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Keywords:

  • bFGF;
  • artificial skin;
  • collagen–gelatin sponge;
  • scaffold;
  • sustained release;
  • wound healing;
  • diabetic mice

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

The objective of this study was to evaluate the ability of a scaffold, collagen–gelatin sponge (CGS), to release basic fibroblast growth factor (bFGF) in a sustained manner, using a pressure-induced decubitus ulcer model involving genetically diabetic mice. We confirmed that CGSs impregnated with a bFGF concentration of up to 50 µg/cm2 were able to sustain the release of bFGF throughout their biodegradation. We prepared decubitus ulcers on diabetic mice. After debriding the ulcers, we implanted CGSs (diameter 8 mm) impregnated with normal saline solution (NSS) or bFGF solution (7, 14, 28 or 50 µg/cm2). At 1 and 2 weeks after implantation, the mice were sacrificed and tissue specimens were obtained. The wound area, neoepithelium length and numbers and total area of newly formed capillaries were evaluated. The CGSs impregnated with NSS became infected and degraded, whereas the CGSs impregnated with 7 or 14 µg/cm2 bFGF displayed accelerated dermis-like tissue formation and the CGSs impregnated with 14 µg/cm2 bFGF produced significant improvements in the remaining wound area, neoepithelium length and numbers and total area of newly formed capillaries compared with the NSS group. No significant difference was observed between the NSS and 50 µg/cm2 bFGF groups. CGSs impregnated with 7–14 µg/cm2 bFGF accelerated wound healing, and an excess amount of bFGF did not increase the wound-healing efficacy of the CGSs. Our CGS is a scaffold that can release positively charged growth factors such as bFGF in a sustained manner and shows promise as a scaffold for skin regeneration. Copyright © 2012 John Wiley & Sons, Ltd.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

We were involved in the development of a bilayered acellular artificial dermis (AD; Pelnac®, Gunze Co. Ltd, Kyoto, Japan) as a biodegradable scaffold, containing an upper layer composed of a silicone sheet and a lower layer made of collagen sponge, by modifying the artificial dermis proposed by Yannas et al. (1980) and Suzuki et al. (1990a). After the AD has been grafted onto a full-thickness skin defect, the collagen sponge was biodegraded and gradually replaced with regenerated dermis-like tissue within 2–3 weeks (Suzuki et al., 1990b). Basic FGF was released during the biodegradation of the CGS.

Artificial dermis has been used in clinical practice for the treatment of full-thickness skin defects caused by severe burns and tumour excision for more than 10 years. However, some problems remain to be solved. Before capillaries have infiltrated the collagen sponge, the artificial dermis is not resistant to infection (Matsuda et al., 1992). Therefore, it is difficult to apply artificial dermis to chronic ulcers, such as decubitus, diabetic and leg ulcers, because of the high probability of infection (Matsuda et al., 1988). Basic fibroblast growth factor (bFGF), which was identified in 1974 (Gospodarowicz, 1974), promotes the proliferation of fibroblasts and capillary formation and accelerates tissue regeneration (Uchi et al., 2009). In Japan, human recombinant bFGF (Fibrast Spray®, Kaken Pharmaceutical, Tokyo, Japan) has been used clinically for the treatment of chronic skin ulcers since 2001, and its clinical effectiveness has been demonstrated (Kawai et al., 2000). Recently, combination therapy involving bFGF and artificial dermis has been reported to accelerate dermis-like tissue formation (Muneuchi et al., 2005; Ito et al., 2005; Akita et al., 2008). In spite of its effectiveness, this combination therapy has not become a standard treatment because bFGF must be applied every day, as it rapidly diffuses away from the site of administration and is also inactivated quickly after its administration (Kawai et al., 2000).

