Clinical trial design
This clinical trial was designed as a 12-week multicenter, randomized, open-label, comparative, investigator-blinded study conducted in three centers (Seoul National University Dental Hospital, Samsung Medical Center, and Chonnam National University), from April 2016 to September 2017. It was registered at ClinicalTrials.gov (ID: NCT02714829) and approved by the Institutional Review Board of the three centers (approval no. CDE16001 in Seoul National University Dental Hospital, 2016-01-035 at Samsung Medical Center, CNUH-2016-140 in Chonnam National University Hospital) and Ministry of Food and Drug Safety of Korea (approval no.: 472).
The protocol included seven visits per subject, and the flow diagram is shown in Fig. 1. At the first visit (screening), candidates were assessed using the following examinations: demographic survey, clinical examinations, physical examination with vital sign checks, medical history, radiographic examination using panoramic radiograph, laboratory test including common blood cell count, blood coagulation test, serum biochemical test, urine analysis, and immune response test for BMP-2. The subjects were selected according to the inclusion and exclusion criteria. At the second visit, tooth extraction and ARP were performed after the subjects were randomized into the test and control groups, and multi-detector computed tomography (CT) was performed to determine the baseline immediately after surgery. The sutures were removed 1 week after the surgery (visit 4). The subjects were observed for 12 weeks following surgery to evaluate their efficacy and safety. Twelve weeks after surgery, CT was performed to evaluate the changes in the alveolar ridge.
Grafted materials and subjects
The graft materials used were rhBMP-2/β-TCP (Novosis Inject Dent, CGBio, Gyeonggi-do, Korea) for the test group and β-TCP (Excelos Inject, CGbio, Gyeonggi-do, Korea) for the control group (Fig. 2). The rhBMP-2/β-TCP used in this study consisted of a carrier and rhBMP-2. The carrier was composed of β-TCP microspheres (diameter, 45–75 μm; porosity, higher than 65 vol.%) with Poloxamer 407 based hydrogel composite and was sterilized with gamma radiation. E. coli-derived rhBMP-2, provided as a lyophilized powder, was added to the carrier in a 2.5 mg/mL rhBMP-2 solution. After 0.5 mg rhBMP-2 was dissolved with 0.2 mL of water, 0.2 mL of rhBMP-2 solution was moved to an empty syringe. The syringe, including 0.2 mL of rhBMP-2 solution, was connected to a syringe containing a 1.5-g carrier using a connector. The contents were mixed by pushing both syringe rods at least ten times and were gathered into one syringe. After detachment of the connector and attachment of the syringe tip, rhBMP-2/β-TCP was grafted into the extraction socket up to the level of the alveolar ridge crest. For the control group, β-TCP, which had the same composition and properties as the carrier used in rhBMP-2/β-TCP, was used.
To be included in this study, subjects needed to meet the following inclusion criteria: (1) patients aged 19 to 80 years who required dental extraction, (2) residual alveolar bone surrounding the tooth: 50% and above of the original alveolar bone height (4 mm and above), (3) no evidence of severe periodontitis, and (4) patients who voluntarily consented to clinical trials and can follow the protocols of the clinical trial. Patients who had already undergone tooth extraction could participate if the extraction was performed within 3 days. The following exclusion criteria were applied: (1) for extraction of the third molar, (2) patients with disease that requires continuous prophylactic antibiotics, (3) patients with major systemic disease, (4) patients who require long-term steroid administration, (5) females who are pregnant or have childbearing potential, (6) patients with severe periodontitis and vertical alveolar bone resorption more than 50%, (7) inadequate oral hygiene, (8) patients who have participated in other clinical trials and received drug treatment or treatment using other medical device within the past 90 days, (9) immune disease including acquired immune deficiency syndrome, (10) patients who recently received periodontal surgery in the target tooth of clinical trial (within 2 months in cases of partial healing such as gingivectomy, periodontal flap surgery, and within 6 months in procedures associated with dental extraction such as guided periodontal tissue regeneration, and guided alveolar bone regeneration), (11) patients who exhibit hypersensitivity to the component included in the grafted materials used in this clinical trial, (12) alcohol use disorder and substance abuse disorder, (13) other patients determined inappropriate for the clinical trial.
