Initial Management of MRONJ Stages I and II, a Clinical Trial With Detection of Salivary Biomarkers
Initial Management of MRONJ Stages I and II, a Clinical Trial With Detection of Salivary Biomarkers
Medication-related osteonecrosis of the jaw (MRONJ) is a serious complication in patients
 receiving antiresorptive therapies, such as Bisphosphonates and Denosumab. It is defined by
 the presence of exposed bone or a fistula that probes bone in the jaws for a period greater
 than 8 weeks in patient with a history of current or past antiresorptive or antiangiogenic
 treatment, and in the absence of prior radiotherapy or maxillary bone metastases. Depending
 on the severity of the disease 4 stages are described.

 On the other hand, although the presence of alterations in the levels of certain biomarkers
 in saliva has been documented in patients with MRONJ compared to healthy patients, its
 applicability in clinical practice is still unknown.

 Until recently, the status quo favored the adoption of a conservative strategy (non-surgical)
 for the initial management of patients with stage I and II. However, in recent years, this
 paradigm has been challenged by multiple authors who report better and more predictable
 outcomes with surgical treatment.

 Based on the hypothesis that patients with MRONJ stage I and II subjected to initial surgical
 treatment have better results than those undergoing conservative (non-surgical) treatment, te
 research group has designed a unicentric, quasi-experimental clinical trial where the
 clinical and radiological outcome at the third month of 2 groups of patients with stages I
 and II MRONJ undergoing non-surgical treatment (Group 1 / control) versus initial surgical
 treatment (Group 2 / intervention) will be compared.

 Also, the investigators hypothesize that the patients with complete resolution of the disease
 will also normalize salivary biomarkers levels unlike those with stable or progressive
 disease, meaning there is a correlation between clinical and biochemical response.
 Accordingly, the levels of specific salivary biomarkers at baseline and at the third month
 will be determined and compared with the clinical outcome.

 After enrollment patients will be instructed and offered both treatment strategies, and
 assigned to the corresponding group according to their choice. Patients in group 1
 (non-surgical) will receive traditional conservative treatment while patients undergoing
 surgical treatment will receive the same guidelines of conservative treatment plus surgery
 according to a specific surgical protocol.
1. Type of study: unicentric quasi-experimental clinical trial

 2. Hypothesis:

 2.1. Patients with MRONJ stage I and II subjected to initial surgical treatment have
 better results than those undergoing conservative (non-surgical) treatment.

 2.2. There is a correlation between clinical and biochemical outcome and the detection
 in saliva of certain biomarkers such as Amino Terminal Crosslinked Telopeptides of Type
 1 Collagen (NTX), Matrix metallopeptidase 9 (MMP-9), Interleukin 1a (IL-1a), Interleukin
 1b (IL-1b), Interleukin 6 (IL-6), Interleukin 17 (IL-17) and Interleukin 36α (IL-36α)
 may be useful to monitor treatment response.

 3. Main Objectives:

 3.1. To compare the clinical and radiological outcome after 3 months of conservative
 treatment versus initial surgical treatment in patients with MRONJ stage I and II.

 3.2. To determine the levels of salivary biomarkers at the beginning and after 3 months
 of treatment while correlating them with the clinical outcome.

 4. Secondary Aims

 -  To determine if there is a relationship between the initial surface of exposed bone
 and the outcome.

 -  To determine if clinical and radiological concordance in the outcome at the third
 month for each group.

 -  To determine the rate of postoperative complications in the surgical group.

 -  To determine if there is a relationship between the number of surgical
 interventions and the outcome.

 -  To determine the percentage of patients undergoing conservative treatment which
 eventually end up requiring surgical treatment due to disease progression.

 -  To determine the elapsed time until complete healing for both groups.

 -  To determine the variables associated with a higher cure rate.

 -  To determine the variables associated with increased risk of disease progression or
 recurrence.