To overcome these problems, we have developed a novel scaffold, collagen–gelatin sponge (CGS), containing a 10 wt% concentration of acidic gelatin that is capable of releasing positively charged growth factors, such as bFGF, for more than 10 days in vivo via the formation of ion complexes between bFGF and gelatin (Takemoto et al., 2008). Human bFGF, which has an isoelectric point (IEP) of 9.6 (Kanda et al., 2011; Artem et al., 2011; Takemoto et al., 2008; Kawai et al., 2000, 2005; Tabata et al., 1999; Muniruzzaman et al., 1998), is ionically complexed with acidic gelatin, which has an IEP of 5.0 (Muniruzzaman et al., 1998). CGS acts in the same manner as a scaffold such as AD, and the bFGF impregnated into the CGS is released during its biodegradation (Takemoto et al., 2008). In our previous study involving normal mouse skin defects, CGS impregnated with 7 µg/cm2 bFGF accelerated dermis-like tissue formation two- or three-fold compared with AD (Kanda et al., 2011). In another study, in which we created full-thickness palatal mucosa defects in beagles, CGS impregnated with 7 µg/cm2 bFGF accelerated the regeneration of the palatal mucosa, induced good levels of neovascularization and produced less wound contracture (Artem et al., 2011). We expect that CGS impregnated with bFGF will prove to be an effective treatment for full-thickness skin defects, including chronic ulcers such as diabetic foot ulcers and decubitus ulcers. In this study, we examined the optimal bFGF dosage with which to impregnate CGS and the release profile of bFGF from CGS after impregnation. Then, we investigated the effectiveness of CGS impregnated with bFGF and the optimal bFGF dosage in an impaired wound-healing model involving genetically diabetic mice with pressure-induced decubitus ulcers.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

Animals and operations

The animals were maintained at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University. The number of animals used in this study was kept to a minimum, and all possible efforts were made to reduce suffering in compliance with the protocols established by the Animal Research Committee of Kyoto University.

Preparation of CGS

We used gelatin isolated from pig dermis with an isoelectric point (IEP) of 5.0 and a molecular weight of 99 000 (Nippi Inc., Tokyo, Japan) and atelocollagen isolated from pig tendons with an IEP of 8.5 and a molecular weight of 300 000 (Nitta Gelatin Inc., Osaka, Japan). CGS was produced according to a production procedure described in Takemoto et al. (2008). CGS with a gelatin concentration of 10 wt% of the total solute was prepared by mixing 3 wt% gelatin solution with 0.3 wt% collagen solution. We then spread a thin layer of silicone paste onto a polyester mesh. Before the silicone paste had dried, the top of the CGS was attached to the silicone paste-covered polyester mesh. As the silicone paste dried, it formed a sheet that adhered to the CGS.

In vitro bFGF release study and degradation rate of CGS

We prepared CGSs of 10 × 20 mm in size and 3.0 mm in thickness. We weighed all of the CGSs (n = 10) and then placed them into 15 ml test tubes (Thermo Fisher Scientific Inc., Osaka, Japan). We prepared distilled water (DW; Otsuka Pharmaceutical, Tokyo, Japan) and distilled water solution containing bFGF (Fibrast Spray) at concentrations of 0.07, 0.14, 0.28 and 0.5 µg/µl and applied 200 µl of each bFGF solution to CGS. Thus, we prepared four bFGF groups, in which CGS was impregnated with 7, 14, 28 or 50 µg/cm2 bFGF and incubated overnight at 4 °C.

We prepared Tris–HCl buffer solution, pH 7.4, containing 4 U/ml collagenase (Collagenase Type A, Sigma-Aldrich Corp. Japan, Tokyo, Japan) and poured 5 ml collagenase solution into the test tubes to dissolve the CGSs at 37 °C. At 1, 2, 4 and 6 h after the degradation, we collected 1 ml of the solution from each of the test tubes and used it to estimate the bFGF concentrations in the four bFGF groups. After collecting the solutions, collagenase was completely removed from the test tubes and the CGSs inside the test tubes were immediately washed with distilled water to stop the enzyme reaction. The CGS were removed from the test tubes and freeze-dried for 12 h to remove any water using a freeze dryer (BRZ350WA, Advantec Toyo Kaisha Ltd, Tokyo, Japan). The CGSs were then weighed in order to calculate the degradation rate of the CGS impregnated with bFGF at each time point.

