Sustained-release form of basic fibroblast growth factor prevents catheter-related bacterial invasion in mice
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《血管的通路杂志》
a Department of Cardiovascular Surgery, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawara-cho, Sakyo-ku Kyoto, 606-8507, Japan
b Department of Laboratory Science, Kyoto University Graduate School of Medicine, Kyoto, Japan
c Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto, Japan
d The Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan
Abstract
Catheter-related infection is a frequent and serious complication. One factor responsible for catheter-related infection is bacterial invasion at the catheter-insertion site. We have shown that the sustained-release form of basic fibroblast growth factor (bFGF) enhances tissue regeneration and angiogenesis in various pathological conditions. The purpose of this study was to determine whether topical use of sustained-release form of bFGF promotes tissue regeneration around the wound and prevents catheter-related bacterial invasion. Fifty-four male mice (C57BL/6) were divided into three groups according to what was implanted subcutaneously on the back (each group, n=18): a Dacron sheet alone (group A), a Dacron sheet and a plain gelatin sheet (group B), and a Dacron sheet and sustained-release of bFGF (50 μg) (group C). Seven days after the implantation, the tissue immediately above the Dacron sheet was inoculated with methicillin-resistant Staphylococcus aureus (MRSA). In histological examinations, group C had a larger granulation tissue area containing a larger amount of collagen tissue and vessels than the other groups. Two days after the MRSA inoculation, the number of MRSA in the Dacron sheet of group C was significantly smaller than the other groups (P<0.01). Pretreatment with sustained-release form of bFGF may prevent catheter-related bacterial invasion.
Key Words: Tissue regeneration; Growth factor; Sustained-release form of bFGF; Catheter-related infection
1. Introduction
Long-term insertion of many catheters for critically ill patients is accompanied by the risk of infection. Once patients develop catheter-related infection, it becomes difficult to treat, which often leads to death or a prolonged hospital stay [1]. The incidence of complications by the infection was reported to be as high as 47% for 123 patients with an indwelling left ventricular assist device (LVAD) cable [2].
Three factors are reported to be responsible for catheter-related infection [3]. One factor pertains to manipulation of catheter hubs at the time of intravenous injection. If the manipulation is not aseptic, bacteria may invade the catheter. Second, microbes hematogenously seed catheters from distant infection sites (e.g. pneumonia, urinary tract infection), leading to catheter-related infection. The third factor reported is bacterial invasion via the wound around the catheter-inserted site of skin. If bacterial invasion from outside at the catheter-insertion site can be minimized, it leads to prevention of catheter-related infection. Among these factors responsible for catheter-related infection, in the present study, we have focused on the third factor, bacterial invasion from outside at the catheter-insertion site.
Basic FGF is a potent mitogen regulating protein that induces angiogenesis [4] and promotes the growth and regeneration of organs and tissues in vivo [5]. We have previously demonstrated the effectiveness of the sustained-release form of bFGF in various pathological conditions [6–9].
We hypothesized that the earlier wound healing at the catheter-insertion site can block catheter-related bacterial invasion mechanically. Thus, the purpose of the present study was to evaluate the effects of topical use of sustained-release form of bFGF on the tissue regeneration and prevention from bacterial invasion.
2. Material and methods
2.1. Preparation of bFGF-incorporating gelatin hydrogel sheet
Gelatin with an isoelectric point of 4.9 was isolated from bovine bone collagen by an alkaline process with Ca(OH)2 (Nitta Gelatin Co., Osaka, Japan). Human recombinant bFGF with an isoelectric point of 9.6 was supplied by Kaken Pharmaceutical Co. (Tokyo, Japan). Gelatin hydrogel sheets were made by the same process as described previously [10]. Sheets were freeze-dried, followed by impregnation with an aqueous solution of bFGF. The prepared hydrogel sheets were square (10x10 mm) and 0.7 mm thick. All experimental processes were conducted under sterile conditions.
