Extracellular vesicle isolation from primary chondrocytes and osteoblasts

Introduction Osteoarthritis (OA) is a disease of the entire joint and the close physical association between subchondral bone and cartilage suggests the existence of biochemical and molecular crosstalk across the bone-cartilage interface, which may even be elevated in OA. It has been demonstrated that extracellular vesicles (EVs) can mediate communication between different cells and regulate recipient cells through bioinformation transfer. The purpose of the present study is to investigate a possible communication between chondrocytes and osteoblasts through EVs.

Methods Chondrocytes (r-ACCs) and osteoblast (r-OBs) were isolated and identified from new-born rats by enzymatic digestion and plastic adherence selection. EVs of chondrocytes were isolated using PEG precipitation and ultrafiltration methods. Nanoparticle tracking analysis determined size distribution and amount of vesicles. Furthermore, Western blot of EV marker proteins identified EVs.

Results We successfully isolated and cultured r-ACCs and r-OBs. Collagen II immunofluorescence was positive in chondrocytes and mineralized nodule formation of osteoblasts was illustrated by von Kossa and Alizarin red staining. Our data revealed that r-ACCs could secret EVs. Different EV isolation techniques (ultracentrifugation, PEG precipitation) were established and efficiencies were compared. Nanoparticle tracking analysis (NTA) indicated a particle distribution ranged from 30 to 200 nm of the majority of EVs. Western blot analysis confirmed exosomal surface markers including CD81, Alix, and TSG101 proteins.

Discussion In the present study, we successfully isolated and identified r-ACCs and r-OBs and r-ACCs secreted EVs. The close connection of cartilage and bone in fracture healing, growth, and joints may allow communication across the osteochondral interface. EVs, as a paracrine pathway, which have been demonstrated to regulate metabolism of both cartilage and bone, may participate in this bone-cartilage crosstalk through transfer of loaded bioinformation. Further experiments will focus on coculture experiments including r-ACCs and r-OBs using the transwell system in vitro and animal experiments in vivo which might give new insights in the interaction between these two cell types and bone-cartilage communication.

Keywords Extracellular Vesicles, Exosomes, miRNA, Cell-cell communication

Korrespondenzadresse Kai O. Böker, UMG Göttingen, Klinik für Unfallchirurgie, Orthopädie und Plastische Chirurgie, Robert-Koch-Straße 40, 37075 Göttingen, Deutschland, E-Mail: [e-mail]

Publication History

Article published online:
08 September 2022