Initial Management of MRONJ Stages I and II, a Clinical Trial With Detection of Salivary Biomarkers

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.

1. 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.