2.2. Animal experiment (Dacron sheet implantation)
Fifty-four male mice (C57BL/6) weighing approximately 20 g were anesthetized with sodium pentobarbital (100 μg/g, intraperitoneally). After a 10-mm horizontal incision in the center of the back, with the mouse in the prone position, the subcutaneous tissue was exposed, and a Dacron sheet (10x10 mm), serving as a test sheet was implanted. They were randomly divided into three groups (each group, n=18): a Dacron sheet alone (group A), a Dacron sheet and a plain gelatin hydrogel sheet (group B), and a Dacron sheet and a gelatin hydrogel sheet incorporating bFGF (50 μg) (group C). The sheets were immobilized with 6-0 polypropylene sutures. The skin was carefully sutured with 6-0 nylon monofilaments, and the skin immediately above the implanted sheets was marked with suture. All animals were treated according to the ‘Guide for the Use and Care of Laboratory Animals’, published by the National Institutes of Health (NIH publication no. 85–23, revised 1985). All of the assessments were done by the investigators who were unknown to the grouping of mice.
2.3. Histological examination of tissue regeneration
Seven days after the implantation of the sheets, eighteen mice were euthanized by intravenous administration of a lethal dose of sodium pentobarbital (each group, n=6). The tissues around the implanted Dacron sheet with skin, subcutaneous tissue, muscle fiber and the Dacron sheet were then sampled and fixed with 10% formaldehyde solution in PBS solution. The transmural sections were stained with Masson trichrome and hematoxylin-eosin for the histological examination. The ratio of granulation tissue area between the Dacron sheet and muscle fiber of total area measured with image analysis software (Scion Image Beta 4.02 Win, Scion Corporation, Frederick, MD) in each of the samples stained with Masson trichrome was calculated. Also five fields were chosen randomly in each of the samples stained with hematoxylin-eosin and the number of capillaries and arterioles (10 to 50 μm in diameter) per unit area (200x200 μm2) was also counted.
2.4. MRSA inoculation
To examine the level of bacterial colonization on transplanted materials, the number of MRSA in the Dacron sheet was counted 2 days after the MRSA inoculation. Seven days after the implantation, thirty-six mice were anesthetized in the same way as described previously and the subcutaneous tissue at the marked site (immediately above the implanted sheets) was inoculated with MRSA (SR3737; 1x106 colony forming units; CFU) (each group, n=12). Two days after the MRSA inoculation, mice were euthanized in the same way as described previously and the Dacron sheet was withdrawn from the subcutaneous tissue.
2.5. Evaluation of bacterial colonization (CFU assay)
The withdrawn Dacron sheets were homogenized with 10 ml of PBS at 15,000 rpm for 5 min by a blender (Ace Homogenizer AM-7 Nihonheiki Kaisha, Ltd, Tokyo, Japan). The homogenates were diluted serially by 10-fold and 0.1-ml samples were plated on normal agar plates (Eiken Chemical Co., Ltd, Tokyo, Japan) and then incubated at 37 °C for 18 h. The number of colonies formed on agar plates was enumerated, and CFU in the original sample was calculated.
2.6. Statistical analysis
Experimental results are expressed as the mean±standard deviation. In multiple comparisons among independent groups in which ANOVA indicated significant differences, the statistical value was determined according to the Bonferroni/Dunn's method. Differences between groups were determined using Student's t-test. All statistical analyses were performed by StatviewTM software (Abacus). A P-value <0.05 was considered significant.
3. Results
There was no death in mice due to surgical manipulation. We found neither disorders, nor complications attributable to the local use of sustained-release form of bFGF.
