This study was approved by the Yenepoya University Ethics committee, Mangalore, India (Approval Number YOEC83/8/3/2014). Fifteen patients who required extraction of a maxillary or mandibular tooth and subsequent single-tooth implant placement and who met the inclusion and exclusion criteria were included in this prospective single-arm clinical study. The patients (4 females and 11 males) had a mean age of 51.3± 14.8 years (range: 27 to 75 years). The site-specific areas and teeth numbers for the study are shown in Table 1.
All patients were systemically healthy at the time of consultation and study inclusion. The reasons for extraction included endodontic treatment failures and advanced caries lesions and tooth fractures. If more than one tooth was extracted (maximum of three teeth), all teeth were treated but only the most anterior tooth with intact socket wall was selected for the study. Standard exclusion criteria for bone grafting procedures were applied including allergy, systemic chronic disease, alcoholism and drug abuse, pregnancy, or nursing mothers. Smokers and patients with any oral tobacco use/habits were excluded. Patients using dentures were also excluded. Patients with acute abscesses or active infections localized in the proximity of the prospective surgical field and those with heavily scarred mucosa at the site and patients who had malignant diseases or other diseases treated with radiotherapy or chemotherapeutic agents (chemotherapy) during the past 5 years were excluded.
Surgical technique
All surgical procedures were performed by one surgeon in this study. The following procedures were planned for all sites. Tooth extraction was performed under local anesthesia without flap elevation (Fig. 1a, b). Periotomes and luxators (Directa, Sweden) were gently used for all extraction procedures. Extraction forceps were only used when the tooth was mobile in the extraction socket. All multi-rooted teeth were sectioned with a Lindemann burr (Komet Inc., Lemgo, Germany) under copious irrigation with sterile saline to minimize extraction trauma. Each root of the multi-rooted teeth was independently mobilized and carefully luxated. Attention was given not to damage the surrounding soft and hard tissues, especially in the buccal aspect. All sockets were thoroughly curetted to remove granulation tissue, followed by irrigation and rinsing with sterile saline. A ball-ended probe was then utilized to explore the buccal plate. All teeth included for this study had an intact buccal and lingual plate (four-wall post-extraction sockets). A biphasic alloplastic in situ hardening bone graft substitute (GUIDOR easy-graft CRYSTAL, Sunstar Suisse SA, Etoy, Switzerland) was used to graft the site according to the manufacturer’s instructions. Attention was given not to overfill the extraction socket to avoid any displacement of the entire graft mass after mechanical irritation during the first phases of healing. A saline-wet gauze was used to further compact the granules and accelerate the hardening of the graft in situ so that after a few minutes the alloplastic bone substitute formed a stable, solid, porous scaffold for the host osseous regeneration (Fig. 1c). An interrupted tension-free nonresorbable 4-0 sutures (Black Silk, Ethicon, Johnson & Johnson, Somerville, NJ, USA) was placed over the filled socket to achieve soft tissue stability (Fig. 1d). All sites were left uncovered without obtaining primary closure in order to heal by secondary intention. The patients did not wear any prosthesis during the healing period.
Antibiotic therapy consisting of 1 g amoxicillin every 12 h for 4 days and mouth rinsing with 0.2% chlorhexidine every 8 h for 10 days were prescribed. The suture was removed 1 week postoperatively. After 3 to 8 months (average 5.2 ± 2 months), the sites (Fig. 2a) were reentered for implant placement. A site-specific full thickness mucoperiosteal flap was elevated to expose the regenerated hard tissue. A bone core biopsy was taken with a minimum depth of 7 mm from the center of the site using a trephine drill with a diameter of 2.3 mm (Komet Inc., Lemgo, Germany) (Fig. 2b). Following the harvesting of the bone sample, the preparation of the bony bed was completed at the same site and a dental implant was placed (Fig. 2b, c) according to the manufacturer’s surgical protocol. Immediately after placement, the initial stability was measured by resonance frequency analysis (Osstell ISQ, Gothenburg, Sweden). For each implant, two ISQ measurements were recorded, palatally (or lingually) and bucally, according to the guidelines of the company. The measurement was repeated after placement of the final restoration 4 months after implant placement. Average of the ISQ measurements was then reported. The mucoperiosteal flap was closed with interrupted nonresorbable 4-0 sutures (Silk, Ethicon, Johnson & Johnson, Somerville, NJ, USA), and Fig. 3 shows the postoperative radiograph of the implants placed in the preserved ridge. Figure 4a shows the second surgery followed by impression making, and Fig. 4b shows implant crowns placed and loaded after 3 months of placement.
Assessment of ridge width changes by cone beam computer tomography
The area of interest (site of socket preservation and grafting) was identified in accordance with the site which was grafted (Fig. 5a–d). Axial correction of the view was performed in conformity with angulation of the alveolar ridge. Contours of the crestal bone were identified, considering apical recession of the alveolar crestal level, if any on buccal or lingual/palatal aspect. Buccolingual/palatal width of the alveolus was then determined 2 mm below the alveolar crestal level before tooth extraction and at the time of implant placement. Ridge width changes were calculated by subtracting preoperative measurements from postoperative ridge width measurements (Fig. 6a–c).
Histological and histomorphometric evaluation
Bone biopsies were harvested using a trephine bur at the site of implant placement. The trephine burs including the bone biopsies were fixed in 4% formalin for 5–7 days, rinsed in water, and dehydrated in serial steps of ethanol (70, 80, 90, and 100%), remaining for 1 day in each concentration. Specimens were then infiltrated, embedded, and polymerized in resin (Technovit 9100, Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer’s instructions. After polymerization, samples were cut in 500-μm sections using a precision cutting machine Secotom 50 (Struers, Ballerup, Denmark). The sections were mounted on acrylic slides (Maertin, Freiburg, Germany) and ground to a final thickness of approximately 60 μm on a rotating grinding plate (Struers, Ballerup, Denmark). Specimens were subsequently stained with Azur II and Pararosanilin (Merck, Darmstadt, Germany), which allowed for a differentiation between graft granules, and preexisting and newly formed bone. Imaging was performed with an Axio ImagerM1 microscope equipped with a digital AxioCamHRc (Carl Zeiss,Göttingen, Germany). Histomorphometric analysis was performed by one observer digitally using the analySIS FIVE software (Soft Imaging System, Munster, Germany). The reference area for the histomorphometric evaluation was the entire area in the biopsy excluding old bone from the defect margins. Values measured in percent of the examined area were taken for remnants of graft material, newly formed bone, and soft tissue/bone marrow (Fig. 7a–c).
Statistics
Data were expressed as means ± standard deviations (SD). A two-sided paired t test was used to assess significance of intra-group longitudinal changes for ridge width changes and changes of ISQ. Statistical significance was set to p < 0.05 for a two-sided test.