Patients and tissue collection
The present study analyzed retrospectively jaw bone samples (n = 70) from 70 patients (MRONJ (BP): n = 30, ORN: n = 15, OM: n = 15, control: n = 10). Patient cohorts and respective samples from this study are identical to those in our previous study [12]. All patients were treated in the Department of Oral and Maxillofacial Surgery of the University Hospital Erlangen between 2007 and 2015. The analyzed jaw bone samples were gathered intraoperatively as part of routine histopathological diagnostics. The control group consisted of patients with histopathologically inconspicuous jaw bone samples (n = 10) which were obtained during dental surgery procedures of teeth with no signs of local infection. The jaw bone probes were fixed in 4% formalin immediately after surgical sampling. Histopathological analysis was performed by the Department of Pathology of the University Hospital Erlangen. Specific clinical disease criteria were checked by the review of medical records and radiographs.
Inclusion criteria for MRONJ (BP) samples were: (1) histopathological confirmation of MRONJ (BP). (2) Evidence of more than 8 weeks of exposed jaw bone. (3) Documented bisphosphonate therapy. (4) No radiotherapy. (5) No therapy with denosumab, bevacizumab, pazopanib, sunitinib, mTOR inhibitors and sorafenib.
Inclusion criteria for OM samples were: (1) histopathological confirmation of OM with evidence of chronic inflammatory processes in the jaw bone. (2) No bisphosphonate therapy. (3) No radiotherapy. Patients with primary chronic OM (non-bacterial cause) were excluded.
Inclusion criteria for ORN samples were: (1) Evidence of devitalized and exposed jaw bone in a previously irradiated field in the absence of local neoplastic processes. (2) No bisphosphonate therapy.
Inclusion criteria for control samples were: (1) no histopathologic evidence of bone disease. (2) No bisphosphonate or local radiation therapy. (3) No medications significantly affecting jaw bone homeostasis. (4) No intraoral inflammation. (5) No relevant periodontitis. (6) No local malignancies. (7) No relevant systemic diseases (e.g., osteoporosis).
For detailed patient data, see Table 1.
Immunohistochemical staining
All formalin-fixed samples underwent decalcification and were embedded in paraffin before being sliced in 3-μm sections using a microtome (RM2165, Leica, Nussloch, Germany). Special microscope slides with improved adhesion were used (SUPERFROST ULTRA PLUS, Gerhard Menzel GmbH, Braunschweig, Germany). The sections underwent dewaxing in xylene and rehydration in graded propanol and distilled water before staining.
Hematoxylin and eosin staining (H&E) was carried out according to standard protocols.
Immunohistochemistry was performed using an automated staining device (Autostainer plus, DakoCytomation, Dako Deutschland GmbH, Hamburg, Germany). Antigen retrieval consisted of section treatment with ethylenediaminetetraacetic acid (EDTA) (dilution 1:100, PMB4-125, Antigen Retrieval Buffer 4, Spring Bioscience, CA, USA) at 66.7 °C for 5 h. The reduction of background staining artifacts was achieved by performing peroxidase-blocking for 5 min (S2023, DAKO REAL, Peroxidase-Blocking Solution, Dako Deutschland GmbH, Hamburg, Germany).
The following primary antibodies were used for protein detection:
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Anti-NFATc1-AK (sc-7294, NFATc1 (7A6), mouse, monoclonal, Santa Cruz Biotechnology, Inc., Heidelberg, Germany). Dilution: 1:50. Incubation time: 20 min.
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Anti-BCL6-AK (HPA004899, Anti-BCL6, rabbit, polyclonal, Atlas Antibodies AB, Stockholm, Sweden). Dilution: 1:50. Incubation time: 20 min.
EnVision Detection System Peroxidase/diaminobenzidine (DAB), Rabbit/Mouse (K5007 HRP/DAB+, Dako Deutschland GmbH, Hamburg, Germany) was used as staining kit. This kit provided a conjugated dextran and a DAB+ chromogen that were used for visualizing the antibody-marked proteins. Hematoxylin was used for nuclear counterstaining (CS700, Dako Deutschland GmbH, Hamburg, Germany). Positive and negative controls were included in each staining series.
Quantitative immunohistochemical analysis
All stained histological sections were scanned and digitalized completely in cooperation with the Institute of Pathology of the University Hospital Erlangen using a Pannoramic 250 Flash III Scanner (3DHISTECH Kft., Budapest, Hungary). Before scanning, the sections were quality-checked under a bright-field microscope (Axioskop, Zeiss, Jena, Germany; at a magnification of 100×–400×). The analysis of the digitalized sections was done via virtual microscopy using CaseViewer version 2.2 (3DHISTECH Kft., Budapest, Hungary). Figure 1 illustrates the method of “whole slide imaging”, that was used in this study. Two visual fields per virtualized section were set within areas with a high probability for the presence of osteoclasts (bone trabeculae, subperiosteal bone, endosteal structures and connective tissue directly adjacent to the bone). If the visual field size exceeded the total section size, only one visual field was used. Areas of pure necrosis were omitted for analysis. Within the visual fields, non-bony medullary areas were marked (regions of interest = ROIs) (Fig. 1c). Any cell counting occurred only within ROIs. Cells were considered osteoclasts if they met the following morphological criteria: (1). Multinuclearity (at least two nuclei). (2). Large cell body (larger than two fused mononuclear cells). (3). Direct contact with bone or proximity to bone. (4). No proximity to granulomatous foci or foreign particles. Area determination within the visual fields was done with Pannoramic Viewer, whereas cell counting was performed with ImageJ (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2014). Section analysis was conducted by two medical students familiar with tissue morphology, IHC-methods and analysis. These students were blinded to the origin of the specimens. Regarding cell counting, inter-individual differences were checked and did not exceed 10%.
Statistical analysis
Statistical analysis was conducted after consultation with the Department of Medical Informatics, Biometry and Epidemiology (IMBE) of the Friedrich-Alexander University Erlangen-Nürnberg.
For quantitative analysis, not only the ratios of osteoclasts to ROI were determined, but also the respective labeling indices (positive osteoclasts of a ROI/all osteoclasts of a ROI) and the ratio of nuclear BCL6+ osteoclasts to cytoplasmic BCL6+ osteoclasts. Results are expressed as the minimum, maximum, average, median, interquartile range (IQR) and standard deviation (SD). Box plot diagrams visualize the respective values.
The Kolmogorov–Smirnov test was used for normal distribution testing. The Mann–Whitney U test was used for statistical hypothesis testing. P-values ≤ 0.05 were considered statistically significant. SPSS 22 (SPSS, IBM, New York, USA) was used for statistics.