The analysis was performed using enzyme-linked immunosorbent assay (ELISA) kits (Human FGF Basic Immunoassay Kit, R&D Systems, Minneapolis, MN, USA). To estimate the concentrations of the bFGF solutions, solutions and standards were assayed in duplicate according to the manufacturer's instructions. The test wavelength of each well was set at 490 nm, using a microplate reader (MTP-450, Corona Electric Co. Ltd, Ibaragi, Japan) and compared to a reference wavelength of 650 nm. The bFGF concentrations were determined by plotting their values on a standard curve. The amount of bFGF released from the CGSs was calculated at each time point.

Pressure-induced ulcer model in diabetic mice

We prepared 40 genetically diabetic mice (9 week-old BKS.Cg + Leprdb/+ Leprdb/Jcl, Clea Japan Inc., Osaka, Japan). All mice had their backs and abdomens shaved and depilated under anaesthesia with diethyl ether (Wako Pure Chemical Industries, Osaka, Japan) and then were positioned on experimental tables. In our previous study, we developed a pressure induced ulcer model using diabetic mice and a pneumatic compressor (Kawai et al., 2005). In this study, 4 h prolonged pressure (2 h × two pressure sessions; 2 h interval between pressure sessions; 500 g/cm2) was loaded onto the area above the femoral trochanters of the mice, using a pneumatically driven compressor for 2 consecutive days (Earth Man AC-20 OL, Takagi Co. Ltd, Niigata, Japan). The air pressure was regulated with a precision regulator, providing a constant pressure level. Five days after the completion of the pressure loading, the area of necrosis was clearly demarcated (Figure 3A).

Impregnation of bFGF into CGS and the implantation of the CGS

We used CGSs of 8 mm diameter and 3 mm thickness. As for the dosage of bFGF, the recommended therapeutic dose of bFGF for chronic ulcers is 1 µg/cm2/day (Uchi et al., 2009). In a diabetic mouse study, the daily application of bFGF produced a similar bell-shaped dose–response pattern with a peak at 1 µg/cm2/day (Okumura et al., 1996). This shows that the effective dosage of bFGF is not very different between humans and diabetic mice. According to the daily bFGF dosage recommendations and a CGS release period of about 10 days, we hypothesized that the optimal bFGF dosage for impregnating the CGSs was in the range 7–14 µg/cm2 for 7 or 14 days. In this experiment, we prepared CGSs containing NSS or one of four different doses of bFGF (7, 14, 28 or 50 µg/cm2), as in the in vitro study, and then incubated them overnight at 4 °C.

The mice were anaesthetized via an intraperitoneal (i.p.) injection of 25 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL, USA) and the inhalation of diethyl ether (Wako). Five days after the completion of the pressure loading, the necrotic tissues were resected and skin defects of 8 mm in diameter were created, using a 8 mm diameter skin punch biopsy tool (Kai Industries, Gifu, Japan) and scissors (Figure 3B). CGSs impregnated with NSS or bFGF solution were implanted into the defects and sutured into the marginal skin wounds with 5–0 nylon sutures (Johnson & Johnson K.K., Tokyo, Japan) (Figure 3C). All wounds were covered with gauze which was fixed in place with adhesive tape (Alcare®, Alcare Co. Ltd, Tokyo, Japan).

Assessment of the wound area and histological assessment of neoepithelization

One and two weeks after implantation, the mice were sacrificed via inhalation of carbon dioxide. After the removal of the silicone sheets, the wounds were photographed, and the wound area was measured using the imaging analyser ImageJ software (v 1.38, National Institutes of Health, USA). The wound area is expressed as a percentage of the original wound area.

The implanted CGS and dermis-like tissue were harvested using scalpels and scissors and sectioned axially. The specimens were then fixed with 20% formalin fluid, paraffin-embedded and sliced into 4 µm thick sections before the sections were stained with haematoxylin and eosin (H&E). Using a light microscope and NIS Elements (Nikon Instruments Co., Tokyo, Japan), the neoepithelium length of each specimen was measured from the innermost hair root of the marginal skin to the end of the neoepithelium on each side of each cross-section at a magnification of × 100.

