Histomorphometric Analysis of Bone Regeneration Using a Dual Layer of Membranes (dPTFE Placed Over Collagen) in Fresh Extraction Sites: A Canine Model

Studies^1–6  have reported that the buccal process of alveolar bone is largely composed of bundle bone (immature bone supplied solely by ligaments and tendons), which makes it more susceptible to resorption DRWE tooth extraction than the lingual/palatal process. It is well documented that extraction of multiple contiguous teeth is associated with a more extensive buccal bone remodeling compared with single-tooth extraction.^4–6  In untreated extraction sockets, buccal bone remodeling may compromise the alveolar ridge width and complicate future oral rehabilitative procedures such as implant therapy.^1–5 

Various alveolar ridge preservation (RP) techniques have been proposed to minimize postextraction alveolar bone remodeling.^4,7–9  Studies have proposed that overbuilding the buccal plate with bone grafting materials minimizes postextraction alveolar bone remodeling.^10,11  The authors hypothesized that adding excessive bone grafting material in the areas where bone resorption is significant (such as buccal and coronal surfaces of the socket) helps compensate the natural bone resorption phenomenon.^10,11  The results showed that buccal overbuilding with excessive graft material is an ineffective technique for RP.^10,11 

Traditionally, collagen membranes are used in guided bone regeneration (GBR) protocols because of their hemostatic, chemostatic, and cell adhesive characteristics;^12,13  however, their fast resorption rate after exposure to the oral environment has raised concerns over their usage in GBR. However, it has been reported that collagen cross-linking increases its biodurability and enables collagen-based membranes to support long-term osteogenic activity while preventing other tissues from invading the defect.^14,15  Cross-linked collagen membranes have been successfully used in GBR procedures.^16,17  Advantages of using high-density polytetrafluoroethylene (dPTFE) membranes in GBR are that they do not require primary closure, they are easy to remove without the need for additional surgical interventions, and the minimize bacterial leakage;^18–20  however, a limitation of dPTFE is that the membrane exhibits poor tissue adhesive properties, which may sequentially jeopardize GBR.²¹ 

The present histologic study in dogs was based on the hypothesis that extraction sockets treated with bone graft and covered with a dual layer of membranes (dPTFE placed over a cross-linked collagen membrane) show significantly higher percentage of trabecular bone and total mineralized tissue compared with those treated with conventional socket augmentation using the GBR concept. The aim of this histologic experiment was to assess the efficacy of using a dual-layer membrane (dPTFE placed over collagen) for RP in fresh extraction sockets.

The study protocol was approved by the research ethics review committee of the Eng. A. B. Research Chair for Growth Factors and Bone Regeneration, 3D Imaging and Biomechanical Lab., College of Dentistry, King Saud University, Riyadh, Saudi Arabia.

Eight healthy female beagle dogs were used. The mean age and weight of the animals were 24 ± 0.83 months and 13.8 ± 0.49 kg respectively. The animals were vaccinated against hepatitis and rabies.

All nonsurgical and surgical procedures were performed under general anesthesia (Pfizer Limited, Sandwich, UK) using ketamine (10 mg/kg body weight) and local anesthesia (AstraZeneca LP for Dentsply Pharmaceutical, York, Pa) with xylocaine (with epinephrine 5 mg/mL).

One week before tooth extraction, supragingival scaling was performed on all dogs using an ultrasonic scaler (NSK, Westborough, Mass). On the day of surgery, intramuscular (IM) injections of amoxicillin (25 mg/kg body weight) (Betamox LA, Norbrook Laboratory Limited, Newry, Northern Ireland) were administered. The animals were draped, and the surgical site was swabbed with an antiseptic solution (Purdue Fredrick Company, Stamford, Conn).

