Poster AA1 - MG63 Derived Matrix Vesicles Differ in Characteristics and Bone Formation Function from Exosomes

Introduction: Extracellular vesicles are cell-free, membrane-bound, organelles, with exosomes (EX) and matrix vesicles (MV) serving as two vesicle types produced by osteoblasts that may contribute to the regulation of bone regeneration. MVs are released by mineralization-competent cells and anchored to the extracellular matrix (ECM). In contrast, EXs are free-floating vesicles. Both contribute to cell signaling via enrichment with microRNA. We hypothesize that EXs and MVs affect osteogenesis differently as they have different microRNA expression. To understand their distinct functions, we characterized each class of vesicle and observed the effects when these vesicles were used to treat cells in vitro.

Materials and

Methods: MVs and EXs were collected from confluent cultures of MG63 osteoblast-like cells via ultracentrifugation of the trypsinized cell layer supernatant and the conditioned media, respectively. The cell pellet (CP) and plasma membranes (PM) of MG63 cells were also collected for comparison. Alkaline phosphatase specific activity (ALPase) was quantified in all fractions. Western blots were performed for integrins, Na-K ATPase, ALIX, HSP70, TSG101, annexin V, CD9, and CD81. MG-63 cells cultured in DMEM containing 10% FBS and antibiotics at confluence were treated with 2.5 µg/well (based on protein concentration) of MVs, EXs, or an equivalent volume of saline to compare the effects on osteoblastic differentiation. At 48 hours, ALPase was assayed in cell layer lysates. Osteocalcin (OCN) and osteopontin (OPN) in the conditioned media were quantified by enzyme linked immunosorbent assay (ELISA) and were normalized to DNA.

Results, Conclusions, and Discussions: MVs and EXs demonstrated significant enrichment of ALPase compared to the PM, with MVs also being significantly greater than EXs. Vesicle markers, CD9 and CD81, were present in MV and EX fractions only. Adhesive proteins, integrins α1, αV, and β3, were expressed in MVs with no expression in the EXs. Endosomal biogenesis markers HSP70 and TSG101 were present in all fractions with higher expression in the MV fractions compared to EXs. PMs and MVs had high expression of Na-K ATPase and ALIX, a calcium ion channel regulator and an endolysosomal regulator protein, respectively. Annexin V, a calcium channel, was present in all fractions in comparable amounts. In the 48-hour cell culture treatment, MVs decreased ALPase and OCN, while EXs increased OPN in comparison to saline. The results validate that MVs and EXs are distinct extracellular organelles, with the highest ALPase in MVs, which is consistent in their role in mineralization. In the western blot, MVs and EXs had different enrichment of integrins, NaK-ATPase, and endosomal proteins. The different location of each vesicle, anchored to the ECM versus free floating, is consistent with their unique characteristics. The variable content of endosomal pathway markers between EXs and MVs suggests similar but ultimately different biogenesis in the cell. The different role of MVs and EXs is further supported by the in vitro data that demonstrate decreased osteoblastic differentiation with MV treatment and increases in OPN with EX treatment, most likely driven by their different microRNA and protein composition. More experiments are needed to further elucidate how and why these two vesicle types exert their separate effects.

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