The correlation between root resorption and some immune parameters in well-controlled type Ι diabetic patients during orthodontic treatment

Background: Type 1 diabetes mellitus is the result of a breakdown in immune regulation that leads to expansion of auto reactive CD 4 and CD 8 T-cells, autoantibody-producing B lymphocytes and activation of the innate immune system, so disturbances of immune system may be the cause and/or associated with diabetes mellitus. Some of these diabetic patients seek an orthodontic care. The orthodontist must remain vigilant as they may be the only health care professional seen by otherwise fit, young patients on a regular basis


Introduction
Type 1 diabetes mellitus is an autoimmune disease that involves the progressive destruction of the insulin-producing beta cells in the islets of langerhans.It is a complex process that results from the loss of tolerance to insulin and other beta-cell-specific antigens.Various genetic and environmental factors have been studied so far, but precise causation has yet to be established.Numerous studies in rodents and human subjects have been performed in order to elucidate the role of B and T cells, which determine the risk of development and progression of diabetes.These studies have demonstrated that while this disease is fundamentally a T-cell-mediated autoimmune response, the development of it results from complex interactions between the adaptive and innate immune systems, with numerous cell types thought to contribute to pathogenesis.Like any complex disease, the variation in severity and incidence of this disease can be attributed to a combination of genetic and environmental factors1,2.Some of these diabetic patients seek an orthodontic treatment which is not restricted for healthy patients.3Orthodontic treatment, like many other interventions, has inherent risks and complications; but the advantages it offers should outweigh any possible damage .One of these adverse effects is root resorption4 , which is a common iatrogenic consequence in the field of orthodontics .With this in mind, orthodontists should take all known measures to reduce its occurrence5,6.
The root resorption may be described as mild, moderate and severe.Usually it is mild and clinically insignificant; however it can occur in large amounts, i.e. loss of over one third of the root length, in some patients7.Close radiographic examination of orthodontically treated individuals show some loss of root length in nearly every patient8,9.The incisors are the most susceptible while the molars seem to be the least affected10.These orthodontically-induced root resorption areas after 7 weeks of treatment, verified histologically, are not visible in periapical radiographs.Thus, using film-based radiography, the diagnosis is uncertain during the first months of treatment.After 5-6 months a reliable radiographic diagnosis of apical root resorption can be performed11.The susceptibility to root resorption may be associated with autoimmune responses against dentine matrix protein , based on evidence that anti-dentine antibodies could be detected in experimental root lesions in mice and in traumatized patients with root resorption 12,13,14.The presence of autoantibodies may not cause root resorption ,where as autoimmune aggression occurs when the tissue antigen are accessible to specific receptors of the immune system and there are costimulatory stimuli15 .During orthodontic treatment ,the compression areas and hyaline necrosis in the periodontium may damage the cementum layer and expose the dentine matrix13 .The resulting inflammation caused by damaged periodontal tissue can result in treatment between the two study groups , there were an increase in the serum level of (IgG ,and IgA) ,and a significant decrease in the serum level of (IgM, C3,and C4 ) in the wearing group comparing with non-wearing group; moreover, a high mean of serum level of IgG and IgA in root resorption grade 1 (R1) than in grade 0 (R0) ,while low mean of serum level of IgM,C3, and C4 were reported in (R1) grade than (R0) grade.Conclusions :There were significant differences in the mean of serum levels of IgG , IgA, IgM ,C3, and C4 between wearing orthodontic appliance and non-wearing groups of well controlled type Ι diabetic patients and also between root resorption grade 1 comparing with root resorption grade 0 in the wearing group .The complement system , which activated by antigen-antibody complexes, is a complex series of some 30 proteins, twothirds of which circulate in the plasma while the remainder are present on cell and tissue surfaces.Complement components are rapidly synthesized and released into the circulation following trauma or inflammation, so its functions includes control of inflammatory reactions and chemotaxis, clearance of immune complexes, cellular activation and antimicrobial defense.21, 22  The previous studies on immunoglobulin levels in diabetic patients have revealed higher levels of IgG, IgA ,and IgM compared with those obtained from healthy controls 23,24 or between diabetic patients of different glycemic controls25, assuming that possible immuneinflammatory abnormalities were the underlying cause for the elevated immunoglobulins in patients with type 1 diabetes mellitus and its complication, and that these abnormalities might have either a role in the genesis of type Ι diabetes and its complications or may themselves be a complication thereof.24,26While in other studies, these immunoglobulins didn't differ between diabetic patients and healthy controls.27,28Other studies on other immune parameters were assessed in type Ι diabetic patients treated with insulin and the control healthy group.The complement C3 and C4, which are the major plasma proteins of the immune system complement pathways.These studies reported that synthesis of serum complement protein C3 increased in response to inflammation and metabolic disorders, diabetes C3 levels elevated in IDDM 29,30 .Zhain et al. 31 reported that serum C3 and C4 of T1DM patients is significantly lower than corresponding value of healthy subjects and the lowest value of C3 was observed in patients with HbA1c >11% .The C3 levels were tended to reduce after improving glycaemia .Insulin has potential anti-inflammatory effects 32 .Insulin closely related with C3 and plays a vital role in alteration of fasting glucose metabolism 33.While in other studies plasma levels of C3 and C4 were nearly normal during insulin treatment in type 1 diabetes.27,28; thus it is of interest to see if there is any difference in the immunoglobulins levels and complemenent components (C3, C4) in type 1 diabetic patients who wearing orthodontic appliances comparing them with non-wearing group and what will happen if these patients suffer from root resorption after 6 months orthodontic treatment.

