Subject selection

This cross-sectional study was conducted in accordance with the recommendations of the Ethics Committee of the National Institute of Traumatology and Orthopedic Research; informed consent was obtained from each subject. One hundred eight Brazilian volunteers, of both sexes, were selected from an outpatient pool during the course of 1 year. Subjects reported their personal and medical histories. They underwent routine consultations in a specialized care center for shoulder and elbow disorders in order to evaluate their shoulder and TMJ conditions. Inclusion criteria for subjects were the following: Brazilian citizen, age >45 years, and no previous surgery or neoplasm in the TMJ or shoulder.

Subjects with a history of trauma, bursitis, rheumatoid arthritis, or autoimmune diseases, chronic use of systemic corticosteroids, hyperlaxity, or who were pregnant were excluded. The control group comprised 30 subjects without pain and with no signs or symptoms of TMD or RCD. Subjects diagnosed as having RCD and/or a TMD were divided into three groups: RCD subjects (TMD-free, n = 16), TMD subjects (RCD-free, n = 13), and TMD/RCD affected subjects (patients with both RCD and TMD, n = 49). The baseline clinical parameters for the subject population are shown in Table 1 .

Diagnosis of temporomandibular disorders

All participants were examined clinically by the same dentist (L.L.B.) according to the Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) Axis I in order to investigate the three groups of TMD: group I, muscle disorder; group II, disc displacement; and group III, arthralgia, arthritis, and arthrosis. The RDC/TMD were used to assess these three groups of TMD using well-validated techniques, including palpation at 20 specified muscle sites. Self-reported symptoms pertaining to jaw impairment and associated pain were also recorded during the evaluation.

Clinical characteristics were noted, which included the following clinical symptoms: ear pain, toothache, burning sensation in the mouth, limited mouth opening, and noises (clicking, crepitation) in the TMJ, as well as diagnosed bruxism. Of the 108 patients examined, 62 showed a TMD.

Diagnosis of rotator cuff disease

Sixty-five subjects were diagnosed with RCD. The diagnosis of RCD was based on the protocol of Motta et al., by clinical examination and imaging of the involved shoulder (radiography and magnetic resonance imaging). Subjects considered to be RCD-free had a negative history for shoulder pain, a negative specific test result for impingement syndrome in a complete physical examination of the shoulders, and an absence of tendinopathy. All clinical evaluations were performed by one of the authors from the specialized care center (M.V.A.).

The records of the orthopedist who carried out the RCD diagnosis and the dentist performing the orofacial examination were independent of one another.

Oestrogen-related receptor β genotyping

DNA from all participants was extracted from buccal cells after vigorously rinsing with 5 ml of saline solution for 60 s, as described previously. A NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to determine the amount and purity of the DNA. Only DNA samples showing A _{260 nm} /A _{280 nm} ratios greater than 1.9 were used.

All procedures included in SNP selection and analysis followed the STREGA reporting recommendations. Linkage disequilibrium relationships and gene structure were considered in order to select the eight SNPs in the ESRRB gene included in this study. The minor allele frequencies reported in the database of the National Center for Biotechnology Information ( ) was >0.12 ( Table 2 ).

Real-time polymerase chain reactions with TaqMan chemistry (Applied Biosystems, Foster City, CA, USA) were used for genotyping of the eight selected SNPs in a thermocycler (PTC-225 Tetrad, Peltier Thermal Cycler; Bio-Rad Life Science, Corston, UK), yielding 1.5 ml/reaction in total. The haplotype analyses were also considered in order to explore SNP associations.

Estradiol level

Endogenous estrogens are a group of steroidal compounds including 17β-estradiol, estrone, and estriol. Because the bioactivity of 17β-estradiol is the greatest of the three in vivo, it was chosen to evaluate the estradiol concentration in serum.

