The Horizontal Ridge Augmentation Using Equine Xenograft and a Collagenated Porcine Cortical Lamina
Condition: Horizontal Ridge Deficiency
Study type: Interventional

Summary: 
      The primary aim of this study is to clinically, radiographically and histologically evaluate
      the lateral bone augmentation of the soft porcine cortical lamina (OsteoBiol®) using
      equine-derived bone particles (OsteoBiol®)
    

Detailed description: 
      The xenograft used in this study was the Gen-Os® by OsteoBiol; The soft lamina (OsteoBiol®)
      used is a 35x35mm medium curved membrane of porcine origins.

      The fixation system used was the Pro-fix™ Precision Fixation System, consisting of
      self-drilling membrane fixation screws of 1.5 mm x 3.0 mm.

      Fifteen patients (1 male, 14 females) aged between 27 and 64 years old were selected from the
      Department of Periodontology of the Faculty of Dentistry, Saint Joseph University Beirut.

      Clinical and radiographic examinations were done prior to any procedure. The edentulous area
      was examined to identify the availability of keratinised mucosa and the possibility of
      implant placement in the mesio-distal and inter-arch planes. Radiographic examination was
      completed using a cone beam computed tomography (CBCT) scan of the affected region, where the
      indication for vertical augmentation was selected and excluded from the study. All the
      patients were given oral hygiene instructions and prophylaxis.

      Each patient received 2g of Amoxicillin one hour prior to surgery. Patients were instructed
      to mouthrinse with chlorhexidine 0.12% gluconate mouthwash for 1 minute. Extra-oral
      disinfection was made using topical chlorhexidine.

      Local and/or regional anaesthesia was obtained using Septanest® (Articaine hydrochloride 4%
      with adrenaline 1:100,000) A mid-crestal incision was performed and extended into the sulcus
      of the adjacent teeth, if present. At least one vertical releasing incision extending beyond
      the muco-gingival junction was made. A muco-periosteal flap of full thickness was raised and
      extended buccally, whereas palatally it was reflected to expose 3 mm of bone. Lingually the
      flap was elevated until reaching the mylo-hyoid line. Decortication was carried out using a
      twist drill with a stop of 3mm of length to ensure a good perfusion of the graft.

      The soft cortical lamina had been soaked in saline sterile water since the beginning of the
      surgery in order to achieve good elasticity and easier manipulation. It was then trimmed with
      sterile scissors and adapted to the recipient site while making sure it was not in contact
      with the surrounding teeth. It was first fixed on the palatal/lingual side. The
      cortico-cancellous heterologous bone mix which was previously hydrated in sterile saline for
      ten minutes, was placed on the ridge in sufficient quantities and covered by the membrane
      while being adapted in the desired shape of the future ridge. The membrane was reclined and
      also fixated on the buccal side for better adaptability and immobilization. In order to
      ensure a tension free closure, the buccal flap was advanced using a periosteal releasing
      incision connecting the vertical incisions thus achieving elasticity of the flap. Precaution
      was taken to identify and carefully isolate the mental nerve from the surrounding tissues
      when present. The lingual flaps were advanced using a blunt instrument to detach the muscular
      insertion of the mylo-hyoid from the lingual flap.

      Once proper elasticity was achieved, horizontal mattress sutures were placed at 4mm from the
      incision line using a non-resorbable polytetrafluroethylene (PTFE) 4/0 monofilament suture.

      They were followed by single interrupted sutures close to the edges of the flap in order to
      create a connective tissue-connective tissue contact, thus creating a barrier to reduce the
      incidence of membrane exposure.

      All patients were instructed to receive an injection of betamethasone dipropionate and
      disodium phosphate directly after the surgery and received 2g of Amoxicillin per day for a
      total of 7 days.

      Post-operative recommendations were clearly written and given to the patient. Chlorhexidine
      was prescribed starting the second day after surgery until suture removal.

      Sutures were removed at 14 days, they were left for another 7 days when found necessary to
      secure the healing.

      At 6 months from the surgery, a CBCT scan of the grafted area was taken and measures were
      made for the choice of the implant diameter and length. In all of the post-operative CBCTs,
      implant placement seemed possible.