Material and Methods
Sixty well-controlled type Ι diabetic patients(HbA1c <8.5) , who were attending to Al-mustansiriya National Diabetes Center from April to October in 2012,were participating in this study.none of these participants reported history of other autoimmune diseases or chronic inflammatory diseases, or the use of steroidal and non-steroidal antiinflammatory drugs for at least one month before sampling.They didn't have history of previous orthodontic treatment or previous trauma of dentition, and they also didn't show clinical or radiogrphical signs of periapical diseases, periapical lesions ,or root resorption before starting this study .Patients with active caries or oral mucosa lesions were excluded.Thirty cases of them(16 male and 14 females) were wearing fixed orthodontic appliance because these patients had class Ι malocclusion (crowding in the upper anterior teeth need to be treated without premolar extraction) by using straight wire fixed orthodontic appliances with 0.018×0.025inch bracket slots ,and the other thirty cases didn't wear orthodontic appliances and considered as the controls(12 male and 18 females).The mean age of study groups was( 15±1 SD)year.The degree of upper central incisor root resorption of all patients were recorded before(T0) and 6 months after(T6) orthodontic treatment through using periapical radiographs (70 kV,10 mA, exposure time 0.7 seconds) with long cone paralleling technique .The most resorbed incisor was considered for analysis .The degree of root resorption was classified using the criteria described by Malmgren et al.(1982)34.Tooth length was measured from the incisal edge to the apex.Root and crown length was measured from the incisal edge to the apex using cemento-enamel junction as the limit.Image distortion was determined by comparing the image length to the real length of a radiopaque object placed on the film, and image distortion between T0 and T6 radiographs was determined by comparing crown length .The maximum acceptable distortion was 5% .Root resorption was graded from 0 to 2, where 0=no discernible root resorption; 1=slight root resorption (less than 2mm); 2 moderate to severe resorption (more than or equal to 2 mm).The level of IgG , IgA, IgM ,C3, and C4 in serum were analyzed after 6 months orthodontic treatment.(5 Ml) of blood samples were collected from well-controlled type Ι diabetic patients of both groups by venipuncture after an overnight fast, allowed to clot ,then these blood samples centrifuged at 30 rpm for 10 minutes to obtain serum samples to asses FBS(fasting blood sugar level) and HbA1c(glycoslyted hemoglobulin)and then stored at -20˚C until using.The quantitative estimation of serum immunoglobulins ( IgG, IgA , and IgM) , and complement components (C3,C4) was done using single radial immunodiffusion (SRID) method as follows35,36:-

1-Principle of the test:
The concentration of serum immunoglobulins ( IgG, IgA , and IgM) ,and complement components (C3,C4) were measured by a single radial immunodiffusion method (SRID) in which equal volumes of reference sera and test samples were added to wells in a garose containing monospecific anti-sera.The sample diffuse radially through this gel and the substance being assayed from precipitin ring with the monospecific antisera.Ring diameter were measured and a reference curve is constructed on a graph paper.
Unknown concentration is determined from the reference standard curve .This work was done in Immunology Unit at Alkaramah teaching Hospital in October 2012.