Immunohistological staining and evaluation of the area and number of newly formed capillaries

Using 4 µm thick paraffin-embedded sections, immunohistological staining with von Willebrand factor was performed to detect newly formed capillaries in the CGSs. After the sections had been dewaxed and rehydrated, they were incubated in phosphate-buffered saline (PBS) with 0.1% trypsin (Vector Laboratories Inc., Burlingame, CA, USA) for 15 min at 37 °C for antigen retrieval. Anti-von Willebrand factor rabbit polyclonal antibody (Dako, Tokyo, Japan) was used as the primary antibody (1:500 dilutions), and EnVision+ Rabbit/HRP (Dako) was used as the secondary antibody. These sections were exposed to DAB (3,3′-diaminobenzidine–4HCl; Dako) for 2 min at room temperature. Counterstaining was performed with haematoxylin.

Digital light micrographs of the sections of the CGSs and dermis-like tissue beyond the muscle layers were taken at a magnification of × 100. In each section, two square areas of 500 µm width and height were chosen from the dermis-like tissues beneath the marginal skin. The area and number of newly formed capillaries in two squares in each section were measured twice. Using a microscope, we measured the epithelium length directly, using NIS Elements. An assessment of neoepithelization and newly formed capillaries was also performed using NIS Elements.

Statistical analysis

All data were analysed using Fisher's protected least significant difference test (Fisher's PLSD) and expressed as mean + standard error (SE); p < 0.05 was accepted as significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

The degradation of CGS and basic FGF release in vitro

The degradation rates of the CGS are shown as percentages compared to their original weight or their collagenase-treated weight (Figure 1). The CGSs were digested by collagenase throughout the degradation period. There were significant differences in the degradation rate at 1, 2 and 4 h after the start of the degradation between the CGS impregnated with 50 µg/cm2 bFGF and those containing 7 or 14 µg/cm2 bFGF, but no significant differences were seen at 6 h after the start of the degradation. The time course of bFGF release from the CGS is shown in Figure 2. No initial burst of bFGF release from CGS was observed; rather, the bFGF was continuously released throughout the degradation of the CGS (Figure 2).

image

Figure 1. Time course of the degradation of CGS by collagenase. CGS were impregnated with 7 µg/cm2 (□), 14 µg/cm2 (×) 28 µg/cm2 (○) or 50 µg/cm2 bFGF (▽); *p < 0.05, **p < 0.01 vs 7 µg/cm2 bFGF; #p < 0.01 vs 14 µg/cm2 bFGF; $p < 0.05 vs 28 µg/cm2 bFGF

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image

Figure 2. Time course of bFGF release from CGS. CGS were impregnated with 7 µg/cm2 (□), 14 µg/cm2 (×), 28 µg/cm2 (○) or 50 µg/cm2 bFGF (▽); *p < 0.05, **p < 0.01 vs 7 µg/cm2 bFGF; #p < 0.05, ##p < 0.01 vs 14 µg/cm2 bFGF; $p < 0.05, $$p < 0.01 vs 28 µg/cm2 bFGF

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Figure 3. (A) Gross appearance of the decubitus ulcers 5 days after the completion of pressure loading. (B) The necrotic tissue was resected. (C) The CGS were implanted into the defect and sutured in place

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Wound area

The gross appearance of the wounds at 1 and 2 weeks after implantation is shown in Figures 4 and 5. One week after implantation, the CGSs in the NSS group were infected, whereas dermis-like tissue had begun to form in the wounds treated with the bFGF impregnated CGS (Figure 4). Two weeks after the implantation, the CGS in the NSS group displayed implantation failure. In contrast, the wound areas covered with CGS containing 7 or 14 µg/cm2 bFGF had markedly reduced and were infection-free, and the wounds treated with 14 µg/cm2 bFGF were almost completely epithelized (Figure 5). In the wounds treated with CGS containing 28 or 50 µg/cm2 bFGF, dermis-like tissue had formed but epithelization had not proceeded as quickly as that seen in wounds treated with CGS containing 7 or 14 µg/cm2 bFGF.

