Heinz Scheuenstuhl

Comparison of fetal, newborn, and adult wound healing by histologic, enzyme-histochemical, and hydroxyproline determinations***

We compared simultaneous healing processes in fetal, newborn, and maternal rabbits using a miniaturized wound cylinder of expanded Gore-Tex tubing. The tubing was placed subcutaneously in fetal and maternal rabbits on day 23 of pregnancy (term = 31 to 32 days), and in 7-day-old newborn rabbits. At specific intervals, the tubing was removed and analyzed for hydroxyproline accumulation, histology, and cellular enzyme-histochemistry. Granulation tissue ingrowth and accumulation of hydroxyproline were each inversely related to age (fetus greater than newborn greater than maternal). The fetus showed an impressive infiltration of macrophages by day 4, fibroblasts by day 7, and a conspicuous lack of neutrophils in all specimens. Newborns and mothers had few cells until day 7, when a mixture of macrophages, neutrophils, and some fibroblasts appeared. We conclude that the fetus heals wounds rapidly by both mesenchymal cell proliferation and collagen deposition, and that these processes are more rapid in fetuses than in newborn or adult animals despite relative fetal hypoxemia.

Lactate and oxygen constitute a fundamental regulatory mechanism in wound healing

For many years, lactate has been known to accelerate collagen deposition in cultured fibroblasts and, without detailed explanation, has been presumed to stimulate angiogenesis. Similarly, hypoxia has been linked to angiogenic effects and collagen deposition from cultured cells. Paradoxically, however, wound angiogenesis and collagen deposition are increased by breathing oxygen and decreased by hypoxia. Lactate accumulates to 4–12 mM in wounds for several reasons, only one of which is the result of hypoxia. Oxygen in wounds is usually low but can be increased by breathing oxygen (without change in lactate). We have reported that lactate elicits vascular endothelial growth factor (VECF) from macrophages, as well as collagen, some heat shock proteins, and VECF from endothelial cells, and collagen from fibroblasts, even in the presence of normal amounts of oxygen. Hypoxia exerts many of these same effects in cultured cells. In this study, we elevated extracellular lactate in wounds by implanting purified solid‐state, hydrolysable polyglycolide. A steady‐state 2–3 mM additional elevation of lactate resulted. With it, there was a significant short‐term elevation of interleukin‐1β, a long‐term elevation of VECF (2×) and transforming growth factor‐β1 (2–3×), a 50% elevation in collagen deposition, and a large reduction of insulin‐like growth factor‐1 (− 90%). We propose that lactate induces a biochemical “perception” of hypoxia and instigates several signals that activate growth factor/cytokine signals while the continued presence of molecular oxygen allows endothelial cells and fibroblasts to reproduce and deposit collagen. The data are consistent with ADP‐ribosylation effects and oxidant signaling. (WOUND REP REG 2003;11:504–509)

Lactate elicits vascular endothelial growth factor from macrophages: a possible alternative to hypoxia

Macrophages respond to various stimuli to produce angiogenic factors but few mechanistic details are known. We examined the effects of hypoxia, lactate and nicotinamide on the expression of vascular endothelial growth factor by cultured macrophages. These agents were chosen because they down‐regulate polyadenosine diphosphoribose levels. Following exposure, conditioned media were analyzed for vascular endothelial growth factor protein. Nicotinamide adenine dinucleotide, polyadenosine diphosphoribose, and vascular endothelial growth factor mRNA were measured in the cellular fraction. Angiogenic capacity of the conditioned media was tested in rabbit corneas and Matrigel implants.

