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Episodic alcohol exposure attenuates mesenchymal stem cell chondrogenic differentiation during bone fracture callus formation.

BACKGROUND
During bone fracture repair, mesenchymal stem cells (MSC) differentiate into chondrocytes and osteoblasts to form a fracture callus. Our laboratory previously reported that alcohol exposed rodents with a surgically created tibia fracture display deficient fracture callus formation and diminished signs of endochondral ossification characterized by the absence of chondrocytes and mature hypertrophic chondrocytes, suggesting alcohol may inhibit MSC differentiation. These findings led to our hypothesis that alcohol exposure inhibits mesenchymal stem cell chondrogenic differentiation within the developing fracture callus.


METHODS
In the present study, we utilized a lineage tracing approach to determine which stage(s) of chondrogenic differentiation may be affected by alcohol exposure. We utilized lineage-specific reporter mice to determine the effects of alcohol on MSC and early and late chondrogenic cell frequencies within the fracture callus. In addition, serially sectioned slides were immunofluorescently and immunohistochemically stained and quantified to determine the effect of alcohol on cell proliferation and apoptosis within the fracture callus of alcohol administered rodents, respectively.


RESULTS
Alcohol-administered rodents had a reduced fracture callus area at 4, 6, and 9 days post-fracture. Alcohol had no effect on apoptosis in the fracture callus at any of the examined timepoints. Alcohol-administered rodents had significantly fewer proliferative cells in the fracture callus at 9 days post-fracture, but no effect on cell proliferation was observed in earlier fracture callus timepoints. Alcohol-administered rodents had reduced Collagen2a1- and Collagen10a1 expressing cells in the developing fracture callus, suggesting that alcohol may inhibit both early chondrogenic differentiation and later chondrocyte maturation during fracture callus development.


CONCLUSION
The data suggest that alcohol might be affecting normal fracture healing through the mitigation of MSC chondrogenic differentiation at Callus site.