Anesth Analg 2005;100:830-834
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000144426.18235.5A
CRITICAL CARE AND TRAUMA
A Single Local Application of Recombinant Human Basic Fibroblast Growth Factor Accelerates Initial Angiogenesis During Wound Healing in Rabbit Ear Chamber
Makiko Komori, MD*,
Yasuko Tomizawa, MD
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Katsumi Takada, MD*, and
Makoto Ozaki, MD*
Departments of *Anesthesiology and Cardiovascular Surgery, School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan
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Abstract
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Local angiogenic therapy with recombinant human basic fibroblast growth factor (rhbFGF) has been used to promote wound healing. To obtain useful information for the development of optimal angiogenic therapy, we chronologically evaluated the effects of a single local application of rhbFGF on angiogenesis in a rabbit ear chamber model of wound healing by observing the subcutaneous vessel bed intravitally. New vessel formation during wound healing was macroscopically and microscopically evaluated for 5 wk. Each rabbit ear chamber received a single dose of 6 µg rhbFGF (treatment B1: n = 13), 18 µg rhbFGF (treatment B2: n = 16), or physiological saline as control (n = 13). At 1 wk the newly vascularized area was significantly larger in groups B1 and B2 than in control. At 2 wk, the vascularized areas in groups B1, B2, and control were similar. At 5 wk, the percentage of rabbits with complete vascularization was significantly larger in group B1 than in control. Capillary density at 5 wk was similar among the three groups. These results suggest that locally applied rhbFGF accelerated angiogenesis during early wound healing in rabbits; however, this effect was transient and no increase in capillary density occurred at the completion of vascularization.
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Introduction
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Basic fibroblast growth factor (bFGF) is a potent angiogenic factor that promotes growth of endothelial cells in vitro and vessel formation in vivo (1–4). The use of bFGF to promote angiogenesis and wound healing may shorten the period of admission to intensive care units. Recombinant bFGF has been used clinically for ischemic hearts (5,6), limbs (7), and decubitus ulcer to promote angiogenesis. However, firm evidence supporting beneficial effects of bFGF on in vivo neovascularization is lacking because the time course of healing after locally applied exogenous bFGF has not been assessed directly. Routine imaging studies, such as angiography and thermography, cannot differentiate preexisting from newly formed vessels. Clinical studies must rely on chronological assessment of cardiac function or on one-time measurement of myocardial capillary density. Furthermore, whether improvement in signs and symptoms, such as decreased frequency or severity of angina (6) and increased walking time (7), is directly attributable to enhanced angiogenesis has been questioned. We chronologically and qualitatively examined the effects of a single local application of recombinant human bFGF (rhbFGF) on wound healing in a punch-out model, using a rabbit ear chamber (REC). We evaluated the effect of protein therapy rather than gene therapy such as treatment with angiogenic growth factor (8,9) on the subcutaneous vessel bed.
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Methods
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The study protocol was approved by the Animal Care Committee of our institution. All animals received humane care in compliance with the Principles of Laboratory Animal Care (National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. The experimental animals were housed at 23°C and were given laboratory chow and tap water ad libitum.
The REC was composed of a disk with a central round table and three peripheral pillars, a cover plate, and a holder ring (10). The disk was designed to leave a cavity measuring 50 µm in thickness and 6.4 mm in diameter between the central round table and the cover plate. New microvessels could grow from the blood vessels of the dermis into the cavity. A cover plate was fixed onto the pillars with the use of a holding ring.
Japanese domestic white male rabbits weighing 2.5–3.5 kg were used. The REC was implanted with the animals during general anesthesia, induced by a bolus IV injection of 30 mg/kg pentobarbital. The rabbits were placed in a holder. One ear of each rabbit was shaved on both sides. Under aseptic conditions, four holes were punched through the cartilage and skin of the distal portion of the ear to accommodate the chamber and three pillars. The sizes and positions of the holes were adjusted according to the REC attachments. The epidermis around the central hole was carefully dissected on both sides of the ear, leaving the subcutaneous tissue intact.
rhbFGF (Kaken Pharmaceutical, Tokyo, Japan) was used. A nebulizer-fitted vial containing 250 µg rhbFGF was reconstituted in 2.5 mL physiological saline. Forty-two rabbits were divided into three groups. In the first group (B1, n = 13), 1 shot (6 µg) of rhbFGF was sprayed onto the ear chamber and surrounding connective tissue at the end of the operation. In the second group (B2, n = 16), 3 shots (18 µg) of rhbFGF were similarly applied at the end of the operation. In the control group (C, n = 13), 0.1 mL of physiological saline was applied at the end of the operation.
