Quantitative ultrasound at the hand phalanges in patients with bisphosphonate-related osteonecrosis of the jaws

Patients with bisphosphonate-related osteonecrosis of the jaws (BRONJ) who received intravenous or oral bisphosphonates (BP) were selected for determination of their bone microarchitecture as a risk predictor of BRONJ development. The diagnosis of BRONJ was made based on clinical and radiographic findings. The control group consisted of healthy patients. All patients underwent quantitative and qualitative ultrasound measurements of bone at the hand phalanges carried out using the DBM Sonic BP. Ultrasound bone profile index (UBPI), amplitude-dependent speed of sound (AD-SoS), bone biophysics profile (BBP), and bone transmission time (BTT) were measured. The BRONJ group consisted of 17 patients (62 ± 4.24; range: 45-82); 10 (58.8%) were male and seven (41.1%) were female, of whom 11 (64.7%) suffered from multiple myeloma, three (17.6%) from osteoporosis, one (5.8%) from prostate cancer, one (5.8%) from kidney cancer, and one (5.8%) from leukemia. Fourteen (82.3%) of them received intravenous BP whereas three (17.6%) received oral BP. Nine (9/17; 52.9%) patients developed bone exposure: two in the maxilla and seven in the mandible. Regarding quantitative parameters, Ad-SoS was low in the BRONJ group, but not significant. The UBPI score was significantly reduced in BRONJ patients with exposed bone when compared to controls (0.47 ± 0.12 vs. 0.70 ± 0.15; p = 0.004). The present study demonstrated that quantitative ultrasound was able to show bone microarchitecture alterations in BRONJ patients, and suggests that these analyses may be an important tool for early detection of bone degeneration associated with BRONJ.


Introduction
Bisphosphonates (BP) are inhibitors of bone resorption and angiogenesis used for the treatment of diseases that affect bone metabolism, since they directly or indirectly inhibit osteoclastic bone resorption. 1,2The most important side effect of these drugs is bisphosphonate-related osteonecrosis of the jaw (BRONJ), whose definition has been recently updated as medication-related osteonecrosis of the jaw (ONJ) since it includes other antiresorptive or antiangiogenic agents than BPs. 3 It is characterized by exposed bone of the jaw without clinical evidence of healing for at least 8 weeks in patients using BPs and without exposure to head and neck radiation therapy or metastatic disease of the jaw. 3,4,5,6,7There is a crucial need to understand the factors involving the pathogenesis of these lesions and to determine which patients may be considered at risk, since these lesions are difficult to control and lead to high morbidity.
Regarding its pathophysiology, BRONJ has been characterized by accumulation of physiological damage in the jaw bones, which results from a marked suppression of normal metabolic turnover. 8,9,10he presence of trauma, including dental invasive procedures and infection, increases the demand for osseous repair that exceeds the capacity of the hypodynamic bone, resulting in bone necrosis. 9In addition, the antiangiogenic effects of BP on the tissues and the presence of comorbid factors, such as immunosuppression and other pathologies, may increase the risk for progression of this condition. 9,10t is necessary to determine the factors that may predispose the patient to the development of osteonecrosis of the jaws.Variables such as age, gender, medications, oral microbiota, preexisting medical conditions, and individual genetic variations need to be investigated.In addition, methods for skeleton assessment may be important to determine early alterations.Quantitative ultrasound (QUS) is a method for estimating bone mineral status through measurements performed at skeletal sites with predominance of cortical bone, such as proximal phalanges. 11QUS measurements provide qualitative and quantitative features of bone related to its microarchitecture or elasticity. 12We hypothesized that QUS could detect skeletal modifications in patients with BRONJ.This study evaluated the bone microarchitecture of patients with BRONJ in order to monitor bone turnover and to determine whether these analyses could predict bone exposures.

Methodology Ethics statement
The study was approved by the Ethics Committee of the Universidade de São Paulo -USP, School of Dentistry of Ribeirão Preto -Brazil (CAAE no.0066.0.138.004-11).All subjects signed a written informed consent for their participation, in accordance with the Declaration of Helsinki.

Patients
Patients presenting with BRONJ (study group) selected from the Surgery Clinics of the School of Dentistry of Ribeirão Preto and from the Clinical Hospital of the Universidade de São Paulo at Ribeirão Preto participated in the cross-sectional study.The diagnosis of BRONJ was made based on the history of BP uses and clinical findings. 7Plain radiographs (panoramic radiographs) were used as adjunctive assessment in the evaluation.The clinical alterations considered were: necrotic exposure of the jaw bones without clinical evidence of healing for at least 8 weeks, oral pains and/or infection signs.The radiographic alterations considered were signs compatible with osteosclerosis, osteolysis, and thickening of the lamina dura, fragmentation of the cortex, thickening of the periosteum, sequestra, and fractures.The control group consisted of healthy patients, pooled from a database at the Teaching Hospital of the Universidade de São Paulo at Ribeirão Preto, without any condition known to interfere with bone metabolism and matched to the study group by age, gender, and race.Subjects were excluded if they had been exposed to head and neck radiation therapy.

