Decellularized extracellular matrix (dECM) hydrogels are engineered constructs that are widely-used in the field of regenerative medicine. However, the development of ECM-based hydrogels for bone tissue engineering requires enhancement in its osteogenic properties. For this purpose, we initially employed bone-derived dECM hydrogel (dECM-Hy) in combination with calcium phosphate cement (CPC) paste to improve the biological and structural properties of the dECM hydrogel. A decellularization protocol for bovine bone was developed to prepare dECM-Hy, and the mechanically-tuned dECM/CPC-Hy was built based on both rheological and mechanical characteristics. The dECM/CPC-Hy displayed a double swelling ratio and compressive strength. An interconnected structure with distinct hydroxyapatite crystals was evident in dECM/CPC-Hy. The expression levels of Alp, Runx2 and Ocn genes were upregulated in dECM/CPC-Hy compared to the dECM-Hy. A 14-day follow-up of the rats receiving subcutaneous implanted dECM-Hy, dECM/CPC-Hy and mesenchymal stem cells (MSCs)-embedded (dECM/CPC/MSCs-Hy) showed no toxicity, inflammatory factor expression or pathological changes. Radiography and computed tomography (CT) of the calvarial defects revealed new bone formation and elevated number of osteoblasts-osteocytes and osteons in dECM/CPC-Hy and dECM/CPC/MSCs-Hy compared to the control groups. These findings indicate that the dECM/CPC-Hy has substantial potential for bone tissue engineering.
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