3.1. Histological assessment of regenerative tissue
Fig. 1 shows histologic sections (stained with Masson trichrome, 20xmagnification) of the tissue around the Dacron sheet 7 days after the implantation. Groups A and B had small granulation tissue area between the Dacron sheet and muscle fiber (Fig. 1A,B). In contrast, group C treated with the sustained-release form of bFGF had larger granulation tissue area containing many collagen tissues and vessels (Fig. 1C). Left panel of Fig. 2 shows the ratio of granulation tissue area to the total area. The ratio of granulation tissue area in group C was significantly higher than the other groups (8.2±2.6%, 9.1±2.4%, and 24.1±1.7% for groups A, B, and C, respectively; P<0.01).
3.2. Histological assessment of angiogenesis
The right panel of Fig. 2 shows the number of blood vessels per unit area, calculated on hematoxylin-eosin stained tissue around the Dacron sheet. The number of both the arterioles and capillaries (10 to 50 μm in diameter) per unit area was increased to a greater extent in group C than in the other groups (13.1±2.1, 12.9±2.6, and 46.2±3.3 vessels per unit area for groups A, B, and C, respectively; P<0.01).
3.3. The number of MRSA in implanted Dacron sheet (CFU assay)
Fig. 3 shows the number of MRSA in the withdrawn Dacron sheet 2 days after the MRSA inoculation (1x106 CFU). The number of MRSA in group C was significantly lower than the other groups ((1.22±0.74)x106, (1.04±0.72)x106, and (1.80±0.34)x105 CFU for groups A, B, and C, respectively; P<0.01). These results suggest that bacterial invasion into the Dacron sheet decreased by approximately 85% in group C treated with the sustained-release form of bFGF compared with control group.
4. Discussion
In the current study, we have demonstrated that a remarkable tissue regeneration took place by using sustained-release form of bFGF in a murine subcutaneously. We also showed that pretreatment with the sustained-release form of bFGF is useful for the prevention of bacterial invasion.
A factor responsible for catheter-related infection is bacterial invasion via the wound around the catheter-inserted site of skin. At the catheter insertion site, bacterial invasion occurs through the space between the skin or subcutaneous tissue and the catheter [3] (Fig. 4a). Our hypothesis was that, if the space between the skin or subcutaneous tissue and the catheter is filled with regenerated tissue especially in a vascular-rich one, bacterial invasion through the space can be minimized, leading to prevention of catheter-related infections (Fig. 4b). Once this wound is completely healed, the risk of catheter-related bacterial invasion should decrease considerably. A novel experimental model is proposed to reproduce this effect. Animals are subcutaneously implanted with a test sheet. Some time later, the skin immediately above the implanted test sheet was inoculated with bacteria (Fig. 5a). The inoculated bacteria invade the test sheet which is a ‘foreign material’. If the tissue is regenerated around the test sheet, bacteria cannot invade the test sheet (Fig. 5b). Although it is hard to evaluate catheter-related bacterial invasion quantitatively under the condition in which a catheter passes through the skin, it is easy to evaluate bacterial invasion in our model. Thus, this model was employed in this study.
MRSA was used as the test bacterium. MRSA is weakly toxic but once its infection occurs in critically ill patients, severe symptoms which are difficult to treat may ensue. MRSA is also known to be one of the major pathogens of catheter-related infections [11]. As an index of bacterial invasion, the number of MRSA cells in the implanted Dacron sheet was counted. In the present study, the number of MRSA in the Dacron sheet decreased to about 15% in mice treated with the sustained-release form of bFGF compared with control group.
It is tempting to speculate on the underlying mechanisms as to why sustained-release form of bFGF was effective for prevention of bacterial invasion in the present study. Both tissue regenerative effects and angiogenetic actions of bFGF might account for the bacterial effects. Since much of the granulation tissue is found microscopically around the Dacron sheet of the treatment arm, tissue regeneration process may occur sufficiently with the sustained-release form of bFGF. We assume that the main beneficial effect of the sustained-release form of bFGF is tissue regenerative action with a mechanical block against bacterial invasion. On the other hand, large numbers of capillaries and arterioles were noted in the mice treated with the sustained-release form of bFGF. Thus, the use of the sustained-release form of bFGF stimulated active neovascularization. Adequate blood supply to tissue from newly formed blood vessels is expected to faciliate immunocompetent cells and some other cells to gather in the local area, leading to suppression of bacterial proliferation. This idea is based on the fact that scalp wounds are free from wound infection because of adequate blood supply. It was reported that skull-mounted pedestals of driveline are free from infection or healing problems [12].
