DOI: 10.5281/zenodo.10177428
title: Micro-CT image of cell-populated collagen scaffold in the aqueous environment (contrasted with PTA)

description: 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.