Person:

Orgill, Dennis

Background: Vacuum-assisted closure induces microdeformations of the wound surface and accelerates healing of complex wounds; however, a thorough understanding of the biology of cellular mechanotransduction is lacking. We hypothesized that fibroblast shape and function can be altered in an in vitro vacuum-assisted closure device.

Methods: A 3-dimensional fibrin matrix with cultured murine fibroblasts and an intervening polyurethane foam was exposed to 125 mm Hg suction and compared with similar wells without suction. We measured fibroblast proliferation and morphology using fluorescence microscopy and gene expression change using real-time reverse-transcriptase polymerase chain reaction at 24, 48, and 72 hours.

Results: Wells exposed to suction induced significant proliferation of fibroblasts and morphologic changes visible by larger, rounder, and notable dendrite-like branching and process extensions. Type 1 collagen alpha 1 (COL1A1), fibroblast growth factor 2 (FGF2, bFGF), and transforming growth factor beta 1 (TGF[beta]1) were all up-regulated after 48 hours of exposure to suction. Smooth muscle actin alpha 2 (Acta2, [alpha]-SMA) was up-regulated after 72 hours.

Conclusions: Microdeformations produced by the combination of polyurethane foam and suction are associated with increased fibroblast proliferation and up-regulation of gene expressions in fibroblasts.

Background: Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. Objective: To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. Methods: Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. Results: VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiologically function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. Conclusions: The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Previous experimental studies to assess the contribution of blood-borne circulating (BBC) cells to cutaneous wound healing have relied on discontinuous pulsing of labeled BBC elements or bone marrow transplant protocols. Such approaches do not allow the examination of stable BBC cells that have matured in a physiologically normal host. We have used a parabiotic murine model for cutaneous wound healing to evaluate the relative contribution of stable populations of peripheral blood cells expressing the green fluorescent protein (GFP) transgene in otherwise normal animals. Circulating cells (mature and immature) expressing the GFP transgene were easily detected and quantified in wounds of GFP− parabiotic twins during all evaluated stages of the healing response. Using multiple antibody probes, the relative contribution of various subsets of BBC cells could be comparatively assessed. In early wounds, some cells expressing mesenchymal epitopes were documented to be of hematopoietic origin, indicating the utility of this model in assessing cell plasticity in the context of tissue regeneration and repair. Application of this approach enables further investigation into the contribution of peripheral blood in normal and abnormal healing responses.