# Extracellular Adenosine Contributes to the Hydrogen Peroxide-Induced Calcification of Cultured Tendon Cells

## TL;DR

Hydrogen peroxide increases calcification in tendon cells, likely through extracellular adenosine and related metabolites.

## Contribution

This study identifies extracellular adenosine as a novel contributor to hydrogen peroxide-induced calcification in tendon cells.

## Key findings

- Hydrogen peroxide significantly enhances calcium deposition in osteogenic media over two weeks.
- Exogenous adenosine and dipyridamole increase mineralization in tendon cell cultures.
- Metabolomic analysis shows hydrogen peroxide boosts extracellular ATP and its metabolites.

## Abstract

Background: Well-known risk factors for soft tissue heterotopic ossification (HO) include aging and mechanical stress, which may be linked to oxidative stress and downstream nucleotide metabolites. Thus, we investigated the involvement of extracellular ATP (ex-ATP) and its metabolites in the oxidative stress-induced mineralization of TT-D6 cells and primary mouse tendon cells. Methods: An osteogenic culture with the intermittent addition of hydrogen peroxide was monitored for two weeks using metabolomic and gene expression analyses. Results: Calcium deposition was significantly enhanced by 0.3 mM hydrogen peroxide in the osteogenic media after 2 weeks, with minimal calcification in its absence. Similar results were observed in a medium transfer experiment using 3-day-old hydrogen peroxide-treated conditioned medium, which led to an increased expression of osterix and alkaline phosphatase. Metabolomic analysis revealed a gradual increase in ex-ATP and its metabolites, including ADP, AMP, and adenosine, in the medium. The metabolite increase was enhanced by hydrogen peroxide after 12 h. Moreover, exogenous adenosine (100 μM) increased mineralization in osteogenic media. Additionally, 1 μM dipyridamole, an inhibitor of equilibrative nucleoside transporter 1 (Ent1), also increased it in response to low-dose (0.1 mM) hydrogen peroxide. Conclusions: The enhanced osteogenic calcification of the tendon cell culture by hydrogen peroxide was associated with an increase in extracellular nucleotide metabolites, especially adenosine, with some evidence of causality.