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Figure 4. Gross appearance of the wounds 1 week after CGS implantation. The wounds were treated with CGS impregnated with NSS (A) or 7 µg/cm2 (B), 14 µg/cm2 (C), 28 µg/cm2 (D) or 50 µg/cm2 bFGF (E). One week after implantation, the CGS impregnated with NSS were infected, whereas dermis-like tissue had begun to form in the wounds treated with the bFGF-impregnated CGS

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Figure 5. Gross appearance of the wounds 2 weeks after CGS implantation. The wounds were treated with CGS impregnated with NSS (A) or 7 µg/cm2 (B), 14 µg/cm2 (C), 28 µg/cm2 (D) or 50 µg/cm2 bFGF (E). Two weeks after implantation, the CGS impregnated with NSS had become infected and degraded. In contrast, the wound areas covered with CGS containing 7 or 14 µg/cm2 bFGF had markedly reduced without infection. The wounds treated with 14 µg/cm2 bFGF had become almost completely epithelialized

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The time course of the remaining wound area is shown in Figure 6. One week after implantation, the wound area in the 14 µg/cm2 bFGF group was significantly smaller than that in the control group. Two weeks after implantation, the wound areas in the 7 and 14 µg/cm2 bFGF groups were significantly smaller than those in the control group and the 50 µg/cm2 bFGF group.

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Figure 6. Time course of the remaining wound area. Wounds treated with CGS impregnated with NSS ( ) or 7 µg/cm2 (□), 14 µg/cm2 (×), 28 µg/cm2 (○) or 50 µg/cm2 bFGF (▽); *p < 0.05, **p < 0.01 vs NSS; #p < 0.05, ##p < 0.01 vs 7 µg/cm2 bFGF; $p < 0.05, $$p < 0.01 vs 28 µg/cm2 bFGF; &p < 0.05, &&p < 0.01 vs 50 µg/cm2 bFGF. One week after implantation, the wound area in the group treated with CGS containing 14 µg/cm2 bFGF was significantly smaller than that in the control group. Two weeks after implantation, the wound areas in the groups treated with CGS impregnated with 7 or 14 µg/cm2 bFGF were significantly smaller than those in the control and CGS with 50 µg/cm2 bFGF groups

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Histological assessment of neoepithelium length

Light microphotographs of the histological sections are shown in Figures 7 and 8. The low-magnification image (×40) shows the neoepithelium, CGS and marginal skin. The high-magnification image (×100) shows neoepithelium formation. The neoepithelium formation was especially marked in the 7 and 14 µg/cm2 bFGF groups.

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Figure 7. H&E-stained sections of wounds 1 week after implantation. The wounds were treated with CGS impregnated with NSS (A) or 7 µg/cm2 (B), 14 µg/cm2 (C), 28 µg/cm2 (D) or 50 µg/cm2 bFGF (E). The low-magnification image (original magnification × 40, left side) shows a whole image of the implanted CGS. The area of remaining CGS is indicated by a broken line. The high-magnification image (original magnification × 100, right side) shows the newly formed epithelium on the left side. Black arrow with solid line indicates the hair root; black arrow with broken line indicates the end of the neoepithelium. The neoepithelium is shown in the upper section as a black line that is closed at both ends

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Figure 8. H&E-stained wound sections 2 weeks after implantation. The wounds were treated with CGS impregnated with NSS (A) or 7 µg/cm2 (B), 14 µg/cm2 (C), 28 µg/cm2 (D) or 50 µg/cm2 bFGF (E). The low-magnification image (original magnification × 40, left side) shows a whole image of the implanted CGS. The black solid squares (500 µm in width and height) show the area of the CGS in which the number and area of newly formed capillaries were investigated. The area of remaining CGS is surrounded by a broken line. The black solid square shows the area used for the immunohistological staining of newly formed capillaries. The high-magnification image (original magnification × 100, right side) shows the newly formed epithelium on the left side. Black arrow with solid line indicates the hair root; black arrow with broken line indicates the edge of the epithelium. The neoepithelium is shown in the upper section as a black line that is closed at both ends

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The time course of neoepithelium length is shown in Figure 9. One week after implantation, the neoepithelial lengths in the 7 and 14 µg/cm2 bFGF groups were significantly longer than those in the control group. Two weeks after implantation, the neoepithelium length in the 14 µg/cm2 bFGF group was significantly longer than those in the control and 50 µg/cm2 bFGF groups.