      On the day of the implant placement, local anaesthesia was made, followed by a mid-crestal
      incision and occasionally a vertical one, allowing access to the fixation screws for their
      removal.

      A biopsy was taken at the site of implant placement using a trephine burr of outer diameter
      of 3.5mm and inner diameter of 2.5mm. The trephine and bone were immersed in 10% buffered
      formaldehyde and fixated for histology. The implants (Straumann®, Bone Level Cylindrical)
      were placed at the corresponding sites of the biopsies, and the choice of a cover or a
      healing screw was made depending on the clinical situation and the primary stability of the
      implant.

      Implant insertion torques were noted as indicated on the implant torque wrench. Periapical
      radio-graphs were taken, the flap was sutured and the patients received a daily dose of 2g of
      Amoxicillin for 7 days and diclofenac potassium inflammatory for pain management. They were
      also notified to mouth rinse with Chlorhexidine 0.12% for 10 days.

      Radiological protocol

      Image acquisition:

      At consultation and six months after the regeneration procedure, patients were scanned with
      the Newton VGI CBCT machine. Imaging conditions were: 110 kv tube voltage; 2.2 to 8.30
      milliampere (mA) tube current; 15 x 15 cm field of view; and 0.3 mm voxel size. Projection
      data were collected with a device rotating 360 degrees around patients over a total
      acquisition time of 18 seconds.

      Evaluation of images:

      Scan data were saved in DICOM (Digital Imaging and Communications in Medicine) format and
      image analysis and measurements were performed using the Blue Sky Plan® (Blue Sky Bio, LLC,
      Grayslake, IL, USA) which provided axial, coronal and sagittal views through multiplanar
      reconstructions of 0.3 mm slices. Axial images were reoriented to occlusal plane when present
      or to palatal plane as a horizontal reference. A panoramic curve was created and
      cross-sectional images perpendicular to that curve were reconstructed at a 1 mm interval.

      Advanced jaw segmentation:

      For each scan, an advanced jaw segmentation technique was realized using the Blue Sky Plan
      software by means of threshold segmentation and contour interpolation. First the region of
      interest (corresponding jaw) was selected on the panoramic view. Second, several axial,
      coronal and cross-sectional slices equally distributed/chosen by the software were used to
      draw the outline of the bone. This created a matrix to the final automatic segmentation step
      by the software to finalize the segmentation data and create a 3D model of the jaw. Finally,
      the outline of the 3D model was checked and adjusted manually on the 2D slices in all the
      planes in cases of over or missing contour. The result was an accurate 3D model of the
      corresponding jaw.

      Virtual implant placement and jaw superimposition:

      On the post-operative CBCT plan, virtual implants were placed in the optimal position
      regarding bone and prosthetic reference when present.

      In order to compare directly the pre and post-operative models, the pre-operative bone model
      was loaded into the post-operative plan an n-point registration technique was used for the
      superimposition of the two models. The outline of each model was visible in a unique color
      for comparison.

      A vestibulo-lingual implant centric section perpendicular to the panoramic curve and parallel
      to the long axis of the simulated implant was used to make all the measurements as follow:

      Horizontal bone width measurements

      For each implant site, pre and post-operative horizontal bone width were measured at 4
      levels. Bone width was calculated from the distance between the most buccal and most lingual
      bone points at each level while being parallel to the simulated implant platform.

        -  H0-T1 and H0-T2: Pre and post-operative horizontal bone width at implant platform level.

        -  H2-T1 and H2-T2: Pre and post-operative horizontal bone width at 2mm apically to implant
           platform.

        -  H4-T1 and H4-T2: Pre and post-operative horizontal bone width at 4mm apically to implant
           platform.

      Vertical bone gain/loss measurements

      For each implant site, pre and post-operative vertical bone gain were measured at 3 levels.
      Vertical bone gain/loss was calculated from the distance between the most coronal
      pre-operative bone points.

      Histology

      Fixation and inclusion:

      Samples were taken at six months after regeneration and were treated with non-demineralized
      histology. They were fixed in LILLIE neutral formalin, diluted to 10% in buffered sodium
      phosphate pH 7.4. The fixation period lasted 3 weeks. The samples were then rinsed under
      running water for 48 hours.

