Abstract
Objective: To test the hypothesis that some metalloproteinases (MMP-2, MMP-9) and inducible nitric oxide synthetase (iNOS) enzymes in dental pulp samples do not vary when subjected to orthodontic treatment.
Materials and Methods: Human dental pulps were taken from male and female patients (N=10; age 10–14 years). A straight wire technique was used with nickel-titanium or steel archwires. The increase of pressure applied on teeth was gradual. Five patients were subjected to premolar extractions after 14 months of treatment and one after 24 months. Samples were Bouin-fixed, paraffin-embedded, and afterwards processed for immunohistochemistry using anti-MMP-2, anti-MMP-9, and anti-iNOS antibodies.
Results: A reduction of MMP-2, MMP-9, and iNOS expression occurred in treated samples. This became more evident with increased treatment time.
Conclusion: The hypothesis is rejected. The reduction of expression of those proteins revealed a time-dependent relationship.
INTRODUCTION
Dental pulp is a special type of loose connective tissue characterized by few cells and abundant amorphous extracellular matrix rich in proteoglycans and glycosaminoglycans and poor in collagen fibers. Mucous tissue is very abundant during the embryonic development, whereas in adult organisms it is present only in the dental pulp, in the umbilical cord, and in the vitreous body.
The cellular types present in the dental pulp include odontoblasts, fibroblasts (which produce the components of the extracellular matrix), mesenchymal cells, and cells of the immune system (macrophages, lymphocytes, leucocytes). Vessels and nerves are also present. The main functions of the dental pulp are structural (synthesis of dentine), trophic (provides the tooth with blood and lymphatic vessels), protective (synthesis of reparative or secondary dentine), and sensory (provides the tooth with nervous innervation).
Some reports assert that the load applied to teeth may cause a loss of pulpal vitality, and others state that the pressure applied does not have these negative effects.1–3 Pulps subjected to orthodontic traction show morphologic and structural changes. There is an increase of vacuolization, extracellular matrix (ECM), and circulatory alterations with an increase of vascularization during the early treatment period and a later reduction of vessel diameter.1,4–7 Moreover, Perinetti et al2 noticed a decreased expression of alkaline phosphatase in the early phases of the treatment and reported a significant difference in the expression of aspartate aminotransferase between treated and control pulps.3 Some authors8,9 investigated the respiratory response of pulps after application of orthodontic forces. and other authors10–12 studied the activity of alkaline phosphatase and inflammatory cytokines in the mature mucous tissue. A very interesting review13 deals with the neural modulation of the inflammatory response in dental tissue after orthodontic movements.
In this study we investigated the expression of MMP-2, MMP-9, and inducible nitric oxide synthetase (iNOS) as parameters of regeneration of tissue matrix and inflammatory reaction after orthodontic treatment.
MMP-2 (matrix Mmetallopeptidase-2, gelatinase A, 72 kDa) is a matrix degradative enzyme that degrades type IV and V collagen, denatured collagen, and elastin. It is produced by several types of cell and its activity is Ca++- and Zn++-dependent.
MMP-9 (matrix metallopeptidase-9, gelatinase B, 92 kDa) is a member of the matrix metalloproteinase (MMP) family involved in the breakdown of the extracellular matrix both in physiological and pathological processes. It degrades type IV and V collagens.
iNOS (macNOS, type II NOS) is the inducible form of nitric oxide synthetase, and it is expressed by macrophages. It is activated by inflammatory signals, such as cytokines and interferon-γ, or oxidative stresses, such as exposure to hydrogen peroxide (H2O2). Few studies have been carried out to elucidate the biology of nitric oxide in dental pulp.14–16 The aim of this study is to report the effects of orthodontic treatment on the activity of these enzymes and determine whether the induced alterations are reversible or not.
MATERIALS AND METHODS
Ten patients (11 to14 years old) with Class II malocclusion and severe to moderate crowding were referred for orthodontic assessment. In four cases, the extraction was performed treatment began; in five cases, teeth were extracted after 14 months of traction. In just one case, extraction was performed 6 months after the end of 24 months of treatment. Treatment consisted of the straight wire technique using nickel-titanium or steel archwires. The treatment always started with 0.12″ nickel-titanium arches followed by wires of increasing diameters (0.14″, 0.16″, 0.18″, 0.16 × 0.16″, 0.16 × 0.22″) with the pressure applied to the teeth increased gradually. This pilot study of MMP-2, MMP-9, and iNOS expression in human dental pulp subjected to orthodontic traction was approved by the appropriate institutional review process.
Dental pulps extracted before the orthodontic treatment were assigned to one of two groups: Group A included pulps extracted after 14 months of traction, and Group B included a single case of a pulp extracted 6 months after the end of a 24-month treatment. Pulps were extracted by incising the tooth longitudinally using a thin diamond bur under a water jet. The cut stopped 2 mm before the pulp cavity to avoid pulp damage. The tooth was split with a cutter, and the pulp was observed in good shape on just one side of the split tooth. The pulp was detached with a sharp instrument and washed in saline.