 5. MATERIAL AND METHODS 5.1. Sample Size Accepting an alpha risk of 0.05 and a beta risk of
 0.2 in a bilateral contrast, 21 patients per group are required to detect a cure rate of
 30% and 70% in the conservative and surgical treatment group, respectively. Adding 10%
 to cover possible experimental losses, the sample increases to 23 per group.

 5.2. Main Variables

 -  MRONJ Stage (1. Stage 1; 2. Stage 2).

 -  Type of treatment (GROUP 1/Control: Conservative Treatment, GROUP 2: Surgical
 Treatment).

 -  Clinical outcome at the third month

 -  Radiological outcome at the third month

 -  Levels of biomarkers in saliva at baseline and at the third month (NTX, MMP-9, IL-1a,
 IL-1b, IL-6, IL-17 and IL-36α). In the case scientific evidence of other relevant
 markers appears during the course of the study they may be included in the study.

 5.3. Secondary Variables 5.3.1. Both groups

 -  Age (years)

 -  Sex (1.Man; 2.Mother)

 -  Diabetes (1. Yes; 2. No)

 -  Medication associated with the disease (1. Oral bisphosphonates, 2. Intravenous
 Bisphosphonates, 3. Denosumab, 4: Antiangiogenic).

 -  Underlying disease for antiresorptive therapy (1. Osteoporosis, 2. Cancer)

 -  Duration of treatment with medication associated with the disease.

 -  Factor that triggers the disease (1. Tooth Extraction; 2. Use of removable prosthesis;
 3. Unclear cause)

 -  Suspension of medication associated with the development of MRONJ (1. Yes, 2. No)

 -  Concomitant treatment with corticosteroids (1. Yes, 2. No)

 -  Concomitant treatment with antiangiogenic drugs (1. Yes, 2. No)

 -  Visual Analogue Scale (VAS) pain in the inclusion visit, and in all follow-up visits.

 -  Exposed bone surface (1. mild: <0,5cm2; 2. Moderate: 0,5-2cm2; Severe:> 2cm2) or number
 of intraoral fistulas otherwise.

 -  Dimension of greater diameter of radiolucent area or sequestrum in the CT or Cone
 Beam-CT scan (cm)

 -  Time elapsed until complete healing.

 -  Recurrence.

 5.3.2. Group 1 (Conservative medical treatment)

 -  Need for surgical treatment due to disease progression

 -  Time elapsed until surgical rescue treatment due to disease progression.

 5.3.3. Group 2 (Surgical treatment)

 -  Postoperative complications.

 -  Number of interventions.

 5.4. Inclusion Visit

 -  Patients will be informed about the different aspects of the study and will be given the
 information sheet to read carefully. Informed consent document will be given to read and
 sign.

 -  A thorough medical history will be collected including data about their socio-family
 environment, psychiatric illnesses and addictions.

 -  Complete and careful physical examination of the head and neck area will be carried out,
 including the measurement of the area of exposed bone in cm2 or, otherwise, the number
 of fistulas. Based on the clinical findings, stage 1 or 2 will be assigned according to
 the AAOMS guidelines.

 -  Once verified that the patient meets the inclusion criteria, patients will be assigned
 to the corresponding group according to their chosen treatment strategy.

 -  Radiological assessment will be carried out with Orthopantomography and CT-scan/Cone
 Beam-CT in all patients.

 -  Both groups will receive the same standard conservative treatment according to our
 protocol, meaning group 1 (non-surgical) will serve as control group.

 -  For patients enrolled in the surgical group, routine preoperative exams will be
 requested and surgical intervention will be scheduled within a maximum period of 3 weeks
 from the inclusion visit following a rigorous and standardized surgical protocol.

 5.4.1 STANDARD PROTOCOL FOR CONSERVATIVE TREATMENT (applicable to both groups)

 -  Soft diet.

 -  Suspension of the use of prostheses or intraoral devices.

 -  Oral hygiene guidelines.

