Study design, participants, and collection of plasma samples
The publication was performed and written according to the CONSORT Guidelines [33] for randomized controlled clinical trials. Blood samples were collected from 25 women who received allogeneic bone grafting material (Maxgraft® Allograft Spongiosa Particle (Botiss Company, Berlin, Germany, part of Straumann Group, Basel, Switzerland)) from male donors (allograft patients, test group) and from 10 women who were treated with autologous graft (autograft patients, control group) for lateral ridge augmentation procedures recruited and treated in a single periodontal office in Hamburg, Germany, between September 2018 to February 2019. All patients were systemically healthy non-smokers and were randomly assigned to one of the parallel groups by a blinded clinician not involved in this study and not involved in the periodontal office by drawing sealed envelopes. Blood samples were collected on the day of surgery preoperatively, immediately postoperatively, at 5 weeks and 4 months after surgery. Plasma was prepared and stored in liquid nitrogen. The mean age of allograft patients was 58 years (range 39–78), and autograft patients were 55 years (range 32–76). The collection of the blood samples and experiments were performed in compliance with the World Medical Association Declaration of Helsinki (version 2008) and were approved by an ethics committee (Hamburg Medical Association, Germany, no. PV5211) and the study was registered with the German Register for Clinical Trials (DRKS No. 00013010). All patients gave their informed consent and all patients completed the study successfully and were available for follow-up visits. No adverse events were recorded.
Surgical procedure
Bone grafting for alveolar ridge augmentation were performed under local anesthesia using Ultracain-DS Forte (Sanofi-Aventis, Frankfurt/Main, Germany). After deflection of a mucoperiosteal flap a cortical perforation was done and allogeneic bone grafting material (test group) or autogenous bone (control group) was inserted. The bone grafts were covered with a collagen membrane (Jason Membrane, Botiss Company) for guided bone regeneration, according to the manufacturer’s recommendations (Botiss Company, Berlin, Germany, part of Straumann Group, Basel, Switzerland). A periosteal releasing incision of the mucoperiosteal flap was performed in order to mobilize the flap for a tension-free primary closure of the surgical site. Flap-fixation was performed using a horizontal and vertical mattress suture with 5.0 Goretex filaments (W. L. Gore & Associates GmbH, Putzbrunn, Germany, Fig. 1). A 2.0% chlorhexidine rinsing solution was administered for post-operative oral hygiene. Post-operative appointments were scheduled after 1–2 days, 2, 6, and 12 weeks. Sutures were removed 2 weeks after augmentation. Two-dimensional radiographs, using the parralleling technique with a Rinn holder (Dentsply-Rinn, 1301 Smile Way, York, PA 17404, USA) were taken immediately following the bone augmentation procedure.
Extraction of circulating cfDNA from plasma samples
Using PaxGene Blood ccf tubes (Qiagen, Hilden, Germany), blood was collected preoperatively and postoperatively (collected on the same day) from 25 allograft and 10 autograft patients. Plasma was prepared by 2 centrifugation steps at 2670 and 10,000g, each for 10 min, and tested for hemolysis. The samples were not hemolytic [30].
Circulating cfDNA was extracted from a total of 120 plasma samples using the QIAamp Circulating Nucleic Acid kit (Qiagen) according to the manufacturer’s instructions. Briefly, 3 mL plasma was mixed with 2.4 ml ACL buffer (containing 0.2 μg/μL carrier RNA) plus 300 μL of proteinase K for 30 s, and incubated at 60 °C for 30 min. After addition of 5.4 mL ACB buffer and incubation on ice for 5 min., the lysate was drawn through a Qiamp Mini column. The column was washed with 600 μL ACW1 buffer, 750 μL ACW2 buffer and 700 μL 100% ethanol. CfDNA was precipitated from the column by 30 μL AVE buffer.
The quantity of the extracted cfDNA was determined spectrophotometrically using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). Briefly, 2 μL cfDNA was diluted in 198 μL HS Reagent solved in HS Buffer (1:200), incubated for 3 min at room temperature and measured on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The fluorometer was calibrated with Standard 1 and 2 of the kit. We repeated the measurement of each sample once and received the same data.
