The clinical trial was approved by the Ethics and Research Committee of Faculty of Medicine, Hasanuddin University, Makassar, Indonesia, with code number 504/UN4.6.4.5.31/PP36/2022. The study was designed according to the principles of the Declaration of Helsinki and will conform to the CONSORT (Consolidated Standards on Reporting Trials) statement [19].

Before participating in the study, all individuals must provide written informed consent, signifying their voluntary agreement to participate. Participants will have the opportunity to seek clarification on any aspects of the study and review all procedures, data collection methods, and data analysis protocols before signing the consent form. The informed consent document will comprehensively outline the research’s purpose, procedures, potential risks and benefits, as well as the confidentiality and security measures pertaining to the collected data. Participants will be explicitly informed of their entitlement to withdraw from the study at any point without experiencing any adverse consequences on their treatment.

Strict privacy and security protocols were implemented, and all study data were stored in a password-protected secure database. Each participant will receive a unique study code, and data sharing with operators and research assessors will be done in an encrypted format that maintains patient privacy. Participants will not be provided with any form of remuneration for their involvement in the study. Nonetheless, they will receive complimentary examination and treatment related to the trial without incurring any costs.

Figure 1 presents a simplified diagram that outlines the study protocol based on the CONSORT diagram. The recruitment of participants and the implementation of the trial will take place at Hasanuddin Dental Hospital, Makassar, Indonesia. This hospital is the primary institution in the designated region that handles the majority of oral surgery cases. To identify eligible participants, all individuals presenting to the Department of Oral and Maxillofacial Surgery with a chief complaint related to the mandibular third molar will undergo screening conducted by the study investigators.

Before enrollment, all participants will be provided with detailed information regarding the risks and potential complications associated with the procedure, such as bleeding, infection, cheek asymmetry, parotid duct injury, potential injury to facial nerve branches, and the unlikely event of nonclosure. Informed consent will be obtained from each participant after ensuring their understanding and agreement.

This prospective control clinical trial will be conducted at a single center and will involve 30 targeted extraction sockets after the removal of impacted mandibular third molars. This case is chosen as it represents a common scenario requiring extraction and often involving the removal of a significant amount of bone, making it an ideal model for studying human alveolar bone loss for socket preservation. The study aims to evaluate the efficacy of a high-purity α-CSH bioceramic (CaSO₄·0.5H₂O), synthesized from calcium sulfate dihydrate (CSD) without the use of chemical reagents, as a bone graft material in the alveolar bone loss case.

The 30 extraction sockets will be randomly assigned into 2 groups: 15 will receive α-CSH as a bone graft, while the other 15 will not receive any graft material. The main end point will be evaluated 6 months into the procedure. During follow-up visits, any adverse events or serious adverse events will be carefully documented, and clinical assessments will be conducted as specified in the “intervention section.” All patients will undergo regular monitoring through clinical examinations, radiographs, lab tests, and any additional necessary procedures. Ultimately, the study will publish a comprehensive report on the feasibility and potential efficacy of the α-CSH approach in promoting bone formation, regardless of the outcome.

To be eligible for participation in this clinical trial, patients must meet specific inclusion criteria, which are as follows: being a healthy male or female; aged 18-35 years old; providing written informed consent to participate in the trial; having an average blood count within normal range; presenting with impacted mandibular third molars that require extraction. The diagnostic indications for tooth extractions include caries, orthodontic, and preventive measures. In particular, based on the relation of the impacted mandibular third molar to the ramus, the impacted teeth will be restricted to Class I and Class II as per the Pell and Gregory classification [20,21]. Patients had to demonstrate good oral hygiene, maintain healthy periodontium, and have a clinical attachment loss score of ≤2 mm and a probing pocket depth (PPD) of <4 mm. Clinical and radiographic images taken before the operation are depicted in Figure 2.

Patients will be disqualified from participating in the study if they meet any of the following exclusion criteria: impacted teeth concerning their position relative to the occlusal plane fall within the Pell and Gregory classification's level C category [20,21]; presence of uncontrolled or untreated periodontal disease; existence of acute abscesses or active infections that occur close to the targeted surgical site; presence of systemic diseases or systemic/local infections; recent receipt of chemotherapy, radiotherapy, immunosuppressant medications, or anticoagulants that could potentially interfere with the healing process; previous administration of bone growth–inducing substances, malnutrition, or current active influenza; regular smoking habit of more than 10 cigarettes per day; being pregnant, breastfeeding, or participating in a fertilization program.

Participants have the right to withdraw from the study at any time and for any reason without facing any negative consequences. In cases where a participant has urgent medical needs, the researcher may choose to exclude them from further participation in the study. Participants who choose to discontinue the study before completing all surgical procedures and follow-up visits will be considered dropouts.

The high-purity α-CSH bioceramic was synthesized from calcium sulfate dihydrate (Acros Organics, Morris Plains, NJ) using a microwave irradiation technique, following the methodology outlined in our previous study [14,17]. In brief, 2 g of CSD powder were mixed with 10 mL of deionized water. Following this procedure, the resulting mixture, along with a magnetic stirring bar, was placed in a 55-ml Teflon pressure tube at approximately 350 pounds per square inch (psi). The mixture was then subjected to microwave irradiation at 800 watts and 160 °C for 10 minutes. After synthesis, the sample was filtered, washed with ethanol, then dried in an oven at 60 °C for 8 hours, and finally ground into a powder.