All surgical and nonsurgical procedures were performed under general anesthesia using intramuscular injections of ketamine (Pfizer Limited) with adjunct buccal infiltration of local anesthesia (AstraZeneca LP for Denstsply Pharmaceutical). Nonsurgical root canal treatment was performed on the bilateral mandibular second premolar (P2), third premlar (P3). and fourth premolar (P4). A No. 2 size round tungsten bur (Brassler, Savannah, Ga) mounted on a high-speed hand piece (Dentsply) was used to prepare the access cavity. Initial instrumentation of the root canals was performed with K-type hand files (JS Dental, Ridgefield, Conn), after which rotary files (Profile, Dentsply, Addlestone, UK) were used for final root canal preparation. The root canals were irrigated with 5.25% sodium hypochlorite and obturated with vertically condensed gutta percha and sealer (Pulp Canal Sealer EWT, SybronEndo, Orange, Calif). Accuracy of root canal length was verified by an apex locator (Root ZX II- Apex Locator, J. Morita USA, Inc, Irvine, Calif) and periapical radiographs. Teeth with periapical lesions, such as periapical granuloma, were excluded.

After 2 months of root canal treatment, none of the teeth showed signs of periapical pathosis. Bilateral mandibular first premolars (P1) were extracted atraumatically. Bilateral mandibular P2, P3, and P4 were hemisected using piezosurgery (Mectron, Columbus, Ohio), and the distal roots were atraumatically extracted using forceps. The distal alveolus was currettaged to stimulate bleeding and eliminate remnants of the periodontal ligaments.

Randomization was performed by picking a paper labeled “group 1,” “group 2,” “group 3,” or “group 4” from a brown bag. In group 1 (the control group), the extraction socket was untreated. In group 2, particulate graft (Puros cancellous particulate allograft, Zimmer Dental, Carlsbad, Calif) was placed in the socket and covered with dPTFE membrane (Cytoplast Barrier Membranes, TXT-200, Osteogenics, Lubbock, Tex). In group 3, particulate graft (Zimmer Dental) was placed in the socket, the buccal plate was overbuilt with excess particulate graft, and the socket was covered with dPTFE membrane (Osteogenics, Lubbock, Tex). In group 4, particulate graft (Zimmer Dental) was placed in the alveolum and covered with a dual layer of membranes (that is, a dPTFE membrane [Osteogenics, Lubbock, Tex ] placed over a collagen membrane [Cytoplast RTM Collagen, Osteogenics Biomedical, Inc, Lubbock, Tex]). In all groups, primary closure was not intended (Vicryl [Polyglactin 910] suture, Ethicon Inc, Somerville, NJ) (Figure 1).

All subjects received IM injections of amoxicillin (Betamox LA, Norbrook Laboratory Limited) (5 mg/kg body weight once a day for 3 days). Plaque control procedures, which included topical application of a 0.2% chlorhexidine digluconate solution (GUM, Chicago, Ill) were performed twice a week for 4 months after surgery. The IM antibiotics (Betamox LA, Norbrook Laboratory Limited) were continued for 3 days after surgery at 25 to 50 mg/kg every 8 hours. After 4 months, all subjects were sacrificed using an overdose of 3% sodium pentobarbitol (Vortech Pharmaceuticals Limited, Dearborn, Mich).

The jaw segments containing the extraction sockets were removed en block using an electric saw (Leica SP 1600, Bannockburn, Ill); and fixed in 10% neutral formalin solution.

The samples and surrounding tissues were washed in saline solution and were immediately fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in 0.15 mol/L cacodylate buffer at 4°C and pH 7.4 to be processed for histology. The specimens were processed to obtain thin ground sections, dehydrated in ascending series of alcohol rinses, and embedded in a glycol (methyl methacrylate) resin. After polymerization, the specimens were sectioned with a high-precision diamond disk at about 150 mm and were ground to about 30 mm. The slides were stained with acid fuchsin and toluidine blue as described earlier.²²  The following descriptive parameters were measured by one examiner (G.I.): (1) buccolingual width of the alveolar ridge was defined as the distance from the outer most point of the buccal bone plate to the outer most point of the lingual bone plate, and the measurement was taken at 2 mm perpendicular from the cementoenamel junction and in the middle of the socket; (2) percentage of bone/socket; (3) percentage of bone marrow spaces; and (4) percentage of nonresorbed bone particles. The measurements were performed with a microscope linked to a video camera/computer and software (Buehler, Lake Bluff, Ill.

