Animal Grouping and allocation:
Six adult mongrel dogs (20-25kg and 3-4 years old) with a fully erupted permanent dentition were enrolled in this experimental study. During the experiment, dogs were fed once per day with a soft food diet. This research was approved by the ethical committee at the Faculty of Dentistry, Ain Shams University, Egypt (Approval number:(FDASU-RecD031603). All procedures were applied according to the international regulations of animal care and use.The experiment was performed in three surgical phases. The first phase is the defect preparation, second is the augmentation phase and the third is surgical reentry phase. Defects were randomly allocated into four groups using balanced block randomization utilizing a computer-generated protocol[*]. In group (1), block graft was covered by a conventional occlusive collagen membrane (Block+CM), while in group (2), four compressed layers of L-PRF membrane were added first before top coverage by occlusive collagen membrane (Block+L-PRF+CM). In group (3), block graft was covered by a perforated collagen membrane (Block+PCM) while in group (4), four compressed layers of L-PRF membrane were added first before top coverage by perforated collagen membrane (Block+L-PRF+PCM).
First surgical phase
Dogs were pre-medicated with subcutaneous Atropine sulphate[†] at a dose of 0.05 mg/kg and intramuscular Xylazine HCl [‡]at a dose of 1mg/kg. Anaesthesia was induced by intravenous Ketamine HCl [§]at a dose of 5mg/kg, and was then maintained by intravenous Thiopental sodium[**] 2.5% solution at a dose of 25 mg/kg. Routine dental infiltration anaesthesia using 1.0-1.8 ml of 4% articaine hydrochloride [††]with 1:100,000 epinephrine was used locally at the surgical site.
In each dog, four standardized saddle-type mandibular defects (mesiodistal width: 10mm, height: 8mm) were planned to be prepared (two defects on each side) on the premolar area. All surgical procedures were performed by one surgeon ( A. K ). A mandibular full-thickness incision was made bilaterally from the canine to the second molar. Mucoperiosteal flaps were raised at the buccal and lingual sides. The mandibular 2nd, 3rd and 4th premolars (P2-4) were splitted buccolengually and extracted (Fig.1 a ). After exposing the alveolar bone, four standardized saddle-type defects including the vestibular and oral aspects of the alveolar ridge were subsequently prepared using a surgical fissure carbide bur in a straight handpiece under copious saline irrigation (Fig.1 b ). Final refinement of the defect to its planned dimension was done by back action chisel. During bone block removal, all attempts were made to standardize the size of all defects using a periodontal probe[‡‡] . The final dimensions of all defects were approximately (8-10 mm) mesiodistally, (6-8mm) apico-coronally, and (12-14 mm) bucco-lingually at the bottom of the defect based on the original ridge dimention. Primary wound closure was achieved by horizontal mattress and simple interrupted sutures using resorbable (5/0) vicryl sutures[§§]. Defects were left for 8 weeks to establish stable chronic defects and create adequate soft tissue for wound primary closure after grafting in the second surgical phase.
Second surgical phase and L-PRF preparation
Following a healing period of 8 weeks, with the same anaesthetic schedule utilized in the first surgical phase, venous blood was collected from the jugular vein in dry glass tubes and centrifuged at low speed (2,700 rpm) for 12 minutes by using PRF specific centrifuge[***] . Three layers were formed: the RBC base layer, acellular plasma top layer and L-PRF clot in the middle. By using a specific box, the L-PRF clots were pressed, and membranes were folded 4 times to be ready for their application [21] .
A mid crestal full-thickness incision and periosteal reflection was performed. The four defects were subsequently exposed and readjusted to their original standardized dimensions using a surgical fissure carbide bur under copious irrigation with sterile saline (Fig.1 c,d ). For each defect, a xenogeneic block graft[†††] was trimmed, adjusted, fitted and secured in place using osteosynthesis screws 12-14 mm in length and 1.5 mm in width with a cross flat head[‡‡‡] (Fig.1 e,f ). All attempts were made to adjust the screw head with the level of the neighbouring bone crest. The lines of demarcation between the block and pristine bone and all voids were grafted by xenogeneic particulate graft of the same block graft origen (Fig.2 a ). The vertical dimension of the augmented defect was measured using a periodontal probe in the middle of the defect in line that coincides with the fixation screw. For group 1, after block tailoring and fixation, occlusive collagen membrane[§§§] was trimmed to the appropriate shape, draped over the ridge to cover the block graft completely and extended beyond the defect margins by approximately 3 mm. In group 2, four compressed layers of L-PRF were adapted over the block graft with 3 mm marginal extension before occlusive collagen membrane over-coverage (Fig.2 b ). Group 3 collagen membrane was perforated using a 25 gauge dental before block top coverage (Fig.2 c ). Interperforation spaces were determined to be ≤ 2mm to avoid rduction in membrane stiffness [22] . In group 4, bone block was covered first by four compressed layers of L-PRF followed by a perforated collagen membrane over-coverage.
Following each surgical procedure, all dogs were given intramuscular injections of Cefotaxime sodium at a dose of 10 mg/kg [****]and Diclofenac sodium at a dose of 1.1 mg/ kg [††††]once daily for five postoperative days[23] .
Third surgical phase of clinical and histomorphometric assessment
Following a healing period of 8 weeks post-grafting, animals were submitted to surgical reentry for clinical and histomorphometric assessment. Mucosal health was inspected for wound closure, oedema, purulence or any area of exposure. Following the same anaesthetic schedule used for the previous 2 surgical phases, a mid crestal full-thickness incision was made in order to expose the area of interest guided by the head of the fixation screw using a back action chisel.
