Natural healing consequences after tooth extraction include three-dimensional (3D) bone remodeling processes with socket resorption and, consequently, ridge atrophy.1 This is a chronic, irreversible phenomenon, which estimates a postextraction reduction of 2.6 to 4.6 mm in width and between 0.4 and 3.9 mm in height.2 Bone resorption continues over time; however, the most significant percentage of alveolar ridge contour loss occurs within the first 3 to 6 months after extraction.3 Moreover, ridge resorption is more pronounced in the mandible than maxilla, and the buccal side loses more tissue volume than lingual, which relocates the ridge to a more palatal/lingual position.4
Aiming to maintain the alveolar ridge architecture and obtain a functional and esthetic rehabilitation, thorough knowledge about the extraction healing process and preservation procedures is essential.5–7 Advantages of socket preservation techniques consist of soft- and hard-tissue maintenance, preservation of a stable ridge volume, and improvement of subsequent treatment outcomes.5–8 Several alveolar ridge preservation (ARP) techniques have been proposed9,10 and include minimally traumatic tooth extraction (eg, flapless instead of flap surgery), guided tissue regeneration (GTR), and immediate grafting of the extraction sockets using particulate bone grafts or substitutes, guided bone regeneration (GBR).11,12
The minimally invasive flapless approach to tooth extraction is known to be a simple and atraumatic method with improved clinical outcomes being reported that include reduced healing time, discomfort, and inflammation.13,14 The possible beneficial effect on limiting the resorptive process through flapless surgery has been investigated in preclinical models by comparing the outcomes with a flapped conventional surgery. Although a few studies have shown slightly less-pronounced bone remodeling of the alveolar ridge process,14,15 others have not reported any significant difference between flapless and flap techniques.16
In addition, GTR procedures, which consist of the coverage of the fresh extraction sockets with a membrane barrier, have been suggested to prevent epithelial down growth, allowing for bone regeneration in a more restrained and protected environment.17,18 Ideally, membranes should be biocompatible, nonimmunogenic, nontoxic, and manageable.19 A wide variety of synthetic and naturally derived, resorbable or nonresorbable membranes are commercially available. Nonresorbable synthetic membranes, such as polytetrafluoroethylene (dPTFE), a stable polymer, chemically and biologically inert, that is able to resist enzymatic and microbiological attack has been indicated for ARP.20 Nonetheless, nonresorbable membranes present limitations such as the need for retrieval after 3 to 4 weeks, especially when left exposed to the oral environment or if unintentional exposure occurs during the healing process.21 A recent systematic review comparing the effects of GTR techniques in nonmolar regions with the untreated control group reported around 1-mm less-dimensional changes favoring GTRs.2 By contrast, the use of grafting materials in fresh extraction sockets has been questioned because contained defects tend to heal with similar regenerative potential relative to GBR augmented sites. The later technique has demonstrated superior amount of tissue regeneration when restoring challenging clinical conditions, as noncontained critical defects, where volume re-establishment is necessary.22,23
Although substantial preclinical and clinical studies have been conducted to determine what the best approach for socket preservation therapy is, data concerning socket preservation therapies are inconclusive in terms of extraction technique and biomaterials used, leading authors of multiple systematic reviews to conclude that no method of ridge preservation emerges clearly superior to another, and that all of them result in some degree of bone loss.2 This study compared different tooth extraction techniques: flapless, flap, and flap + dPTFE and its effects on healing pattern evaluated by histological/metric changes using a controlled and reproducible study model. The postulated null hypotheses of this study were as follows: (1) Different tooth extraction techniques, flapless, flap, and flap + dPTFE, would not influence bone formation; and (2) different tooth extraction techniques, flapless, flap, and flap + dPTFE, would not influence socket dimensional stability.
Materials and Methods
The research protocol was submitted to and approved by the local Bioethics Committee for Animal Research from Ecole Nationale Veterinaire Alfort, Paris, France.
Preclinical In Vivo Model
Twelve adult beagle dogs in good health condition were used in the experiment. The animals were allowed to acclimate for 1 week before surgery.
All animals were preanesthetized with an intramuscular administration of atropine sulfate (0.044 mg/kg) and xylazine chlorate (8 mg/kg); then, general anesthesia was obtained after an intramuscular injection of ketamine chlorate (15 mg/kg). During surgical procedure, the animals inhaled O2 and were maintained on an intravenous infusion of saline.
