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Toxins in cigarette smoke prevent stem cells from becoming cartilage
Date:3/3/2008

A toxic pollutant spread by oil spills, forest fires and car exhaust is also present in cigarette smoke, and may represent a second way in which smoking delays bone healing, according to research presented today at the annual meeting of the Orthopaedic Research Society in San Francisco.

In 2005, researchers from the University of Rochester Medical Center identified one ingredient in smoke, nicotine, that delays bone growth by influencing gene expression in the two-step bone healing process: stem cells become cartilage; cartilage matures into bone. In the current study, some of the same researchers found that a second smoke ingredient, the polyaromatic hydrocarbon benzo(a)pyrene (BaP), also slows bone healing, but in a different way.

Smoking has been shown to delay skeletal healing by as much as 60 percent following fractures. Slower healing means a greater chance of re-injury and can lead to chronic pain and disability. The obvious solution is for smokers to quit when they get hurt, but studies show that just 15 percent can.

Our results provide the first evidence that BaP prevents stem cells from becoming cartilage cells as part of healing, said Regis J. O'Keefe, M.D., Ph.D., chair of the Department of Orthopaedics and Rehabilitation at the Medical Center and a study investigator. These findings extend our understanding of the impact of cigarette smoke on a process that is critical to fracture repair. Perhaps down the road we will be able to speed bone healing among smokers in more than one way.

Study Details

Gene expression is the process by which instructions encoded in genes are followed for the building of proteins, the workhorses that make up the bodys organs and carry its signals. In the current study, polymerase chain reaction (PCR), a technique that measures gene expression levels, revealed the genetic changes caused by exposure to BaP in mouse stem cells.

Among the many factors that influence gene expression are transcription factors, proteins designed to direct genes to create more or less of a protein. One such factor is Sex Determining Region Y-box 9 (SOX-9), required for the transition of stem cells into cartilage cells. The PCR results show that BaP in cigarette spoke interferes with SOX-9 expression in mesenchymal stem cells, blocking their conversion into cartilage cells. When this group of stem cells is free to differentiate, the newly formed cartilage cells immediately begin manufacturing collagen 2, the tough, fibrous protein framework for cartilage. Along with interfering with SOX-9, BaP was also found to reduce levels of type II collagen gene expression.

Past studies had shown that stem cells involved in cartilage formation contain proteins known to react with BaP called aryl hydrocarbon receptors. The current results suggest that BaP binding with these receptors may suppress SOX-9 activity, reducing the number of stem cells that turn into cartilage cells and the amount of collage produced. No one knows what such receptors are doing in these cells in the first place, but one theory has it that they signal cellular machinery to metabolize toxins.

The study compared the effect of BaP versus that of cigarette smoke extract, a substance representing all the ingredients in cigarette smoke. The hope was to confirm BaP as the specific cause of the observed effect on SOX-9. Results indeed suggest BaP alone may responsible for this specific mechanism of healing delay, since its effect was equal to the extract.

In addition measuring gene expression levels, researchers also conducted tests to show the effect of BaP visually. When newly differentiated cartilage cells begin to produce collagen in a culture dish, little mounds or nodules of collagen can be visualized using a stain. Staining experiments captured images showing BaP to completely inhibit collagen nodule deposition.

Along with OKeefe, the Medical Center effort was led by Ming Kung, Donna Hoak, HsinChiu Ho, Edward Puzas and Michael Zuscik, all within the Department of Orthopaedics at the Medical Center.

"Smoking reduces the rate at which the two sides of a fracture come together," said Michael Zuscik, Ph.D., associate professor in the Department of Orthopaedics and Rehabilitation at the Medical Center. "We believe this new research will establish for the first time the mechanisms by which polyaromatic hydrocarbons interfere with the healing process.


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Contact: Greg Williams
Greg_Williams@urmc.rochester.edu
University of Rochester Medical Center
Source:Eurekalert

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