There are some limitations to the current study. First, the environments for catheter-related infection, which catheters pass through the skin in the clinical setting, may be different from the experimental model reported here. Second, we used mice as our experimental model, which may be stronger against the infection than humans. Further investigation with a larger animal model that has similar anatomical, physiological and immunological features to human patients is necessary.
In conclusion, the results reported here suggest that the use of sustained-release form of bFGF in the catheter-insertion site may facilitate tissue regeneration and thus may block the route of bacterial invasion. This method may provide a useful means of preventing catheter-related infection caused by bacterial invasion from outside without systemic effects. We are now in the process of making an application for clinical trials.
Acknowledgements
The authors would like to express their sincere appreciation to Dr Shigeru Amano for the histological study, Mr Yu Kimura, and Ms Shiho Yuzawa for the animal surgery.
References
Rello J, Ochagavia A, Sabanes E, Roque M, Mariscal D, Reynaga E, Valles J. Evaluation of outcome of intravenous catheter-related infection in critically ill patients. Am J Respir Crit Care Med 2000;162:1027–1030.
Poston RS, Husain S, Sorce D, Stanford E, Kusne S, Wagener M, Griffith BP, Kormos RL. LVAD Bloodstream infections; therapeutic rationale for transplantation after LVAD infection. J Heart Lung Transplant 2004;22:914–921.
Mermel LA. Prevention of intravascular catheter-related infections. Ann Intern Med 2000;132:391–402.
YanagisawaMiwa A, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C. Salvage of infracted myocardium by angiogenic action of basic fibroblast growth factor. Science 1992;257:1401–1403.
Rafkin DB, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 1989;109:1–16.
Iwakura A, Tabata Y, Miyao M, Ozeki M, Tamura N, Ikai A, Nishimura K, Nakamura T, Shimizu Y, Fujita M, Komeda M. Novel methods to enhance sternal healing after harvesting bilateral internal thoracic arteries with use of basic fibloblast growth factor. Circulation 2000;102:III-307–311.
Yamamoto T, Suto N, Okubo T, Mikuniya A, Hanada H, Yagihashi S, Fujita M, Okumura K. Intramyocardial delivery of basic fibloblast growth factor-impregnated gelatin hydrogel microspheres enhances collateral circulation to infracted canine myocardium. Jpn Circ J 2001;65:439–444.
Sakakibara Y, Nishimura K, Tambara K, Yamamoto M, Lu F, Tabata Y, Komeda M. Prevascularization with gelatin microspheres containing basic fibloblast growth factor enhances the benefits of cardiomyocyte transplantation. J Thorac Cardiovasc Surg 2002;124:50–56.
Ueyama K, Bing G, Tabata Y, Ozeki M, Doi K, Nishimura K, Suma H, Komeda M. Development of biologic coronary artery bypass grafting in a rabit model: revival of a classic concept with modern biotechnology. J Thorac Cardiovasc Surg 2004;127:1608–1615.
Tabata Y, Nagano A, Ikada Y. Biodegradation of hydrogel carrier incorporating fibloblast growth factor. Tissue Eng 1999;5:127–138.
Ferretti G, Mandala M, Di Cosimo S, Moro C, Curigliano G, Barni S. Catheter-related bloodstream infections, part II: specific pathogens and prevention. Cancer Control 2003;10:79–91.
