The Influence of Different Pore Sizes of Beef Bone Scaffold Material on the Initial Colonization of Oral Microbiota

1、Initial Colonization of Oral Microbiota and Sample CollectionTen volunteers (equally numbers men and women) were randomly selected for oral health examination (21±2 years). The oral health examination was completed by one person and included vestibular, dental, periodontal, mucosal and pharyngeal areas of the mouth, all of which were required to be free of disease. All volunteers had not received any treatment in the last month, and female volunteers were confirmed to not be pregnant. All volunteers consented to participate in this sampling, and this experiment was approved by the ethics committee of Northwest National University. One milliliter of saliva was collected using a nonirritating method, and the saliva collected from each person was immediately and thoroughly mixed with 10 ml of saline and later dispensed into 30 3 ml centrifuge tubes containing 2 ml each. Ten mixtures were selected and added to 1 piece of calcined adult cow bone to form group C. Ten mixtures were selected and added to 1 piece of calcined fetal cow bones to form group W. The same weight and numbers of nanohydroxyapatite powder were used as the bone pieces to form group F. All samples were incubated at 37°C for 6 h. After incubation, the materials were removed and dried.2、High-throughput Sequencing and AnalysisThe dried material was crushed and first preserved and lysed in TE buffer (25 mM Tris HCl, 10 mM EDTA, pH 8.0) solution. Then, DNA was extracted using the Qiagen Stool Mini Kit (Tiangen Biochemical Technology (Beijing) Co., LTD, Beijing, China) following its instructions. High-throughput sequencing was carried out by Beijing Ovison Gene Technology Co., Ltd., Beijing, China. The V3-V4 region was amplified. The amplification system conditions were as follows: 9 μl of deionized water, 5 μl of 5× PCR buffer, 5 μl of 5× PCR GC high hydroxyapatite, 2 μl of dNTPs (2.5 mM), 2 μl of template (200 ng/μL), 0.25 μl of TaKaRa DNA amplification enzyme (5 U/μL) and 1 μl of each primer (10 μM). (10 μM) in 1 μl. The PCR conditions were as follows: predenaturation at 98°C for 5 min, start of 27 cycles, denaturation at 98°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 5 min. Three microliters of PCR product was subjected to electrophoresis in a 2% agarose gel and sequenced on the MiSeq high-throughput sequencing platform, using 2x250 bp. OTU clustering analysis was performed with QIIME v.1.5.0 software, using a 97% similarity level, in the Human Oral Microbiology Library (http://www.homd.org/).3、The data listed here are all the taxon in this papper including: phlym, family, order,class and genus. All the analysis here were based on these data.4, If different software is used and different standards are set for this data, the analysis results will be different. Therefore, if in doubt, please contact the corresponding author in time.