Dimensional ridge preservation using epithelialized palatal free graft or platelet rich fibrin following tooth extraction: An experimental study in minipigs Buasod.K 1, Suttapreyasri.S 1, Leepong.N 1, Pripatnanont.P 1 1 Dept of oral & maxillofacial surgery, Prince of Songkla University, Thailand *Corresponding author. E-mail: kantheeraa@yahoo.com Abstract Objective: To evaluate the potential of platelet-rich fibrin and/or epithelialized palatal free graft compared to blood clot to preserve alveolar ridge after tooth extraction in minipigs. Materials and Methods: In six minipigs, the maxillary and mandibular 2 nd and 4 th permanent premolar (P2 and P4) were extracted. The treatments 1) Epithelialized palatal free graft (FGG), 2) Platelet-rich fibrin (PRF), 3) combination of FGG and PRF, and 4) blood clot (control) were placed in the fresh extraction sockets by rotated in a cyclic permutation. Radiological examination was performed to compare at initial time, 2, 6 and 12 weeks. Evaluation of bone height and bone density was performed using Image Pro Plus 5.0 software. After 6 and 12 weeks, the animals were sacrificed and bone specimens were harvested. Decalcified specimens were sectioned at bucco-lingual plane, representing the central part of socket, and stained with hematoxylin and eosin for histological and histomorphometric examination. Results: Clinical and radiographic data showed that PRF, FGG, and combination groups displayed contour shrinkage at buccal aspect of 1.07±0.19, 1.14±0.69, and 1.50±0.61 mm respectively. In control group, the buccal shrinkage showed mean value of 1.30±1.03 mm (P>0.05). After 12 weeks, mean bone height reduction of the PRF, FGG, and combination groups were 1.11, 1.88, and 1.79 mm respectively, while the mean bone height reduction of control was 1.57 mm (P>0.05). Two weeks after extraction, PRF group presented a statistically greater bone density than control. After 12 weeks, there were a statistical difference between all experimental groups and control. Moreover, all experimental groups also demonstrated lower significant difference between buccal and lingual intersection at 12 weeks post extraction. The percentage of newly formed bone in PRF, FGG, and combination groups were changed from 42.31±4.69 to 52.00±21.20, 42.72±9.17 to 42.00±7.08, and 42.06±4.98 to 42.55±2.39 respectively comparing to control (39.65±6.13 to 42.74±2.39), though no significant was shown. Conclusions: PRF demonstrated early higher bone density during first 2 weeks, while FGG and combination of PRF and FGG resulted in increasing bone density and reducing of marginal bone resorption after 12 weeks. PRF alone may be suitable for short term ridge preservation. Keywords: ridge preservation; platelet rich fibrin; epithelialized palatal free graft. Introduction Following extraction of the tooth, a dimensional loss of bone height and bone width is a natural occurrence during the healing phase. The resorption of bone walls of the extraction sites occurred in two overlapping phases. During the first phase, the bundle bone was resorbed and replaced with woven bone, resulting in vertical reduction of the alveolar crest. The second phase, resorption had occurred from the outer surfaces of both bone walls. This remodeling pattern causes a horizontal resorption that may also induce a further vertical reduction of the buccal bone[1]. The remodeling of alveolar bone at the extraction site always decreases ridge volume and deforms the ridge configuration, which consequently impairs placement of dental implants in the ideal positions[2] and orthodontic movement of the tooth posteriorly[3]. Several studies have proposed various ridge preservation techniques and graft materials following tooth extractions, aiming to preserve the bone and acquiring for healthy soft tissue[4]. Although the gold standard is the autogenous bone graft, it may be considered an excessive or aggressive method for such small contained defects. Studies have clearly proven the reliability and functionality of using either allografts or xenografts, which avoids an additional surgical site for bone harvesting. However, the materials pose only osteoconduction property and still need to combine with autogenous bone when using in a large defects.