Jarvik R, Westaby S, Katsumata T, Pigott D, Evans RD. LVAD power delivery: a percutaneous approach to avoid infection. Ann Thorac Surg 1998;65:470–473.(Keiichi Hirose, Akira Mar)
b Department of Laboratory Science, Kyoto University Graduate School of Medicine, Kyoto, Japan
c Department of Microbiology, Kyoto University Graduate School of Medicine, Kyoto, Japan
d The Institute for Frontier Medical Science, Kyoto University, Kyoto, Japan
Abstract
Catheter-related infection is a frequent and serious complication. One factor responsible for catheter-related infection is bacterial invasion at the catheter-insertion site. We have shown that the sustained-release form of basic fibroblast growth factor (bFGF) enhances tissue regeneration and angiogenesis in various pathological conditions. The purpose of this study was to determine whether topical use of sustained-release form of bFGF promotes tissue regeneration around the wound and prevents catheter-related bacterial invasion. Fifty-four male mice (C57BL/6) were divided into three groups according to what was implanted subcutaneously on the back (each group, n=18): a Dacron sheet alone (group A), a Dacron sheet and a plain gelatin sheet (group B), and a Dacron sheet and sustained-release of bFGF (50 μg) (group C). Seven days after the implantation, the tissue immediately above the Dacron sheet was inoculated with methicillin-resistant Staphylococcus aureus (MRSA). In histological examinations, group C had a larger granulation tissue area containing a larger amount of collagen tissue and vessels than the other groups. Two days after the MRSA inoculation, the number of MRSA in the Dacron sheet of group C was significantly smaller than the other groups (P<0.01). Pretreatment with sustained-release form of bFGF may prevent catheter-related bacterial invasion.
Key Words: Tissue regeneration; Growth factor; Sustained-release form of bFGF; Catheter-related infection
1. Introduction
Long-term insertion of many catheters for critically ill patients is accompanied by the risk of infection. Once patients develop catheter-related infection, it becomes difficult to treat, which often leads to death or a prolonged hospital stay [1]. The incidence of complications by the infection was reported to be as high as 47% for 123 patients with an indwelling left ventricular assist device (LVAD) cable [2].
Three factors are reported to be responsible for catheter-related infection [3]. One factor pertains to manipulation of catheter hubs at the time of intravenous injection. If the manipulation is not aseptic, bacteria may invade the catheter. Second, microbes hematogenously seed catheters from distant infection sites (e.g. pneumonia, urinary tract infection), leading to catheter-related infection. The third factor reported is bacterial invasion via the wound around the catheter-inserted site of skin. If bacterial invasion from outside at the catheter-insertion site can be minimized, it leads to prevention of catheter-related infection. Among these factors responsible for catheter-related infection, in the present study, we have focused on the third factor, bacterial invasion from outside at the catheter-insertion site.
Basic FGF is a potent mitogen regulating protein that induces angiogenesis [4] and promotes the growth and regeneration of organs and tissues in vivo [5]. We have previously demonstrated the effectiveness of the sustained-release form of bFGF in various pathological conditions [6–9].
We hypothesized that the earlier wound healing at the catheter-insertion site can block catheter-related bacterial invasion mechanically. Thus, the purpose of the present study was to evaluate the effects of topical use of sustained-release form of bFGF on the tissue regeneration and prevention from bacterial invasion.
2. Material and methods
2.1. Preparation of bFGF-incorporating gelatin hydrogel sheet
Gelatin with an isoelectric point of 4.9 was isolated from bovine bone collagen by an alkaline process with Ca(OH)2 (Nitta Gelatin Co., Osaka, Japan). Human recombinant bFGF with an isoelectric point of 9.6 was supplied by Kaken Pharmaceutical Co. (Tokyo, Japan). Gelatin hydrogel sheets were made by the same process as described previously [10]. Sheets were freeze-dried, followed by impregnation with an aqueous solution of bFGF. The prepared hydrogel sheets were square (10x10 mm) and 0.7 mm thick. All experimental processes were conducted under sterile conditions.