One 5-ml blood sample was taken from 41 subjects in the total sample group after 8 h of overnight fasting (eight controls, nine RCD, four TMD, and 20 TMD/RCD affected subjects). The blood was centrifuged and the serum samples stored at −20 °C. In order to exclude the influence of hormone variables on estradiol levels in the women, the use of contraceptives, hormone replacement therapy, menopause status, and menstrual cycle status (normal length menstrual cycles (26–35 days duration) or luteal phase of the menstrual cycle (22–25 days based purely on self-report of the first day of menses)) were recorded. For pre-menopausal women, blood was taken only within the first 7 days from the first day of menstrual flow, since during this period the oestrogen level is not under the influence of the physiology of menstruation.

Assays were performed concurrently on serum specimens from cases and controls. All laboratory personnel were blinded to the subjects’ status. A competitive chemiluminescent immunoassay performed using the ACS-180 automated immunoassay system (Bayer Diagnostics Corp., Tarrytown, NY, USA) was used to measure total estradiol in the serum samples.

The normal values for estradiol were determined according to the manufacturer’s instructions: menopause, <40 pg/ml; non-pregnancy, 27–433 pg/ml; and men, <47 pg/ml. Taking into account the hormonal influence and the natural difference between men and women for serum estradiol levels, subjects were classified as having a normal level, below normal level, or above normal level.

Surface electromyography

The activity of the muscles was recorded by surface electromyography (EMG) on the right and left side masseter, anterior temporal, sternocleidomastoid, trapezius, and deltoid muscles in a representative sample, including 12 subjects from the control and TMD/RCD affected groups. EMG signals were obtained using a four-channel module (EMG System do Brasil Ltda, São José dos Campos, SP, Brazil). Surface active bipolar electrodes with 10 mm of inter-electrode distance and Ag 99.9% (3 M Brazil, Sumaré, São Paulo, Brazil) were used, along with a disposable monopolar electrode, used as a reference (Ag/AgCl; 3 M Brazil) and placed on the forearm.

Patients were instructed to remain seated in a chair, feet apart, shoulders relaxed, and hands resting on their thighs, with their heads in the Frankfort plane parallel to the ground. The electrode attachment sites were cleaned with a cotton ball soaked in 70% alcohol to reduce the impedance between the skin and electrodes, as per the SENIAM (Surface ElectroMyoGraphy for the Non-Invasive Assessment of Muscles project) recommendations. EMG signals were sampled at 1000 Hz, band-pass from 20 to 500 Hz. The signals were recorded for 10 s each. In all tests, a 5-s period was selected (the two initial and three final seconds of the EMG signal were discarded). The signal processing was performed using specific routines carried out in WinDaq software (WinDaq/HS; DATAQ Instruments Inc., Akron, Ohio, USA) and was based on the protocol of Lauriti et al., calculating the average value and standard deviation from the root mean square (RMS).

Masseter and anterior temporal muscles

Evaluations of the masseter and anterior temporal muscles were carried out at rest and at maximum clenching effort (MCE). To avoid direct occlusal contact, a strip of Parafilm M (Bemis Company, Inc., Oshkosh, WI, USA) was folded five times and arranged bilaterally in the molar region, based on the protocol of Lauriti et al.

Sternocleidomastoid muscle

EMG of the sternocleidomastoid muscle was recorded by placing an electrode along a line drawn from the sternal notch to the mastoid process, at one-third the distance from the mastoid process. Subjects were asked to turn their head and neck to maximum rotation.

Trapezius muscle

EMG of the upper trapezius muscle was recorded by placing an electrode along a line joining the acromion and C7, at one-third the distance from the acromion process. These steps were based on a protocol by Chowdhury et al. Subjects had to raise their shoulders to their maximum position in order to analyze trapezius activity.

Deltoid muscle

EMG activity in the deltoid muscle was measured under complete arm abduction with electrodes positioned on the greatest tonus.