Determination of the sample size
The sample size was determined based on previous studies conducted to compare the two groups according to the inclusion of BMP-2 (weighted mean difference, − 0.72 mm; standard deviation, 1.10 mm) [20, 21]. A minimum of 74 subjects (37 subjects in each group) were required to detect a difference between the two groups with 80% power and an alpha value of 0.05. Finally, assuming a dropout rate and serious protocol violation rate of 15%, the total required number of enrolled subjects was 88 (44 subjects in each group).
Randomization and blinding
Subjects were randomly assigned to the test or control groups with a 1:1 allocation ratio by the randomization code, which was generated using the SAS PROC PLAN procedure (SAS Institute Inc., Cary, NC, USA), using a stratified block randomization method. An individual randomization envelope was prepared and sent to the principal investigator of each clinical trial center. The investigator could only know the assigned group after the registration of the subject was completed.
Although the blinding was not applied to the subjects and investigators in this clinical trial, two external independent evaluators were blinded to the group subjects when evaluating the CT data after the end of the clinical trial.
Surgical procedures
The surgical procedure was performed during the second visit (Fig. 3). After local anesthesia, tooth extraction was performed carefully with minimal damage to the surrounding alveolar bone, and the remaining granulation tissue and infected tissue were removed. For each group, the allocated graft material was applied to the extraction socket up to the crestal level of the remaining alveolar bone. The amount of grafted rhBMP-2/β-TCP or β-TCP can be different depending on the size of each extraction socket, but the concentration of rhBMP-2 in the grafted β-TCP was kept the same by making the mixing ratio the same before grafting. After the absorbable barrier membrane (Remaix, Matricel GmbH, Herzogenrath, Germany) was placed on the extraction socket to cover the graft material, wound closure was performed with 4-0 Vicryl (Ethicon, Somerville, NJ, USA) at the level of approximation of the buccal and lingual/palatal flaps.
Efficacy outcome
In this study, the primary efficacy outcome was the changes in the alveolar bone height compared with the baseline at 12 weeks after tooth extraction and ARP, and the secondary efficacy outcome was the changes in the alveolar bone width compared with the baseline at 12 weeks after tooth extraction and ARP. The alveolar bone width was measured at 75%, 50%, and 25% of the extraction socket length (ESL). CT data taken at baseline and 12 weeks after surgery were superimposed based on stable anatomic structures that did not change during the follow-up period using OnDemand3D software (Cybermed, Seoul, Korea). The measurement method was determined based on previous studies that analyzed the changes in the alveolar bone after ARP using CT data [10, 20, 22]. Landmarks and linear measurement parameters used to evaluate changes in the alveolar bone height and width in this study are shown in Fig. 4. Line A was the line passing through points a and b, where points a and b were defined as the most superior point of the lingual and buccal alveolar bone, respectively. Point c was the point that bisects line A, and point d was the apex of the extraction socket. The line passing through points c and d was defined as line B. Line C was set as the line perpendicular to line B and passing through point d. The height of the alveolar bone immediately after surgery was measured as the distance from point c to point d. The width of the alveolar bone was measured at the points where the alveolar bone height was divided into quarters. To measure the changes of the alveolar ridge 12 weeks after surgery, the reference lines B and C, reference lines for 25%, 50%, and 75% ESL, and point d were transferred to the CT images taken 12 weeks after surgery. Point a’ and b’ were marked as the most superior point of the lingual and buccal alveolar bone, and line A’ was defined as the line passing through points a’ and b’. Point c’ was the intersection of line A’ and line B. By measuring the distance from point c’ to point d, the alveolar bone height at 12 weeks after surgery was measured. The width of the alveolar bone was measured at the points where the alveolar bone height was divided into quarters.
Safety assessment
Adverse events were collected from visit 2 to the end of the clinical trial. When an adverse event occurred, the symptoms, onset date, resolution date, degree of severity, and suspected causal relationship with the grafted material were recorded. All adverse events were classified as preferred term by system organ class according to the MedDRA (Medical Dictionary of Regulatory Activities). The degree of severity was divided into three levels: mild, moderate, and severe. Mild referred to symptoms that caused minimal discomfort and were easily tolerated without interfering with normal daily life, and moderate referred to symptoms that significantly interfered with normal daily life. Severe referred to symptoms that made normal daily life impossible. The causal relationship was evaluated considering the following factors: (1) evidence that the graft material was used, (2) time sequence of use of the graft material and occurrence of adverse events, (3) cases that were most likely to be explained by the use of the graft material than other causes, (4) resolution of the symptoms of the adverse event after removal of the graft material.