Study design
A cross-sectional study was conducted from June 2011 to January 2012.After clinical examination, radiomorphometric indices were used to analyze alveolar bone loss: cortical width (CW), panoramic mandibular index (PMI) and degree of alveolar crest resorption (M/M ratio).CW is the thickness of the mandibular inferior cortex; PMI is the ratio between the thickness of the mandibular cortex and the distance between the mental foramen and the inferior mandibular cortex.The thickness of the cortex was divided by the distance from the inferior margin of the mental foramen to the inferior border of the mandible.For the M/M ratio, the total mandibular height was divided by the height from the center of the mental foramen to the inferior border of the mandible. 13,14All measurements were made in millimeters on the same computer program (Image J, NIH; Bethesda, USA).When the mental foramen was visible bilaterally, the measurements were done bilaterally and the final result is an average between both; when only one foramen was visible, the measurements were done only on that side.
After clinical and radiographic examinations, all patients underwent double sequential examination of proximal phalanx metaphysis (II-IV) of the non-dominant extremity, in which soft tissue, three qualitative parameters, and one quantitative parameter were analyzed.These examinations were carried out using the DBM Sonic BP (IGEA; Carpi, Italy), which consists of an electronic caliper with two ultrasound probes (emitter and receiver) recording the ultrasound modifications through the phalanx.In all double measurements, the caliper was positioned at the distal metaphysis of the proximal phalanx of the last four fingers (II to IV) of the non-dominant hand.The probes were positioned on the mediolateral phalangeal surfaces using the phalanx head as reference point.The measurements were obtained by automation and the final result of each patient showed the mean of 96 measurements.This methodology informs, by a specific window on the display, whether the technical process is correct and, if not, it automatically blocks measurement acquisition.To restart the measurements, it is necessary to reposition the caliper.This technology forces the recording of the zero reference level for soft tissues, preceding the measurements of the four distal phalanges (to increase the precision of this exam).At the end of the recording, it was possible to evaluate the coefficients of intra-rater and inter-rater reliability.The UBPI, AD-SoS, BBP, BTT, and QUS-phalanges slices were measured. 11

Statistical analysis
The qualitative and quantitative parameters had normal distribution among all the patients.Comparisons between the groups were performed using the unpaired Student's t test for two groups and the analysis of variance (ANOVA) for three or more.The Chi-square test on 2×2 contingency tables provided the values for the assessment of the association among categorical variables (gender, age, DBM parameters) and the presence of BRONJ.These analyses were performed using the SAS software (Statistical Analysis System -SAS ® 9.0 software; San Diego, USA).The data were reported as means and standard deviations (SD), and the level of significance was set at 5% in all analyses.

Patients
The demographic characteristics and medical history of the subjects with BRONJ are shown in Table 1

BRONJ
Nine (9/17; 52.9%) patients developed frank exposed osteonecrosis of the jaw (Stage 2), and it occurred in three (3/9; 33.3%) cases without apparent precipitating events, and in six patients (6/9; 66.6%) with history of tooth extraction (the lesion occurred at the same site of previous tooth extraction); the mandible was affected in seven cases (7/9; 77.7%) and the maxilla in two cases (2/9; 22.2%).Eight patients (8/17; 47.05%) presented subclinical bone alterations (osteolysis and sclerosis) classified as a non-exposed variant of BRONJ (Stage 0), 15,16 and these bone alterations were diagnosed by routine panoramic radiographs.Three (3/8; 37.5%) patients presented this variant in the mandible and maxilla, four (4/8; 50%) presented it only in the mandible, and one (12.5%)presented it only in the maxilla.The average time from dental intervention or the beginning of the symptoms to the first examination was 8 months.

Bisphosphonate therapy
The average time of treatment with bisphosphonates until the occurrence of first symptoms of BRONJ was 23.1 months for intravenous bisphosphonate administration (range 2-63 months) and 60 months for oral bisphosphonates (range 48-72).

Bone microarchitecture analysis
The trends of quantitative and qualitative parameters presented by BRONJ patients and control subjects are shown in Table 2. Regarding quantitative parameters, the BTT score was similar in the three groups, and the AD-SoS score was lower in the BRONJ group compared to the control (Figures 1 to 3); however, no significance was obtained.The UBPI score was significantly reduced in BRONJ patients with exposed bone when compared to controls (0.47 ± 0.12 vs 0.70 ± 0.15; p = 0.004) (Table 2).