Macroscopic photographs of the ear chamber were taken once per week for 5 wk as described previously (11). All observations of the ear chamber were made without anesthesia or stress; the rabbit remained secured in an animal holder. The filming required 1 to 2 min and was accomplished without difficulty.
To analyze the vascularized area and capillary density, the photographs of the ear chamber were digitized with the use of the graphic software package NIH Image version 1.62 (National Institutes of Health, Bethesda, MD) on a personal computer. The area covered by vascularized granulation tissue in the chamber was macroscopically estimated by counting the pixels of the area on a digitized REC image. Capillary areas were estimated from the digitized images at 1, 2, and 5 wk. Capillary density at 5 wk was calculated by dividing the capillary area by the chamber area.
Completion of vascularization in the REC was defined as complete filling of the observation field of the REC with capillaries and newly formed tissue without bleeding, effusion, infection, or tissue detachment. The percentage of rabbits with complete vascularization was analyzed with the 
2 test with Bonferroni correction for multiple comparison. One-way analysis of variance and Tukey-Kramer multiple comparisons were used to determine the statistical significance of differences in vascularization area within groups and between groups. A two-tailed 5% difference was considered significant. The results are expressed as means ± sd.
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Results
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Complete vascularization in the REC was observed at week 5 in 10 of 13 rabbits (77%) in group B1, 7 of 16 (44%) in group B2, and 4 of 13 (31%) in the control group. The percentage of rabbits with complete vascularization was significantly larger in group B1 than in control (P < 0.05). Thereafter, we focused on rabbits with complete vascularization at 5 wk.
Figure 1 shows typical vascular findings of wound healing in the RECs in the three groups. Capillary infiltration was observed at 1 wk in groups B1 and B2.
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Figure 1. Chronological changes of the rabbit ear chamber to week 5. Values were determined at 1-wk intervals for rabbits in group B1 (a), group B2 (b) and control (c). Bar: 1 mm.
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At 2 wk, new vessels were formed throughout the chamber in group B1 with a massive area of bleeding; group B2 was characterized by a circular area of dense bleeding. In the control group, capillary infiltration was initially sporadic; vessel formation was irregular at this stage. The time to completion of vascularization was 3 wk in group B1, as compared with 4 wk in group B2 and the control group.
At 1 wk in group B1, the connective tissue surrounding the chamber was clear without hematoma, as shown in Figure 2. Many branching capillaries extended from disrupted vessels. In the observation window, fine capillaries extended and infiltrated from the surrounding tissue. In the controls, the connective tissue surrounding the chamber was opaque because of residual hematoma, with few capillaries (Fig. 2).
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Figure 2. Magnified images of rabbit ear chambers at 1 wk for a group B1 rabbit (a) and a control rabbit (b). In group B1, branching capillaries extending from disrupted vessels, set against a clear background, can be seen in the connective tissue surrounding the chamber. Fine capillaries extend and infiltrate into the observation window (a). In the controls, the surrounding tissue was opaque, with few capillaries (b). Bar: 1 mm.
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At 1 wk, the newly vascularized area was significantly larger in groups B1 (10.68 ± 3.11 mm2) and B2 (11.08 ± 2.66 mm2) than in control (4.43 ± 2.04 mm2) (both P < 0.01). The difference between groups B1 and B2 was not significant (Fig. 3). At 2 wk, all 3 groups had similar vascularized areas (22.28 ± 5.77, 21.71 ± 3.74, and 15.18 ± 6.09 mm2) (Fig. 3).
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Figure 3. Comparison of newly vascularized area at 1 wk (a) and 2 wk (b) after punch-out. The newly vascularized area was significantly greater in groups B1 and B2 than in control at 1 wk (both P < 0.01). At 2 wk, the vascularized areas were similar in groups B1, B2, and control.
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Capillary density at 5 wk was 11.23% ± 3.00% in group B1, 10.95% ± 4.03% in group B2, and 11.35% ± 2.80% in the control group. These differences were not significant (Fig. 4).
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Discussion
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The beneficial effects of a single application of exogenous bFGF, as compared with multiple applications (12) or sustained release (13), remains a matter of debate. The optimal method of application, such as intraoperative intramyocardium injection (5), intracoronary injection through a catheter (6), intraarterial infusion to limbs (7), or direct application to target sites (12), is also controversial. Our study showed that a single local application of rhbFGF had discernible effects on wound healing and angiogenesis for a limited period of time. Our results suggest that rhbFGF sprayed in a liquid form remained at the wet wound and initially promoted angiogenesis; subsequently, a natural healing process ensued. Consequently, the final vascular density in the rhbFGF-treated groups was similar to that of the control group and treatment was not associated with an abnormal increase in capillaries. Our findings imply that the demonstration of bFGF-induced therapeutic angiogenesis requires the establishment of an optimal application technique, dose, and dosage form, as well as measurement of the rate of angiogenesis.