2.2. Animal experiment (Dacron sheet implantation)
Fifty-four male mice (C57BL/6) weighing approximately 20 g were anesthetized with sodium pentobarbital (100 μg/g, intraperitoneally). After a 10-mm horizontal incision in the center of the back, with the mouse in the prone position, the subcutaneous tissue was exposed, and a Dacron sheet (10x10 mm), serving as a test sheet was implanted. They were randomly divided into three groups (each group, n=18): a Dacron sheet alone (group A), a Dacron sheet and a plain gelatin hydrogel sheet (group B), and a Dacron sheet and a gelatin hydrogel sheet incorporating bFGF (50 μg) (group C). The sheets were immobilized with 6-0 polypropylene sutures. The skin was carefully sutured with 6-0 nylon monofilaments, and the skin immediately above the implanted sheets was marked with suture. All animals were treated according to the ‘Guide for the Use and Care of Laboratory Animals’, published by the National Institutes of Health (NIH publication no. 85–23, revised 1985). All of the assessments were done by the investigators who were unknown to the grouping of mice.
2.3. Histological examination of tissue regeneration
Seven days after the implantation of the sheets, eighteen mice were euthanized by intravenous administration of a lethal dose of sodium pentobarbital (each group, n=6). The tissues around the implanted Dacron sheet with skin, subcutaneous tissue, muscle fiber and the Dacron sheet were then sampled and fixed with 10% formaldehyde solution in PBS solution. The transmural sections were stained with Masson trichrome and hematoxylin-eosin for the histological examination. The ratio of granulation tissue area between the Dacron sheet and muscle fiber of total area measured with image analysis software (Scion Image Beta 4.02 Win, Scion Corporation, Frederick, MD) in each of the samples stained with Masson trichrome was calculated. Also five fields were chosen randomly in each of the samples stained with hematoxylin-eosin and the number of capillaries and arterioles (10 to 50 μm in diameter) per unit area (200x200 μm2) was also counted.
2.4. MRSA inoculation
To examine the level of bacterial colonization on transplanted materials, the number of MRSA in the Dacron sheet was counted 2 days after the MRSA inoculation. Seven days after the implantation, thirty-six mice were anesthetized in the same way as described previously and the subcutaneous tissue at the marked site (immediately above the implanted sheets) was inoculated with MRSA (SR3737; 1x106 colony forming units; CFU) (each group, n=12). Two days after the MRSA inoculation, mice were euthanized in the same way as described previously and the Dacron sheet was withdrawn from the subcutaneous tissue.
2.5. Evaluation of bacterial colonization (CFU assay)
The withdrawn Dacron sheets were homogenized with 10 ml of PBS at 15,000 rpm for 5 min by a blender (Ace Homogenizer AM-7 Nihonheiki Kaisha, Ltd, Tokyo, Japan). The homogenates were diluted serially by 10-fold and 0.1-ml samples were plated on normal agar plates (Eiken Chemical Co., Ltd, Tokyo, Japan) and then incubated at 37 °C for 18 h. The number of colonies formed on agar plates was enumerated, and CFU in the original sample was calculated.
2.6. Statistical analysis
Experimental results are expressed as the mean±standard deviation. In multiple comparisons among independent groups in which ANOVA indicated significant differences, the statistical value was determined according to the Bonferroni/Dunn's method. Differences between groups were determined using Student's t-test. All statistical analyses were performed by StatviewTM software (Abacus). A P-value <0.05 was considered significant.
3. Results
There was no death in mice due to surgical manipulation. We found neither disorders, nor complications attributable to the local use of sustained-release form of bFGF.
3.1. Histological assessment of regenerative tissue
Fig. 1 shows histologic sections (stained with Masson trichrome, 20xmagnification) of the tissue around the Dacron sheet 7 days after the implantation. Groups A and B had small granulation tissue area between the Dacron sheet and muscle fiber (Fig. 1A,B). In contrast, group C treated with the sustained-release form of bFGF had larger granulation tissue area containing many collagen tissues and vessels (Fig. 1C). Left panel of Fig. 2 shows the ratio of granulation tissue area to the total area. The ratio of granulation tissue area in group C was significantly higher than the other groups (8.2±2.6%, 9.1±2.4%, and 24.1±1.7% for groups A, B, and C, respectively; P<0.01).