The following measurements were assessed by a blind examiner (AMA) who was blinded about the type of intervention and not involved in any other part of the study:
Measuring the amount of vertical graft loss (VGL) was performed using a periodontal probe to measure the mean of the exposed parts of the osteosynthesis screw at their four different sides ( mesial, distal buccal, lingual ) (Fig.2 f ). The vertical bone gain (VBG) was calculated by subtracting the exposed part of the screw from the vertical dimension of the middle part of the block graft that was measured during surgery. For horizontal augmentation assessment, a sliding bone calliper [‡‡‡‡]was used at sex reference points(Fig.2 e ). Two points at the middle (coincide with screw), two mesial (3 mm apart from screw) and 2 distal (3mm apart from screw). The coronal three points (3mm from the top of the defect) were used for the assessment of the mean horizontal dimension coronally, and the apical points (6 mm from the top of the defect) for the assessment of the mean horizontal dimension apically.
By the end of the 3rd surgical phase, all dogs were sacrificed by an overdose of thiopental sodium. Individual blocks containing the fixation screw and the surrounding hard tissues were fixed in 10% formaldehyde followed by decalcification via immersion in EDTA 17% solution for about 100 days. Finally, the specimens were dehydrated in a series of graded ethanol solutions. Blocks were cut in a buccolingual plane using a diamond band saw fitted into a precision slicing machine. Two histological slides were obtained from the central part of the augmented area marked by screw (central slides). Sections were subsequently reduced to a thickness of about 50 µm using a cutting–grinding device and stained with haematoxylin and eosin (H&E) stain and Masson's trichrome (MTC) stain.
Histomorphometric analyses and microscopic observations were performed by an experienced investigator masked to the specific experimental conditions (ESA). The percentages of new bone, marrow space, and remaining graft particles were assessed. Also, the amount of mature bone and immature tissues was inspected by using (Masson trichrome stain). For image acquisition, a colour CCD camera[§§§§] was mounted on a binocular light microscope[*****]. Digital images with different magnifications were evaluated using an image analysis software program[†††††]. Since the quantity of newly formed bone could vary along the height of the block graft, the area of interest was divided into two parts taking the fixation screw as a reference point. The upper area corresponds to the first three threads of the fixation screw (new bone - periosteal side), and the next three threads represent the apical bone area (new bone - native bone side).
For every specimen, two sections were obtained with four randomly selected fields within each section making a total of eight measurements for every specimen with their mean value representing the final value used in statistical analysis. A calibration procedure was initiated for the image analysis software and revealed that repeated measurements of different sections were similar at ˃95% level.
Statistical analyses
For the sample size calculation, the power analysis was performed using the G Power System. [‡‡‡‡‡]The criterion for significance was set at α = 0.05 (type I error) and β= 0.20 (type II error). The resultant sample size was 5 dogs (defects) per group. By calculating an attrition rate of 10%, the final sample size was 6 animals (defects) per group. The power of this study was kept at 80%. [24]. The mean and standard deviation values were calculated for each group in each test. Data were explored for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests, data showed parametric (normal) distribution. Clinical outcome variables included vertical graft loss (VGL), vertical bone gain (VBG), horizontal ridge dimension coronally, horizontal ridge dimension apically and total horizontal ridge dimension (mean value of 6 point measurements of ridge width). Histological outcome variables included the percentage of new bone, marrow spaces, and remaining graft particles after image analysis. New bone-in upper (periosteal side) and apical (native bone side) were analyzed separately. Also, the amount of mature bone and immature tissues was calculated and analyzed. One-way ANOVA followed by Tukey post hoc test was used to compare more than two groups in non-related samples. Paired t-test was used to compare two groups in related samples. The significance level was set at P ≤ 0.05. Statistical analysis was performed with IBM® SPSS® Statistics Version 20 for Windows [§§§§§].
[*] (Excel; Microsoft, Redmond, USA)
[†] (Atropine sulphate 1%®, ADWIA, Egypt)
[‡] (Xylaject 2%®, ADWIA, Egypt)
[§] (Keiran®, EIMC Pharmaceuticals Co., Egypt)
[**] (Thiopental sodium®, EPICO, Egypt)
[††] (Artinibsa 40mg/mL, Inibsadental, Spain)
[‡‡] (CP15; Hu-Friedy Co., Chicago, IL, USA).
[§§] (Assucryl,Assut sutures, Switzerland)
[***] (IntraSpin System, Intra-Lock, USA)
[†††] (Bio-Gen Block, Bioteck, Arcugnano (Vicenza) – Italy)
[‡‡‡] ( Aesculap, Inc. Hazelwood, USA )
[§§§§] (Hypro -sorb, Bioimplon GmbH, Friedrich, Germany)
[****] (Cefotax 250mg vial®, T3A Co., Egypt)
[††††] (Voltaren 75 amp®, Novartis Co., Egypt)
[‡‡‡‡] (Medesy Sri, industrial area, Italy)
[§§§§] (Color View III, Olympus, Hamburg, Germany)
[*****] (Olympus BX50, Olympus, Germany)
[†††††] (Image J 1.49b, NIH, USA)
[‡‡‡‡‡] (G Power, Ver. 3.192 copyright 1992-2014, Düsseldorf, Germany)
[§§§§§] (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp, USA)