There was no significant difference in wound healing between groups B1 and B2 at 1 week, although some animals in group B2 had capillary bleeding. Such bleeding might have been caused by weak walls of newly formed capillaries, over-acceleration of angiogenesis, or other factors. Capillary bleeding in group B2 was associated with delayed angiogenesis and a slow rate of complete vascularization. In a previous study using a REC model and a cotton-type collagen hemostat as a scaffold, well-developed, newly formed capillaries showing accelerated angiogenesis without bleeding were observed (14). Angiogenesis is influenced by many factors, including cells, extracellular matrix, and cytokines, as well as the combination and balance of these factors.
One week after bFGF treatment, hematoma had regressed completely and branching capillaries extending from disrupted vessels were observed against a clear background of connective tissue. In contrast, hematoma persisted in the controls. During angiogenesis, erythrocytes are probably removed through vessels in connective tissue because blood cells may slip into vessels during healing (15). Perhaps the rate of angiogenesis in the controls was slower than that in the groups applied exogenous bFGF, resulting in the persistence of opaque hematoma and blood cells in connective tissue.
Topical use of bFGF accelerates wound healing in association with fibroblast mitosis and angiogenesis. Experimentally, purified bFGF accelerates healing in a duodenal ulcer model (16). In our study, early angiogenesis after injury was accelerated in group B1, without infection, suggesting that rhbFGF promoted wound healing. This is supported by a more rapid rate of complete vascularization in group B1 than in the control group at 5 weeks.
The species-specificity of bFGF is probably not strong, as rhbFGF promoted vascularization in rabbits in our study. Other studies have shown that bovine bFGF applied to human epidermis is effective for the treatment of second-degree burns (12). Exogenous bFGF may act for only a limited time. In the present study, local application of a single dose significantly promoted angiogenesis at 1 week, and at 2 weeks the vasculature of the treated animals was nearly indistinguishable from that of the untreated controls.
Angiogenic studies should include quantitative assays of newly formed vessels and provide direct proof of neovascularization in response to treatment (17). The process of neovascularization from sprouts to established vessels should be clearly demonstrated in such studies because sprout formation can be observed with the use of properly selected animal models and equipment. However, neovascularization has only been indirectly investigated. In cardiology, for example, improved cardiac performance, reduced infarct size, angiographically visible collateral vessels, increased density of cardiac capillaries on histological examination, and color analysis on thermographic examination provide indirect evidence of angiogenesis.
Animal models using invasive monitoring systems have been used for angiogenic research. These models may not permit accurate evaluation of the effect of exogenous bFGF because biophylaxis occurs in conditions such as severe ischemia of the heart and limbs. Moreover, the effects of exogenously applied cytokines may be difficult to discern in ischemic models. Endogenous bFGF is produced from endothelial and other cells in response to stimulation caused by conditions such as injury (18). Studies using a rat gastric ulceration model have shown that endogenous bFGF produced from omentum is as effective as exogenous bFGF (19). In a rabbit ischemic limb model, exogenous bFGF was injected IM in ischemic limbs (20); hence the target lesion was subjected to the stress of severe ischemia caused by ligating the leg vessels as well as the stress caused by IM injection. Such factors represent important limitations that may bias the results of studies attempting to assess the therapeutic activity of exogenous bFGF in animal models. The REC wound healing model that we used is relatively noninvasive and thereby places minimal stress on the experimental animals. We believe that it allowed the effect of exogenous bFGF to be evaluated even in the presence of endogenous bFGF.
In conclusion, our study showed that a single local application of rhFGF accelerated angiogenesis associated with wound healing in a punched-out area of the rabbit ear. This angiogenesis-promoting effect was transiently seen during early angiogenesis, and there was no increase in capillary density at the completion of vascularization. Angiogenesis is a delicate phenomenon that is influenced by environmental factors, model selection, and exogenous factors.
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Footnotes
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Supported, in part, by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology (#15591661).
Accepted for publication August 23, 2004.
Author correspondence and reprint requests to Makiko Komori, MD, Department of Anesthesiology, School of Medicine, Tokyo Women’s Medical University, 8–1, Kawada-cho, Shinjuku-ku, Tokyo, 162–8666, Japan. Address e-mail to komorim{at}jj.openbit.net.
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Abstract
Introduction