The pulps were fixed in Bouin solution and paraffin embedded. The sections, 7 μm thick, were processed for immunohistochemistry with anti-MMP-2, anti-MMP-9 and anti-iNOS antibodies. Mouse anti human MMP-2 monoclonal antibody (Chemicon, Chemicon International, Temecula, Calif) (1:800 dilution), rabbit anti-mouse MMP-9 full-length polyclonal antibody (Chemicon) (1:100 dilution), and rabbit anti-iNOS/ NOS type II polyclonal antibody (BD Biosciences, Bedford, Mass) (1:25 dilution) were used. The immunohistochemistry was performed using DakoCytomation EnVision (Dako North America, Carpinteria, Calif) + System-HRP (AEC) kit from Dako, following the manufacturer's instructions. The kit used allows the antigen-antibody complex to be detected as brownish granules.
The immunohistochemical specimens were examined using a Leica Laborlux S Microscope (Leica Microsystem GmbH Wetzlar, Germany) with a Nikon DSL2 photo digital system (Nikon Corp, Tokyo, Japan). Each sample was analyzed with a double-blind system and two different operators. Moreover, the results were compared to an image analysis obtained from digital TIF files acquired with the multispectral system.17,18 To apply this method, we made sequential shots using CoKin (Cokin SAS, Rungis Cedex, France) filters to obtain all the different color spectra. Adobe Photoshop CS2 (Adobe Systems Inc, San Jose, CA) with image analysis tools was used to elaborate images.19,20 Choosing the spectrum related to AEC, we converted the image color profile from RGB to CMYK. Then we chose the yellow channel because the literature indicates that it has the best linear response to color intensity and thus to protein expression.21 The quantification of colorimetric staining has been represented using a score with values from 1+ to 5+.21–23 Numerical data obtained from image analysis has been statistically elaborated using the t-test to evaluate the significance.
RESULTS
Immunohistochemical results are summarized in Table 1 and a graphic representation of the image analysis data is provided in Figure 1.
Group A
Group A consisted of pulps not subjected to any treatment expressed MMP-2 and MMP-9 (Figure 2). The right margin of the section is represented by the odontoblastic epithelium, in which cells were positive for MMP-2 (++). Some positive cells were also found in the subodontoblastic region (+). Note that the reactivity is more intense in the small vessels that spread inside the pulp (+++). Immunoreactivity for MMP-9 showed the same features as MMP-2. Furthermore, the positive cells were inside the pulp parenchyma (Figure 3). In addition, iNOS reactivity was observed in the odontoblastic epithelium and in the subodontoblastic region, but it was a weak reactivity (+) except for a few cell clusters in the subodontoblastic region, which showed a more intense staining (++++) (Figure 4).
Control human dental pulp MMP-2; brown stain around vessel walls, 10× (arrow)
Control human dental pulp MMP-9; brown stain around vessel walls, 20× (arrow)
Control human dental pulp iNOS; group of cells positive to iNOS in the subodontoblastic region, 20× (arrow)
Control human dental pulp iNOS; group of cells positive to iNOS in the subodontoblastic region, 20× (arrow)
Group B
In Group B, immunohistochemistry showed that MMP-2 was weakly expressed by odontoblasts (+). These cells were alos on the subodontoblastic region that was positive for MMP-2 (+). Sporadic cells showed that the immunoreaction was scattered in the pulp parenchyma (Figure 5). Vessel walls were positive for MMP-2 (+++) (Figure 6). Immunoreactivity for MMP-9 was localized in the endothelium of vessel walls (Figure 7) and in the odontoblasts (Figures 8, 9), where the staining appeared particularly intense (++++). The iNOS expression was weak in most of the tissue, and its reactivity was very intense in the vessel walls (+++++) (Figure 10).
14 months traction dental pulp MMP-2; sporadic cells and vessel walls are positive; (A) Immunohistochemistry; (B) Multispectral analysis, 40×
14 months traction dental pulp MMP-2; sporadic cells and vessel walls are positive; (A) Immunohistochemistry; (B) Multispectral analysis, 40×
14 months traction dental pulp MMP-2; vessel walls are positive, 10× (arrow)
14 months traction dental pulp MMP-9; endothelial cells are positive, 20× (arrow)
14 months traction dental pulp MMP-9; endothelial cells are positive, 20× (arrow)
14 months traction dental pulp MMP-9; odontoblasts are positive 20× (arrow)
14 months traction dental pulp MMP-9; positive odontoblasts; (A) Immunohistochemistry; (B) Multispectral analysis, 40×
14 months traction dental pulp MMP-9; positive odontoblasts; (A) Immunohistochemistry; (B) Multispectral analysis, 40×
14 months traction dental pulp iNOS; weak positive parenchyma but intense staining in the vessel walls 10× (arrow)
14 months traction dental pulp iNOS; weak positive parenchyma but intense staining in the vessel walls 10× (arrow)
The Single Pulp from a Tooth Extracted 6 months After the End of the Treatment
Immunoreactivity was very weak for MMP-2 (+) (Figure 11), MMP-9 (Figure 12), and iNOS (Figure 13). Morphologic analysis of these samples showed altered pulp architecture. Vacuoles with irregular borders were visible in the tissue, and this morphology could be related to the disappearance of the remodeling activity of the MMP-2 and MMP-9 enzymes.
