Phosphate buffered saline (pbs)

All the soft tissues of the proximal half of the left femur were removed, freshly prepared 10% neutral buffered formalin (Sinopharm Chemical Reagents) was used to fix the bone for 24 h. The bone was then rinsed with 0.01 M phosphate buffered saline (Sinopharm Chemical Reagents). After that, the bone was decalcified by 20% formic acid (Sinopharm Chemical Reagents) for 6 days, with the formic acid changed daily. Then the bone was rinsed with 0.01 M phosphate buffered saline, and dehydrated with 75, 80, 95, and 100% ethanol (Sinopharm Chemical Reagents) successively, followed by treating 15 min in xylene (Sinopharm Chemical Reagents) twice. After that, the bone was soaked in a 60^°C paraffin bath for 3 h and embedded overnight. On the next morning, 5 μm thick cross sections were prepared on a polylysine-treated glass slide. The glass slide was dried, followed by hematoxylin and eosin staining (Sinopharm Chemical Reagents) and changes in bone structure were examined under a light microscope (Leica, Wetzlar, Germany).

Neutral protease, papain, chitosan, bovine insulin, myoglobin, and tryptophan were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Tryptone, yeast extract, Agar, NaCl, glycerin, K₂HPO₄·3H₂O (AR), sorbitol, MgSO₄·7H₂O (AR), trisodium citrate, MgCl₂ (AR), kanamycin, ampicillin, peptone, maltose, sodium acetate, calcium sulfate, acetic acid, Triton X-100, phosphate-buffered saline, normal saline, trichloroacetic acid, and other reagents (AR) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).