Statistical analysis

Data entry and the statistical analysis were implemented with Stata 11.1 (StataCorp, College Station, TX, USA). The sample included all subjects attending for routine consultation at the Center of Shoulder and Elbow Surgery of the National Institute of Traumatology and Orthopedics who fulfilled the inclusion criteria. The χ ² test was used to assess the significance of differences in nominal variables (expressed as frequencies and percentages), including the frequencies of genotypes and alleles, between cases and controls. After the Shapiro–Wilk test for continuous variables was applied (variables expressed as the mean and standard deviation), analysis of variance was performed with the Student t -test or Mann–Whitney test, depending on whether the distribution was normal or non-normal, respectively. All genetic analyses were performed just after fitting for Hardy–Weinberg equilibrium. The odds ratio (OR) and 95% confidence interval (95% CI) were used to calculate the risk associated with alleles and genotyping. Multivariate logistic regression explored variables with statistical significance in the univariate analysis. Multiple comparisons were corrected by Bonferroni correction ( http://www.quantitativeskills.com/sisa/calculations/bonfer.htm ). The program ARLEQUIN (v.20; http://anthro.unige.ch/arlequin ) was used to calculate linkage disequilibrium and haplotypes. Values of P < 0.05 were considered to have statistical significance.

Clinical findings: TMD and RCD incidence

Of the total 410 subjects evaluated during 1 year at the specialized care center for shoulder and elbow disorders, 108 were included in this study. There were 80 (74%) women and 28 (26%) men, with a mean age of 57.2 ± 8.3 years. The control group consisted of 30 subjects, 22 (73%) women and eight (27%) men, with a mean age of 55 ± 7.8 years. No difference was found among test groups (RCD, TMD, and TMD/RCD affected) when compared to the control group for age, sex, smoking habit, alcohol consumption, systemic disease, diabetes, hypothyroidism, and the use of calcium supplementation, analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), bisphosphonates, and muscle relaxant drugs.

The χ ² test revealed a higher prevalence of white ethnicity in the TMD ( P = 0.003), RCD ( P = 0.03), and TMD/RCD affected ( P < 0.001) groups. On the basis of OR calculation, the risk associated with those of white ethnicity having both diseases (OR 5.9; 95% CI 1.9–18.5) was six times higher than in control subjects. Additional details regarding the demographics of the subjects are shown in Table 1 .

During the clinical TMJ and shoulder evaluations on the subjects, the incidence of TMD was 57.4% and RCD was 60.2%. From the total patients with TMD ( n = 62), 79.0% ( n = 49) were diagnosed with RCD. The χ ² Fisher’s test showed a marked difference in RCD incidence in patients with and without TMD ( P < 0.001). Subjects with TMD were seven times more susceptible to RCD (OR 7.0; 95% CI 2.7–18.4) than subjects with a healthy TMJ.

No difference was detected among the groups with TMD according to the RDC/TMD Axis I diagnosis. From the total of 62 subjects diagnosed with TMD, 24 showed a muscle disorder (group I), three had disc displacement (group II), and five were classified with a degenerative local disease (group III); more than one diagnosis was identified in 30 of the subjects ( Table 3 ).

Genetic association study

The results for the eight SNPs in the ESRRB gene are provided in Table 4 . As indicated by the χ ² test, statistically significant associations were observed. Allele and genotype frequencies for SNPs were within Hardy–Weinberg equilibrium in all groups. All evaluations considered the control group as the reference for statistical calculation.

Table 4

ESRRB genotype and allele distribution in all subjects.