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Figure 9. Time course of neoepithelium length. Wounds treated with CGS impregnated with NSS ( ) or 7 µg/cm2 (□), 14 µg/cm2 (×), 28 µg/cm2 (○) or 50 µg/cm2 bFGF (▽); *p < 0.01 vs NSS, #p < 0.01 vs 50 µg/cm2 bFGF. One week after implantation, the neoepithelial lengths in the wounds treated with 7 µg/cm2 or 14 µg/cm2 of bFGF were significantly longer than that of the control group. Two weeks after implantation, the neoepithelium length of the wounds treated with 14 µg/cm2 bFGF was significantly longer than those of the control group and the group treated with 50 µg/cm2 bFGF

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Evaluation of newly formed capillaries in the wounds

Light microphotographs of newly formed capillaries stained with von Willebrand factor are shown in Figure 10. The number of capillaries in the bFGF-impregnated group was significantly larger than that in the NSS group, although no significant difference was observed among the bFGF-impregnated groups (Figure 11). The capillary areas in the bFGF-impregnated groups treated with 7, 14 or 28 µg/cm2 bFGF were significantly larger than those in the control (almost 20 times larger) and 50 µg/cm2 bFGF groups (almost eight times larger). No significant difference was observed between the control and 50 µg/cm2 bFGF groups (Figure 12).

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Figure 10. Immunohistological staining of CGS 2 weeks after implantation. The newly formed capillaries were immunostained with von Willebrand factor (original magnification × 200). CGS with NSS (A), 7 µg/cm2 bFGF (B), 14 µg/cm2 bFGF (C), 28 µg/cm2 bFGF (D) and 50 µg/cm2 bFGF (E). Black arrowheads indicate capillaries

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Figure 11. The number of newly formed capillaries immunostained with von Willebrand factor (2 weeks after implantation). CGS treated with NSS or 7 µg/cm2, 14 µg/cm2, 28 µg/cm2 or 50 µg/cm2 bFGF; *p < 0.01 vs NSS. The number of newly formed capillaries in the groups treated with CGS impregnated with bFGF was significantly larger than that in the control group

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Figure 12. The area of newly formed capillaries immunostained with von Willebrand factor (2 weeks after implantation). CGS treated with NSS or 7 µg/cm2, 14 µg/cm2, 28 µg/cm2 or 50 µg/cm2 bFGF; *p < 0.01 vs NSS; #p < 0.01 vs 50 µg/cm2 bFGF. The area of newly formed capillaries in the group treated with CGS impregnated with 7 µg/cm2 bFGF was significantly larger than those in the control group and the group treated with CGS impregnated with 50 µg/cm2 bFGF. The areas of newly formed capillaries in the groups treated with CGS impregnated with 14 µg/cm2 or 28 µg/cm2 bFGF were significantly larger than that in the control group

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

Chronic skin ulcers can result from diabetic neuropathy, pressure sores, venous insufficiency, peripheral vascular disease, infectious disease or acute surgical wounds in cases in which the healing process is disturbed. Chronic skin ulcers have a significant impact on public health through increased disability, morbidity and mortality, all of which increase the cost of healthcare (Ho et al., 2005). In patients suffering from these conditions, diabetic foot ulcers are a leading cause of hospitalization and amputation. Many advanced technologies, such as wound dressings, topical ointments, enzymatic debridement compounds and hyperbaric oxygen therapy, have been developed to improve the treatment of chronic skin ulcers (Heyneman et al., 2008; Dunn and Dolianitis, 2008; McCallon and Hurlow, 2009; Collier, 2009; Baroni et al., 1987; Oriani et al., 1990; Kessler et al., 2003; Ong, 2008). Recently, due to advances in tissue-engineering and cell-culture techniques, cultured skin substitutes have been used in the treatment of diabetic ulcers, pressure ulcers and venous leg ulcers (Karr, 2008; Ohara et al., 2010; Cervelli et al., 2010). Negative pressure wound therapy, such as vacuum-assisted closure (VAC) therapy (KCI, TX, USA), involves the delivery of intermittent or continuous subatmospheric pressure, thereby providing an occlusive environment in which wound healing can proceed under moist, clean and sterile conditions (Labanaris et al., 2009; Nather et al., 2010). In addition, various growth factors, such as basic FGF (Fu et al., 2002; Kurokawa et al., 2003), PDGF-BB (platelet-derived growth factor; Bhansali et al., 2009), EGF (epidermal growth factor; Kim et al., 2010) and platelet-rich plasma (PRP; Cervelli et al., 2009; Kathleen et al., 2010) have been used for the clinical treatment of chronic wounds.

