Molecular Markers Associated with Bone Deterioration in the Saliva of Patients with Bisphosphonate Related Osteonecrosis of the Jaw (BRONJ)

Bisphosphonate related osteonecrosis of the jaws (BRONJ) is an adverse sequelae of therapy most commonly associated with nitrogen containing bisphosphonates.  These medications are often used in the treatment of osteoporosis as well as metastatic neoplasms.  Bisphosphonates are potent inhibitors of osteoclasts, which play an integral role in bone homeostasis.  Molecular markers related to bone resorption and formation have been used to evaluate bone turnover in various diseases associated bone loss.  These markers have been shown to correspond well with regards to response to treatment, discontinuation of treatment and disease progression.  Peptide-bound collagen cross-links N-telopeptide (NTX) and C-telopeptide (ICTP) are indicative of bone resorption when not bound together.  Bone specific alkaline phosphatase (B-AP) is representative of bone deposition.  All of these markers have been measured in serum as well as urine.   

We hypothesize that the same molecular markers that are found in the blood to evaluate bone turnover can be found in the saliva.  If these markers can be predictably detected in saliva of subjects diagnosed with BRONJ, saliva can be employed as a diagnostic tool to evaluate patients suspected of having a diagnosis of BRONJ or at risk of developing BRONJ.

A retrospective study of patients with a history of IV bisphosphonate use and a diagnosis of BRONJ were asked to participate in this study.   IRB approval was obtained and informed consent was obtained from each participant. The study involved collection of patient saliva for molecular analysis to detect N-telopeptide (NTX) and alkaline phosphatase (B-AP).  Patients were instructed to be NPO for 8 hours prior to collection of approximately 2 tablespoons of saliva.  Medical data and radiographic data were also reviewed to confirm the diagnosis of BRONJ.  After collection of specimens from 12 patients diagnosed with BRONJ and 12 patients with no risk factors for BRONJ were obtained, the specimens were frozen and stored.  At the time of processing the samples were thawed, centrifuged and processed using the Osteomark NTX ELISA assay as well as the Ostase B-AP ELISA assay to evaluate for levels of NTX and B-AP in each sample.  All samples were processed three times to assure accurate results according to the protocol set forth by the manufacturer.

NTX and B-AP were detected in samples of saliva of patients with a diagnosis of BRONJ as well as in patient with no risk factors for BRONJ. NTX levels were found to be consistently lower in the saliva samples of those patients with a diagnosis of BRONJ when compared to controls.  B-AP levels were found to be consistently higher in the saliva samples of patients with a diagnosis of BRONJ when compared to saliva samples collected from controls.

Molecular markers related to bone resorption and formation have been used to evaluate bone turnover in various metabolic conditions and diseases associated with bone loss.  Traditionally these molecular markers have been identified in the serum and the urine.  In this study we have identified a difference in the levels of N-telopeptide (NTX) and alkaline phosphatase (B-AP) in the saliva of patients with BRONJ when compared to controls.  Saliva may be a less invasive medium for collection to diagnose, stage and guide treatment decisions of BRONJ in the future.  The detection of molecular markers in saliva as a medium for diagnosis and management of BRONJ deserves further scientific investigation in the literature.  The use of saliva as a diagnostic fluid medium offers an inexpensive clinical diagnostic tool that is non-invasive and easily accessible

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