Abstract
The goal of the present clinical study was to evaluate new bone formation in human extraction sockets augmented with enamel matrix derivatives (EMD) and Bio-Oss Collagen. Patients with symmetrical single-rooted teeth in the bilateral quadrants of the upper jaw condemned for extraction participated in this study. Following extraction, the sockets (20 sockets) were randomly augmented using either EMD or Bio-Oss Collagen. After 3 months of healing, bone biopsies were obtained and prepared for histological analyses. Dental implants were then placed. Implant stability quotient (ISQ) readings were obtained for each implant at the time of surgery and at 1 and 3 months postoperatively. The mean new bone formation was 34.57 ± 25.67% in the EMD sites and 28.80 ± 16.14% in the Bio-Oss Collagen sites. There was no significant difference between the groups. The ISQ values were significantly higher for the implants placed in the EMD sites at the first and third months, but no significant differences were observed in the ISQ values for the implants placed in the Bio-Oss Collagen sites. The augmentation of the extraction sockets with EMD or Bio-Oss Collagen leads to similar behaviour in bone regeneration.
The fresh extraction socket in the alveolar ridge represents a special challenge in everyday clinical practice. Numerous studies have demonstrated that following tooth extraction, undisturbed wound healing will lead to the loss of ridge volume and changes in the ridge shape. The delicate marginal portion of the buccal bone wall frequently contains larger amounts of bundle bone than the lingual wall. Bundle bone is a tooth-related tissue that disappears following tooth loss. This healing process results in various complications, including the lack of available alveolar bone for implant placement, an unfavourable crown–implant ratio, and aesthetic problems.
Socket preservation is a procedure in which autogenous bone, graft materials, or biological agents are placed in the socket of the extracted tooth at the time of extraction. Socket preservation is technique-sensitive, is not 100% successful, and can be unpredictable. Different materials have been tested, with many shown to be efficacious compared with the natural healing of the socket. Because of their excellent biocompatibility, deproteinized bovine bone grafts have been widely used in bone grafting. Many studies have reported the use of deproteinized bovine bone grafts in various types of bone deficiencies, with clinically successful results. Recent data have indicated that the application of bovine bone mineral embedded in 10% highly purified porcine collagen in fresh extraction sockets in dogs minimized the resorption of the original alveolar bone and allowed adequate new bone formation within the extraction socket. Histomorphometric studies describing the early healing events of bovine bone minerals in human extraction sites are limited to two human studies on the physiology of bovine bone mineral embedded in 10% highly purified porcine collagen (Bio-Oss Collagen) in human extraction sockets.
Enamel matrix derivatives (EMD) have been used in clinical treatment procedures to promote periodontal regeneration. They are derived from embryonic enamel of a porcine origin, and their use is based on the high degree of homology between porcine and human enamel proteins. EMD is thought to mimic the role of enamel matrix proteins in cementogenesis during nascent root development. EMD has attracted interest because of its effect on osteogenic gene expression and cell adhesion.
Cardaropoli et al. reported that during the first week of healing following tooth extraction, the periodontal ligament (PDL) adjacent to the bundle bone of the socket walls maintained its vitality. Lekic et al. suggested that mesenchymal cells from the PDL may participate in the healing of the socket wound. EMD may facilitate PDL cell attachment and growth. Moreover, the attachment of PDL cells to the EMD appears to generate an intracellular signal to increase their proliferation and the secretion of various autocrine growth factors. In particular, transforming growth factor-beta 1 (TGF-β1) has been shown to affect osteoblast proliferation, chemotaxis, and extracellular matrix deposition. EMD has been reported to stimulate the production of TGF-β1.
Our present knowledge suggests that EMD possesses an inductive stimulating effect, whereas the Bio-Oss Collagen more passively supports bone deposition. Based on these observations, the application of EMD in extraction sockets may promote new bone formation. The goal of the present clinical study was to evaluate new bone formation in human extraction sockets augmented with EMD and Bio-Oss Collagen.
Materials and methods
Patient selection
All patients were systemically healthy, not pregnant, and had never smoked. The subjects were not taking any regular medications that have been associated with a compromised bone healing response. All patients had at least two symmetrical single-rooted teeth to be extracted and replaced with endosseous implants. Patients with severe periodontitis, active periodontal lesions, or severely resorbed sockets with a remaining height of less than 5 mm were excluded from the study.
The study protocol had institutional review board approval and all subjects were asked to provide informed consent to participate after a detailed explanation of the procedures and objectives of the study. After the initial periodontal therapy, the surgical operations were started.