Lactate stimulates endothelial cell migration

The significance of the high lactate levels that characterize healing wounds is not fully understood. Lactate has been shown to enhance collagen synthesis by fibroblasts and vascular endothelial growth factor (VEGF) production by macrophages and endothelial cells. VEGF has been shown to induce endothelial cell migration. However, it has not been shown whether accumulated lactate correlates with the biological activity of VEGF. Therefore, we investigated the effect of lactate on migration of endothelial cells. Human umbilical vein endothelial cells and human microvascular endothelial cells were cultured to subconfluent monolayers in standard six‐well tissue culture plates. Following a 24‐hour serum starvation, cells were treated with the indicated concentrations of l‐lactate. Cell migration was assessed using a modified Boyden chamber. VEGF protein in the cell culture supernatant was measured by enzyme‐linked immunoassay. Lactate‐enhanced VEGF protein synthesis in a time‐ and dose‐dependent manner. Lactate added into the bottom well did not stimulate cellular migration from the upper well. However, lactate when added together with endothelial cells to the bottom well of the Boyden chamber increased cellular migration in a dose‐dependent manner. This effect was blocked by anti‐VEGF and by cycloheximide. Lactate enhances VEGF production in endothelial cells, although lactate, itself, is not a chemoattractant. We conclude that the lactate‐mediated increase in cellular migration is regulated by VEGF.

Mild hypothermia during halothane-induced anesthesia decreases resistance to Staphylococcus aureus dermal infection in guinea pigs

Because various immune functions are impaired at temperatures only 1° to 3° C less than normal, we tested the hypothesis that mild hypothermia during anesthesia impairs resistance to dermal infections. Guinea pigs were anesthetized for 6 hours with 1% inspired halothane. Their core temperatures were maintained at either 39° C (normal for guinea pigs, n = 12) or 36° C (n = 12). Two hours after induction of anesthesia, three doses each of Staphylococcus aureus (108, 107, and 106 organisms) were injected intradermally at nine sites on each animals back. Core temperatures were not controlled after recovery from the anesthetic, and animals in each group were maintained in the same environment. Four days after anesthesia, each injection site was excised to obtain a count of viable bacteria. Subcutaneous oxygen partial pressure values, averaged over time, were 53 ± 3 mm Hg (mean ± SEM) in the hypothermic group and 62 ± 4 mm Hg in the normothermic group (p = 0.06). Capillary perfusion, as assessed by laser Doppler flowmetry, was comparable in the two groups. One day after injection of 108 bacteria, the area of induration was 89 ± 11 mm2 in the hypothermic group but only 61 ± 6 mm2 in the normothermic group (p < 0.05). On postanesthetic day 4, the area of induration was 72 ± 6 and 59 ± 6 mm2 in the hypothermic and normothermic groups, respectively (p > 0.05). After inoculation with 108 bacteria, the fraction recovered was 1.0 ± 0.2 in the hypothermic groups and 0.6 ± 0.2 in the normothermic group (p < 0.05). After inoculation with 107 and 106 bacteria, the fraction recovered was less than 0.2, and no difference was found between the hypothermic and normothermic animals. Thus mild hypothermia during halothane‐induced anesthesia slightly impairs resistance to dermal infection.

An experimental model to determine the effect of irradiated tissue on neutrophil function

Complications of irradiated tissue include infections and impaired healing. Although fibrosis and hypovascularity contribute, a cellular mechanism has not been identified. This study examines the effect of radiation (10 to 30 Gy) on neutrophil function in a rabbit wound cylinder model. At 3 to 12 weeks after radiation, subcutaneous wound cylinders were implanted in both irradiated and control fields in 19 rabbits. Wound neutrophils were subsequently assayed for phagocytosis (3H-labeled Staphylococcus aureus assay), superoxide production (cytochrome c reduction assay), and surface Mac-1 expression (flow cytometric assay using MHM 23 monoclonal antibody). Phagocytosis of 3H-labeled S. aureus was significantly lower in neutrophils from irradiated fields compared with controls at 6 and 12 weeks after radiation (6.5 versus 18.9 bacteria per neutrophil at 12 weeks; p = 0.027). Stimulated neutrophils from irradiated tissue could not increase superoxide production or Mac-1 expression as much as controls, with differences increasing as postirradiation time increased. The diminished phagocytosis, superoxide production, and Mac-1 expression provide a cellular mechanism that may account for susceptibility to infection and poor healing in irradiated tissues.

Examination of expanded polytetrafluoroethylene wound healing models.

