A comparative study on fabrication techniques of gelable bone matrix derived from porcine tibia

Recently, several types of native tissues have been enzymatically digested to prepare hydrogels that have natural-mimic extracellular matrix (ECM) proteins, architecture, and biologic activities. However, the residual detergents and salts remaining in the hydrogel may cause some undesirable effects on compatibility, functionality, and bioactivity of the material. In this study, we enzymatically digested the demineralized and decellularized bone matrix (DDBM) and adopted two common methods that included dialysis against distilled water and acetone precipitation for sample desalting. Efficiency in salt removal, protein preservation, gelation ability, and in vivo biocompatibility and function were compared to the DDBM digest without a desalting treatment. After lyophilization, the dialyzed, precipitated, and non-desalted DDBM digests all exhibited cotton-like texture and were water-soluble; however, only the precipitated DDBM digest could be gelled. We also found that the method of acetone precipitation could effectively remove salt from the DDBM digest while preserving of multiple proteins from the native bone and internal porous structure. A total of 57 proteins were identified by mass spectrometry in the precipitated DDBM digest and the majority of these proteins are critical to overall protein assembly, scaffold structure and stability, and cell-activities. Additionally, the precipitated DDBM digest possessed enhanced biocompatibility and osteointegration in repairing a cranial bone defect in Sprague-Dawley (SD) rat. In conclusion, the soluble, biodegradable, and biocompatible natures of the precipitated DDBM digest allow its usage in bone tissue engineering as a protein carrier because of its resemblance to native bone-like protein composite and operative flexibility.