Pediatric Temporomandibular Articulation Ankylosis: OPG and RANKL Gene Expression Analysis

André da Silveira Braune1, Patrícia Cataldo de Felipe Cordeiro2, Letícia Ladeira Bonato3, Ricardo Lopes Cruz1, Jamila Perini1, Valquiria Quinelato2*, Jose de Albuquerque Calasans-Maia2 and Marco Bernardo Cury Fernandes1 1National Institute of Traumatology and Orthopedics, Brazil 2Orthodontics Department, Universidade Federal Fluminense, Brazil 3School of Dentistry, Universidade Federal Fluminense, Brazil


Introduction
Temporomandibular Joint Ankylosis (TMJA) is defined by bone and/or fibrous fusion of joint surfaces associated with limitation of masticatory function, nutritional imbalances and psychosocial changes [1][2][3]. Additionally, TMJA has a direct influence on craniofacial development due to the impairment of structural growth sites such as the mandibular condyle [4]. Specifically, craniofacial deformities are still related to dental malocclusions [3] and obstruction of airways, resulting in increased risk of recurrent infections and obstructive sleep apnea [5].
TMJA frequently occurs in the first decade of life [6] and severity varies with the degree of structural involvement [7]. The etiology of TMJA is classified as multifactorial, including facial trauma [8], poorly treated otitis [9] and neoplasms [1]. Additionally, molecular, genetic factors related to bone tissue also play an important role in the development of TMJA [7,10,11].
Bone structural integrity is established by bone remodeling processes in which synthesis and resorption occur simultaneously. This tissue homeostasis is mediated by osteoblasts and osteoclasts signaled by various molecular factors [7] such as osteoprotegerin (OPG), the kappa B nuclear factor activating receptor (RANK) and its ligand RANK (RANKL). The osteoblastic lineage regulates osteoclastic activity via the RANK/RANKL/OPG signaling pathway [12]. RANKL is produced by osteoblasts and binds to RANK present in osteoclast precursor cells stimulating differentiation and osteoclastic activity. The interaction between RANKL and its RANK receptor is controlled by osteoprotegerin (OPG), which inhibits the binding of RANKL to RANK, competing with it for the same ligand, and thus acts as a protective factor against bone resorption [7,12].
The biological basis of the etiology of TMJA is still unknown, which limits clinical practice and is commonly directed to multiple surgical treatments from childhood to adulthood [8]. Thus, the identification of genetic biomarkers associated with TMJ ankylosis and bone metabolism is a promising approach for personalized diagnosis and treatment. Currently, temporomandibular disorders and TMJA have already been related to genetic alterations of the RANK/OPG/RANKL triad in local manifestations [13] and in association with other arthropathies [14], confirming the systemic and primordial role of the molecular triad. However, the molecular and genetic mechanism associated with TMJA has not yet been elucidated, as well as genetic factors related to predisposition to ankylosis development in children. In this context, the present study aimed to evaluate the association between the presence of Temporomandibular joint ankylosis (TMJA) and the expression profile of the RANKL and OPG genes.

Materials and Method
The clinical and laboratory protocol of the pilot study was approved by the Research Ethics Committee (CEP) of the National Institute of Traumatology and Orthopedics Jamil Haddad (INTO), under number 401.481, on September 20, 2013. All included research participants, or their legal representative, agreed and signed the informed consent and/or informed consent prior to the conduct of the research.

Sample characterization / surgical protocol for biological material collection
Eleven Children diagnosed with TMJ ankylosis attending the Maxillofacial Skull Surgery sector at INTO were evaluated during the period from November 2013 to March 2017. The inclusion criteria of the study were individuals between 8 to 17 years of age requiring surgical treatment of TMJ ankylosis.
Prior tomographic recordings were performed to assess coronary process hypertrophy and true bone fusion in the TMJ ankylosis group. Surgical access was performed through a preauricular incision with temporal extension and bone tissue samples not committed by ankylosis (mandibular branch and/ or coronoid process) and ankylotic block were collected during surgery treatment of TMJA, coronary process hypertrophy and/or mandibular extractor installetion. Thus, only four Children (n=4) needed for surgical therapy and they were included in this study.
The control group consisted of adult individuals without pathological alterations in the ATM, no history TMJA, who underwent mandibular surgery treatment. Thus, Jawbone tissue samples (jaw branch, jaw body) were collected from five adults during jaw surgical treatment (trauma, extraction of mandibular third molar included and jaw dental implant installation). The health history, identification of past trauma and infections prior to the manifestation of TMJ ankylosis were evaluated through anamnesis form and dental clinical examination.

Processing of biological samples
All tissue samples collected were sent to the Jamil Haddad National Institute of Traumatology and Orthopedics (INTO) Clinical Research Center for immediate processing by washing with sodium phosphate buffer (PBS), mechanical grinding. The Bone fragments were subsequently digested with 1% collagenase (Sigma-Aldrich, St. Louis, MO) for 2 hours at 37 °C. Harvested cells were centrifuged at 300g for 10 minutes and resuspended in Dulbecco's modified essential medium (low glucose DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum selected by batch (FBS, Gibco). A total of 1.0 x 10 5 cells were plated in 75cm 2 flasks and allowed to grow to 70% confluence in a humidified atmosphere of 5% CO 2 and 37 °C. Osteoblasts were subsequently harvested by enzymatic digestion with 0.125% trypsin and 0.78mM EDTA (both from Sigma-Aldrich) and expanded to passage.