Quantitative PCR of Alu elements
In a 10-μl reaction, 1 μl of 1:5 diluted DNA was mixed with 5 μl SYBR Green qPCR Mastermix (Qiagen) and the following 0.25 μl 10 mM Alu115 and Alu247 primer sets:
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Alu115 primer forward: 5′-CCTGAGGTCAGGAGTTCGAG-3′
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reverse: 5′-CCCGAGTAGCTGGGATTACA-3′
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Alu247 primer forward: 5′-GTGGCTCACGCCTGTAATC-3′
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reverse: 5′-CAGGCTGGAGTGCAGTGG-3′
The Alu115 and Alu247 primer sets amplified PCR products at sizes of 115 and 247 bp, respectively. A negative control without any template was also performed on each plate. The reactions were run on an MJ Research PTC-200 Peltier Thermal Cycler (Global Medical Instrumentation, Ramsey, MN, USA). The following real-time PCR program was carried out: 1 cycle at 95 °C for 15 min; 40 cycles at 95 °C for 30 s, 64 °C for 30 s and 72 °C for 30 s. Subsequently, a melting curve analysis was carried out as followed: 95 °C for 15 s, 60 °C for 15 s and heating up to 95 °C in 20 min and holding the temperature for 15 s. Mean values were calculated from triplicate reactions.
CfDNA integrity was calculated as a ratio of concentrations of long to short cfDNA fragments of Alu elements (Alu247/Alu115).
Caspase assay
For measurements of the activity of caspase 3 and 7, the Caspase Glo®3/7 assay (Promega, Mannheim, Germany) was carried out. The Caspase Glo®3/7 assay is based on the cleavage of the DEVD sequence of a luminogenic substrate by the caspases 3 and 7 which results in a luminescent signal. To draw a standard curve from 5 × 10−3 down to 5 × 10−6, dilution steps in a ratio 1:2 were carried. Briefly, 1 μl caspase solution (100 U/μl, Enzo Life Sciences AG, Lausen, Switzerland) was twice diluted in 100 μl enzyme dilution buffer (50 mM HEPES, pH 7.4, 100 mM sodium chloride, 0.5% CHAPS, 1 mM EDTA, 10% glycerol and 10 mM dithiothreitol). Then, this 0.01 U/μl caspase solution was 11 times serially diluted in a ratio of 1:2. In each case, 50 μl of these standards as well as the plasma samples were mixed with 50 μl Caspase-Glow reagent (Promega) and incubated on a shaker at 200 rpm for 1 h. On a 96 well-plate, the samples were measured on a Glomax Luminometer 20/20 (Thermo Fisher Scientific) for 1 s. A blank reaction was used to measure background luminescence.
Exosome assay
For measurements of the exosome concentrations, the ExoQuantTM Overall Exosome Capture and Quantification assay, a double sandwich ELISA using an antibody against the exosomal marker CD63 (BioVision, Milpitas, California, USA), was carried out according to the manufacturer's instructions. For stabling the calibration curve, lyophilized exosomes were solved in 100 μl of deionized water and 100 μl of PBS to reach a final volume of 200 μl per vial. Standard dilutions were prepared directly in the strips by using the exosome solution to perform six two-fold serial dilutions with PBS. The standard concentrations were 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 μg. Then, 100 μl plasma diluted to 1:1 in PBS was loaded on a 96-well plate pre-coated with proprietary pan-exosome antibodies enabling specific capture of exosomes. The plate was incubated at room temperature while shaking (2–3 rotations/s) for 30 min, and subsequently, at 4 °C overnight. After washing, 100 μl of mouse anti-human exosome Detection Antibody solution (diluted to 1:500 in Sample Buffer) was added to each well and incubated at room temperature while shaking for 15 min and subsequently at 4 °C for 2 h. After washing, 100 μl of rabbit anti-mouse IgG HRP-conjugated secondary antibody solution (diluted to 1:2000 in Sample Buffer) was added to each well and incubated at room temperature while shaking for 15 min and subsequently at 4 °C for 1 h. After washing, 100 μl of Substrate Chromogenic Solution was added to each well and incubated at room temperature in the dark for 10 min. The reaction was stopped by adding 100 μl of Stopping Solution to each well. For colorimetric detection, the absorbance at 450 and 570 nm was measured within 10 min on a Microplate reader (Tecan, Männerdorf, Switzerland).