Prior to the surgical procedures, a preoperative panoramic x-ray and cone-beam computed tomography (CBCT) scan will be conducted to evaluate the tooth position and the surrounding bone structure. A complete blood count (CBC) will also be performed to ensure the safety of all patients undergoing surgical procedures and receiving bone grafts. Furthermore, the PPD score will be measured using a periodontal probe (BPWHO: Single, Osung, South Korea) to evaluate the periodontal condition. Patients exhibiting a significant amount of calculus will be referred for scaling and root planning procedures. Skilled oral and maxillofacial surgeons will carry out the interventions following standardized operating procedures. Video demonstrating the clinical and surgical procedures will be presented during the initial calibration meeting to ensure consistency across all operators.

The extraction of mandibular third molars will be carried out in a sterile environment under local anesthesia, minimizing damage to both the soft tissue (gingiva) and the hard tissue up to the crest of the bone. The surgery will adhere to the principle of minimally invasive techniques, using a split technique. To preserve the integrity of the marginal alveolar bone, an elevator will be used carefully to avoid any harm to the bone margins. Initially, a triangular access flap was created, with a releasing incision made on the tuberosity and another distal to the second molar. Then, the third molars will be split, and extraction will be performed using forceps or a dental elevator. After removing the tooth, a thorough sharp curettage will be performed to eliminate any remnants of the periodontal ligament and other soft tissues. Special attention will be given during the procedures to protect the socket walls and interproximal papillae from damage.

The fresh extraction sockets will be treated differently based on the assigned groups. In the experimental group, α-CSH material will be used to fill the socket, while no bone graft material will be applied in the control group. The amount of α-CSH used will depend on the size of the socket. Using a periosteal elevator, the material will be carefully placed and gently packed into the extraction site until it reaches the top of the socket. The graft material will be applied up to 2 mm apical to the soft tissue edge, with a maximum total dose of 3 g per patient. A membrane will be used to cover the upper side of the socket, and primary wound closure will be achieved using absorbable sutures.

Postoperatively, patients will be prescribed appropriate antibiotics and pain relievers for a duration of 7 days. They will be provided with instructions to follow a soft food diet, gargle after meals, and limit physical activities for the first 3 days following surgery. Clinical follow-up examinations will be conducted at 7 days and 14 days after the procedure. Control assessments will include panoramic radiography and CBCT scans within 3 and 6 months after the surgery to monitor the progress of bone formation.

Adverse events encountered during the study will be classified as serious or nonserious based on severity using the classification system established by the World Health Organization [22]. Participants who experience any adverse events will receive comprehensive medical care to address their needs. Any changes in the participant’s health status that occur between

the screening examination and the initial administration of α-CSH or associated procedures will be documented in their medical records. In the event of a severe adverse event, the principal investigator will be promptly notified within 24 hours of its occurrence. If the severe adverse event is determined to be associated with severe toxicity or infection at the transplant site, the experiment will be immediately halted to ensure participant safety.

As this study focuses on feasibility rather than achieving statistical significance, formal power calculations were not conducted prior to its initiation. Instead, the emphasis lies on assessing safety, feasibility, and generating preliminary data.

With no prior data on the use of this bone graft material in humans, the sample size was determined based on practical considerations, including resource availability, feasibility of recruitment within the study timeline, and ethical concerns regarding participant risk exposure. The aim was to enroll a sample size, allowing for meaningful observations and providing valuable preliminary insights into safety and efficacy.

Furthermore, following a common rule of thumb for pilot studies, which typically suggest around 30 participants, we decided on a sample size of 30 extractions, with 15 extraction sockets in each group. The number of sockets per patient varies based on individual clinical circumstances. This sample size was deemed appropriate for assessing safety, feasibility, and generating preliminary efficacy data.

The allocation of participants into the treatment and control groups will be determined through randomization. This process will be conducted using a secure computer system for central randomization. Each patient will be assigned a unique study code, and their data will be provided to the operators and research evaluators in a format that preserves patient confidentiality through coding. When evaluating the data, the radiologist and the statistician will be blinded to the treatment.

The study team will receive detailed guidelines and responsibilities. The medical professionals within the research team will collect the required information as outlined in Table 1. Training sessions will be conducted for the entire research team to ensure consistent data collection at each study visit. The clinical assessors and investigators will receive the data in a format that maintains patient confidentiality through coding. The participants will be monitored for a maximum duration of 6 months. The data manager will prioritize the confidentiality and security of the participant's data.

Statistical analysis will be performed using SPSS 16.0 (IBM Corp). Student t test will be used to analyze the difference between groups and different follow-up points. The statistical significance level will be set at a P value of .05 or lower.

Any significant changes that arise during the course of the trial will be promptly reported to the ethical committee and the relevant authority. This ensures the participant’s safety and well-being, maintains the trial’s integrity, and upholds its scientific value.

In order to ensure the safety of participants and monitor any potential long-term adverse effects of the α-CSH graft material, secondary follow-up will be conducted for all participants at 1 and 2 years. This extended follow-up period will allow for thorough evaluation and observation of any potential late-onset complications or concerns related to the use of α-CSH.