Statistical analysis was performed using statistical software (SPSS version 18.00, SPSS Inc, Chicago, Ill). The descriptive analyses of each histomorphometric parameter in each dog was obtained after analyzing 3 sections per site, 50 μm apart for each position. Significant differences among the groups were determined using one-way analysis of variance. For multiple comparisons. s Bonferroni post hoc test was used. P values < .05 were considered statistically significant.

The buccolingual widths of the alveolar ridge in groups 1, 2, 3 and 4 were 5.27 ± 1.22 mm, 5.81 ± 1.11 mm, 6.41 ± 0.99 mm, and 6.64 ± 1.64 mm, respectively. The buccolingual width of the alveolar ridge was significantly higher among sockets in group 4 (6.64 ± 1.64 mm) compared with those in group 1 (5.27 ± 1.22 mm) (P < .05).

The amount of bone in each socket was significantly higher among extraction sockets in group 4 (92.50% ± 10.40%) compared with those in group 1 (34.00% ± 19.35%) (P < .001), group 2 (43.00% ± 29.41%) (P < .001), and group 3 (56.50% ± 25.01%) (P < .001). These results are shown in Table.

The percentage of bone marrow spaces in groups 1, 2, 3, and 4 were 54.42% ± 17.97%, 50.64% ± 26.51%, 35.64% ± 22.70%, and 7.50% ± 10.40%, respectively. The difference was significance between all the groups (P < .001).

The remaining nonresorbed bone particles were counted and calculated per socket. The first group had no bone graft material, so it was not included. The remaining particles for groups 2, 3, and 4 were 13.0% ± 4.2%, 14.80% ± 4.49%, and 10.50% ± 3.34%, respectively. No significance difference was found between the groups (P < .633).

In group 1 (at 10×), mandibular cortical bone surrounded by oral mucosa was observed in the area of the first premolar tooth with periodontal ligament surrounding the apical portion of the canine. Mature compact bone with large amounts of osteons was also be observed (Figure 2a and b).

In group 2, osteons cells and cortical bone with mature compact and trabecular bone were observed (Figure 3a). At 40× magnification, particulate graft, compact bone, and mature bone were evident on the buccal surface along with newly formed bone and small bone marrow spaces (Figure 3b). At 40× magnification, newly formed bone with small bone marrow spaces and cement lines were observed between preexisting bone and newly formed bone (Figure 3c). At the same magnification (40×), no residual particles of biomaterial were detected inside the bone defect (Figure 3d).

In group 3, cortical, compact and mature bone were observed with fibrointegrated residual particulate graft particles next to oral mucosa (Figure 4a through c). At 100× magnification, fibrointegrated particulate graft particles with dense connective tissue in tight contact with particulate graft were observed (Figure 4d through f). At 40× magnification, newly formed bone with small bone marrow spaces was observed. Cement lines were observed between preexisting bone and newly formed bone (Figure 4a through f).

In group 4, at 10× magnification (Figure 5a), a large amount of trabecular bone was observed in the socket site. At 40× magnification, significant mature remodeled bone was observed (Figure 5b). Higher magnification showed evidence of remaining particles and evidence that modeling and remodeling were taking place (Figure 5c and d). At another view, the bonding between newly formed bone and native bone was evident (Figure 5e and f).

The histologic results showed that RP (in fresh extraction sites) using a dual layer of membranes (dPTFE placed over collagen membrane) enhances the buccolingual width of the alveolar ridge compared with regenerative protocols performed using either a single layer of membrane or no barrier membrane. In addition, the present experimental histologic results also demonstrated that the amount of new bone formed in extraction sockets in group 4 was significantly higher than that formed in groups 1, 2, and 3 and that the number of nonresorbed bone particles was significantly less among extraction sockets in group 4 compared with groups 2, 3, and 4. Furthermore, scanning electron microscopy results by Yun et al²⁰  showed that a dual layer of membrane (dPTFE placed over collagen membrane) is an effective way for RP around immediate implants.