Bilateral maxillary premolars (1st–3rd) were the selected area for the experiment; and, regardless of the group, exodontia was performed using modern techniques to minimize trauma to alveolar bone walls and to assure that all socket walls were preserved. All experimental groups were nested within the same animal to maximize statistical power and minimize number of animals. The surgical approach was randomly assigned for each area based on the experimental group: (1) flapless—premolars were hemisected with the use of fissure burs. No incisions or flap elevations were made. The roots were carefully removed with elevators and forceps; (2) flap—intrasulcular incisions were made along the roots of premolars, and buccal/lingual full-thickness flaps were elevated. Flap elevation was extended to a level beyond the mucogingival line and disclosed the alveolar crest and the marginal 3 to 4 mm of the buccal and lingual bone walls. The roots were hemisected with the use of fissure burs and carefully removed with elevators and forceps. Flap advancement was achieved through periosteal release to allow for tension free wound closure; and (3) flap + dPTFE, surgical extraction identical to flap group + sockets covered with textured, high density polytetrafluoroethylene (Osteogenics Biomedical, Inc., Lubbock, TX) (dPTFE) nonresorbable membrane. Membranes were trimmed to fully cover the 2 sockets and fixed by the placement of mini screws at both buccal and palatal flanges; also, they were not removed throughout the experiment. Flap advancement was achieved through periosteal release to allow for tension free wound closure. For all groups, the wounds were sutured with 4-0 PTFE sutures (Osteogenics Biomedical, Inc.), providing primary soft-tissue closure. Postoperatively antibiotics penicillin (20.000 UI/kg) and analgesics ketoprofen (1 mL/5 kg) were given for a period of 48 hours. The animals were kept in cages with free access to water and feed with moistened balanced dog chow. Animals were euthanized at 1 week (n = 6) and 4 weeks (n = 6) by means of anesthesia overdose.
Histologic Preparation and Histomorphometric Analysis
Individual bone blocks consisting of experimental groups (hard and soft tissues) were harvested and processed. The bone blocks were kept in 10% buffered formalin solution for 24 hours, washed in running water for 24 hours, and gradually dehydrated in a series of ethanol solutions ranging from 70% to 100%. Finally, the samples were embedded in a methacrylate-based resin (Technovit 9100; Heraeus Kulzer, Wehrheim, Germany) following manufacturer's instructions. The blocks were then cut in a buccopalatal direction, perpendicular to socket inclination, into slices (300-μm thickness) with a precision diamond saw (Isomet 2000; Buehler, Lake Bluff, IL). Then, the slices were glued to acrylic slides using an acrylate-based glue, and a 24-hour setting time was established.24 The embedded blocks were then cut and ground (Metaserv 3000, Buehler) to a final thickness of approximately 100 μm by means of a series of SiC abrasive papers under water irrigation (400, 600, 800, 1200, and 2400). Thereafter, the samples were stained with Stevenel's Blue and Van Giesons' Picro Fuschin (SVG) stains and scanned through an automated slide scanning system and specialized computer software (Aperio Technologies, Vista, CA).
Histological observation and histomorphometric evaluation were conducted, and the amount of bone formation (%) and socket total area (mm2) were quantified by means of a computer software (Image J; National Institutes of Health, Bethesda, MD, Leica Application Suite, Leica Microsystems).25,26 The inflammatory infiltrate content was ranked using a specialized computer software (Aperio Technologies) as follows:27
A 0 to 5 scale was used to assess inflammatory cells content at the surgical site and/or membrane/host tissue interface. A 0 indicated no inflammation, while 5 indicated a significant amount of inflammation.
The data regarding the amount of bone formation and ranking of inflammatory infiltrate content have shown distinguishable variances in the study of the dependent variable (Levene test, all p < 0.25). Amount of bone formation differences was examined through the Friedman nonparametric test, and ranking of inflammatory infiltrate content through the Kruskal-Wallis nonparametric test. The data are presented as a function of median and quartiles. Socket total area has shown indistinguishable variances (Levene test, all p > 0.25), and the data were statistically evaluated through a linear mixed model with fixed factor of time (1 and 4 weeks) and group (flapless, flap, and dPTFE) and a random intercept. A post hoc comparison of the means was performed using least significant difference (LSD) test. The data are presented as the mean values with the corresponding 95% confidence interval (CI). The analyses were accomplished using SPSS (IBM SPSS 23, IBM Corp., Armonk, NY).
All surgeries were uneventful, and healing process evidenced no signs of infection and/or inflammation throughout the study period. Animals recovered consciousness after 0.5 hours after surgery, and no complications, such as membrane exposure, were recorded during healing time. Clinically healthy soft-tissue appearance was observed. Clinical evaluation at the time of sacrifice depicted soft-tissue closure for all experimental groups.
Statistical analysis of amount of bone formation is presented as a function of median and quartiles in Figure 1. Data evaluation regarding to time revealed significant difference between 1 and 4 weeks in vivo, 0% (0%–0.1%) and 32.1% (16.5%–48.7%), respectively (collapsed over group) (Fig. 1, A) (p = 0.001). Nonetheless, no significant difference was evidenced between flapless, 5.4% (0.1–15.2), flap, 9% (0%–27.2%), and flap + dPTFE groups, 8.5% (0%–39.8%) (collapsed over time) (p = 0.996; Fig. 1, B). When bone formation was evaluated as a function of both, time and group, all groups at 4 weeks in vivo presented higher amount of newly bone compared with 1 week (p = 0.001; Fig. 1, C).
