Fibroblast-Derived Decellularized Extracellular Matrix as a Bioactive Substrate for Osteoblast Activation

The extracellular matrix (ECM) plays a critical role in regulating cell behavior and is increasingly incorporated into biomaterial design. However, the reproducible generation of cell-derived ECM remains challenging due to variability in decellularization methods. This study refines a detergent-based protocol for preparing fibroblast-derived ECM and evaluates its effects on primary human osteoblasts (HOBs). Modifications to detergent exposure and processing conditions enhanced DNA removal (up to 98.3%) while preserving protein content, with residual DNA predominantly below 100 bp. Proteomic analysis was included as a descriptive, contextual reference and is not directly linked to functional results. Functional assays demonstrated that fibroblast-derived ECM modulated osteoblast responses in a concentration-dependent manner. A concentration of 1.25 mg/mL increased alkaline phosphatase activity and significantly increased RUNX2 levels, indicating enhanced early osteogenic signaling. Mineralization assessment at day 14 using Alizarin Red S and von Kossa staining revealed increased calcium and phosphate deposition in dECM-treated cultures compared with controls, suggesting progression toward matrix mineralization. Overall, the fibroblast-derived dECM provides a biologically active substrate that supports osteoblast function and osteogenic responses. Further studies are required to fully establish its osteogenic potential.