1.1 Sample size calculation
The sample size was calculated using PASS 15.0 software (NCSS, LLC, Utah, USA). The statistical design was based on comparisons of the bone absorption rate between the PRF, CGF, PPP and control groups. The analysis module One-Way-Analysis of Variance F-Tests in the Means was used. According to the results of the preliminary experiment, the mean bone absorption rate in the PPP, CGF, PRF and control groups was 14%, 13%, 16%, and 39% respectively. The standard deviation was set as 12%. Statistical significance was set as α = 0.05, with four groups, statistical power of 0.9, and a group allocation ratio of 1: 1: 1. With these parameters, the sample size needed for the current study was six in each group (Fig. 1).
1.2 Animals and study design
All of the research protocols used in study were approved by the ethical committee of Southwest Medical University, Luzhou, China (Certificate number 201906-1). A randomized controlled study was conducted according to the ARRIVE guidelines . Healthy male New Zealand White rabbits weighing 2.0–2.5 kg (average 2.2 kg) and aged 8–12 weeks each were used in this study. All animals were purchased from the Department of Animal Science Central of Southwest Medical University and were taken good care of by professional laboratory technicians. They were housed in a temperature (22 ± 2°C) and humidity (55±5%) controlled room under a 12/12 h light/dark cycle and kept in separate cages, with free access to food and water. After 2 weeks of observation, the experimental treatment was carried out.
1.3 Preparation of autologous PPP, CGF, and PRF
9 ml venous blood from the ear veins of each rabbit were drawn and collected into sterile vacuum tubes without additive (Greiner BioOne, Kremsmünster). The samples were immediately put into a Medifuge MF200 (Silfradent srl, Forlì, Italy), and centrifugation was carried out according to a preset procedure: acceleration for 30 seconds, then 2 minutes at 2700 rpm, 4 minutes at 2400 rpm, 4 minutes at 2700 rpm, 3 minutes at 3000 rpm, deceleration for 36 seconds, and stop . This process separated the samples into three layers: a red blood cell (RBC) layer that covered the lower part of the tube, a CGF layer that covered the middle part and a PPP layer that covered the upper part (Fig. 2). The PPP was activated for experimental use with 10% calcium chloride. The CGF and activated PPP gel were thus collected for experimental use.
Based on a previously described protocol , 9 ml venous blood was collected into a centrifuge tube without any anticoagulant. After centrifuging immediately for 10 min at 3,000 rpm, the whole blood separated into two layers, the lower layer being the RBC layer, and the upper layer being the PRF layer (Fig. 2).
1.4 Surgical procedure
The animals were randomly and evenly divided into four groups. Three groups received PPP, CGF and PRF gel respectively, while the remaining control group did not receive any implant material. They all received an intramuscular injection of penicillin (800,000 units three times daily) for 3 days postoperatively. Intravenous injection of 30 mg/kg sodium pentobarbital (Sigma, St. Louis, MO, USA) through the ear margin was used for general anesthesia. After the anesthetic had taken effect, the gingiva was separated with a periosteal elevator, then the teeth were loosened with the elevator, and after that the bilateral mandibular anterior teeth were extracted. All the above procedures were performed by the same oral and maxillofacial surgeon. Subsequently, the materials were severally implanted into the tooth extraction sockets in the experimental groups (Fig. 3). After that, the extraction sockets were carefully sutured and closely observed for avoiding infection. Thereafter, at 2, 4 and 8 weeks after tooth extraction, three rabbits were randomly selected from each group and euthanized with an overdose of pentobarbital sodium. The bilateral mandible was taken as the specimen for subsequent analysis.
1.5 Radiographic analysis
All animals were scanned twice by cone beam computed tomography (CBCT I and CBCT II), once after tooth extraction surgery, and the other after euthanasia, when the alveolar bone around the mandibular anterior teeth was immediately removed for scanning. All sectional images were obtained by the same radiologist using a Kodak 9500CBCT scanner (Carestream Health, Rochester, NY, USA), with the following settings: exposure at 5.0 mA and 120 kV for 9.6 s and axial slice thickness 0.2 mm. The results were processed and analyzed by the same radiologist (who was blinded to the group allocation) using image analysis software (CS Imaging Version 126.96.36.199.d, Carestream Health, Rochester, NY, USA). Changes in alveolar bone width (ABW) and alveolar bone height (ABH) were observed. Three sections were selected for each CBCT to measure the height and width respectively, and each section was randomly measured three times. ABW was measured using the method of Chen et al . Measurements were performed on cross-sectional slices in the apical, median, and coronal third of the socket. ABH was measured using a method described previously by Liu et al . Measurement was carried out on three sagittal planes, namely the buccal plane of the extraction socket, the lingual plane, and the middle plane of the first two planes. The changes in ABW and ABH were expressed by the measured value of CBCT before tooth extraction (CBCT I) minus the measured value of CBCT after euthanasia (CBCT II).
1.6 Histological analysis
After fixing in 10% paraformaldehyde solution for 48 h, the samples were demineralized in 10% EDTA solution (North Tianyi Chemical Reagent Co. Ltd., Tianjin, China) for 5 weeks, washed, dehydrated, and paraffin embedded (Paraplast; Kendall Healthcare, Mansfield, MA, USA), parallel to the long axis of the tooth, comprising a continuous section in the buccal and lingual direction with a section thickness of 5 µm, and then stained with hematoxylin and eosin(H&E). Masson’s trichrome stain was carried out in the same way. The above processes were performed by a histological technician who was blinded to the experimental protocols. An optical microscope (Olympus BX43, Olympus Corporation, Tokyo, Japan) with a magnification of × 200 was used for observation, and a digital camera installed on the microscope was used to obtain images.
1.7 Real-time quantitative polymerase chain reaction (RT-qPCR)
Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of two markers of osteogenic genes: a differentiation marker of early osteoblasts — alkaline phosphatase (ALP), and a differentiation marker of late osteoblasts — bone morphogenetic protein-2 (BMP-2). After sacrificing the rabbits, bone tissue was obtained from the tooth extraction sockets and immediately stored in liquid nitrogen and ground in a mortar. A chloroform-free RNA extraction kit (BioTeke, Beijing, China) was used to extract total RNA from the samples. Then, according to the manufacturer's instructions, 1 µg of RNA was reverse transcribed into cDNA, using ReverTra Ace qPCR RT Master Mix (TOYOBO, Japan), and stored at -20°C before use. The cDNA was used as the template for real-time quantitative polymerase chain reaction (RT-qPCR). The total volume of the amplification reaction system was 20 µl, including 6 µl primers, 12 µl of QuantiNova STBR Green PCR (Qiagen, Germany), 1.5 µl cDNA and 2.5 µl ddH2O. The primers were purchased from Sangon Biotech Co. (Shanghai, China). The sequences were: 5′-TCCCACTTTGTCTGGAACCG-3′ and 5′-TCCTGTTCAGCTCGTACTGC-3′ for ALP, 5′-AGGAAGCTTTGGGAGACGAC-3′ and 5′-AAGTGGGTCACTTCCACCAC-3′ for BMP, and 5′-GTGGCATCCTGACGCTCAAGTAC-3′ and 5′-AAGCTCGTTGTAGAAGGTGTGGTG-3′ for β-actin.
1.8. Statistical analysis
Data were analyzed using IBM SPSS statistical package 22.0 (IBM Co., Chicago, USA). Categorical variables are presented as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with the Student–Newman–Keuls (SNK) comparison test was employed to detect differences among different groups. A significance level of 0.05 was chosen.