The object of this animal study was to examine and further develop the expanded polytetrafluoroethylene wound healing model. The goal was to increase its potential for assessing wound healing by increasing yield, reducing variability, establishing the elements of a standard technique, and further testing its ability to detect variations of healing which have clinical significance. Expanded polytetrafluoroethylene implants of various dimensions and fabrications and several implantation and sterilization techniques were compared in rats. Hydroxyproline, DNA, and protein deposition into the expanded polytetrafluoroethylene implants as parameters for wound healing were assessed. Additionally, a 4 cm skin incision for tensile strength assessment was created. Wound healing was assessed under normal and corticosteroid‐impaired healing conditions. The highest yield of collagen was found in the stiffer fabrication of expanded polytetrafluoroethylene with the larger pore size and after the more traumatic implantation technique of incisional placement. Variability was unaffected by fabrication, implantation technique, indexing by various geometric dimensions of the implant, sterilization, or sampling techniques. Variability was the same in the individual animals as in groups of animals. The expanded polytetrafluoroethylene method also detects the influence of antiinflammatory corticosteroids and reflects the tensile strength of incisional wounds made in other sites in the same animal.

INDUCTION OF NEUTROPHIL MAC-1 INTEGRIN EXPRESSION AND SUPEROXIDE PRODUCTION BY THE MEDICINAL PLANT EXTRACT GOSSYPOL

Gossypol is present in antiinflammatory poultices made from the medicinal treeThespesia populnea. Isolated human neutrophils exposed to 3–20μM gossypol for 15–90 min were assayed in vitro for superoxide production and surface expression of Mac-1 (CD11b/CD18). Gossypol increased superoxide production in a time- and concentration-dependent fashion consistent with a moderate, delayed respiratory burst. Surface Mac-1 expression was increased within 15 min by 3–5μ M gossypol, resulting in a 14-fold increase over controls and a threefold greater increase over that produced by PMA. Staurosporine failed to block gossypol induction of superoxide and Mac-1, while EDTA inhibited induction of Mac-1 only, implicating a calcium-dependent mechanism. Gossypol increased intracellular calcium to peak levels, but1 in a delayed fashion as compared to FMLP. These findings demonstrate that gossypol is a highly potent stimulant of Mac-1 expression and suggest at least two protein kinase C-independent pathways of neutrophil activation. The resultant exhaustion of neutrophils may account for the antiinflammatory properties of plants containing gossypol.

A quantitative in vitro study of fibroblast and endothelial cell migration in response to serum and wound fluid

Chemoattractant activity for irradiated and nonirradiated rabbit skin fibroblast and bovine aortic arch endothelial cells was assayed in rabbit wound fluid and sera using a modification of the agarose well method originally described for polymorphonuclear leukocytes. Both serum and wound fluid contained chemoattractants for fibroblasts and endothelial cells. Fibroblast migration was decreased by 70 to 80% when the serum or wound fluid was heated to 56 degrees C for 30 min while endothelial cell migration was reduced by 50 to 60%. Platelet-poor plasma-derived serum had no directive effect on the migration of either cell type.

Effect of growth hormone replacement on wound healing in healthy older men

The secretion of growth hormone, an important anabolic agent, declines with aging. We hypothesize that growth hormone levels (measured as insulin‐like growth factor‐1) correlate with postoperative tissue repair in otherwise healthy, elderly persons. The goal was to determine whether growth hormone supplementation can improve wound healing in this circumstance. We conducted a randomized controlled double‐blind trial of 6 months of growth hormone replacement or placebo in 28 healthy older men (>69 years of age) with low baseline plasma insulin‐like growth factor‐1. Growth hormone doses were adjusted to elevate insulin‐like growth factor‐1 to levels expected in younger adults. Wound healing was tested by implanting 10 cm expanded polytetrafluoroethylene porous tubes for 10 days, then measuring the content of collagen (as hydroxyproline), DNA, and total protein. Hydroxyproline content was 15% greater in the wounds of the growth hormone group (n = 13) compared with the placebo group (n = 15), (4.52 ± 0.94 versus 3.92 ± 0.78 µg/cm; p = 0.04). Therefore, healthy older men who took growth hormone had enhanced reparative collagen deposition during the wound healing process. This action may be clinically useful after selected surgery or trauma in the elderly.