3.2. Histological assessment of angiogenesis
The right panel of Fig. 2 shows the number of blood vessels per unit area, calculated on hematoxylin-eosin stained tissue around the Dacron sheet. The number of both the arterioles and capillaries (10 to 50 μm in diameter) per unit area was increased to a greater extent in group C than in the other groups (13.1±2.1, 12.9±2.6, and 46.2±3.3 vessels per unit area for groups A, B, and C, respectively; P<0.01).
3.3. The number of MRSA in implanted Dacron sheet (CFU assay)
Fig. 3 shows the number of MRSA in the withdrawn Dacron sheet 2 days after the MRSA inoculation (1x106 CFU). The number of MRSA in group C was significantly lower than the other groups ((1.22±0.74)x106, (1.04±0.72)x106, and (1.80±0.34)x105 CFU for groups A, B, and C, respectively; P<0.01). These results suggest that bacterial invasion into the Dacron sheet decreased by approximately 85% in group C treated with the sustained-release form of bFGF compared with control group.
4. Discussion
In the current study, we have demonstrated that a remarkable tissue regeneration took place by using sustained-release form of bFGF in a murine subcutaneously. We also showed that pretreatment with the sustained-release form of bFGF is useful for the prevention of bacterial invasion.
A factor responsible for catheter-related infection is bacterial invasion via the wound around the catheter-inserted site of skin. At the catheter insertion site, bacterial invasion occurs through the space between the skin or subcutaneous tissue and the catheter [3] (Fig. 4a). Our hypothesis was that, if the space between the skin or subcutaneous tissue and the catheter is filled with regenerated tissue especially in a vascular-rich one, bacterial invasion through the space can be minimized, leading to prevention of catheter-related infections (Fig. 4b). Once this wound is completely healed, the risk of catheter-related bacterial invasion should decrease considerably. A novel experimental model is proposed to reproduce this effect. Animals are subcutaneously implanted with a test sheet. Some time later, the skin immediately above the implanted test sheet was inoculated with bacteria (Fig. 5a). The inoculated bacteria invade the test sheet which is a ‘foreign material’. If the tissue is regenerated around the test sheet, bacteria cannot invade the test sheet (Fig. 5b). Although it is hard to evaluate catheter-related bacterial invasion quantitatively under the condition in which a catheter passes through the skin, it is easy to evaluate bacterial invasion in our model. Thus, this model was employed in this study.
MRSA was used as the test bacterium. MRSA is weakly toxic but once its infection occurs in critically ill patients, severe symptoms which are difficult to treat may ensue. MRSA is also known to be one of the major pathogens of catheter-related infections [11]. As an index of bacterial invasion, the number of MRSA cells in the implanted Dacron sheet was counted. In the present study, the number of MRSA in the Dacron sheet decreased to about 15% in mice treated with the sustained-release form of bFGF compared with control group.
It is tempting to speculate on the underlying mechanisms as to why sustained-release form of bFGF was effective for prevention of bacterial invasion in the present study. Both tissue regenerative effects and angiogenetic actions of bFGF might account for the bacterial effects. Since much of the granulation tissue is found microscopically around the Dacron sheet of the treatment arm, tissue regeneration process may occur sufficiently with the sustained-release form of bFGF. We assume that the main beneficial effect of the sustained-release form of bFGF is tissue regenerative action with a mechanical block against bacterial invasion. On the other hand, large numbers of capillaries and arterioles were noted in the mice treated with the sustained-release form of bFGF. Thus, the use of the sustained-release form of bFGF stimulated active neovascularization. Adequate blood supply to tissue from newly formed blood vessels is expected to faciliate immunocompetent cells and some other cells to gather in the local area, leading to suppression of bacterial proliferation. This idea is based on the fact that scalp wounds are free from wound infection because of adequate blood supply. It was reported that skull-mounted pedestals of driveline are free from infection or healing problems [12].
