This split-mouth, double-blind randomized clinical trial was conducted in accordance with the Helsinki Declaration at the Department of Periodontology, Isfahan University of Medical Sciences. The methodological workflow is illustrated in Figure 1. The Ethics Committee of Isfahan University approved the research under registration number IR.MUI.MED.1398.107. This study was registered in the Iranian Registry of Clinical Trials under the number IRCT20110109005570N11. Written informed consent was signed by all patients, and participation was voluntary.

The primary outcomes were vertical and horizontal dimensional changes (bone loss) in postextraction sockets between the test and control groups. The secondary outcomes were changes in trabecular thickness, and the tertiary outcomes were the width and thickness of keratinized gingiva.

The inclusion criteria for patient selection were as follows: at least 18 years of age, requiring single-root tooth extraction, and presenting a minimum of 10 mm vertical bone height without invading adjacent critical structures. The exclusion criteria included smokers, pregnant patients, systemic disease, periapical or acute periodontal infections, bone wall fracture at the time of tooth extraction, and statin use for hypercholesterolemia.

The sample size was calculated to be 15 patients assuming α = 0.05, β = 20%, and a study power of 80%. Clinical examination was performed to select individuals with at least two single-root teeth requiring extraction in different quadrants and not adjacent to each other. In this split-mouth clinical trial, in each patient, one dental socket was selected as the case and the other socket as the control. Randomization for selecting the socket in each patient was done by sealed envelope. The envelopes were numbered, and each socket was assigned a number. The pathologist, patients, and data analysts were blinded to the allocated arm.

The ATV gel was synthesized by mixing 1.2% ATV powder (Amin Pharmaceutical Company, Isfahan, Iran) with 30% pistachio resin and 2% oleo-gum-resin. It was then mixed with polyethylene glycol and gelation materials to become injectable. Oleo-gum has disinfecting properties and solidifies in the presence of blood, filling the socket. It can also serve as a matrix for ATV and enable its sustained release within 6 weeks. The homogeneity of the formulation was assessed macroscopically after 48 h (presence of palpable folliculated particles and also in terms of color and transparency) and microscopically after 24 and 48 h, 1 week, and 1 and 3 months to ensure homogeneity. The pH of the compound was also measured at 48 h to 3 months after synthesis. Stability against centrifugal force, thermal stability, freezing and melting temperatures, rheological properties, viscosity, dispersion, microbial contamination, and the release rate of ATV were all evaluated using respective methods.

The same surgeon (N.H.) conducted all surgical procedures. After signing informed consent forms, all patients underwent oral and dental examination and received the required periodontal treatments to optimize oral conditions for wound healing. Patients received oral prophylaxis 1 week preoperatively and were instructed to rinse with 0.12% chlorhexidine mouthwash for 1 min twice daily. For the surgical procedure, local anesthesia (lidocaine 2%, Aburaihan Pharmaceutical Co., Tehran, Iran) was first administered, and atraumatic tooth extraction was performed using a fine periotome and forceps. The width (KGW) and thickness (KGT) of keratinized gingiva were measured with a periodontal probe (Williams Probe, Hu-Friedy) perpendicular to the bone before flap elevation. The sockets were thoroughly curetted, irrigated with sterile saline, and inspected for perforations, fenestration, or dehiscence. Thereafter, the mucoperiosteal flap was reflected 5 mm beyond the alveolar crest on the buccal side, and a titanium pin was fixed 4 mm apical to the crest of the ridge. The height of available bone was measured from the crest of the edentulous ridge to the opposing landmark (titanium pin) with a periodontal probe. The titanium pin served as a reference for assessing the vertical dimension of bone and as a guide for bone core harvesting. The width of available bone was measured between the facial and lingual plates at the crest of the alveolar socket using a caliper. Then, 0.5 ml of ATV gel was randomly chosen to fill the sockets and subsequently covered with a collagen membrane (Collprotect membrane, Botiss). In the control socket, only the resorbable membrane was placed. The membrane was first hydrated in sterile saline for 5 min and trimmed to completely cover the extraction socket, extending 3 mm beyond it. The flap was then repositioned.

No attempt was made to cover the socket with the flap. In case of membrane exposure into the oral cavity, it was left exposed. Horizontal cross-mattress resorbable sutures were used to stabilize and fix the membrane. Patients were prescribed 500 mg amoxicillin three times daily for 10 days, as well as analgesics and 0.12% chlorhexidine twice daily for 4 weeks. Sutures were removed 2 weeks after surgery. Patients were recalled for implant placement after 2 months. Following administration of local anesthesia, all clinical parameters were measured as described earlier. KGW and KGT were measured with a probe. A mucoperiosteal flap was elevated, alveolar width was measured with a caliper at the crest of the ridge, and bone height from the crest of the ridge to the titanium pin was measured with a periodontal probe.

A trephine with a 3 mm internal diameter and 6 mm length was used to obtain a core sample of bone. Harvested bone cores were immediately placed in 10% neutral buffered formalin. The osteotomy was then widened for implant placement according to standard protocol. Patients were followed according to standard clinical protocols until completion of implant restoration.

The biopsy specimens were decalcified in 5% nitric acid for 1 week, sectioned, and the margins marked with India ink. They were subsequently transferred into a tissue processor, immersed in 10% formalin, and subjected to ascending concentrations of alcohol (60%, 70%, 80%, 90%, and 100%). They were immersed in xylol and embedded in paraffin blocks after 13–17 h. The paraffin blocks were sectioned into 3.5-μm slices by a microtome, deparaffinized in xylol, dehydrated in descending concentrations of alcohol, and stained with hematoxylin and eosin. Slides were inspected under a light microscope (Nikon E400, Japan) at magnifications of ×100 and ×400 to determine the percentage of osteogenesis (woven, cancellous, and lamellar bone). The thickness of bone trabeculae was measured by histomorphometric analysis. All measurements were made by a single expert pathologist.

In histological studies, the percentage of bone formation (woven, spongy, and lamellar) and, in histomorphometry studies, bone trabecular thickness was measured.

No interim analyses were performed, and no stopping guidelines were established.

Alveolar ridge dimensions (height: from the crest of the edentulous ridge to the opposing landmark [titanium pin] and width: between the facial and lingual plates at the crest of the alveolar ridge) were measured immediately after extraction and before implant placement. No statistical differences between the case and control groups were noted [Tables 1 and 2].

The percentage of lamellar bone formation and trabecular thickness in the experimental group was greater than in the control group, but the differences were not significant [Table 3, Figures 2 and 3].

Evaluation of keratinized gingival width after 2 months showed that in the experimental group, the mean keratinized gingival width increased from 5.33 ± 1.45 to 7.33 ± 1.83 mm (P = 0.000). In the control group, the mean keratinized gingival width increased from 5.53 ± 1.40 to 7.46 ± 1.45 mm 2 months after the intervention (P = 0.000). In both the groups, the mean keratinized gingival thickness increased after 2 months, but there was no significant difference between the two groups (P = 0.347).

This research study received financial support from the Vice-Chancellor for Research at Isfahan University of Medical Sciences (Grant No: 398107).

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.