Micro-CT image of cell-populated collagen scaffold in the aqueous environment (contrasted with PTA)

A dataset of the collagen scaffold populated with the 3T3 cells scanned in the aqueous environment using Bruker Skyscan 1276 machine (Bruker, Belgium). Scaffold production The processes of obtaining and working with collagen scaffolds were conducted in an isolated environment under sterile conditions. The collagen sponge matrix was manufactured at the Center for Collagen Innovation within the Institute of Regenerative Medicine at Sechenov University and provided to us for experimental purposes. In order to obtain the collagen, the authors utilized animal-derived materials sourced from the tendons of large horned cattle. To do this, the tendons were cleaned of excess tissues, cut into pieces with a thickness of 0.5-1 cm, and sequentially treated for 12 hours in a 0.5 M NaCl solution. Subsequently, the mass was homogenized in a 0.83 M acetic acid solution. The resulting suspension was hydrolyzed with 0.24% pepsin for 2 days, after which 1 M NaOH was added to adjust the pH to 7.5, halting the hydrolysis process. The suspension was precipitated with a 12% NaCl solution, the resulting precipitate was redissolved in 0.02 M acetic acid, and then dialyzed. To obtain collagen porous matrices (sponges), the obtained solution was neutralized using 0.1 M NaOH until a pH of 7-7.5 was reached, and the resulting suspension was lyophilized at -40°C for 2 days. Subsequently, the collagen matrix was cut into cubes with sides measuring 0.5 cm. These cubes were placed in 15 ml test tubes filled with 70% ethyl alcohol for sterilization. The test tubes were then placed on a shaker and left in the refrigerator at +4°C for 24 hours. Afterward, the collagen matrices were removed from the alcohol and rinsed five times with 0.9% NaCl. Following the alcohol rinse to confirm the absence of toxicity, an elution test, adapted following the ISO 10993 protocol, was conducted. To obtain collagen cube extracts, they were incubated in a cell culture medium at a volume of 1 ml per sample for 24 hours at 37°C. The 3T3 cell culture was passaged, with 5000 cells seeded in each well of a 96-well plate. After 24 hours, the cells were treated with extract at a volume of 200 µl per well and left in the incubator at 37°C for 24 hours. The following day, extracts were collected, and AlamarBlue reagent (Invitrogen, Waltham, MA, USA) was added according to the manufacturer's instructions to assess the metabolic activity of the cells. Serial dilutions of sodium dodecyl sulfate (SDS) were used as the positive control. Fluorescence intensity was measured using a Victor Nivo spectrofluorimeter (PerkinElmer, Waltham, Massachusetts, USA) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Cell seeding After confirming the absence of cytotoxic effects, collagen sponges were seeded with the NIH 3T3 cell line at a density of 50,000 cells per sample (cubes of collagen sponge measuring 0.5 cm per side). Staining technique Fixed specimens in 10% formalin with PBS were washed after 24 hours with distilled water and after that placed in 3% phosphotungstic acid dissolved in distilled water for 24 hours and kept on the rotary shaker at room temperature. After staining, samples were washed and stored in distilled water at 5 °C. Image acquisition and reconstruction A plastic tube filled with distilled water containing the contrasted sample was placed on the sample holder in a SkyScan 1276 micro-CT (Bruker, Kontich, Belgium) and were scanned at 3 μm voxel resolution with 70 kV voltage and 200 uA source power and an aluminum filter with 1 mm of thickness. The rotation was set to 360° around the vertical axis of the sample, with two middle frames for each 0.2° angle step. After scanning, the data were reconstructed using Bruker's NRecon software. During reconstruction, the ring artifact reduction value was set to 20% and the beam hardening correction value to 30%. After that, samples were exported as a series of 16-bit TIFF images which could be opened in the specialized software.本数据集为搭载3T3细胞（3T3 cells）的胶原支架（collagen scaffold）数据集，所有样品均在水相环境下使用比利时布鲁克（Bruker）公司Skyscan 1276型设备完成扫描。 支架制备流程 胶原支架的获取与处理均在无菌隔离环境中进行。胶原海绵基质由谢切诺夫大学再生医学研究所胶原创新中心制备，并向我们提供用于本实验。本次实验的胶原蛋白提取采用大型有角牛肌腱来源的动物材料：先将肌腱去除多余组织，切割为厚度0.5~1 cm的片段，先后在0.5 M氯化钠溶液中处理12小时；随后将物料在0.83 M乙酸溶液中均质化，所得悬浮液用0.24%胃蛋白酶水解2天，之后加入1 M氢氧化钠将pH调至7.5以终止水解反应。悬浮液经12%氯化钠溶液沉淀，所得沉淀物重新溶解于0.02 M乙酸中，随后进行透析。 为制备胶原多孔基质（海绵），使用0.1 M氢氧化钠中和所得溶液至pH为7~7.5，随后将悬浮液在-40°C下冻干2天。 随后将胶原基质切割为边长0.5 cm的立方体，置于装有70%乙醇的15 ml试管中灭菌。将试管置于摇床，于+4°C冰箱中静置24小时。之后取出胶原基质，用0.9%氯化钠溶液冲洗5次。 为确认样品无细胞毒性，在酒精冲洗后参照ISO 10993标准方案开展洗脱试验：将胶原立方体样品置于细胞培养基中，每样品1 ml体积，于37°C孵育24小时以获得胶原提取物。将3T3细胞传代后接种至96孔板，每孔接种5000个细胞；24小时后向每孔加入200 μl提取物，置于37°C培养箱中孵育24小时。次日收集提取物，按照制造商说明书加入阿尔玛蓝试剂（AlamarBlue，Invitrogen，美国马萨诸塞州沃尔瑟姆）以评估细胞代谢活性。以十二烷基硫酸钠（SDS）的系列稀释液作为阳性对照。使用Victor Nivo型分光荧光计（珀金埃尔默，美国马萨诸塞州沃尔瑟姆）在激发波长530 nm、发射波长590 nm条件下测定荧光强度。 细胞接种 确认无细胞毒性后，向胶原海绵接种NIH 3T3细胞系（NIH 3T3），接种密度为每样品（边长0.5 cm的胶原海绵立方体）50000个细胞。 染色流程 将样品用10%福尔马林固定，经磷酸盐缓冲液（PBS）洗涤后，24小时内用蒸馏水冲洗，随后置于3%磷钨酸蒸馏水溶液中，在室温摇床上孵育24小时。染色完成后，对样品进行冲洗并置于5°C蒸馏水中保存。 图像采集与重建 将装有经染色样品的蒸馏水溶液置于塑料试管中，固定于SkyScan 1276型显微CT（micro-CT，布鲁克，比利时孔蒂奇）的样品架上，以3 μm的体素分辨率、70 kV管电压、200 μA源电流，搭配1 mm厚铝滤片进行扫描。扫描时样品沿垂直轴旋转360°，每0.2°角度步长采集2幅中间帧图像。 扫描完成后，使用布鲁克NRecon软件重建数据。重建过程中，环形伪影抑制参数设为20%，线束硬化校正参数设为30%。随后将数据导出为一系列16位标记图像文件格式（TIFF）图像，可通过专业软件打开。