A major clinical problem with the use of AD is their low resistance to infection when applied to chronic ulcers. We have attempted to experimentally impregnate AD with antibiotics or sulphadiazine silver to prevent infection; however, the clinical application of these techniques has not been actualized because there is a danger of the emergence of antibiotic-resistant bacteria or silver toxicity (Matsuda et al., 1991; Kawai et al., 2001). Recently, the combination of AD and the daily application of bFGF was reported to accelerate granulation tissue formation during the treatment of uninfected chronic ulcers (Muneuchi et al., 2005; Ito et al., 2005; Akita et al., 2008). We previously reported that AD containing bFGF-impregnated gelatin microspheres (MS) sustained the release of biologically active bFGF and accelerated angiogenesis and promoted dermis-like tissue formation (Tabata et al., 1999; Kawai et al., 2000, 2005). This therapy did not require the daily application of bFGF; however, the injection of MS into AD is complicated and time consuming. Our CGS is a scaffold that can sustain the release of positively charged growth factors, such as bFGF, PDGF-BB and transforming growth factor (TGFβ; Takemoto et al., 2008). As for the optimal dosage of bFGF to impregnate CGSs, it is reported that bFGF forms a polyion complex with acidic gelatin at a bFGF:gelatin molar ratio of 1:1 (Muniruzzaman et al., 1998). Therefore, our CGSs were able to sustain a bFGF concentration of > 60 µg/cm2, depending on the gelatin content of the CGSs. In our previous study, we achieved the sustained release of bFGF from CGSs impregnated with 20 µg/cm2 bFGF. In this study, we confirmed that CGSs are able to sustain bFGF at concentrations in the range 7–50 µg/cm2 during its biodegradation (Figure 2).

It has been reported that the dose–effect relationship of bFGF is bell-shaped (Okumura et al., 1996; Motomura et al., 2008; Uchi et al., 2009). In a clinical study of diabetic foot ulcers, Uchi et al. (2009) reported that the bFGF showed a bell-shaped dose–response pattern with a peak at 1 µg/cm2 bFGF/day. In our previous studies, we compared the wound-healing processes induced by various bFGF concentrations. In our previous study, in which we created skin defects in C57BL mice, CGSs impregnated with 7 µg/cm2 bFGF accelerated dermis-like tissue formation the most, and CGSs impregnated with 14 µg/cm2 bFGF had the second strongest effect. However, the application of CGSs impregnated with 50 µg/cm2 bFGF did not accelerate wound healing (Kanda et al., 2011). In another study, in which palatal mucosal defects were created in beagles, CGSs impregnated with 7 µg/cm2 bFGF also accelerated the tissue regeneration the most, and 14 µg/cm2 bFGF had the second strongest effect (Artem et al., 2011). According to these results, we considered that a bFGF concentration in the range 7–14 µg/cm2 would be optimal for accelerating wound healing.

