Engineered Milk-Derived Extracellular Vesicles Ameliorate Bone Defects

Osteoblast-targeting milk-derived extracellular vesicles (denoted as DPS-MEVs) were synthesized to evaluate their therapeutic potential for bone defects. The osteoblast-targeting ability of DPS-MEVs was evaluated through in vitro internalization assays. The effects of DPS-MEVs on osteoblasts were subsequently assessed using CCK-8 assays and RT-qPCR. The mechanism by which DPS-MEVs promote osteogenesis was investigated through transcriptome sequencing. A skull defect model was constructed using sixteen male C57BL/6 mice (aged 8 weeks), which were divided into four groups (n = 5). The experimental protocol was approved by the Ethics Committee of XX University. Tissue repair was analyzed using hematoxylin and eosin (HE) and Masson staining, and osteogenesis-related factors were detected via immunohistochemical staining. We observed that the SDSSD peptide combined effectively with osteoblasts, and DPS-MEVs promoted MC3T3-E1 cell proliferation and upregulated osteogenic factors Alp, Opn, and Runx2 (p < 0.05). DPS-MEVs enhanced osteogenesis through mitochondrial OXPHOS, mediated by KLF4 regulation of Ndufa4 expression. In vivo, DPS-MEVs demonstrated significant osteogenic potential, with HE, Masson, and immunohistochemical staining revealing superior bone repair and higher expression of ALP, RUNX2, and OPN compared to controls (p < 0.05). DPS-MEVs promoted osteoblast proliferation and differentiation, enhanced osteoblast OXPHOS activity, and facilitated osteoblast differentiation through KLF4 regulation of Ndufa4 expression. In vivo experimental studies demonstrated that DPS-MEVs enhanced bone formation in skull defects.