Fig. 2: Fabrication and characterization of demineralized bone paper (DBP).

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.