Statistical analysis of socket total area is presented as a function of mean and 95% CI in Figure 2. Socket total area evaluated as a function of time revealed significantly decreased area after 4 weeks in vivo (Fig. 2, A). Also, the flap + dPTFE group (16.4 ± 2.2 mm2) showed higher total area than flap (12.7 ± 2.5 mm2) and flapless (12.8 ± 2.4 mm2) (data collapsed over time) (p = 0.046; Fig. 2, B). Similarly, when data were evaluated as a function of both time and group, flap + dPTFE socket total area (16.4 ± 3.3 mm2) was significantly higher than flapless (11.2 ± 3.7 mm2) (p = 0.019) and flap (10.3 ± 3.7 mm2) (p = 0.042) at 4-week time point, and no significant difference when compared with flap + dPTFE 1 week (16.4 ± 3.0 mm2) was demonstrated (p = 0.993; Fig. 2, C).
Survey optical micrographs for all groups are presented in Figure 3. Histological sections revealed no bone formation after 1 week in vivo with no difference in the socket total area between groups. Nonetheless, after 4 weeks in vivo, new bone formation was observed for all groups without significant difference, as confirmed in the histomorphometric analysis. The images indicated that the newly formed tissue within the socket comprising woven bone demonstrating that normal healing mechanisms were not impaired regardless of surgical technique. Additionally, lower socket total area was seen for 4 weeks flapless and flap groups when compared with flap + dPTFE at 4 weeks and all groups at 1 week (Fig. 2, C).
Higher magnification microscopy of each group is depicted in Figures 4–9. Socket buccal plate contour, both in thickness and in height, was similar between groups at 1-week time point (Figs. 4–6). However, more pronounced bone remodeling was observed after 4 weeks in vivo (Figs. 7–9). Flapless and flap groups presented noticeable soft-tissue migration inside the socket (Figs. 4, 5, 7, and 8) in contrast to no soft-tissue presence within the flap + dPTFE group that evidenced more cervical bone formation (Figs. 6 and 9). In addition, while buccal plate of flapless and flap groups showed evident remodeling, flap + dPTFE depicted noticeable bone formation between the outer part of the buccal plate and dPTFE membrane (Figs. 4–9).
Widespread inflammatory infiltrate content can be observed in the socket of all groups (Fig. 10, A–F), and it is concentrated to regions in proximity with the membrane for flap + dPTFE groups (Fig. 10, C and F). No significant difference between groups was observed according to ranked inflammatory infiltrated content; however, a borderline absence of statistical difference indicated a trend to a decrease in the inflammation process after 4 weeks in vivo regardless group (p = 0.051; Fig. 10, G).
Numerous clinical conditions can lead to tooth loss such as extensive carious lesions, periapical pathology, dental fracture, and periodontal disease. In addition, as the alveolar process is a tooth-dependent tissue, several dimensional changes during socket healing unavoidably occur, and subsequent rehabilitation treatment may be compromised.1,2,4,28,29 Although the resorption process is not completely inevitable, several ARP techniques have been recommended to maintain socket architecture.9,10 This study compared different methods of ARP: flapless minimally traumatic exodontia, minimally traumatic exodontia with a mucoperiosteal flap, and minimally traumatic exodontia with the use of a mucoperiosteal flap and a nonresorbable membrane (flap + dPTFE), evaluated histomorphometrically.
The first null hypothesis that the different experimental techniques would not influence bone formation was accepted; histometric analysis of amount of bone formation within socket evidenced significant more pronounced percentage of new bone after 4 weeks in vivo for all groups when compared with 1 week; however, no significant difference between groups was observed. The second hypothesis that socket dimensional stability would not be influenced by tooth extraction techniques was rejected since total area was significantly lower after 4 weeks in vivo for all groups but for the flap + dPTFE, the only group that maintained socket architecture over healing time.
