Journal: Nature Communications

Article Title: Functional and analytical recapitulation of osteoclast biology on demineralized bone paper

doi: 10.1038/s41467-023-44000-9

Figure Lengend Snippet: A (i) A bovine femur was cut into blocks and cleaned with a chloroform-methanol solution to dissolve residual fat. (ii) The compact bone block was demineralized in a 1.2 N of hydrochloric acid (HCl) solution under the cyclic pressure (0.1 Hz at 4 bar) for 5–7 days while daily replacing the solution. (iii) X-ray scanning was used to check for demineralization status. If residual minerals exist, the demineralization process is continued. (iv) The fully demineralized bone piece was cryo-sectioned. (v) A typical DBP is larger than 2 × 2 cm with a 20 µm thickness. B Representative microscopic images of DBP from routine sample preparations that show notably different collagen patterns depending on the cutting direction. C The biochemical integrity of collagen in DBP was confirmed by (i) fluorescent dye-conjugated collagen hybridizing peptides (CHP) that specifically bind to denatured collagen fibrils and (ii) second harmonic generation (SHG) imaging that visualizes intact collagen fibrils under multiphoton microscopy. Heated DBP with denatured collagen was used as a positive control. Representative images from 3 independent experiments. D DBP was decellularized with 1% sodium dodecyl sulfate (SDS) and decellularization was confirmed by the nucleus (DAPI) staining ( n = 10 independent samples). E Cross-sectional images of DBP with three different thicknesses and corresponding optical transparency based on an empty well ( n = 3 independent experiments). F Characteristic mechanical stiffness of DBPs with three different thicknesses ( n = 3 independent experiments). G Images of biopsy-punched DBP (D = 6 mm) placed in a 96-well plate. DBPs were stained with a red dye to visualize. The data are presented as mean ± standard deviation. P -values are derived from unpaired two-tailed t -tests. Related source data are provided as a Source Data file.

Article Snippet: Mineralized collagen fibers by osteoblasts were imaged using a resonant scanning multiphoton microscope (Nikon A1MP) with a 25× objective lens via second harmonic generation, excited at 810 nm.

Techniques: Blocking Assay, Imaging, Microscopy, Positive Control, Staining, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Functional and analytical recapitulation of osteoclast biology on demineralized bone paper

doi: 10.1038/s41467-023-44000-9

Figure Lengend Snippet: A Primary osteoblasts from DsRed reporter mice were harvested from femoral bone chips, expanded on TCP, and cultured on 5 different substrates. The red mouse image has been obtained from Clker.com and flyclipart.com. B Representative fluorescent microscopy images of the nucleus (blue: DAPI) and actin (green: Phalloidin) after 1-week of culture and measured cell alignment angles on different substrates ( n = 40 independent samples). C A comparison of OB growth after 1 week of culture on different substrates ( n = 10 independent samples). D Fluorescent microscopy images of osteoblasts (DsRed) and mineral (green: calcein) show rapid and homogenous mineral deposition on DBP compared to TCP. E 3D multiphoton microscopy images of remineralized-DBP by osteoblasts after 1-week of culture (red: actin-phalloidin, green: mineral-calcein, blue: collagen-second harmonic) show mineral deposition in DBP collagen. F Time-course measurement of mineral deposition with calcein staining for 7 days on TCP and DBP ( n = 8 independent samples). G Transmission microscopy images of DBP, remineralized-DBP (R-DBP), and bone discs (BD), and quantitative comparison of their transmittance ( n = 3 independent experiments). The data are presented as mean ± standard deviation. P -values are derived from unpaired two-tailed t -tests. ns not significant ( p > 0.05). Related source data are provided as a Source Data file.

Article Snippet: Mineralized collagen fibers by osteoblasts were imaged using a resonant scanning multiphoton microscope (Nikon A1MP) with a 25× objective lens via second harmonic generation, excited at 810 nm.

Techniques: Cell Culture, Microscopy, Comparison, Staining, Transmission Assay, Standard Deviation, Derivative Assay, Two Tailed Test