Wound healing kinetics in extraction sites is a tendency for soft tissue invagination and the formation of fibrous tissue in the coronal one third of the socket[5]. The principles of guided bone regeneration demonstrated to prevent invagination of oral epithelium into socket, favoring the repopulation of the socket with cells with bone regenerating potential and leading to more complete bone fill[6]. Sealing extraction sites with autogenous soft tissue grafts enables optimal preservation of ridge topography. The term epithelialized palatal graft (FGG) was used by Sclar to described the transfer of gingival tissue included epithelialized tissue harvested from the palate[7].the palatal area is often used and can generally supply adequate donor site. The advantages over other methods include predictability, color match with the adjacent tissues and minimal postoperative sequelae. The soft tissue seal is a free soft tissue graft. Blood supply from the underlying bed to the free gingival graft was considered the key for success[8]. Platelet-rich fibrin (PRF) is known as a rich source of autogenous cytokines and growth factors [9-11]. PRF can be considered as a healing biomaterial that consists of platelets, leukocyte, and cytokines, and the presence of circulating stem cells within a polymerized fibrin network. The clinical applications of PRF was divided into 4 highly specific aspects of healing: angiogenesis, immune control, harnessing the circulating stem cells, and wound protection by epithelial cover[12]. PRF may be considered as a useful material for ridge preservation. The aim of this study was to evaluate the potential of platelet-rich fibrin and/or epithelialized palatal free graft compared to blood clot for preserving alveolar ridge after tooth extraction in minipigs. Materials and Methods Animals The study protocol was approved by the Committee for Animal Research, Prince of Songkla University. Six 15-months-old minipigs weight 45-60 kg were used. The animals were kept and fed with pig food in a daily amount equivalent to 2% of the animal weight and water ad libitum. The alveoli created in the minipig jaws were randomly divided into 4 groups: Group I alveoli sealed with FGG; Group II alveoli filled with PRF; Group III alveoli filled with PRF and sealed with FGG; Group IV alveoli filled with blood clot and allowed to heal spontaneously (control group). All experiments and measurements were done by one examiner (B.K.). Surgical procedures During the examinations and surgical procedures the animals were sedated by intramuscular injection with Azaperone (Stresnil, 2mg/kg weight). Anesthesia was induced with an intravenous bolus of Tiletamine/zolazepam (Zoletil, 2.5 mg/kg weight). Before surgery, the animals were given 1 g of prophylactic amoxicillin (Vetrimoxin, 15mg/kg weight) intramusculary and continuing for 3 days. The single dose of metamizol (Dipyrone, 5mg/kg weight) was administered intramuscularly for post operative analgesic. The animal was checked daily at first week for signs of infection and fed a standard diet and water ad libitum until the date of sacrifice. The incisions were made in the crevice region of the 2 nd permanent premolar (P2) and 4 th permanent premolar (P4). The papillae were remained entirely intact. The P2 and P4 of each quadrant in both maxillary and mandibular jaws were extracted to create the four alveoli. Each group of material was assigned alternately to each site by rotating in a cycle permutation order. Group I: Alveoli sealed with FGG (Figure 2A) The FGG 2- to 3-mm thick was obtained from the palate using a 15C scalpel blade. Each graft was slightly larger in diameter than the socket orifice. The FGG were held in place by five to six simple sutures passing through the surrounding gingiva with resorbable suture material (Vicryl 4-0). Group II: Alveoli filled with PRF (Figure 2B) While the alveoli were prepared, 10-ml of autologous whole blood was collected from femoral vein (from forelimb) and transferred into 10 ml sterile glass test tube without anticoagulant. Immediately test tube was centrifuged using Hettich Zentrifugen centrifuge EBA 20 (Andreas Hettich GmbH & Co.KG, Germany) for 10 minutes at 3000 rpm. A fibrin clot was then obtained in the middle of the tube, just between the red corpuscles at the bottom and acellular plasma (PPP) at the top (Figure 1A). The PRF clot was collected and cut from red corpuscles with scissor, then made a second cut to yield two vertical halves of PRF (Figure 1B) for two extraction sockets. The PRF was held in place by one figure eight suture with resorbable suture material (Vicryl 4-0). Group III: Alveoli filled with PRF and sealed with FGG (Figure 2C) The alveoli were filled with PRF and covered with the FGG according to the technique mention earlier. Group IV: Alveoli exclusively with blood clot The alveoli were filled with natural blood clot and allowed to heal spontaneously. Figure 1. Blood processing with centrifugation for PRF(A), and the dot line represents the vertical cut to separate PRF into halves of PRF(B).