To assess the safety of the application of the graft material, clinical examinations, vital signs, and laboratory tests, including common blood cell count, blood coagulation test, serum biochemical test, and urine analysis, were performed with the immune response test of BMP-2 and monitoring of adverse events. Vital signs, including the systolic and diastolic blood pressure, pulse rate, and body temperature were measured at each visit prior to other scheduled tests. Immune response tests for BMP-2 were performed at visits 1, 4, and 7. At visits 4 and 7, evaluations were only conducted for the test group. The BMP-2-antibody in the serum was analyzed by enzyme-linked immunosorbent assay with purified rabbit anti-human BMP-2 (RHF913; Antigenix America, NY, USA). Healing status was assessed using a visual analog scale for postoperative pain, healing grade evaluated by the investigator, and white blood cell count. When measuring the visual analogue scale, the subject evaluated the degree of pain in the range of 0 to 10, and it was judged that the higher the value, the more severe the pain. The healing grade was evaluated by dividing the healing degree of the surgical site into 5 grades from visit 4 to visit 7: grade 0, absence of inflammation; grade 1, mild inflammation (partial involvement); grade 2, mild inflammation (entire involvement); grade 3, moderate inflammation; grade 4, severe inflammation [23]. White blood count was checked at visit 4 and visit 7 for objective evaluation of postoperative infection.
Statistical analysis
Statistical analysis was performed using SAS software (SAS Institute, Cary, NC, USA). In the comparison of demographic data between the two groups, the two-sample t test or Wilcoxon’s rank-sum test was performed for continuous variables, and the chi-square test or Fisher’s exact test was conducted for categorical variables. When a missing value occurred in the analysis, it was treated as missing without correction.
To compare the change in alveolar bone height at 12 weeks after tooth extraction and ARP between the test and control groups, an analysis of covariance (ANCOVA), which adjusted the baseline as well as the trial center (a stratification factor), was conducted. When the lower limit of the 95% two-sided confidence interval for the differences between the test and control groups estimated in this model was greater than 0, the test group was determined to be superior to the control group. Additionally, a paired t test or Wilcoxon’s signed-rank test was conducted to evaluate whether the decrease in alveolar bone height in each group was statistically significant. Least-square mean (LSM), which was corrected for the baseline and institution, standard error, two-sided 95% confidence interval corresponding to the LSM difference in contrast to the control group, and the p value were presented for each group.
To evaluate whether the decrease in alveolar bone width at 75%, 50%, and 25% ESL in each group at 12 weeks after surgery was statistically significant, a paired t test or Wilcoxon’s signed-rank test was conducted, and ANCOVA analysis, which adjusts the baseline as well as the trial center (a stratification factor) was conducted to evaluate the significance of the difference between groups. Additionally, LSM, standard error, two-sided 95% confidence interval corresponding to the LSM difference in contrast to the control group, and the p values were presented for each group.
Safety assessment was conducted on all subjects who underwent tooth extraction and the application of the graft material. Subjects enrolled in this clinical trial but dropped out before the extraction and application of the graft material were excluded from the safety assessment. The ratio of subjects who experienced adverse events and the corresponding 95% confidence interval was presented for each treatment group. To test whether there was a difference in the incidence rate of adverse events between the two groups, Fisher’s exact test was conducted. All adverse events were organized according to severity and relationship with the graft material. For the continuous variables in the laboratory test, vital signs, and healing status assessment, differences between two visits were tested using a paired t test or Wilcoxon’s signed-rank test, and comparisons between the two groups were evaluated using a two-sample test or Wilcoxon’s rank-sum test. In terms of the categorical variables, McNemar’s test was conducted to evaluate the differences between two visits for each group, and the chi-square test or Fisher’s exact test was conducted to compare the differences between the groups.