Discussion
The diagnosis of BRONJ is established based on the history of BP uses and on clinical signs and symptoms. 7,9,16,17,18,19,20Imaging exams should be used as adjunctive assessment in the evaluation of patients as they provide useful information about alterations in bone morphology that may be detectable by radiographs, computerized tomography (CT) scan, and magnetic resonance imaging (MRI). 12,21,22  Radiographs may be negative for early signs of BRONJ, and CT and MRI are also not useful to detect initial mineral bone loss in early cases. 23Previous studies demonstrated that CT and MRI are adequate for evaluating more significant bone involvement; 22 however, in most cases, these exams show nonspecific findings, resembling those of a chronic osteomyelitic process, with predominant signs of osteosclerosis and periosteal reaction.Despite nonspecific findings, bone scintigraphy has been better at detecting early inflammatory processes than other methods. 23,24onsidering that the diseased bone goes far beyond the limits of the clinically exposed bone areas 10,12 and that the imaging findings may not reflect the true bone architecture, it seems reasonable to evaluate the bone microarchitecture of patients with BRONJ through the QUS method, as reported in the present cross-sectional study.
Osteonecrosis seems to be time-and dose-dependent and increased cumulative doses and long-term BP treatment are considered important risk factors for BRONJ development. 9,24However, some studies 4,25 have reported that BRONJ may develop after few months of BP therapy, as demonstrated in the present study (Table 1; cases IAL and GJS).Local and systemic factors, such as oral microbiota, smoking, underlying medical conditions, previous or simultaneous therapies with glucocorticoids and/or immunosuppressive drugs might promote an additional risk for BRONJ, but the real impact of these factors remains to be determined. 17Prospective studies are needed to further elucidate BRONJ pathogenesis, and the search for methods that can determine which patients are at risk is important.Thus, bone microarchitecture analysis through QUS may be an adjuvant method for determining the risk for the development of bone necrosis.
Ultrasound measurement provides information on both bone structure and resistance to mechanical solicitations, permitting the detection of reduced bone mineral content. 26,27 halangeal QUS evaluates bone condition through sonographic parameters st r ict ly dependent on bone biome ch a n ica l properties. 28,29BTT demonstrates cortical thickness and can discriminate different patterns of bone disease regardless of bone density; 29 AD-SoS mostly reflects bone density and elasticity, being marginally influenced by structural changes in the bone; 28 and UBPI provides information about the quality of bone mass. 29In the present study, BRONJ patients presenting frank bone exposure showed reduced UBPI scores compared to BRONJ patients presenting subclinical findings and control patients (Table 2).Moreover, most of the patients with subclinical BRONJ (62.5%) presented an inadequate UBPI profile compared to the control group (4/17; 23.5%), and this result suggests high bone mass deterioration in these patients.Radiomorphometric indices in panoramic radiographs also allow for quantitative assessment of bone mineral loss in BRONJ patients.The PMI was found to be altered in 43% of the patients when compared to the normal standards. 302][23] There is a need to determine predictive exams that may indicate early bone pathology in the absence of clinical ONJ, and bone microarchitecture analysis may be useful for that purpose.Future additional analyses may be required to further assess the impact of QUS as a predictor of BRONJ development.In this study, QUS was not performed before BP treatment; thus, it was not possible to compare the effect of BP on bone microarchitecture.Additionally, serial analyses in BP users without BRONJ were not performed, so early skeletal changes could not be detected.The relatively small number of subjects included in this study may also be a limitation, considering the large number of BP users in the population.Furthermore, given that underlying diseases may alter bone mass, as demonstrated by previous studies, 25,26,28 QUS could allow assessing the influence of underlying diseases on the bone tissue.

Conclusion
The present study demonstrated that QUS was able to show bone microarchitecture alterations in patients with BRONJ and suggested that these analyses may be an important tool for early detection of bone degeneration associated with BRONJ.

Figure 1 .
Figure 1.Biophysical bone profile of a control patient: presence of attenuation and inversion of the pulses compatible with age (74 years old).The value of AD-SoS > 2.040 m/s classifies the patient as having normal bone quantity; patient does not present bone fracture risk.

8 )
Different letters in the same row mean statistical difference (Chi-square test; p < 0.05).

Table 1 .
Demographic and clinical data of BRONJ patients.

Table 2 .
Gender, age, and microarchitecture profile in BRONJ subjects with and without exposed bone and in controls.