Figure 2. Clinical appearance of immediate extraction alveoli filled with FGG (A), PRF (B); empty socket; control(c). At the end of each designated healing period 6 and 12 weeks, the animals were sacrificed by induction of deep anesthesia followed by withdrawal of the entire blood volume. The maxilla and mandible were harvested and the alveolar ridges were cut in a block of 1.5 cm in height above the sites for histological preparation. The harvested blocks were fixed in 10% formalin, decalcified in EDTA, dehydrated in increasing concentrations of ethanol, embedded in paraffin and cut in bucco-lingual plane. The sections were stained in hematoxyline-eosin and examined in the light microscope (Axiostar, Carl Zeiss, Germany). Clinical observation During follow up period of 6 and 12 weeks, the alveoli dimensions were recorded using periodontal probe (Hu-Friedy, IL, USA). Mean values and standard deviations were calculated for the mid bucco-lingual (B-L) width, the buccal and lingual concavity of buccal plate, and occluso-gingival concavity. Radiographic observation Immediately after surgery, 2, 6, and 12 weeks post operative, the serial periapical radiographs of the experimental sites were obtained to evaluate the bone height and bone density. Radiograph was taken digitally using portable x-ray (NOMAD, Aribex Inc., Utah, USA) with digital sensor. Individual bite block was used to control the vertical distance (Figure 3A). The image analysis software (Image Pro Plus 5.0, Media Cybernetics, MD, USA) was used to evaluate bone height and density. Figure 3. The bite block for periapical radiographic with occlusal acrylic jig used for positioning the x- ray(a) and the 5 standardized line for determining alveolar height (B). To perform the bone height analysis, 5 standardized lines beginning in a base line were used In the maxillary alveolus, horizontal base line was drawn perpendicular to a line located below marginal ridge of the 3 rd permanent premolar (P3) 11 mm and 13 mm for P2 and P4 alveolus respectively. In mandibular alveolus it was drawn perpendicular to a line located below the cemento-enamel junction of the 3 rd permanent premolar (P3) 11 mm and 13 mm for P2 and P4 alveolus respectively. Once the horizontal base line was drawn, 5 vertical lines were drawn perpendicular to the base line and ended at the bone level visualized by one examiner (K.B.): bone height- 1 (H1) was located in the mesial extremity of alveolus; bone height-2 (H2) was located in the distal extremity of alveolus; bone height-3 (H3) was located in the center of the alveolus; bone height-4 (H4) was located in the middle between H1 and H3; bone height-5 (H5) was located in the middle between H2 and H3 (Figure 3B and Figure 4). Bone density of the alveolus area was evaluated through densitometry variations of grayscale, which varied from 0 to 255 (transparent to opaque). The area was 10 mm located from cementoenamel junction of the P3 to the bottom of the root socket of extracted tooth, including the alveolus area, extending from the apex to the bone crest of the P3 or the 1 st permanent molar (M1)(Figure 5). Histological and histomorphometric analysis Images were obtained using a light microscope at a magnification of 5X, associated with a camera. Digital images were evaluated using a software program Image Pro Plus 5.0 (Media Cybernetic, Silver Springs, MD, USA). To evaluate: (A) The area fraction (%) of newly formed bone within 2 mm vertical from the most superior new bone that examiner could see. (B) The height of the cortical bone walls was determined (Figure 6): an axis line started from the most superior of alveolus soft tissue contour to the center of the socket (C C) to separate the buccal and lingual/palatal (maxilla) compartments. Subsequently, horizontal lines (L and B) perpendicular to C C were drawn to connect the most coronal portions of the buccal and lingua/palatal bone crest to C C. The vertical distance between the buccal and lingual (palatal) intersections with C C was measured and expressed in millimeter.