In this study, as expected, CGSs impregnated with 7 or 14 µg/cm2 bFGF accelerated dermis-like tissue formation, and CGSs containing 14 µg/cm2 bFGF produced a significant reduction in the remaining wound area compared with CGSs impregnated with 7 µg/cm2 bFGF. The remaining wound area, neoepithelium length and area of newly formed capillaries in the wounds treated with CGSs containing 50 µg/cm2 bFGF were significantly inferior to those of wounds treated with CGS containing 7 or 14 µg/cm2 bFGF. It has been reported that treatment with an excess amount of bFGF prolonged wound closure in diabetic mice and prevented keratinocyte proliferation in vitro (Okumura et al., 1996; Motomura et al., 2008). CGS impregnated with 50 µg/cm2 bFGF released a persistently high dose of bFGF throughout their biodegradation, which might have inhibited the formation of the neoepithelium and capillaries. Our results regarding the degradation rate of CGSs suggest that 50 µg/cm2 bFGF inhibits the activity of collagenase. This inhibition might prolong the biodegradation of CGSs and hence bFGF release in vivo, and so have an adverse effect on wound healing.

Our CGSs containing bFGF showed resistance to infection. Some combination therapies have been reported to solve the problem of the lower resistance of AD to infection. Combination therapy involving AD (Integra®, Integra Life Sciences Corp., Plainsboro, NJ, USA) and the VAC therapy system has been reported to be effective at increasing granulation tissue formation (Molnar et al., 2004; Pollard et al., 2008). This therapy does not require daily treatment, but does need a specialized pump to maintain a constant negative pressure, and the patient's movements are restricted by the device. As another combination therapy, AD seeded with autologous or allogeneic cultured fibroblasts has been reported to be effective at accelerating granulation tissue formation. The fibroblasts contained in the AD release various growth factors and extracellular matrix molecules and accelerate wound healing (Ohara et al., 2010). Kuroyanagi et al. (2001) reported that spongy collagen containing allogeneic fibroblasts was an effective therapy for patients with intractable skin ulcers, including burns, venous ulcers and autoimmune disease. Although tissue-engineered substitutes are attractive, some problems remain, such as the possibility of disease transmission, unfavourable immune and local inflammatory reactions and their high cost, the latter of which is very important from a clinical perspective. Tissue-engineered skin substitutes, such as Dermagraft® (Advanced Tissue Sciences Inc, USA), Transcyte® (Advanced Tissue Sciences) and Aprigraf® (Organogenesis Inc., Canton, MA, USA, and Novartis Pharmaceuticals Corp., USA), are expensive, and access to these treatments is limited in many parts of the world (Eran et al., 2006). CGS is cheap compared with AD, and bFGF is cheaper than treatment with the VAC therapy system or living cells. The procedure for our novel combination therapy involving CGS and bFGF is simple, i.e. bFGF is sprayed onto the CGS just before their application.

Basic FGF has not been authorized for use in most countries. The acidic gelatin contained in CGS can sustain the release of not only bFGF but also other positively charged growth factors, such as PDGF-BB and TGFβ, found in PRP (Hong et al., 2000; Kanematsu et al., 2004). PRP can be prepared from a patient's blood and platelet lysate (PL) produced from donated platelets (Mirabet et al., 2008). When bFGF can not be used, combination therapy involving CGS and autologous or allogeneic PRP is an alternative.

Recently, tissue engineering has been recognized as a newly emerging biomedical technology for regenerating and repairing body defects, using various combinations of cells, scaffolds and growth factors (Tsuji-Saso et al., 2007). Collagen sponge scaffolds are one of the most common scaffolds, and it has been reported that collagen scaffolds treated with growth factors are effective at regenerating various kinds of tissues, such as the dermis, epidermis, fat, bone and cartilage (Langer, 2007; Kimura et al., 2010; Nishizawa et al., 2010). Our CGS, which is capable of the sustained release of positively-charged growth factors, is useful as a scaffold for tissue engineering.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

CGS impregnated with bFGF at concentrations in the range 7–14 µg/cm2 accelerated wound healing in decubitus ulcer models involving diabetic mice, and an excess amount of bFGF did not increase their wound-healing efficacy. Our CGS is a novel scaffold that can sustain the release of positively-charged growth factors, such as bFGF. Combination therapy involving CGS and bFGF or PRP is a promising strategy for the treatment of chronic skin ulcers.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References

This work was supported by a grant from the Japan Science and Technology Agency.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgement
  9. References
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