Pouya Dastouri

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy

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 physiological 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.

Mast cells are required in the proliferation and remodeling phases of microdeformational wound therapy.

Background: Mast cells are important in numerous inflammatory processes. They are also mechanosensitive and likely play an important role in wound healing. The authors hypothesized that mechanical alteration of the wound environment with a distributed suction device could link mast cells to the healing cascade. Methods: Controlled uniform full-thickness wound surface microdeformations were induced by suction combined with an open-pore polyurethane foam (microdeformational wound therapy) in mast cell–deficient WWv mice and their mast cell–sufficient littermates. Wound healing parameters were assessed in the inflammatory, proliferative, and remodeling phases of healing. Results: Wound tissue granulation, cell proliferation, blood vessel sprouting, and collagen maturation were found to be mast cell–dependent throughout the proliferating and remodeling stages of healing. Conclusion: Mast cells are critical in the robust granulation tissue response seen in microdeformational wound therapy and in the modulation of the remodeling phase of wound healing.

Foam pore size is a critical interface parameter of suction-based wound healing devices.

Background: Suction-based wound healing devices with open-pore foam interfaces are widely used to treat complex tissue defects. The impact of changes in physicochemical parameters of the wound interfaces has not been investigated. Methods: Full-thickness wounds in diabetic mice were treated with occlusive dressing or a suction device with a polyurethane foam interface varying in mean pore size diameter. Wound surface deformation on day 2 was measured on fixed tissues. Histologic cross-sections were analyzed for granulation tissue thickness (hematoxylin and eosin), myofibroblast density (&agr;-smooth muscle actin), blood vessel density (platelet endothelial cell adhesion molecule-1), and cell proliferation (Ki67) on day 7. Results: Polyurethane foam–induced wound surface deformation increased with polyurethane foam pore diameter: 15 percent (small pore size), 60 percent (medium pore size), and 150 percent (large pore size). The extent of wound strain correlated with granulation tissue thickness that increased 1.7-fold in small pore size foam–treated wounds, 2.5-fold in medium pore size foam–treated wounds, and 4.9-fold in large pore size foam–treated wounds (p < 0.05) compared with wounds treated with an occlusive dressing. All polyurethane foams increased the number of myofibroblasts over occlusive dressing, with maximal presence in large pore size foam–treated wounds compared with all other groups (p < 0.05). Conclusions: The pore size of the interface material of suction devices has a significant impact on the wound healing response. Larger pores increased wound surface strain, tissue growth, and transformation of contractile cells. Modification of the pore size is a powerful approach for meeting biological needs of specific wounds.

Analysis of Nerve and Neuropeptide Patterns in Vacuum-Assisted Closure-Treated Diabetic Murine Wounds

Background: Reestablishment of the peripheral nervous system occurs in parallel with wound healing. With accelerated wound healing seen with the vacuum-assisted closure device, the authors studied its effects on nerve fiber regeneration, nerve sprouting, and the stimulation of neuropeptides and neurotrophins. Methods: A vacuum-assisted closure device was applied to a full-thickness diabetic mouse wound using continuous or cyclical modes and compared with foam dressing or occlusive dressing controls, using 10 mice per group. Nerve fibers, substance P, calcitonin gene-related peptide, and nerve growth factor were analyzed using two-dimensional immunohistochemistry and real-time reverse-transcriptase polymerase chain reaction. Results: A significant increase in dermal and epidermal nerve fiber densities and in substance P, calcitonin gene-related peptide, and nerve growth factor expression was seen in vacuum-assisted closure–treated wounds. Cyclical treatment mode correlated with the largest increase in granulation tissue production, wound surface microdeformations, and a slightly faster wound closure rate. Conclusions: This study suggests that vacuum-assisted closure therapy can modulate nerve fiber and neuropeptide production in the wound. Optimized kinetics of vacuum-assisted closure application may provide an opportunity for clinicians to further improve wound healing in denervated wounds such as pressure sores and diabetic foot ulcerations.

Waveform modulation of negative-pressure wound therapy in the murine model

Background: Negative-pressure wound therapy applied with a porous foam interface has been shown to accelerate granulation-tissue formation when a cyclic application mode of suction is applied, but the optimal waveform has not been determined. The authors hypothesized that changes in the suction waveform applied to wounds would modulate the biological response of granulation tissue formation. Methods: A vacuum-assisted closure device (Kinetic Concepts, Inc., San Antonio, Texas) was applied to full-thickness wounds in 48 male diabetic mice (C57BL/KsJ-Lepr db), which were treated with six different waveforms: square waveforms of 125 mmHg of suction for 2 minutes, alternating with 50 mmHg of suction for 2 minutes, 5 minutes, or 10 minutes; triangular waveform with a 7-minute period oscillating between 50 and 125 mmHg; and static suction at 125 mmHg or static suction at 0 mmHg (occlusive dressing). Wounds were quantitatively evaluated for granulation tissue thickness as well as the number of proliferating cells and the number of blood vessels of the newly formed granulation tissue. Results: At 7 days, the continuous and triangular waveforms induced the thickest granulation tissue, with high rates of cellular proliferation and blood vessel counts compared with square wave and occlusive dressing control wounds. Decreasing square waveform frequency significantly increased granulation tissue thickness, cellular proliferation, and blood vessel counts. Conclusions: Waveform modulation has a significant effect on granulation tissue formation, angiogenesis, and cellular proliferation in excisional wounds in diabetic mice. The rapid change in pressure seen in our square wave model may be detrimental to granulation tissue formation.

Controlled induction of distributed microdeformation in wounded tissue via a microchamber array dressing

Mechanical stimuli are known to play an important role in determining the structure and function of living cells and tissues. Recent studies have highlighted the role of mechanical signals in mammalian dermal wound healing. However, the biological link between mechanical stimulation of wounded tissue and the subsequent cellular response has not been fully determined. The capacity for researchers to study this link is partially limited by the lack of instrumentation capable of applying controlled mechanical stimuli to wounded tissue. The studies outlined here tested the hypothesis that it was possible to control the magnitude of induced wound tissue deformation using a microfabricated dressing composed of an array of open-faced, hexagonally shaped microchambers rendered in a patch of silicone rubber. By connecting the dressing to a single vacuum source, the underlying wounded tissue was drawn up into each of the microchambers, thereby inducing tissue deformation. For these studies, the dressings were applied to full-thickness murine dermal wounds with 200 mmHg vacuum for 12 h. These studies demonstrated that the dressing was capable of inducing wound tissue deformation with values ranging from 11 to 29%. Through statistical analysis, the magnitude of the induced deformation was shown to be a function of both microchamber height and width. These results demonstrated that the dressing was capable of controlling the amount of deformation imparted in the underlying tissue. By allowing the application of mechanical stimulation with varying intensities, such a dressing will enable the performance of sophisticated mechanobiology studies in dermal wound healing.