Bioreactor for application of subatmospheric pressure to three-dimensional cell culture.

Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) is a highly successful and widely used treatment modality for wound healing, although no apparatus exists to monitor the effects of subatmospheric pressure application in vitro. Such an apparatus is desirable to better understand the biological effects of this therapy and potentially improve upon them. This article describes the development and validation of a novel bioreactor that permits such study. Tissue analogues consisting of 3-dimensional fibroblast-containing fibrin clots were cultured in off-the-shelf disposable cell culture inserts and multi-well plates that were integrated into the bioreactor module. Negative pressure dressings, commercialized for wound therapy, were placed on top of the culture, and subatmospheric pressure was applied to the dressing. Cultures were perfused with media at controlled physiologic wound exudate flow rates. The design of this bioreactor permits observation of the culture using an inverted microscope in brightfield and fluorescence modes and sustained incubation of the system in a 5% carbon dioxide atmosphere. This closed-system mimics the wound micro-environment under VAC NPWT. Matrix compression occurs as the subatmospheric pressure draws the dressing material down. At the contact zone, surface undulations were clearly evident on the fibroblast-containing tissue analogues at 24 h and appeared to correspond to the dressing microstructure. The bioreactor design, consisting of sterilizable machined plastics and disposable labware, can be easily scaled to multiple units. Validation experiments show that cell survival in this system is comparable with that seen in cells grown in static tissue culture. After application of VAC NPWT, cell morphology changed, with cells appearing thicker and with an organized actin cytoskeleton. The development and validation of this new culture system establishes a stable platform for in vitro investigations of subatmospheric pressure application to tissues.

Peritoneal fluid: a potential mechanism of systemic neutrophil priming in experimental intra-abdominal sepsis.

BACKGROUND: Recent studies suggest that peritoneal fluid (PF) may be an important mediator of inflammation. The aim of this study was to test the hypothesis that PF may drive systemic inflammation in intra-abdominal sepsis by representing a priming agent for neutrophils. METHODS: PF was collected 12 hours after the initiation of intra-abdominal sepsis in swine. Naive human neutrophils were primed with PF before treatment with N-formyl-Met-Leu-Phe or phorbol 12-myristate 13-acetate to elucidate receptor-dependent and receptor-independent mechanisms of neutrophil activation. Flow cytometry was used to quantify neutrophil surface adhesion marker expression of integrins and selectins and superoxide anion production. Additionally, proinflammatory cytokines were quantified in PF. RESULTS: PF primed neutrophils via receptor-dependent and receptor-independent mechanisms. There were significant increases in the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α in PF correlating with the development of intra-abdominal sepsis. CONCLUSIONS: PF represents a priming agent for naive polymorphonuclear cells in intra-abdominal sepsis. This may be secondary to increased levels of proinflammatory cytokines. Strategies to reduce the amount of PF may decrease the systemic inflammatory response by reducing a priming agent for neutrophils.

A novel mechanism for neutrophil priming in trauma: potential role of peritoneal fluid.

BACKGROUND: We sought to determine the effect of peritoneal fluid from a novel animal model of abdominal compartment syndrome (ACS) on the proinflammatory status of polymorphonuclear leukocytes (PMNs) and monocytes. We hypothesize that peritoneal fluid is a potential priming and/or activating agent for PMNs/monocytes. METHODS: ACS was induced in female Yorkshire swine, and peritoneal fluid was collected at the time of decompressive laparotomy. Naïve PMNs/monocytes were primed and/or activated with peritoneal fluid, phosphatidylcholine (PAF) plus peritoneal fluid, peritoneal fluid plus n-formyl-met-leu-phe (fMLP), and peritoneal fluid plus phorbol 12-myristate 13-acetate (PMA). Activation was determined by surface marker expression of integrins (CD11b an CD18) and selectins (CD62L). Additionally, proinflammatory cytokines in peritoneal fluid were analyzed. RESULTS: Peritoneal fluid did not activate PMNs but increased CD11b expression on monocytes. When used as a primer for fMLP- or PMA-induced activation, peritoneal fluid significantly increased CD11b and CD18 expression on PMNs and monocytes. Peritoneal fluid collected at 6 and 12 h post decompressive laparotomy had similar effects. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) levels were increased in peritoneal fluid. CONCLUSION: Peritoneal fluid represents a primer for PMNs/monocytes and seems to act through receptor-dependent and receptor-independent pathways. Strategies to reduce the amount of peritoneal fluid may decrease the locoregional and systemic inflammatory response by reducing priming and activation of neutrophils/monocytes.

V.A.C. Therapy normalizes vascular response of injured tissue in full-thickness wounds in rabbits.

V.A.C. Therapy applied to a cutaneous wound was hypothesized to alter vascular regulation in underlying tissue. Two full-thickness wounds were created on the dorsum of 7 New Zealand albino rabbits. One site was treated with V.A.C. Therapy; the other, with saline-moistened gauze. Local perfusion levels in the muscle were measured using laser Doppler flowmetry. Using vena caval occlusion, blood pressure was decreased progressively from baseline under vasonormal conditions and during systemic infusion of a vasodilator and a vasoconstrictor. The pressure-flow relationships for the carotid and V.A.C. Therapy-treated wound sites showed 3 distinct curves, with vasodilator curves shifted upwards and vasoconstrictor curves downwards relative to vasonormal curves (P < 0.05). By contrast, vasodilator curves in the saline-gauze treated sites were indistinguishable from vasonormal curves. Thus, in a rabbit model, application of V.A.C. Therapy to cutaneous wounds normalized the vascular response to vasoactive drugs in a manner similar to undisturbed carotid circulation.