RNA extraction and gene expression analysis
Total RNA was isolated from osteoblasts (1x10 6 ) using Trizol® reagent (Invitrogen™, Life Technologies, NY, USA) according to the manufacturer's protocol. Samples were treated with DNase to digest genomic DNA using DNase DNA-free (Ambion por Invitrogen™). RNA integrity was confirmed on 1.2% agarose gel electrophoresis stained by electrophoresiswith Nucleic Acid Gel Stain® (Invitrogen™). RNA purity was confirmed with spectrophotometric absorbance at a ratio of 260/280 Nanodrop® 1000, Thermo Scientific, Wilmington, DE, EUA). Only RNA samples with a 260/280 ratio above 1.7 were included in this study. The samples were stored at -80 ºC and properly cataloged.

Statistical analysis
The Shapiro-Wilk test was used to evaluate the distribution between the variables. The Kruskal-Wallis test was used for comparison between groups, including gene expression analysis after calculation by the 2-ΔΔCT method. P values <0.05 were considered statistically significant. Statistical analyzes were performed using the software Graphpad Prism 7 (GraphPad Software, Inc. La Jolla, CA, EUA) and Excel.

Results
The Test group was consisted of 4 children (n=4) the average age of the participants was 12.5±2.06 years and 3 (75.00%) subjects reported a history of previous infection in the neonatal period in the health history and dental clinical evaluation ( Table  1). The average age of the control group was 34.66±13.02 years, gender was three (60%) men and two (40%) women (Table 1). The expression level values of the OPG and RANKL genes in the test (ankylotic block and Bone) and control groups did not show normal distribution pattern by the Shapiro-Wilk test. Thus, Kruskal-Wallis test was used at a significance level of 0.05 to compare the levels of mRNA of the OPG and RANKL genes between the groups ( Table 2).

Gene expression values based on calculation of 2-ΔΔCT
The gene expression OPG/RANKL ratio showed no significant differences among the control, ankylotic block and Bone groups (p=0.72) (Figure 1) as well as the OPG mRNA gene expression was compared between the groups did not show significant difference (P=0.50) (Figure 2).

Discussion
The biological basis of Temporomandibular joint ankylosis (TMJA) has not yet been completely elucidated, however it is known that there is a deregulation of the bone homeostasis process and maintenance of the normal anatomical architecture of the TMJ, culminating in the formation of a continuous bone block characteristic of TMJA [13]. Bone homeostasis is influenced by individual factors and is directly related to the function of the RANK/RANKL/OPG glycoprotein molecular triad [9,13].
Temporomandibular joint ankylosis (TMJA) represents a challenge in clinical practice [15] with high relapse rates and the need for additional surgical interventions throughout the craniofacial growth process [1]. In the present study, the mean age of the test group was 12.5±2.06 years and three children (75.00%) underwent previous surgery to treat TMJA throughout childhood.
The aim of this study was to evaluate the association between the presence of TMJA and the RANKL and OPG mRNA levels. Thus, a low RANKL expression was observed in the test group compared to control group (p=0.01), suggesting that the differentiated repair pattern does not occur only in areas of the ankylotic block, which highlights the systemic alteration of the pattern of remodeling that favors the bone synthesis. There was no significant difference between control and Ankylotic block groups (P=0.44).
The involvement of this triad (RANK/RANKL/OPG) in the pathogenesis of TMJA has been previously investigated [13] and a reduced RANKL/OPG ratio was observed in ankylotic block samples compared with the control group, suggesting that osteoclast deficiency may be an important factor for the development of Temporomandibular joint ankylosis. When osteoclast stimulation is impaired and resorptive capacity is deficient, the process of bone remodeling will be impeded and lead to a progressive increase in bone density [13]. However, the present study did not show significant differences in gene expression of the gene expression OPG/RANKL ratio showed no significant differences among the groups (p=0.72).
The specific role of genes in the etiology of TMJA is still unknown, but in association with other etiological factors may favor the development of the disease. Local trauma and poorly treated infections are reported as major causes of childhood TMJA [16]. In the present sample, three (75.00%) participants in the experimental group had a previous history of infection. However, the high frequency of such factors in childhood is not directly related to the rare manifestation of the disease in the population. Structural imbalance occurs only in individuals genetically predisposed to bone apposition behaviour [7,10].
The genetic influence as the etiological basis of TMJA has been demonstrated by studies of genetic polymorphisms. The ANKH gene polymorphism was associated with TMJA [10]. Additionally, OPG gene polymorphism (rs2073618) was presented as a potential marker associated with the risk of TMJA manifestation [7]. Specifically, plasma OPG levels were associated with intronic ANKH genetic polymorphisms [17]. The results of these studies strengthen the findings of this pilot study.
Identification of genetic biomarkers for pediatric TMJA is a promising approach for personalized diagnosis and treatment. In the present pilot study, low RANKL expression in test group when compared whit control (p=0.01), signaling a possible change in the bone homeostsis from children with temporomandibular joint ankylosis that favors the bone synthesis. RANKL expression also has been researched in other pediatrics diseases: Type 1 diabetes mellitus (T1DM), Alkaptonuria, Osteogenesis imperfecta, 21-Hydroxylase deficiency and Prader-Willi syndrome. All diseases presented RANKL upregulation, but the Hemophilia A presented normal or Increased levels of RANKL [18]. Unlike TMJA, these pathologies present reduced bone mass.
However, the need for molecular investigations in the pathogenesis of TMJA is highlighted, elucidating etiological factors, and directing specific therapies. In pediatric individuals, individualized genetic therapeutic approaches promise to reduce relapses and additional surgical interventions throughout the craniofacial growth process.
Despite being a pilot study, this research shows some relevance for evaluating a rare disease with great psycho-social impact on the lives of children with condylar ankylosis. The TMJA affects 1:2173.91 children in a study that evaluated 21.720 children over a one-year period [6].