SNP	Genotypes	Control ( n = 30)	RCD ( n = 16)	TMD ( n = 13)	TMD/RCD affected ( n = 49)	Healthy TMJ with RCD P -value (OR; CI) a	TMD without RCD P -value (OR; CI) a	TMD/RCD affected P -value (OR; CI) a
rs4903399	CC–CT–TT	14–13–2	14–2–0	6–6–1	36–12–1	0.031	0.9	0.07
	CT+TT	15	2	7	13	0.009 (0.1; 0.0–0.8)	0.8 (1.0; 0.24–4.91)	0.02 (0.3; 0.1–0.9)
	C	41	30	18	84	0.01 (6.2; 1.2–42.2)	0.2 (2.3; 0.5–11.6)	0.02 (2.4; 1.0–5.9)
	T	17	2	3	14
rs1077430	CC–CT–TT	6–14–9	4–9–3	2–5–6	11–21–17	0.6	0.6	0.8
	CT+TT	23	12	11	38	0.5 (0.7; 0.1–4.1)	0.6 (1.4; 0.2–12.3)	0.8 (0.9; 0.2–3.1)
	C	26	17	9	43	0.4 (1.3; 0.5–3.6)	0.3 (0.6; 0.2–1.8)	0.9 (0.9; 0.4–1.9)
	T	32	15	17	55
rs2860216	CC–CT–TT	3–14–12	3–4–9	2–8–3	4–16–29	0.2	0.5	0.3
	CT+TT	26	13	11	45	0.4 (0.5; 0.0–3.7)	0.6 (0.6; 0.0–6.4)	0.7 (1.3; 0.2–7.6)
	C	20	10	12	24	0.7 (0.8; 0.3–2.3)	0.3 (1.6; 0.5–4.6)	0.1 (0.6; 0.2–1.3)
	T	38	22	14	74
rs10132091	CC–CT–TT	8–16–5	4–7–5	2–5–6	4–25–20	0.5	0.1	0.02
	CT+TT	21	12	11	45	0.8 (1.1; 0.2–5.7)	0.3 (2.1; 0.3–17.2)	0.02 (4.2; 1.0–19.4)
	C	32	15	9	33	0.4 (0.7; 0.2–1.8)	0.08 (0.4; 0.1–1.2)	0.008 (0.4; 0.2–0.8)
	T	26	17	17	65
rs1676303	CC–CT–TT	1–7–21	2–9–5	1–1–11	4–17–28	0.02	0.4	0.3
	CT+TT	28	14	12	45	0.2 (0.2; 0.0–4.0)	0.5 (0.4; 0.0–17.3)	0.4 (0.4; 0.0–4.1)
	C	9	13	3	25	0.007 (3.7; 1.2–11.4)	0.6 (0.7; 0.1–3.2)	0.1 (1.8; 0.7–4.7)
	T	49	19	23	73
rs745011	CC–CT–TT	9–13–7	7–9–0	3–2–8	12–26–11	0.09	0.05	0.7
	CT+TT	20	9	10	37	0.3 (0.5; 0.1–2.4)	0.5 (1.5; 0.2–8.9)	0.5 (1.3; 0.4–4.3)
	C	31	23	8	50	0.0 (2.2; 0.8–6.2)	0.5 (0.3; 0.1–1.1)	0.7 (0.9; 0.4–1.8)
	T	27	9	18	48
rs4903419	AA–AG–GG	20–8–1	13–3–0	9–4–0	32–17–0	0.5	0.7	0.3
	AG+GG	9	3	4	17	0.3 (0.5; 0.0–2.6)	0.9 (0.9; 0.1–4.9)	0.7 (1.1; 0.4–3.5)
	A	48	29	22	81	0.3 (2.0; 0.4–10.1)	0.8 (1.1; 0.2–4.9)	0.9 (0.9; 0.3–2.5)
	G	10	3	4	17
rs6574293	AA–AG–GG	0–11–18	0–5–11	0–1–12	0–10–39	0.6	0.04	0.09
	AG+GG	29	16	13	49	–	–
	A	11	5	1	10	0.6 (0.7; 0.2–2.8)	0.06 (0.1; 0.0–1.4)	0.1 (0.4; 0.1–1.3)
	G	47	27	25	88

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

1. Clinical and radiological outcomes of transoral endoscope-assisted treatment of mandibular condylar fractures
2. Three-dimensional evaluation of healing joint morphology after closed treatment of condylar fractures
3. Platform-switching implants and bone preservation: a systematic review and meta-analysis
4. Recurrence rate following treatment for primary multicystic ameloblastoma: systematic review and meta-analysis
5. Mitoxantrone as a contributing factor in medication-related osteonecrosis of the jaws
6. Efficacy of a single dose of low-level laser therapy in reducing pain, swelling, and trismus following third molar extraction surgery

Stay updated, free dental videos. Join our Telegram channel

Join