Figure 4. Measurements of bone height using the image analysis software (Image Pro Plus 5.0, Media Cybernetics, MD, USA) in the maxilla (A, B, C) and in the mandible (D, E, F), immediately after tooth extraction(a, D), after 2 weeks (B, E) and after 6 weeks (C, F). Figure 5. Measurements of bone density in the mandible, using the image analysis software (Image Pro Plus 5.0), immediately after the tooth extraction (A), after 2 weeks (B) and after 6 weeks (C). Figure 6. Schematic drawing representing the location where the histomorphometric measurement were performed. Statistical analysis All data were presented in mean and standard deviations. One-way analysis of variance (ANOVA) was applied to detect differences among groups where the data were normally distributed. Paired T-Test was used to analyze the different between the 2 times interval. The statistical analysis was performed using SPSS software version 13.0, SPSS, Chicago, IL, USA). The significance level is P< 0.05. Results Clinical observations All alveoli healed uneventfully. Overt signs of soft tissue inflammation (swelling and redness) were seen during the first week of healing. After 2 weeks, soft tissue healing proceeded normally but appeared to be more rapid in the experimental groups (FGG, PRF and FGG+PRF) than in the control group. The granulation tissue covering socket orifice in PRF looked to be more mature but was not covered by epithelium (Figure 7). After 6 weeks, all sites were clinically completely healed. Alveolar ridge resorption both bucco-lingual (B-L) and occlusogingival (O-G) were clearly observed and continued until 12 weeks (Table1). The B-L contraction was marked on buccal side and was progressed rapidly in FGG, FGG+PRF and control group. Radiographic evaluation Bone height analysis: Table 2 showed the results of bone height as measured from 5 locations (H1-H5) at immediate after extraction, 2, 6, 12 weeks after the extractions All the treatment groups represented the same pattern of bone remodeling, which was decreased most in 6 weeks after extraction and decreased gradually at 12 weeks after extraction. The PRF group a mean bone loss of 0.80 mm and 1.49 mm at 2 and 6 weeks respectively were reported. Interestingly, after 12 weeks, a bone gain of 1.18 mm and mean overall bone loss of 1.11 mm were recorded. The FGG group, a mean bone loss of 0.92 mm and 0.73 mm at 2 and 6 weeks respectively were reported. After 12 weeks, a mean bone height loss of 0.22 mm and mean overall bone loss of 1.88 mm were measured. In FGG+PRF group a mean bone loss of 0.84 mm and 1.35 mm at 2 weeks and 6 weeks respectively were reported. After 12 weeks, a mean bone gain of 0.67 mm and mean overall bone loss of 1.57 mm were measured.
Figure 7. Clinical appearance of healing socket at 2, 6 and 12 weeks postextraction. Table 1. Means and standard deviations of alveolar ridge width at 6 and 12 weeks No statistically significant differences were found between experimental groups (P>.05). Bone density analysis: Table 3 showed the results of bone densitometry between the groups at immediate, 2, 6, and 12 weeks after the extraction. In the control group the mean bone density was gradually decline from 1.12 to 0.95 and 0.75 at 2, 6 and 12 weeks post extraction respectively. In the PRF group the mean bone density was increased significantly in first 2 week before slightly declined and stable at 6 and 12 weeks. Histological observations After 6 weeks, all groups were characterized by the presence of complete keratinized mucosa. The crestal region of buccal bone wall was resorbed with partially replaced by woven bone and was apical to its lingual bone walls. In center of crestal region, the PRF groups were comprised mainly of woven bone while provisional matrix (PM) was filled generalized throughout in the FGG group. The FGG+PRF and control group was filled with newly formed woven bone that penetrated by PM and vessels. All groups, the central and apical portions of the socket were dominated by bone marrow especially in FGG and control groups. After 12 weeks, the coronal portion of the socket of all of experimental groups showed histologically similar to the control group which presented of a hard tissue bridge that sealed the coronal part of the socket. The marginal bridge was continuous with the buccal and lingual bone wall that comprised mainly of woven with small area of lamellar bone. The central and apical portions of all groups were dominated by bone marrow except the PRF group included of newly formed woven bone and small lamellar bone. Also, the crestal region of buccal bone wall was resorbed apical to its lingual bone wall except the PRF group (Figure 8). Histomorphometric analysis Alteration of the height of the bone crest during healing : At 6-week interval, the buccal bone crest was found to be located on the average 2.04±1.48mm apical to the lingual crest. The vertical distance between buccal and lingual crest in PRF, FGG and FGG+PRF were 1.55±0.65mm, 1.89±0.99mm and 1.46±0.64mm apical to the lingual crest respectively
