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American Association of Plastic Surgeons
20. Collagen Organization not Myofibroblasts Contract Wounds
Kurtis Moyer, MD, H. Paul Ehrlich, PhD, Donald R. Mackay, MD.
Penn State Hershey Medical Center, Hershey, PA, USA.

An increasing body of evidence indicates that collagen organization and not myofibroblast contraction is responsible for wound contraction.
PURPOSE: To define the role of myofibroblasts in wound healing. Myofibroblasts are identified by α smooth muscle actin stress fibers.
METHODS: 1.5 cm square wounds were made on the backs of 5 rats and the edges and corners tattooed. Wound contraction was measured. At 1,2 and 3 wks the rats were sacrificed and the wounds examined histologically for collagen organization (Sirius red stain and polarized light microscopy) and myofibroblasts/alpha smooth muscle actin(SMA). A second group of 5 rats were given 2mg/l vanedate in drinking water.
In an in vitro model Fibroblast Populated Collagen Lattices (FPCLs) were constructed from soluble collagen, human dermal fibroblasts and serum containing medium. Free floating FPLCs, released at 1 hour, contract fully in 2 days. Attached delayed released FPCLs, released at day 4, contract fully in 2 hours. The latices were examined histologically as described above as well as immuno-staining for Death Associated Protein (DAP kinase)
In a third experiment fibroblasts were plated at low density (50 fibroblasts/sq mm) and high density(500fibroblasts/sq mm) and the cells examined histologically as described. Vanadate was added to a second group of confluent fibroblasts.
RESULTS: The in vivo experiments showed that by day 7 the open wounds contracted by >50%. The addition of Vanadate eliminated myofibroblasts in these wounds (no α SMA staining) for the entire 3 week period but did not affect wound contracture. Collagen was more organized in the vanadate treated group.
Free floating FPCLs showed highly organized collagen and no SMA staining whereas attached delayed released FPCLs had poorly organized collagen and cells that stained for SMA .
All fibroblasts plated at low density expressed SMA, and were myofibroblasts when they reached confluence. In contrast high density plated fibroblasts were morphologically smaller rounded cells and did not stain for SMA. The addition of vanadate to confluent low density plated fibroblasts eliminated SMA expression.
DAP kinase receptors were demonstated on myofibroblasts that expressed SMA but were absent from fibroblasts that did not stain for SMA.
Myofibroblasts appear in wounds on day 7 when wound contraction is already advanced. Vanedate eliminates myofibroblasts but does not affect wound contraction and enhances collagen organization.
Tension appears to be one of the mechanisms for the expression of SMA and the appearence of myofibroblasts.
It is proposed that myofibroblasts are associated with fibroblasts entering apoptosis. DAP kinases are a family of kinases that phosphorylaties myosin light chains, which leads to SMA expression. DAP kinases also induce programmed cell death.
The triggers for generating SMA in stress fibers is when tissue is place under tension and/or when apoptosis is induced through a DAP kinase.
Myofibroblasts may then counteract transient changes in tension within granulation tissue and also prepare cells to enter apoptosis however the myofibroblast is not necessary for wound contraction.

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