The Horizontal Ridge Augmentation Using Equine Xenograft and a Collagenated Porcine Cortical Lamina: A Clinical, Radiographic and Histological Prospective Study
Phase: NA
Conditions: Horizontal Ridge Deficiency
Sponsor: Saint-Joseph University

The horizontal ridge augmentation using equine xenograft and a collagenated
porcine cortical lamina membrane:
A clinical, radiographic and histological prospective study
Study Protocol
Date: April 4, 2017

Materials and methods
1. Biomaterials
1.1.
Equine xenograft
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.
1.2.
Cortical lamina
The soft lamina (OsteoBiol®) used is a 35x35mm medium curved membrane of
porcine origins. Its clinical indications as described by the manufacturer: 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.
1.3.
Fixation
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.

2. Study design
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,
XXXXXX
2.1.
Inclusion criteria
Confining with the inclusion criteria, all the selected patients were systematically
healthy, had good oral hygiene (FMPS and FMBS <20%). When presenting for
implant placement and upon examination, the ridges had deficiencies in width
(<4 mm, Cawood and Howell class IV)  which did not allow correct implant
placement. Therefore, horizontal bone augmentation procedures were proposed.
All areas needing GBR, maxilla or mandible, posteriorly or anteriorly were
included in this study.
2.2.
Exclusion criteria
Exclusion criteria were the following: pregnant and lactating women, patients
suffering from a systematic disease, patients on bisphosphonates, smokers (>10
cigarettes/day) and patients needing vertical augmentations.
Patients were given a thorough description of the procedure with a highlight on
the risks, after which they received a consent form to sign before any procedure.
Ethical  approval  for  this  study  was  obtained  from  the  Scientific  Research
Commission (USJ- 2017- 107).
2.3.
Pre-surgical preparations
Clinical and radiographic examinations were done prior to any procedure. The
concerned 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 concerned 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.
2.4.
Surgical procedure
Local  and/or  regional  anaesthesia  was  obtained  using  Septanest® (Articaine
hydrochloride 4% with adrenaline 1:100,000), depending on the concerned area. 
A mid-crestal incision using a 15C blade (Swann-Morton®) 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 and lingually the flap was
elevated until reaching the mylo-hyoid line, using the Buser and the Prichard
periosteal  elevators  (Hu-Friedy®, USA). A  Rhodes  back action  (Hu-Friedy®,
USA) was used to debride the concerned crest from any fibers and connective
tissue debris (Fig. 10).
Decortication was perfomed using a twist drill with a stop of 3mm of length
(Meisinger® 203S) to ensure a good perfusion of the grafted site (Fig. 11).
    
Figure 1: Incision line (a) and flap elevation (b).
b
a

    
Figure 2: Measuring the area to be grafted (a) and decortication (b).
    
Figure 3: Twist drill used for decortication (a) and the cortical lamina soaked in saline water (b).
The soft cortical lamina had been soaked in saline sterile water since the start of
the surgery in order to achieve a good elasticity and easier manipulation (Fig. 12).
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. The cortical lamina
was first fixed on the palatal side (Fig. 13). The cortico-cancellous heterologous
bone mix (Gen-Os®, Osteobiol) which was previously hydrated in sterile saline
for 10 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 immobilisation.  Bone particles  were added from the lateral  sides  when
necessary to insure a good volume under the membrane (Fig. 14a).
b
a
b
a

  
Figure 4: Membrane fixated (a) and reflected (b) palatally.
  
Figure 5: Membrane fixated buccally (a) and horizontal mattress and single interrupted sutures (b).
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.  It  was  followed  by the  brushing  technique  when  the
elasticity was not sufficient. Precaution was taken to identify and carefully isolate
the mental foramen from the surrounding tissues when present. The lingual flaps
were advanced using a blunt instrument (e.g. Prichard) that would 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 PTFE 4/0 monofilament
suture (Cytoplast™).
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 (Fig. 14b)
The  vertical  incisions  were  also  closed  with  interrupted  resorbable  sutures
(Novosyn® 5/0).
All patients were instructed to receive an injection of betamethasone dipropionate
and disodium phosphate (Diprofos Injection- 2mL ampoule) directly after the
surgery and received 2g of Amoxicillin per day for a total of 7 days.
b
a
b
a

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.
Patients were told not to wear their prosthesis when present for a month at least,
after that the prostheses were relined with a soft liner and were worn only
occasionally.
Healing was uneventful in all cases except two where an exposure occurred along
with suppuration. They were treated with an extended dose of Amoxicillin (2
weeks) and with chlorhexidine rinsing. In both cases the membrane was not
removed and re-epithelization occurred after seven to ten days.
2.5.
 Implant placement
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. 
The crest was debrided from any soft tissue remnants and 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  (Meisinger®).  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. In most of the cases,
a 2 mm of bone structure was established buccally while positioning the implant.
Implant insertion torques were noted as indicated on the implant torque wrench.

Periapical radiographs were taken, the flap was sutured and the patients received
a daily dose of 2g of Amoxicillin for 7 days and diclofenac potassium (Cataflam ®
50mg) inflammatory for pain management.  They were also notified to mouth
rinse with Chlorhexidine 0.12% for 10 days.
Figure 6: View of the crest after biopsy (a) and implant placement in the mandible (b).
a
b

3. Radiological protocol
3.1. Image acquisition
At consultation and six months after the regeneration procedure, patients were
scanned with the Newtom VGI CBCT machine. Imaging conditions were: 110 kv
tube voltage; 2.2 to 8.30 mA tube current; 15 x 15 cm field of view; and 0.3mm
voxel size. Projection data were collected with a device rotating 360 degrees
around patients over a total acquisition time of 18 seconds.
3.2. 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 0.3mm
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.
3.3. 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.
3.4.
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:
3.4.1.  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.

3.4.2. 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.
Figure 7: Bone superimposition and measurements at the site of implant placement.
H6
H4
H0
H2

4. Histology
4.1.
Fixation and inclusion 
Samples were taken at six months after regeneration and were treated with non-
demineralized histology. At the time of sampling, the samples were fixed in LILLIE
neutral formolin, 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
concentration and for 48 hours in each bath, in ethanol 70°, 80°, 90°, 95°, 100°, 100°,
100°, then the clarification, allowing the penetration of methacrylate, in 2 successive
xylene baths of 24 hours each.
4.2.
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 Jm. 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. 
4.3.
Staining
The sections were stained with Giemsa-Paragon and basic fushin.
Giemsa will give cells and nuclei the colour blue, and Paragon will stain bone in red.

5. Histomorphometry
The qualitative observation of the sections was done under a digital microscope
(Keyence  digital  microscope  VHX-6000)  with  normal  and  polarised  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.