Table 2. Measurements of mean change in bone height of assigning location comparing between the groups. = The different of mean change between time interval Table 3. Results of bone densitometry comparison between the groups at the investigated stages. Statistically significant P-values (0.018) * and P-values (0.045) ** with ANOVA for difference in means between groups Statistically significant P-values (0.014) # and P-values (0.006) ## with ANOVA for difference in means between groups Figure 8. Overview of the histological sections of the extraction sites at 6 and 12 weeks of healing. (Arrows, the crestal region of buccal or lingual bone wall; B,buccal; Li,lingual. H&E staining, original magnification x5). Figure 9. Histogram describing the vertical distance between the buccal and lingual intersection with C-C. All the investigated stages the buccal crest was consistently apical of its lingual counter part. * Statistically significant P-values (0.021) with ANOVA for difference in means between control and FGG ** Statistically significant P-values (0.020) with ANOVA for difference in means between control and PRF # Statistically significant P-values (0.042) with ANOVA for difference in means between control and FGG+PRF
Figure 10. Histogram describing the mean percentage of new bone formation at 6 and 12 weeks. At 12-weeks, the buccal bone crest was consistently located apical of its lingual counterpart. The distance between buccal and lingual crest in PRF group was 0.85±0.42mm, while the corresponding distances in FGG, FGG+PRF and control groups were 0.86±0.41mm,1.02±0.96mm and 2.48±1.38mm apical to the lingual crest respectively. There was a significant vertical distance between buccal and lingual intersection between control and all experimental groups (Figure 9). New bone formation measurement: The histomorphometric evaluation of the percentage of new bone from the PRF, FGG, FGG+PRF, and control groups returned a mean ± SD valued of 42.31±4.69, 42.72±9.17, 42.06±4.98, and 39.65±6.13 % respectively, 6 weeks after extraction (Figure10). These indices turned to 52±21.20, 42±7.08, 42.55±2.39, and 42.74±2.39% respectively, 12 weeks after extraction. No statistically significant difference between groups was found in either of the observed parameters (P=0.841, P=0.272). Discussion The finding from the present study disclosed that PRF clinically did allows early healing of soft tissue for covering socket orifices in the first 2 weeks, but not in alveolar ridge shape at 6 and 12 weeks comparing with the control. The use of FGG with or without PRF did not show neither faster soft tissue healing, nor better alveolar ridge dimension when compared to the control group, especially at 6 weeks after extraction. The marked collapse of buccal bone walls were demonstrated at 6 weeks and progressed until 12 weeks. In histological sections, the coronal portion at 6 weeks of the PRF socket showed mainly comprised of woven bone while the FGG+PRF and control groups were contained woven bone penetrated with PM. In contrast with FGG group generalized PM occurred. At 6 and 12 weeks of all groups except the PRF showed a large marrow space filled the central and apical portions of the healed sockets. This corresponded to previously published studies [13,14] explained the healing of non load carrying tissue such as the extraction socket, once the cortical bridge had formed, there was apparently no obvious demand for mineralized tissue in the space. However in the PRF group these portions occupied by more mature mineralized tissue. This finding was corresponded to the radiographic analysis which shown that the alveoli filled with PRF presented greater bone density in first 2 week then declined and stable at 12 weeks. These data may be explained by previous studies that PRF, a natural fibrin containing various kinds of bone healing cytokines[15] can protect growth factors from proteolysis[16] and prolong the release of growth factors and delayed the peak of releasing. PRF reached the peak levels of TGF-ß1 and PDGF-AB at the time point of day 14[17]. The ingrowth, proliferation and differentiation of osteoblasts occurred during the initial 14 days[18]. It is thus deduced that PRF could stimulate bone regeneration better. The histomorphometric evaluated for the percentage of new bone demonstrated that there is no statistically significant among the experimental and the control group. However, the PRF group tendency showed the higher percentage of new bone in 12 weeks. In agreement with previous clinical studies reported that following tooth extraction the vertical bone loss became more pronounced on the buccal than on the lingual bone wall[19]. The crestal region of the buccal bone plate is often exclusively composed of bundle bone while the lingual bone plate consists of a combination of bundle bone and lamella bone. This may explain the marked reduction of height in particular at the buccal aspect. In this study the vertical bone loss in the control group was constantly increased from 6 to 12 weeks. In contrast to all experimental groups which the degree of the vertical bone loss was decreased. That was clearly demonstrated that PRF, FGG or combination of both is significantly preserved the crestal bone wall. Regarding to bone healing, PRF accelerated the process of bone healing. The marginal bone height demonstrated less bone resorption and overall new bone formation. This result was confirmed by the highest bone density of extraction site in PRF group at 2 weeks, more mature bone in histologic section at 6 and 12 weeks, and more new bone formation at 12 weeks. Conclusion From the presented study it may be concluded that PRF not only accelerated soft tissue healing, but also increased bone density during first 2 weeks and prolonged until 12 weeks. Whereas FGG and PRF+FGG resulted in late increasing bone density after 12 weeks. Application of PRF for socket preservation will give the maximum benefit for shortterm ridge maintenance. PRF can stimulate faster healing of soft and hard tissue at the extraction site.
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