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.

1、口腔微生物群初始定植与样本采集
随机选取10名年龄为(21±2)岁的健康志愿者，男女各半，接受口腔健康检查。本次检查由同一名医师完成，涵盖口腔前庭、牙齿、牙周、黏膜及咽部区域，所有受检部位均需无病变。所有志愿者近1个月内未接受任何口腔治疗，女性志愿者经确认未妊娠。所有志愿者均签署知情同意书同意参与本次采样，本实验已通过西北民族大学（Northwest National University）伦理委员会审批。采用无刺激采集法获取每名志愿者的唾液1 mL，采集后立即将唾液与10 mL生理盐水充分混匀，随后将混合液分装至30支容量为3 mL的离心管中，每管装入2 mL混合液。选取10份混合液，加入1块煅烧成年牛骨，记为C组；另选取10份混合液，加入1块煅烧牛胎骨，记为W组；设置纳米羟基磷灰石（nanohydroxyapatite）粉末组（F组），其粉末用量与上述骨块的重量、数量一致。所有样本均置于37℃条件下孵育6 h，孵育结束后取出材料并进行干燥处理。
2、高通量测序（High-throughput Sequencing）与数据分析
将干燥后的材料进行粉碎，首先使用TE缓冲液（25 mM Tris-HCl、10 mM EDTA，pH 8.0）进行保存与裂解。随后采用Qiagen粪便DNA提取试剂盒（Qiagen Stool Mini Kit，购自北京天根生化科技有限公司，中国北京），严格按照试剂盒说明书完成DNA提取。高通量测序由北京奥维森基因科技有限公司（北京，中国）承接完成。对V3-V4区域进行扩增，扩增反应体系配置如下：去离子水9 μL、5×PCR缓冲液5 μL、5×PCR GC高羟基磷灰石、2 μL dNTPs（2.5 mM）、2 μL模板DNA（200 ng/μL）、0.25 μL TaKaRa DNA聚合酶（5 U/μL）以及上下游引物各1 μL（10 μM）。PCR扩增程序设置如下：98℃预变性5 min；随后进行27个循环，每个循环包括98℃变性30 s、50℃退火30 s、72℃延伸30 s；最后72℃终延伸5 min。取3 μL PCR扩增产物进行2%琼脂糖凝胶电泳检测，随后在MiSeq高通量测序平台上以2×250 bp的模式开展测序。使用QIIME v.1.5.0软件对测序数据进行操作分类单元（OTU，Operational Taxonomic Unit）聚类分析，聚类相似度阈值设为97%，比对数据库采用人类口腔微生物组数据库（Human Oral Microbiology Library，http://www.homd.org/）。
3、本研究涉及的所有分类单元数据涵盖门（phylum）、科（family）、目（order）、纲（class）及属（genus）水平，所有分析均基于上述数据进行。
4、若针对该数据集采用不同软件或设置不同分析标准，所得分析结果可能存在差异。因此，若存在疑问，请及时与通讯作者联系。