Study design and participants
The present prospective single-center randomized controlled trial was approved by the institutional ethics committee at the Electric Power Teaching Hospital of Capital Medical University, Beijing, China (Approval ref No.: ky-2018-037-02), and the study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. Thirty-six participants who underwent extraction of the upper molars were recruited from the Department of Stomatology, Electric Power Teaching Hospital of Capital Medical University during the period from June 2018 to August 2020.
Informed consent forms were signed by all included participants, and the participants were randomly assigned to three groups: grafted with CGFs combined DBBM and further covered with CGFs membranes (CGFs/DBBM group), grafted with DBBM alone and covered with collagen membranes (DBBM group), Control group with no grafting procedure and spontaneous healing.
The randomized sequence was generated using SPSS 26.0 (IBM, USA) and concealed from the study clinician. The assignments were revealed to the clinician on the day of treatment.
Inclusion and exclusion criteria
The inclusion criteria were as follows: (1) Presence of upper molars that are not restorable; (2) Preoperative CBCT indicated the presence of at least one adjacent tooth and three bone walls at the extraction site; (3) Periodontal tissues are healthy with no signs of severe periodontal diseases; (4) Signed the informed consent form voluntarily. The exclusion criteria were: (1) Acute periapical periodontitis; (2) Moderate to severe absorption of mesial and distal bone plates; (3) History of local radiotherapy for head and neck regions in the last 5 years; (4) History of using high-dose steroids and drugs that affect bone metabolism; (5) Excessive drinking and smoking (> 10 cigarettes/day); (6) Pregnant or breastfeeding women; (7) Uncontrolled diabetes, serious mental illness, or any other systemic diseases.
Presurgical treatment
All routine laboratory investigations were assessed before the procedure to avoid further complications during the trial. Clinical examination and supragingival scaling were performed for all of the patients 1 week prior to the surgical procedure. CBCT (KaVo 3D eXam®, KaVo, Germany) scans were taken with a scan time of 8.9 s, 120 kV, 5 mA, and 0.25 mm slice thickness for each patient before surgery.
Preparation of CGFs
Four 9 ml vacuum tubes without anticoagulants were used to collect the patients’ venous blood, which was immediately processed in the rotating cylinders of a centrifugal accelerator (Medifuge®, Silfradent, Italy). The cylinders were accelerated for 30 s, centrifuged at 2700 rpm for 2 min, 2400 rpm for 4 min, 2700 rpm for 4 min, and 3000 rpm for 3 min, and decelerated for 36 s to stop. The test tube was divided into three layers. The top layer consisted of serum, the middle layer consisted of CGF gel, which was a light yellow gelatin structure, namely the fibrin layer (the main carrier of CGFs) and the bottom layer comprised of red blood cells (RBCs) and platelets. There were a lot of growth factors at the junction between the fibrin layer and the RBCs layer. The fibrin layer and the junction of the fibrin and RBCs layers were reserved in a container with diluted antibiotics for further use.
Surgical procedure
The surgical procedure was performed under local anesthesia (4% articaine hydrochloride with 1:100,000 epinephrine tartrate). The tooth was extracted using a minimally invasive method and the socket was debrided for complete removal of the inflammatory granulation tissues. Then the patients were randomly assigned to one of three groups according to a concealed randomization envelope. For the CGFs/DBBM group, CGF gels were cut into small pieces and mixed with DBBM (Bio-Oss®, Geistlich, Switzerland) at a 1:1 ratio (volume fraction). Then the mixtures were grafted into the sockets, covered with CGF membranes, and sutured with 4-0 monofilament nylon sutures (Unik, Taiwan, China). For the DBBM group, the sockets were grafted with DBBM alone, covered with collagen membranes (Bio-Gide®, Geistlich, Switzerland), and sutured with 4-0 monofilament nylon sutures. For the control group, the sockets were filled with autologous blood coagulum and left open for spontaneous healing (Fig. 1).
Patients were instructed to take antibiotics (Cefaclor Capsules, North China Pharmaceutical, China) three times a day for 5 days, analgesics (Ibuprofen, Tianjin Smith Kline & French Laboratories. Ltd, China) if necessary, and rinse twice a day with 0.2% Chlorhexidine (South China Pharmaceutical, China). All patients were recalled for check-up and suture removal (CGFs /DBBM and DBBM group) after 7–10 days.
All patients were recalled after 8 months, and a second CBCT scan was performed using the same settings. The implant surgery was performed under local anesthesia (4% articaine hydrochloride with 1:100,000 epinephrine tartrate). An incision was made over the middle of the alveolar crest and the mucoperiosteal flap was elevated for access. A core of bone about 4–6 mm long was obtained (CGFs /DBBM and DBBM group) using a 2.8 or 3.3 mm internal diameter trephine (Helmut Zepf, Germany), immediately placed in 10% neutral buffered formalin, and sent to the Department of Pathology, Electric Power Teaching Hospital of Capital Medical University. Implants with appropriate dimensions were placed using routine processes and postoperative instructions were given to the patients.
Radiographic analysis
The CBCT measurements were performed by an experienced investigator. Pre and postoperative CBCT images from the same patient were superimposed using a 3D image processing software (Invivo 5, Anatomy, USA). Afterward, the superimposed images were transformed to a two-dimensional format. The coronal plane in the center of the extraction site in mesiodistal direction was designated as the C-section. The coronal plane parallel to the C plane and 2 mm from the mesial bone wall was designated as the M-section. The coronal plane parallel to the C plane and 2 mm from the distal bone wall was designated as the D-section. Reference lines were drawn at the bottom of the maxillary sinus on the C-, M-, and D-sections, respectively. Marks were placed along the contour of the alveolar bone under the reference lines in the pre and postoperative images. Alveolar bone areas in each section of pre and postoperative images could be measured by the software, and then recorded for further statistical analysis (Fig. 2).
Histological analysis
All of the bone cores were decalcified, embedded in paraffin, and cut into 5 μm sections. The sections were stained with hematoxylin and eosin (HE). The images were observed under a light microscope at various magnifications (× 40 and × 100). The total area of the bone core in the × 40 visual field was defined as the region of interest (ROI). The percentage of newly formed bone (NB) and residual materials (RM) in the ROI in CGFs/DBBM and DBBM group were calculated using image analysis software (Image Pro Plus 6.0, Media Cybernetics, USA) for further statistical analysis.
Statistical analysis
The data were analyzed statistically with SPSS version 26.0 software. The enumeration data were expressed as frequency and percentage. The Fisher exact test was used for comparisons between the groups and the measurement data were expressed as the mean ± standard deviation (Mean ± SD). The Shapiro–Wilk test was used to estimate whether the data followed a normal distribution and homogeneity was assessed with the homogeneity of variance test. If the data followed a normal distribution with the same variance, the t test and one-way ANOVA were used to compare the intra-and inter-group parameters, respectively, and the least significant difference (LSD)-t test was used for post-hoc test. For non-normal distribution, the Kruskal–Wallis H test was used. All the data were measured twice by the same person every 7 days. A P value less than 0.05 was considered statistically significant.