2-The procedure:-
Before starting the procedure, the plates were opened and left for 5 minutes at room temperature for evaporation of any water (if present in the wells due to storage at 4˚C).(5µl) of each serum sample was dispensed by Hamnilton syringe into one well of each plate (containing 16 wells) for three classes of immunoglobulins and two types of complement components.The plates were left opened for (10-20) minutes then covered and left at (10˚ -20)˚C for three to four days for precipitin ring to be formed.The diameter at each immune precipitating using formed around each well was measured in millimeter by immune viewer and the concentration of each class of immunoglobulins and complement level was calculated from standard curve.All results were in mg/dl for serum concentration of immunoglobulins , and complement components.

Statistical analysis:
The data were entered and analyzed on SPSS version-20, and were summarized using frequency and proportions.Chisquare test ,T-test and paired t-test were used for assessing significance of association .P-value of equal or less than 0.05 was used as the level of significance.37,38

Results
The results of this study showed that there were no statistical significant differences between the two study groups according to age and gender as seen in Table -1.The results also showed that there were significant statistical differences between the two study groups(wearing and nonwearing groups) in the mean serum level of study immune parameters (IgG ,IgA, IgM ,C3), there were an increase in the serum level of (IgG ,IgA ) , and a decrease in the serum level of (IgM, C3,and C4 )in the wearing group comparing with nonwearing group as shown in Table -2.Regarding the statistical analysis of association between the two study groups and root resorption grades by using chi square ,it reported (36.7% ) root resorption grade 1(R1) and (63.3%) no root resorption grade 0 (R0) in the wearing group, with no root resorption (100% R0) in the non-wearing group as seen in Table -

Discussion
Disturbances of immune system may be the cause and/or associated with diabetes mellitus.Type 1 Diabetes mellitus is the result of a breakdown in immune regulation that leads to expansion of autoreactive CD4 and CD8T cells, autoantibody-producing B lymphocytes and activation of the innate immune system. 39,40he innate and adaptive immune responses are components of an integrated system of host defense in which numerous cells and molecules function cooperatively .The mechanisms of innate immunity provide an effective initial defense against infections .However, many pathogenic microbes have evolved to resist innate immunity , and their elimination requires the more powerful mechanisms of adaptive immunity.The innate immune response to pathogen also stimulates adaptive immune responses and influences the nature of the adaptive responses which consist of lymphocytes and antibodies .The adaptive immune response is initiated by the recognition of foreign antigens by specific lymphocytes which producing antibodies in response to exposure to these foreign structures .21,22 The antigen -antibody complexes activate the complement system , which is a system of serum and cell surface proteins ,that interact with one another and with other molecules of the immune system to generate important effectors of innate and adaptive immune responses .The complement system serves as an important mediator of the humoral responses by amplifying the response and converting it into an effective defense mechanism to destroy the invading pathogen , participating in the inflammatory responses ,opsonization of antigen, and clearance of immune complexes .21,22 In the present study ,it can be seen that these type Ι diabetic patients who wearing orthodontic appliance have high serum levels of IgG,and IgA, and low serum levels of IgM,C3,and C4 comparing with non wearing group and also in root resorption grade 1 (R1) comparing with root resorption grade 0(R0) , suggesting that tooth movement may be considered an inflammatory process ,thus antibodies levels of (IgG and IgA)increased ,while the level of IgM decreased due to their short life because it is the first antibody appearing in response to initial exposure to ) 15 20 ( 1 ….The correlation between 111 antigen then decreased after 5-6 days from exposure 21 .These findings agreed with exposure the patients for pathological root resorption, the presence of antibodies against dentine antigens, causing increased serum IgG, and low levels of IgM suggests that an autoimmune reaction may present and induce of primary immune response.14 The orthodontically induced inflammatory root resorption is a inflammatory process that is extremely complex and composed of various disparate components including forces, tooth roots, bone, cells, surrounding matrix, and certain known biologic messengers 41 .It is therefore not surprising to find that terms such as individual susceptibility 42,43 , genetics 44 , and systemic factors 45 are being discussed when damage is evident after otherwise successful orthodontic treatment.The degree of resorption can be very variable, highlighting the importance of individual susceptibility over and above other risk factors 46 .It was hypothesized that susceptibility to root resorption may be associated with autoimmune responses against dentine matrix proteins, based on evidence that anti-dentine antibodies could be detected in experimental root lesions in mice and in traumatized patients with root resorption 12,13,14 .Immune responses can influence the resorption of calcified tissues through interactions among immune and clast cells or through the production of cytokines and other mediators that modulate local inflammatory responses 19,20 .
Orthodontic forces induce an inflammatory cell infiltration on periodontal tissues that produce signals and cytokines for differentiation and activation of clast cells 47,48,49,13,50,51.
The chronic inflammatory process may aid the presentation of autoantigens to the immune system and the breakdown of immunological tolerance 52,53 .Leading to Migration of immunocompetent cells to the periodontal ligament, such as lymphocytes, plasma cells, and antigenpresenting cells (macrophages and dendritic cells), has been reported during orthodontic movement and stimuli for all non specific immune response including immunoglobulins and complement 54,17 .Low serum levels of C3,C4 can be also found in high level in the wearing group comparing with non wearing group and in serum of patients with root resorption grade 1 (R1) comparing with root resorption grade 0(R0),suggesting that the complement concentration can be useful in diagnosis and monitoring progress of inflammation.When low concentrations are present indicate increased consumption or decreased synthesis of the complement components.The decrease of serum levels of C3,C4 indicated that innate and adaptive immunity are declined in type 1 diabetic patients 55,56 .On the other hand, the decrease of serum C4 level pointed out the existence of immune complex diseases which clearly observed in type 1 diabetes mellitus 57