      The dehydration of the samples was carried out in alcohol baths of increasing concentrations
      for 48 hours, then the clarification, allowing the penetration of methacrylate, in 2
      successive xylene baths of 24 hours each.

      Cutting technique:

      The blocks were cut under irrigation and at slow speed with an Exact saw (Cutting machine
      EXACT-APPARATEBAU Nordersted, Germany), so as to take cuts of at least 80 μm. These cuts were
      subsequently reduced in thickness with the Exact abrasion system. The polishing was carried
      out with abrasive paper discs of decreasing granulometry making it possible to reduce the
      thickness of the cuts automatically to the desired value. The cuts were separated into S
      (superficial), M (median) and P (profound) cuts, the superficial ones being the cuts facing
      the periosteum.

      Staining:

      The sections were stained with Giemsa-Paragon and basic fuchsin. Giemsa will give cells and
      nuclei the color blue, and Paragon will stain bone in red.

      Histomorphometry:

      The qualitative observation of the sections was done under a digital microscope (Keyence
      digital microscope VHX-6000) with normal and polarized light visualisations. For histological
      quantification, an optical microscope was used (Olympus BX 60, Olympus Corporation, Tokyo,
      Japan) connected to a digital camera (Olympus E330), along with the software Image J/ Fiji.

      It was first calibrated by measuring the scale bar present on the image. The image was made
      into black and white (Type: 8-bit). The scale was set by measuring the length of the scale
      bar in pixels and by setting its known distance in millimetres. It was then set as 'Global'
      for all the images with the same scale.

      The total area of the concerned section was measured. Then the bone and osteoid volume were
      quantified using the Bone Volume Mask and by using the 'wand tool' to select all the black
      areas. The percentage of bone and osteoid matrix in each section was consequently calculated.

      Statistical analysis:

      The statistical package software for social sciences (SPSS for Windows, Chicago, IL, USA,
      version 25.0) was performed for statistical analysis of the data. The alpha error was set at
      -p-value<0.05. Frequency and percentage were utilized to describe categorical variables. Mean
      and standard-deviation were used for continuous variables.

        -  Repeated measure analysis of variance with two within-subjects factors (Time: Baseline
           and six months; distance: 0mm, 2mm, 4mm, and 6 mm) was performed to compare the mean
           bone level within groups. It was followed by univariate analyses and Bonferroni post hoc
           tests.

        -  Repeated measure analysis of variance with one within-subjects factor (distance: 0mm,
           2mm, 4mm, and 6 mm) followed by Bonferroni post hoc tests was used to compare the mean
           horizontal gain.

        -  Repeated measure analysis of variance with one within-subjects factor (level: buccal,
           median, lingual) followed by Bonferroni post hoc tests was executed to compare the mean
           vertical gain.

        -  One sample t tests were used to compare the mean vertical gain with a theoretical value
           "0'' that supposed the absence of gain.

        -  Student t tests were performed to compare continuous variables between two groups.

        -  Pearson correlation coefficients were calculated to assess the relationship between
           continuous variables.

        -  Kruskal-Wallis tests were used to compare histomorphometric measurements between
           different levels.
    

Intervention name(s): Gen-Os®; The soft lamina (OsteoBiol®)
Intervention type(s): Device
Intervention description(s): The xenograft used in this study was the Gen-Os® by OsteoBiol; as described by the manufacturer, it is a carbonated nanocrystal bone mineral and collagen of natural heterologous origin, obtained by the treatment of cortical bone tissue of equine origins. Its granulometry ranges between 0.25 to 1 mm and it is described as being slightly radiopaque.
The soft lamina (OsteoBiol®) used is a 35x35mm medium curved membrane of porcine origins. Its clinical indications as described by the fabricator: a co-adjuvant for the reconstruction or the partial or complete recovery of lost bone portions, fillers of non-infected, non-sclerotic and well blood bedewed bone defects.
Intervention model: Single Group Assignment
Intervention model description: Fourteen patients (1 male, 13 females) aged between 27 and 64 years old were selected from the Department of Periodontology of the Faculty of Dentistry, Saint Joseph University Beirut.
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: osteogenesis; xenograft; membrane