Regarding the utilization of a mucoperiosteal flap or a flapless extraction approach, previous studies are controversial, and no unequivocal conclusions may be drawn on their potential advantages on bone formation and ridge preservation.13 Hypothetically, more atrophy should be seen after flap elevation because of a disruption of the blood supply to the buccal and/or palatal socket walls. Although a few studies evidenced less-pronounced bone remodeling for tooth extraction followed by flap elevation,14,15 another study has demonstrated no significant difference on bone formation and ridge preservation between flap and flapless approaches.16 The latter, also supported by our results, may be attributed to the short-term healing evaluated in this study and/or the soft-tissue primary closure obtained that may have favored primary healing as previously reported by Darby et al.5 Despite the absence in significant differences in both bone formation within socket and socket total area after flap and flapless tooth extraction, it has been previously suggested that flapless tooth extraction may present clinical advantages such as periosteum preservation, decreased surgical time, lower patient discomfort, and earlier establishment of normal oral hygiene procedures after the surgery.30
When both minimally traumatic exodontia approaches, flapless and flap, were compared with the use of dPTFE membrane placed under a mucoperosteal flap, the latter presented significantly higher results according to socket total area after healing period (an indicator of alveolar ridge maintenance). These findings are in agreement with systematic reviews available in the literature that concluded that covering the socket with a membrane, regardless its composition reduces bone structural changes after tooth extraction.2,9 A previous clinical study evaluated the use of dPTFE membrane in 276 extraction sockets, demonstrating predictable preservation of soft and hard tissues after histological analysis.31 Rehabilitation is usually simplified when ridge architecture is maintained since an adequate three-dimensional (3D) soft- and hard-tissue volume allows for dental implants placement in a prosthetically driven and biomechanically favorable position.32
Furthermore, micrographs depicted that while buccal plate of flapless and flap groups evidenced more bone remodeling, the flap + dPTFE group demonstrated noticeable new bone formation outside the buccal plate. It is well understood that shortly after tooth extraction, bundle bone is rapidly absorbed and replaced by woven bone, while alveolar bone is absorbed throughout life.1 This resorption/modeling process results in a reduced ridge volume, mainly at the expense of the buccal plate because its crestal portion merely comprises bundle bone.1 The results of the current study show that although coverage of the socket with a membrane does not completely prevent bone resorption, it has the ability to attenuate this process by maintaining space in the presence of osteogenic tissue, thereby enabling not only bone healing but also new bone formation to occur simultaneously.
Given the bone formation levels, alveolar ridge maintenance, and low inflammatory infiltrate levels detected at 4 weeks in vivo for the dPTFE group relative to others, the presence of the membrane throughout the course of the experiment secured by microscrews on both buccal and palatal aspects, along with primary soft-tissue closure, presented no detrimental effect to the results. Primary closure was used in this in this study because of the difficulty of maintaining hygienic conditions with exposed membranes in the canine model. However, when nonresorbable membranes such as textured dPTFE are used in clinical settings where primary closure is not typically achieved over extraction sites, removal at 21 to 28 days has been suggested to prevent plaque accumulation and associated inflammatory response, potential increase in membrane exposure with concomitant risk of gingival dehiscence, and possibly infection underneath the membrane.20,21 Future studies are warrented to determine whether similar histologic and dimensional results can be expected when dPTFE membranes are left partially exposed instead of completely covered by mucoperiosteal flaps.
Although histologic analysis provides excellent information on healing dynamics and quantification of tissue remodeling, it has the inherent limitations of the assessment of few 2D sections at specific healing periods. Hence, future studies should consider using 3D imaging techniques, such as computerized tomography and computerized microtomography that successfully support a linear evaluation of hard tissue (re)modeling and, also, the calculation of morphometric indices. Within the limitations of the current study, the coverage of fresh sockets with polytetrafluoroethylene (dPTFE) membranes after minimally traumatic exodontia seems to be a promising method to maintain alveolar ridge architecture when compared with flap and flapless approaches.33
Although dimensional changes unavoidably occur after tooth extraction, extraction socket coverage with polytetrafluoroethylene (dPTFE) membrane after minimally traumatic exodontia seems to be a superior method of ARP when compared with flap and flapless approaches. Despite, the amount of bone formation within socket was not influenced by any method employed as an experimental group in this study, we observed an appositional bone development on the buccal plate for the dPTFE group.
The authors claim to have no financial interest, either directly or indirectly, in the products or information listed in the article. PGC has served as a scientific consultant to Osteogenics.
The research protocol was submitted to and approved by the local Bioethics Committee for Animal Research from Ecole Nationale Veterinaire Alfort, Paris, France (protocol number B940473).
Roles/Contributions by Authors
P. G. Faciola Pessôa de Oliveira: performed the experiments, data analysis, and manuscript review. E. T. Pedroso Bergamo: performed the experiments, data analysis, and manuscript review. D. Bordin: performed the experiments and manuscript review. L. Arbex: performed the experiments and manuscript review. D. Konrad: performed the experiments and manuscript review. L. F. Gil: study design and data analysis. R. Neiva: study design, experimental design, and manuscript writing. N. Tovar: performed the experiments and manuscript review. L. Witek: performed the experiments, data analysis, and manuscript review. P. G. Coelho: study design, surgical procedures, data analysis and statistical evaluation, and manuscript writing.
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