 -  Topical antiseptics: in the form of mouthrinses with 0.12% chlorhexidine after every
 meal and clorhexidine gel over the exposed bone or fistula.

 -  Systemic antibiotics: for patients in stage 2 in which active acute infection is
 detected with Amoxicillin/Clavulanic Acid 875/125 mg every 8 hours for 2 weeks. Those
 allergic to penicillin will receive clindamycin 300 mg every 8 hours for 2 weeks or
 Levofloxacin 500 mg / day for 2 weeks. Systemic antibiotics treatment can be prolonged
 indefinitely until infection and symptoms are controlled.

 5.4.2. SPECIFIC PROTOCOL FOR SURGICAL TREATMENT

 -  Wide mucoperiosteal flaps and complete surgical excision of necrotic bone together with
 a mucosa margin of at least 2 mm.

 -  Removal of dental pieces included in the diseased area and regularization of bony
 margins avoiding leaving sharp edges or spicules.

 -  Secure a two layered waterproof closure without tension (simple or with local flaps).

 -  Samples will be sent for Pathological and microbiological analysis.

 -  Stitches removal after two weeks

 -  Postoperative systemic antibiotic following the mentioned protocol until stitches
 removal.

 5.4.3. SALIVA COLLECTION PROTOCOL

 -  Patients must avoid rinsing and ingestion of solids or liquids for at least 1 hour prior
 to collection.

 -  The collection will take place between 8 and 10 am.

 -  Five minutes after an oral rinse with distilled water, 5 ml of unstimulated full saliva
 from the lower lip will be collected.

 -  The samples will be processed within the first hour of collection of the sample by
 centrifuging at 2,600 x g for 15 minutes at 4 ° C. The supernatant will be collected and
 stored at -80 ° C until the biomarkers are analyzed.

 5.4.4. SUCCESSIVE VISITS

 -  Controls will be carried out 2 weeks after the inclusion visit and then monthly until
 the third month when the outcomes will be determined and the second sample of saliva
 will be collected. Long term follow-up will continue every three to six months depending
 on patient characteristics and desires.

 -  At each visit, the following information will be recorded: symptoms with VAS for pain;
 physical examination of head and neck; presence of exposed bone and surface in cm2 or
 number of fistulas otherwise. If complete mucosal healing is found, the time elapsed
 since the start of treatment will be recorded.

 -  Patients in GROUP 1, may develop disease progression refractory to conservative
 management. In that case, the second saliva sample will be collected before the third
 month and previous to surgical treatment.

 -  Patients of GROUP 2 with persistent disease, recurrence or significant worsening will
 undergo revision surgery if accepted.

 -  Radiological follow-up will be carried out with orthopantomography and CT scan/Cone
 Beam-CT at the third month.

 5.4.5. STATISTICAL ANALYSIS Normality tests and graphs will be used to determine if the
 variables follow a normal distribution. Variables with normal distribution are expressed as
 mean ± standard deviation and those without normal distribution are expressed as median and
 interquartile range. To evaluate the differences between groups, the student's t test or the
 Mann-Whitney U test will be used for quantitative variables; and the chi-square test or
 Fisher's exact test for qualitative variables.

 The correlations will be examined by the Spearman or Pearson rank correlation. Paired or
 related sample tests will be used to assess intragroup differences. A value of p <0.05 will
 be considered an indicator of a significant difference.
Inclusion Criteria:

 - Diagnosis of stage I or stage II medication related osteonecrosis of the jaws (MRONJ)
 according to the 2014 AAOMS position paper.

 Exclusion Criteria:

 -  Coexistence of any medical or social condition that prevents proper adherence to
 treatment or follow-ups (dependent patients without adequate socio-familiar support,
 drug addiction, severe psychiatric illness, terminal disease with life expectancy less
 than 3 months).

 -  Coexistence of Brown's Tumor, Paget's Disease or other bone diseases with the
 exception of Osteoporosis or bone metastases.