but with a very clear contoured aspect, as a result of a thin smear layer (Figures 3, 4 and 6 Left). For sample group B (excavation performed with polymer burs), the dentinal surface appearance is vaguely irregular, globular due to the presence of a massive quantity of smear layer (Figure 5, 6 Right).

Figure 3. (Left) AFM morphology and (Right) linear profile topography for a sample from group A.

Scan surface range is 150x150µm.

Figure 4. (Left) AFM 3D mapping of the surface and a selected line profile (Right) for a sample from group A. Scan surface range is 60x60µm.



Figure 5. (Left) AFM mapping and line profile (Right) for a sample from group B.

Scan surface range is 60x60µm.

Figure 6. High resolution (10x10 µm) AFM topography for a sample from group A (Left)

and a sample from group B (Right).

From quantitative point of view, these morphological differences are better revealed when taking into account the roughness RMS values that were obtained from sectional analysis measurements of our samples and the algorithms in the Nova AFM software (Figure 7).

1 2 3 4 5 6 7 8 9 100

50

100

150

200

250

300

350

400

RM

S (n

m)

Sample number from group A and B

A B

Figure 7. RMS values for roughness for two samples from group A (black) and group B (red).

The results are presented for increasing ordered RMS values. The average RMS for the entire batch A is 332 nm, while for batch B is 230 nm.



Dentinal surface roughness excavated using metal instruments (carbon steel burs) is clearly increased relative to the surface where we employed polymer burs for our samples. Fascinating new materials with particular characteristics may be obtained in a laboratory cold plasma reactor and their interaction with e.g. bi-stable phases can be extremely interesting, while the plasma treatment is demonstrated to be capable of transforming existing physical and chemical properties of cured samples (D. STAICU & al. [27]; A.L. IONESCU & al. [28]; A.L. IONESCU & al. [29]; V. BARNA & al. [30]; V. BARNA & al. [31]; V. BARNA & al. [32]; C. NASTASE & al. [33]). A particularly important application of plasma is represented by the surface treatment / curing (mechanical and biological) that can be performed for various materials placed in a plasma flux. In the same time, a more aggressive plasma treatment may induce physical and geometrical changes to the respective surface. The miniaturization process available also for close to atmospheric pressure plasma generators opens a fresh perspective for great future applications, even in the medical (including dentistry) arena (H. RAUSCHER & M. PERUCCA [34]; A. FRIDMAN & G. FRIEDMAN [35]). The electro-mechanical effect of the plasma jet (formed in particular by ions) may be further amplified if the incident angle with respect to the normal to the surface is large. This can be explained by the appearance of some supplementary compression and shear forces that lead to micrometrical scale changes at the sample surface (while enhancing non-homogeneities of the substrate). Other changes of physical properties such as adhesion forces magnitude to the surface (anchoring energy) may also be modified via plasma treatment. The plasma treatment for our samples (Figure 8) shows the influence of the plasma jet incidence angle to the sample surface; at low incidence angles the clearly noticeable effect is represented by a decrease in the surface roughness (C. BORCIA & al. [36]; G.J. HAN & al. [37]) while at incidence angles greater than 60 degrees we notice a tendency of removing the smear layer. An interesting point for the future is the understanding the way that the plasma reactor discharge power affects these processes.

Figure 8. Optical microscope investigations of a plasma treated sample from group 1. Incidence angle is 70

degrees, discharge current is 0.01A, vapour pressure 0.1 torr. (Left) Plasma treatment for 1 minute. The smear layer is removed and a small number of dentin tubules orifices can be observed. (Right) Plasma

treatment for 5 minutes. The smear layer is almost completely removed and a large number of dentin tubules orifices are exposed.

The implications of removing the smear layer are related to obtaining a hybrid layer able to provide superior adhesion of the restorative material to the dental tissue. Yet, further research investigations are required about the possibilities of clinical application of this method to remove the smear layer.



5. Conclusions Dentin sample surfaces prepared by two methods of excavation were examined by

means of the optical microscopy and Atomic Force Microscopy (AFM) techniques, and the significant morphological differences between them were highlighted. Ultrastructural different topographical characteristics are important in terms of obtaining a proper dentin adhesion, in order to achieve a proper sealing of restoration to dental tissues. The mechanical rotary method that uses carbon steel or carbide drills for the preparation of the cavities is highly utilized because it shows clear advantages: high efficiency, reduced working time, relatively low cost price of the instruments, highly proven clinical efficiency. It is very useful especially in the regions with high masticatory load, like incisal or oclusal areas. In these particular areas, the minimally invasive concept of the cavity preparation needs to meet the necessity of a proper resistance of the dental restauration (A.A. ŞTEŢIU & al. [38]). The highest risk is over-preparation of the cavity and removal of the sound tissue unaffected by the carious process (M. CORTES & al. [39]). Excavation of the altered dentin using polymer burs, which are self-limiting, is an innovative method that ensure the principle of the minimal invasive modern dentistry, implying that excision has to be strictly limited to the infected tissue (healthy dental structures must not be affected in any way during cavity preparation). Unfortunately, the use of this method/materials is still restricted, due to the increased working time required for the excision of altered dentin at low rpm, and because of the relatively higher costs associated with the disposable polymer burs. In contrast to the classic preparation with rotary carbon steel drills, the use of the polymeric burs can lead more frequently to the situation of incomplete cavity preparation. This means that more residual caries were found after using this method of dentin excavation. Also, a minimally dimension and depth of the dental cavity is needed, in order to achieve the best mechanically properties of the modern restorative materials (A.A. ŞTEŢIU & al. [40]). In some clinical cases, the use of the polymer burs only is not enough and the modern techniques can be combined to design the shape and the necessary depth for a proper restauration (A. FRĂŢILĂ & al. [41]). On the other hand, it should be mentioned that the use of the polymer burs is more easily accepted by the patient, because the associated pain is diminished and there is often no need for local anesthesia (K.L. ALLEN & al. [42]). The possibility of pulp damage and even accidentally pulp exposure is also low, even in medium and deep cavities situations (M. TOLEDANOA & al. [43]). Based on the results obtained in this study we can say that further research (both in-vitro and in vivo) about the structural aspects and clinical perspectives are necessary on the long term, for a more accurate global assessment of the advantages and disadvantages these two (conventional carbon steel burs and polymeric burs) dentin excavation methods have to offer. 5. Aknowledgements For this research article, the authors have equal contributions. We acknowledge the productive scientific discussions with Conf. Dr. Valentin Barna. The research activities were supported from the RO PN-II-RU-TE-2014-4-2412 grant. References [1] A. de ALMEIDA NEVES, E. COUTINHO, M.V. CARDOSO, P. LAMBRECHTS, V. VAN MEERBEEK.

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