Extraction of miRNAs
MiRNAs were extracted in 100 μl plasma supplemented with 150 μl lysis buffer (Thermo Fisher Scientific, Vilnius, Lithuania) and 50 μl PBS (Life Technologies) by using the TaqMan miRNA ABC Purification Buffer Kit (Thermo Fisher Scientific). According to the manufacturer’s instructions, the miRNAs were bound to 80 μl anti-miR beads using the TaqMan miRNA ABC Purification Bead kit Human panel A (Thermo Fisher Scientific). To avoid technical variability, 2 μl of 1 nM synthetic non-human cel-miR-39 were added as an exogenous spike in control.
Reverse transcription
Reverse transcription was carried out using a modified protocol of TaqMan MicroRNA Reverse Transcription kit (Thermo Fisher Scientific). The reaction contained 4.0 μl RT Primer Pool of miR-484, cel-miR-39 (reference miRNAs) and miR-21, miR-27a, miR-218 (miRNAs of interest) diluted in 1:100 Tris–EDTA, 0.2 μl dNTPs with 100 mM dTTP, 2.0 μl (50 U/μl) MultiScribe Reverse Transcriptase, 1 μl 10 × RT Buffer, 0.127 μl (20 U/μl) RNase Inhibitor and 2 μl miRNAs. The conversion of miRNAs into cDNA was carried out at 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min on an MJ Research PTC-200 Peltier Thermal Cycler (Global Medical Instrumentation, Ramsey, Minnesota, USA).
Preamplification of cDNA
To increase input cDNA, a pre-amplification step of cDNA was included. For TaqMan PCR analyses of miR-21, miR-27a and miR-218, cDNA of the reference miR-484 and cel-miR-39 was also preamplified. Here, 1 μl cDNA was preamplified in 5 μl TaqMan PreAmp Master Mix (Thermo Fisher Scientific) and 1.5 μl specific PreAmp primer pool. The reaction was run on a MJ Research PTC-200 Peltier Thermal Cycler (Global Medical Instrumentation): 1 cycle at 95 °C for 10 min, 55 °C for 2 min, 72 °C for 2 min; 16 cycles at 95 °C for 15 s, 60 °C for 4 min; and a final cycle 99.9 °C for 10 min. A negative control without any templates was included from the starting point of reverse transcription, too.
TaqMan real time PCR analyses of miR-21, miR-27a and miR-218
For quantitative real-time PCR, the TaqMan miRNA assays (Thermo Fisher Scientific) for miR-484 and miR-39 (reference miRNAs), and miR-21, miR-27a miR-218 were used. In a 10 μl-reaction, 0.25 μl preamplified cDNA were mixed with 5 μl TaqMan Universal PCR Master Mix and 0.5 μl TaqMan MicroRNA Assay. Quantitative real-time PCR reaction was performed at 95 °C for 10 min. and in 40 cycles at 95 °C for 15 s and 60 °C for 60 s, on a C1000 Touch real-time PCR device (Bio-Rad, Hercules, California, USA).
Data normalization of miRNA data
As there is no consensus on a reference miRNA for data normalization, we chose miR-484 for plasma quality control and cel-miR-39 for inter-individual variability of the efficiency of our procedures as an endogenous and exogenous reference gene, respectively, to normalize our miRNA data. The obtained data of the miRNA expression levels were calculated by the ΔCt method as follows: ΔCt = mean value Ct (reference cel-miR-39 + miR-484) − mean value Ct (miRNA of interest). The relative expression data were 2(ΔCt) transformed in order to obtain normal distribution data. The confidence of 2(ΔCt) data were verified by amplification curves and Ct confidence (0–1, whereby 1 refers to the highest confidence). Our data showed a Ct confidence of 0.95. Values below 0.95 were discarded.
Statistical analysis
Statistical analyses were performed using the SPSS software package, version 24.0 (SPSS Inc. Chicago, IL, USA). Statistical differences in the measured data were calculated using ANOVA with Tukey's HSD test for all pairwise comparisons that correct for experiment-wise error rate. Two-sample comparisons were performed using Student's t-test for equal or unequal variance where appropriate. Due to the small size of the variables, the Holm-Bonferroni method was not employed for multiple test correction. Missing data were handled by pairwise deletion. A p-value ≤ 0.05 was considered as statistically significant. All p-values are two-sided (Additional file 2).