There are some limitations to the current study. First, the environments for catheter-related infection, which catheters pass through the skin in the clinical setting, may be different from the experimental model reported here. Second, we used mice as our experimental model, which may be stronger against the infection than humans. Further investigation with a larger animal model that has similar anatomical, physiological and immunological features to human patients is necessary.
In conclusion, the results reported here suggest that the use of sustained-release form of bFGF in the catheter-insertion site may facilitate tissue regeneration and thus may block the route of bacterial invasion. This method may provide a useful means of preventing catheter-related infection caused by bacterial invasion from outside without systemic effects. We are now in the process of making an application for clinical trials.
Acknowledgements
The authors would like to express their sincere appreciation to Dr Shigeru Amano for the histological study, Mr Yu Kimura, and Ms Shiho Yuzawa for the animal surgery.
References
Rello J, Ochagavia A, Sabanes E, Roque M, Mariscal D, Reynaga E, Valles J. Evaluation of outcome of intravenous catheter-related infection in critically ill patients. Am J Respir Crit Care Med 2000;162:1027–1030.
Poston RS, Husain S, Sorce D, Stanford E, Kusne S, Wagener M, Griffith BP, Kormos RL. LVAD Bloodstream infections; therapeutic rationale for transplantation after LVAD infection. J Heart Lung Transplant 2004;22:914–921.
Mermel LA. Prevention of intravascular catheter-related infections. Ann Intern Med 2000;132:391–402.
YanagisawaMiwa A, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C. Salvage of infracted myocardium by angiogenic action of basic fibroblast growth factor. Science 1992;257:1401–1403.
Rafkin DB, Moscatelli D. Recent developments in the cell biology of basic fibroblast growth factor. J Cell Biol 1989;109:1–16.
Iwakura A, Tabata Y, Miyao M, Ozeki M, Tamura N, Ikai A, Nishimura K, Nakamura T, Shimizu Y, Fujita M, Komeda M. Novel methods to enhance sternal healing after harvesting bilateral internal thoracic arteries with use of basic fibloblast growth factor. Circulation 2000;102:III-307–311.
Yamamoto T, Suto N, Okubo T, Mikuniya A, Hanada H, Yagihashi S, Fujita M, Okumura K. Intramyocardial delivery of basic fibloblast growth factor-impregnated gelatin hydrogel microspheres enhances collateral circulation to infracted canine myocardium. Jpn Circ J 2001;65:439–444.
Sakakibara Y, Nishimura K, Tambara K, Yamamoto M, Lu F, Tabata Y, Komeda M. Prevascularization with gelatin microspheres containing basic fibloblast growth factor enhances the benefits of cardiomyocyte transplantation. J Thorac Cardiovasc Surg 2002;124:50–56.
Ueyama K, Bing G, Tabata Y, Ozeki M, Doi K, Nishimura K, Suma H, Komeda M. Development of biologic coronary artery bypass grafting in a rabit model: revival of a classic concept with modern biotechnology. J Thorac Cardiovasc Surg 2004;127:1608–1615.
Tabata Y, Nagano A, Ikada Y. Biodegradation of hydrogel carrier incorporating fibloblast growth factor. Tissue Eng 1999;5:127–138.
Ferretti G, Mandala M, Di Cosimo S, Moro C, Curigliano G, Barni S. Catheter-related bloodstream infections, part II: specific pathogens and prevention. Cancer Control 2003;10:79–91.
Jarvik R, Westaby S, Katsumata T, Pigott D, Evans RD. LVAD power delivery: a percutaneous approach to avoid infection. Ann Thorac Surg 1998;65:470–473.(Keiichi Hirose, Akira Mar)