Various explanations may be posited for this. First, because collagen and dPTFE membranes exhibit advantageous properties (such as optimal behavior toward soft tissue responses and optimal durability respectively), it is postulated that using these membranes (collagen and dPTFE) as a dual layer augments greater new bone formation than when each membrane type is used individually. Second, it may also be proposed that placement of a dPTFE augments new bone formation by stabilizing the graft at the defect site. Moreover, bacterial count has been reported to be significantly lower on the inner aspect of the dPTFE membrane than the outer surface.²⁰  This suggests that the dPTFE membrane provides a reasonably bacteria-free environment to the underlying collagen membrane that may in turn facilitate its chemostatic and cell adhesive properties. The present histologic study supports the scanning electron microscope results reported by Yun et al.²⁰  However, further studies are warranted to identify the microbial species that may be associated with extraction barrier membranes placed in fresh extraction sites.

Various bone grafting materials with different densities are used in RP procedures.^23–25  It is hypothesized that the bone mineral density of the bone graft material used during RP protocols may influence the overall outcome of the procedure. In the present experiment, a standardized bone particulate graft was used in all extraction sockets, which indicates that the outcome was chiefly associated with the dual-layer technique and not the type of particulate graft used. An interesting finding in the present experiment was that the percentage of nonresorbed bone particles was significantly lower in extraction sockets in group 4 than in groups 2, 3, and 4. A possible explanation is that placement of a dual layer of membrane helps stack the bone minerals, thereby preventing dispersion of the bone matrix. This may in turn have helped augment the bone regeneration compared with the other groups, where early resorption and collapse of collagen membrane may resulted in reduced bone formation and the increased number of nonresorbed bone mineral particles.

Tissue biotypes have been associated with the outcomes of periodontal surgical interventions.^26–28  Thin soft-tissue biotypes have been associated with thinner underlying alveolar bone and angular bone defects compared with thick soft-tissue biotypes.^26,27  In a recent clinical study, Le and Borzabadi-Farahani²⁷  investigated the relationship between buccal/labial bone thickness and crestal labial soft-tissue thickness around dental implants. The results demonstrated a significant relationship between the amount of buccal bone and thickness of soft tissues. As canine models exhibit a thin periodontal tissue biotype, it is tempting to speculate that the outcomes of bone regenerative protocols may also be compromised compared with those performed in clinical scenarios. However, further studies are warranted in this regard.

The present experiment has a few limitations. It is noteworthy that in the present experiment, all walls of the extraction sockets were intact. Therefore, we hypothesized that the presence of an osseous defect or a pathological lesion within or around the extraction socket may jeopardize new bone formation. Likewise, clinical studies^29–32  have shown that systemic disease (such as poorly controlled diabetes mellitus and acquired immune deficiency syndrome) and tobacco habits (such as cigarette smoking and tobacco chewing) jeopardize the alveolar bone and negatively influence the outcome of periodontal therapy. Thus, it is tempting to speculate that the efficacy of novel RP techniques (such as those described in the present study) may be compromised in persons who are immunocompromised and those who smoke tobacco. Researchers have also reported that the severity of periodontal disease increases with advancing age.²⁶  Therefore, the outcomes of ridge preservative techniques in elderly persons may be compromised compared with those in younger patients. However, further studies are required in this regard.

Within the limits of the present histologic investigation, it is concluded that ridge preservation using a dual layer of membrane after tooth extraction enhances the alveolar ridge width compared with when a single layer of membrane is used.

Abbreviations

dPTFE

high-density polytetrafluoroethylene

GBR

guided bone regeneration

RP

ridge preservation

The project was supported by King Saud University, Deanship of Scientific Research, GFBR Research Chair. This study is registered at the College of Dentistry Research Center (CDRC No: FR 0026), King Saud University, Riyadh, Saudi Arabia.