Surgical procedures
Tooth extraction and socket augmentation
All surgeries were performed by one operator (EAA). Following gentle tooth extractions without flap elevation, the fresh sockets were debrided to remove all soft tissue. A sharp No. 15 surgical blade was used to sever the dento-gingival connective tissue fibres. A periotome and the appropriate dental forceps were used to minimize the surgical trauma to the surrounding tissue. To achieve a forceless extraction, a slow, gentle rotational-pulling force was preferred, until the periodontal ligament fibres were torn completely. All extraction sockets had to be intact (four walls). The gingival walls at the socket orifice were gently de-epithelialized. The extraction sites were randomly assigned to the EMD (Straumann Biologics Division, Waltham, MA, USA) group or the Bio-Oss Collagen (Geistlich Pharma AG, Wolhusen, Switzerland) group. The treatment procedure (EMD or Bio-Oss Collagen) at each site was determined by the toss of a coin. Before application, the EMD, which was stored in a refrigerator at 2–8 °C, was warmed at room temperature for 15 min. The EMD was then applied to the entire socket, starting from the most apical area. Once the Bio-Oss Collagen was placed into the socket, it was compressed without excessive force. Primary coverage of the socket was achieved using a mucosal punch graft harvested from the palate. A suitable site for graft harvesting was chosen distal of the rugae of the palate with a distance of 4–5 mm from the gingival margin. With the selected punch, a free gingival graft of 2–3 mm thickness was cut and removed using an all-around ball-attached scalpel blade. The harvested graft was placed on top of the socket orifice and sutured to the marginal gingiva of the extracted teeth with six interrupted sutures ( Fig. 1 ).
Postoperative systemic antibiotics and 0.12% chlorhexidine mouthwash were prescribed. The sutures were removed 10 days after the surgery.
Re-entry surgery and implant placement
After 3 months of healing, a surgical re-entry procedure was performed. Following the application of local anaesthesia, crestal and intrasulcular incisions were made, and mucoperiosteal flaps were reflected to allow access to the alveolar ridge.
A total of 18 biopsies (nine Bio-Oss Collagen and nine EMD) were harvested for histological evaluation. A trephine bur with a 2.0-mm internal diameter was used to take the core biopsies, which were approximately 8 mm in length. Specimens were marked to identify the coronal and apical ends. The bone cores were coded and fixed in a 10% neutral buffered formalin solution. After removal of the cores, an additional osteotomy was performed, and the dental implants (Standard 4.1, 10 mm, Institut Straumann AG, Switzerland) were installed ( Fig. 2 ). Implant stability quotient (ISQ) readings were obtained for each implant at the time of surgery before flap closure using an Osstell Mentor instrument (Institut Straumann AG, Switzerland). The implant stability measurements were re-evaluated at the first and third postoperative months.
The patients were rehabilitated with both fixed and removable implant-supported prostheses. The prosthetic rehabilitation was started 3 months after the implant placement.
Histological and histomorphometric analysis
Overall, 18 cylindrical bone cores were decalcified with 5% formic acid for 2 weeks, sectioned into 5-μm thick sections, and stained with haematoxylin and eosin (H&E) and Van Gieson Trichrome (Bio-Optica) stains. The sections were evaluated by light microscopy to quantify the bone content using 40× magnification. The specimen examination was performed blindly by two investigators (BS and BY).
Photographs were taken using a digital camera (Nikon Coolpix 4500), and the images were saved on a computer. Computer-assisted histomorphometric measurements of the newly formed bone were obtained using the Leica Q Vin version 3 program.
Statistical evaluation
Data analysis was performed using SPSS version 11.5 software (SPSS Inc., Chicago, IL, USA). The data were expressed as the mean ± standard deviation. The differences among more than two repeated measurements were evaluated by Friedman test, and the Wilcoxon signed-rank test was used to determine differences between the EMD and Bio-Oss Collagen groups. When the P -value from the Friedman test was significant, determining those times that differed from the others, non-parametric multiple comparison tests were used. Pearson’s correlation coefficients were also calculated to determine the degree of the associations between continuous data. A P -value of less than 0.05 was considered statistically significant. For all possible multiple comparisons, the Bonferroni correction was applied to control for type I errors.
Statistical evaluation
Data analysis was performed using SPSS version 11.5 software (SPSS Inc., Chicago, IL, USA). The data were expressed as the mean ± standard deviation. The differences among more than two repeated measurements were evaluated by Friedman test, and the Wilcoxon signed-rank test was used to determine differences between the EMD and Bio-Oss Collagen groups. When the P -value from the Friedman test was significant, determining those times that differed from the others, non-parametric multiple comparison tests were used. Pearson’s correlation coefficients were also calculated to determine the degree of the associations between continuous data. A P -value of less than 0.05 was considered statistically significant. For all possible multiple comparisons, the Bonferroni correction was applied to control for type I errors.
Results
Ten patients (four males and six females, aged 40–58 years) with symmetrical single-rooted teeth in the bilateral quadrants of the upper jaw condemned for extraction were included in this randomized clinical study.
Clinical observations
All experimental sites healed uneventfully after the tooth extraction and socket augmentation. Four weeks after healing, punch grafts were well integrated. Only two grafts in one patient were necrotic. In all cases, the grafted sites were able to support the implant placement.
Histological assessments
One patient with a necrotic punch graft was excluded from the study. All specimens were free of inflammatory cells.
All sections revealed new bone formation in all specimens ( Fig. 3 ). The total tissue area was calculated, as was the total bone area (bone area/total area). The amount of newly formed bone was 34.57 ± 25.67% in the EMD sites and 28.80 ± 16.14% in the Bio-Oss Collagen sites. In the EMD group, the new bone formation was more obvious at the apical region of the specimen. In the Bio-Oss Collagen group, new bone was observed surrounding the remaining Bio-Oss Collagen particles, which was mainly observed in the coronal area. The within-group comparison revealed no significant difference between the coronal and apical sections for the EMD group ( P = 0.354) and Bio-Oss Collagen group ( P = 0.613) in terms of new bone.
