CATEGORY 64 AUSTRALASIAN DENTIST Scanning electron microscopy The samples were fixed in 3% glutaraldehyde for 24 h, dehydrated through increasing concentrations of ethanol, and immersed in hexamethyldisilane (Sigma-Aldrich) 50% for 10 min and 100% for 3 x 10 min, before being aspirated dry and evaporated dry overnight. They were mounted on metal stubs and sputter coated with 20 nm gold film. The samples were imaged in a JEOL 6480 LV scanning electron microscope (Japan electron optics laboratory, Japan) with a voltage of 10 kV and a viewing distance of 20 mm. Microbiome analysis Genomic DNA was extracted using a combination of proteinase K (SigmaAldrich) and lysozyme (Sigma-Aldrich) digestion followed by phenol/chloroform extraction and ethanol precipitation as previously described (Jacombs et al., 2012). Extracted DNA was resuspended in 100 μL TE (10 mm Tris-HCl pH 8.0 and 1 mm EDTA) buffer. DNA quality and concentration of each sample were checked by Nanodrop 2000 (Agilent Technologies Inc. Santa Clara, CA, USA). Bacterial tagencoded FLX amplicon pyrosequencing (bTEFAP) was commercially performed by Molecular Research Lab (MR-DNA) using the Titanium platform (Roche) as previously described (Dowd et al., 2008). The V1-V3 regions of the the16S rRNA gene were sequenced and used to identify and quantify the different bacterial species in dental samples. Pyrosequencing data were analyzed by QIIME software (Werner lab, NY, USA) (Lawley and Tannock 2017). After removing low-quality and ambiguous sequences, all the remaining reads were de-multiplexed and chimeric reads were removed by chimera slayer using default parameters. Operational taxonomic units were assigned against a curated green genes database (McDonald et al., 2012). RESULTS Patient 1 Case Report Clinical History and SEM Patient 1 presented for dental implant therapy with a terminal diseased dentition in both arches. Only the mandible was treated with a full hybrid prosthesis, the maxilla received a conventional full upper denture (Fig. 2). Following extractions and a healing period of three months, 5 inter-foraminal implants were placed in the mandible using a two-stage protocol. The right-side anterior mandible presented as a block of osteosclerotic bone which obstructed osteotomy preparation at 2000 rpm ad modum Brånemark. Sufficient penetration was obtained to secure bone samples for CLINICAL SEM and microbiome analysis. Implants were placed to the right and left of the sclerotic block. A “wound gape” presented at suture removal on day 10, extending for 1.5 cm corresponding to the mesiodistal arch length of bone exhibiting severe sclerotic change (Fig. 3). At second-stage surgery, the osteosclerotic block had become radiographically demarcated to the point of sequestration. The presence of what appeared to be a refractory pathologic biofilm and the presence of persisting severe sclerotic change and sequestration provided a provisional diagnosis of chronic osteomyelitis (Figs. 4-5). SEM images of bone samples taken at implant installation from the sclerotic block (Fig. 5) provided visual confirmation of bacteria and bacterial biofilm. Figure 4 indicates the sclerotic demarcation of the sequestrating lesion, which was a segment of dead, sclerotic bone tissue caused by osteomyelitis (Cierny, 2011). Osteosclerotic bone persisted in a three-month apparently healed mandibular edentulous ridge at implant installation. SEM images revealed cocci bacteria covered in maturing Extracellular Polymeric Substance (EPS). A post-surgical wound gape persisted above this reactive sclerotic site until surgical debridements and closure of a blood-filled space. Clinical outcomes after surgical debridement It followed, therefore, that a mandated surgical debridement should be pursued as curative therapy. A block resection was undertaken to punctate point bleeding beyond the sclerosis on all exposed surfaces, without vertical progression to the inferior border of the mandible. Two excisions of reduced dimensions had proved unsuccessful in promoting wound closure prior to this. It was expected that the apical section that remained of the sclerotic lesion would have received sufficient red cell and antibiotic perfusion to heal satisfactorily without structurally endangering the integrity of the mid-line of the mandible by inclusion in the resection (Fig. 6). A review of the site 12 months following surgical debridement drilling (Fig. 7), with blood-fill and closure and antibiotic administration before and after surgery, revealed satisfactory bone regeneration with no apparent consequences for adjacent implants. No bone graft or exclusion membrane was used (Cierny, 2011). Retained apical section of the sclerotic lesion was more vaguely demarcated and Fig. 2: Terminal, diseased dentition on a presentation for mandibular implant therapy. Note significant bony degeneration. Location A: Radiolucent osteolytic lesions, location B: Radiopaque osteosclerotic lesions Fig. 3: Devitalised bone below soft tissue “wound gape” with texture and color similar to ivory. Location A is a devitalized ivory bone. The arrow corresponds to the length of the wound gape. Fig. 4: Bone samples were taken for SEM analysis (locations H and I) at implant installation. (Location L) indicates sclerotic demarcation of the sequestrating lesion. Radiograph was taken 4-months after the placement of five implants Fig. 5: SEM images of samples taken from location H (Fig. 5A) and I (Fig. 5B) in Fig. 4. BF: Biofilm. Scattered cocci (green highlight). Cocci (red highlight) with biofilm EPS. Fig. 6: Surgical defect in anterior mandible. Location A: Blood-filled and closed following Regenerative Surgical forage Drilling Debridements (RSD) of the sclerotic lesion. Location B: Apical section of the lesion was not debrided out of concern for the anatomical size of the infra-bony defect and possible pathologic fracture. Severe sclerotic bone as a pathologic biofilm biomarker (Walenkamp, 1997), treated by multiple small drill surgical forage reaming debridements, capillary perfusion, blood fill, and immediate closure.
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