Conclusion
There were significant differences in the mean of serum levels of IgG , IgA, IgM ,C3, and C4 between wearing orthodontic appliance and non-wearing groups of well controlled type Ι diabetic patients and also between root resorption grade 1 comparing with root resorption grade 0 in the wearing group.
antigen -presenting cells16 and can also induce the expression of costimulatory molecules that favor lymphocyte activation 17which are the primary cells of the immunologic system, and developed one of the most sophisticated defense mechanisms in the biological system18; The resorption of mineralized tissues by clast cells is influenced by cytokines and costimulatory molecules produced by lymphocytes19,20.The immune modulation of T and B lymphocyte responses to dentine has been observed in other inflammatory diseases where clasts play a pivotal role, and this phenomenon induces bone destruction19,20.T lymphocytes appear in the form of two subpopulations of opposite regulative function: T-helper and T-killer of cytotoxic cells.T-helper lymphocytes stimulate the transformation of B lymphocytes into plasma cells.The plasma cells are in turn responsible for the formation of antibody which is a type of glycoprotein molecule, also called immunoglobulin (Ig) that binds to antigen, often with a high degree of specificity and affinity.Every individual has millions of different antibodies (IgA, IgG, IgM, IgD, and IgE), each with a unique antigenbinding site.Secreted antibodies perform various effectors' functions, including neutralizing antigen, promoting leukocyte dependent destruction of microbes ,and activating complement.21

Table - 1
: Distribution of study groups according to gender Mean level of study parameters in both wearing and non-wearing orthodontic appliance groups .

Table - 3
: Distribution of study groups according to root resorption grades.Radunovic V, Kvinnsland I H ,Kvinnsland S , Jonsson R .immunocompetent cells in rat periodontal ligament and their recruitment incident to experimental orthodontic tooth movement .European Jornal of Oral Sciences 1997;105:36-44.

Table - 4
: Mean of study variables according to the grade of root resorption in wearing orthodontic appliance group Kurol J, Owman Moll P, Lundgren D. Time related root resorption after application of controlled continuous orthodontic force.Am J Orthod Dentofac Orthop.1996;110:303-310.44-Harris EF, Kineret SE, Tolley EA.A heritable component for external apical root resorption in