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The effects of preosteoblast-derived exosomes on macrophages and bone in mice
Sema S Hakki1,2, Lena Batoon1, Amy J Koh1
1School of Dentistry, Periodontics and Oral Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Abstract:
The effect of preosteoblast-derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated in vitro, and bone formation studies were performed in vivo in mice. Preosteoblastic MC3T3-E1 clone 4 (MC4) cell-derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre-labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro- and anti-inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1β, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro-inflammatory 'M1-like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For in vivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro-CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post-injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast-derived exosomes enhanced bone formation by influencing macrophage responses.
Insights
Preosteoblast-derived exosomes (MC4exo) promote bone formation by modulating macrophage polarization and osteoclast activity in mice. These exosomes enhance bone mineral density and trabecular bone volume, suggesting therapeutic potential for bone regeneration.
Area of Science:
- Biomedical Sciences
- Cell Biology
- Regenerative Medicine
Background:
- Exosomes are crucial mediators of intercellular communication.
- Preosteoblasts release exosomes that may influence bone remodeling.
- Understanding exosome-osteoblast-macrophage interactions is vital for bone tissue engineering.
Purpose of the Study:
- To evaluate the effect of preosteoblast-derived exosomes (MC4exo) on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) in vitro.
- To investigate the in vivo bone formation capacity of MC4exo in mice.
- To elucidate the role of MC4exo in macrophage polarization and osteoclastogenesis.
Main Methods:
- Characterization of MC4exo using particle tracking, TEM, and Western blot.
- In vitro assessment of MC4exo's effect on BMMΦ polarization (cytokine expression) and osteoclast differentiation (TRAP staining).
- In vivo study involving intratibial injection of MC4exo in mice, followed by micro-CT analysis and histological evaluation (TRAP, OCN, OSX, F4/80 staining).
Main Results:
- MC4exo treatment induced a pro-inflammatory M1-like macrophage polarization.
- MC4exo enhanced RANKL-induced osteoclast differentiation in vitro.
- In vivo, MC4exo administration increased diaphyseal bone mineral density and trabecular bone volume fraction, accompanied by reduced bone marrow macrophages and osteoclasts.
Conclusions:
- Preosteoblast-derived exosomes significantly enhance bone formation in vivo.
- MC4exo influences macrophage polarization and osteoclast activity, contributing to increased bone mass.
- These findings highlight the potential of preosteoblast exosomes as a therapeutic agent for bone regeneration.