24 months traction dental pulp MMP-2; very weak immunoreactivity, 10× (arrow)
24 months traction dental pulp MMP-9; negativity of all structures, although very weak positivity in some vessels, 20× (arrows)
24 months traction dental pulp MMP-9; negativity of all structures, although very weak positivity in some vessels, 20× (arrows)
24 months traction dental pulp iNOS; few groups of cells are weakly positive, 10× (arrows)
24 months traction dental pulp iNOS; few groups of cells are weakly positive, 10× (arrows)
The results of the statistical analysis are summarized in Table 2. Statistical analysis indicated that MMP-2 expression showed a significant (P < .05) reduction in the odontoblastic region and in the vessel wall of treated pulps (at 14 months and 24 months of treatment) compared with the control group, but the difference was not significant in the submucosal region.
MMP-9 decreased significantly in the treated pulps (at 14 and 24 months of treatment; P < .05) both in the odontoblastic and submucosal regions. The iNOS difference of expression between the treated and the control samples was not significant in any of the areas investigated.
DISCUSSION
One patient was subjected to premolar extraction 6 months after the 24 months of traction to reduce the overjet (orthodontic camouflage). This patient refused extracts of premolars at the beginning of the treatment. However, at the end of the treatment the patient was dissatisfied with the outcome because of the wide overjet and asked for retreatment with extractions. We want to highlight the peculiarity of this case, which we have included in our study, even though it is unique. It is obvious that it is not easy to get pulps from teeth whose treatment was already achieved. This patient was a particular situation and our study was carried out using the aforementioned three dental pulps under different conditions.
In the present work we investigated the expression of MMP-2, MMP-9, and iNOS enzymes in pulp subjected to orthodontic treatment. Brackets were fitted on the teeth, and the nickel-titanium wire was inserted in the suitable slot and tied with an elastic band. All the samples were subjected to the same treatment (even if for different time periods) with the aim of reducing variability and using parameters and experimental conditions that were as constant as possible. The clinical outcome was alignment of teeth.
We based our study of dental pulps on the morphology of the tissue (hematoxylin-eosin staining), on the immunoexpression of MMP-2, MMP-9, and iNOS and on the multispectral analysis of data.
As shown in the results, metalloproteinases are expressed by the dental pulp up to 14 months into treatment. Pulps subjected to 24 months of treatment and extracted 6 months after the end of the treatment expressed a very low level of MMP-2 and MMP-9, which suggests a condition of metabolic modification. This was confirmed by morphologic observation, which shows a dental pulp with evident signs of structural modification, empty spaces within the cells and no regeneration of the ECM.
Other authors who studied the effects of orthodontic traction on dental pulp focused their attention on the mineralized tissues, for example, measuring the thickness of dentin after treatment. They observed an increase in the pre-dentin thickness corresponding with the peak of tooth movement1,24 or odontoblast degeneration.25 Other authors have revealed the presence of pulpal microcalcifications after only 1 month of treatment.26 Our results are not in agreement with data from other authors who reported that a pulp subjected to orthodontic treatment does not show any metabolic changes. However, it is possible that these results refer to orthodontic treatments with different characteristics (forces and orthodontic techniques) and thus to different dental movements. Nevertheless these data do not unambiguously demonstrate a pulp disorder whereas our data do.
All the studies performed until now are not enough to demonstrate without any doubts that orthodontic treatment could induce pulpal death. Thus, more investigations are necessary, and it could be interesting to extend these investigations to a larger number of pulps and molecules involved in the physiology of dental pulp tissue. From this point of view our study contributes to the comprehension of orthodontic treatment effects.
CONCLUSIONS
A significant reduction of MMP-2 and MMP-9 protein expression is related to the length of orthodontic treatment. Moreover, the sample of dental pulp extracted 6 months after the end of the treatment shows very low MMP-2 and MMP-9 expression and altered morphologic features, which suggests that the decrease of metalloproteinase's activity after orthodontic traction represents an impediment to the regeneration of the ECM and the restoration of dental pulp structure.
Damages caused by the orthodontic traction do not seem to be reversible.
iNOS expression, typical of inflammatory conditions, was observed both in control dental pulp and in the samples subjected to orthodontic forces; The iNOS activity in this tissue could be explained as a response to extractive stresses.