The symbiotic effect of osteoinductive extracellular vesicles and mineralized microenvironment on osteogenesis

The increasing prevalence of bone‐related diseases has raised concern about the need for an osteoinductive and mechanically stronger scaffold‐based bone tissue engineering (BTE) alternative. A mineralized microenvironment, similar to the native bone microenvironment, is required in the scaffold to recruit and differentiate local mesenchymal stem cells at the bone defect site. Further, extracellular vesicles (EVs), pre‐osteoblasts' secretome, contain osteoinductive cargo and have recently been exploited in bone regeneration. This work developed a cell‐free and mechanically strong interpenetrating network‐based scaffold for BTE by combining the action of osteoinductive EVs with a mineralized microenvironment. The MC3T3 (a pre‐osteoblast cell line) is used as a source of EVs and as the target population. The optimal concentration of MC3T3‐EVs was first determined to induce osteogenesis in target cells. The osteoinductive potential of the scaffold was estimated in vitro by osteogenesis‐related markers like the alkaline phosphatase (ALP) enzyme and calcium content. The MC3T3‐EVs cargo was also studied for osteoinductive signals such as ALP, calcium, and mRNA. The findings of this work indicated that MC3T3‐EVs at a 90 μg/mL dose had significantly higher ALP activity than 0 μg/mL (1.47‐fold), 10 μg/mL (1.41‐fold), and 30 μg/mL (1.39‐fold) EV‐concentration on day 14. Further combination of the optimum dose of EVs with a mineralized microenvironment significantly enhanced ALP activity (1.5‐fold) and mineralization (3.36‐fold) as compared to the control group on day 7. EV cargo analysis revealed the presence of calcium, the ALP enzyme, and the mRNAs necessary for osteogenesis and angiogenesis. ALP activity was significantly boosted in the EV‐containing target cells as early as day 1, and mineralization began on day 7 because MC3T3‐EVs carry ALP enzymes and calcium as cargo. When osteoinductive EVs were combined with an osteoconductive mineralized microenvironment, osteogenesis was significantly enhanced in target cells at early time points. The interaction between osteoinductive EVs and the mineralized milieu facilitates the process of osteogenesis in the target cells and suggests a potential cell‐free strategy for in vivo bone repair. A potential “cell‐free” scaffold for bone tissue engineering could be created by the synergistic action of osteoinductive EVs and a mineralized milieu for improved osteogenesis and angiogenesis.