Inhibitory Effects of a Biodegradable Gelatin Hydrogel Sponge Sheet on the Progression of Experimental Abdominal Aortic Aneurysms
Article Outline
We investigated the effects of a biodegradable gelatin hydrogel sponge sheet (GHSS) or GHSS incorporating basic fibroblast growth factor (GHSS + bFGF), which could prolong the effects of bFGF, on the progression of experimental abdominal aortic aneurysms (AAAs). Experimental AAAs were induced in male Sprague-Dawley rats by intra-aortic elastase infusion. The rats were divided according to the following treatments: (1) untreated, (2) GHSS alone, (3) GHSS incorporating 100 ng, 1 μg, and 10 μg of bFGF. GHSSs were placed over the elastase-infused aortas. After 14 days, the GHSS alone group and the three groups with GHSS + bFGF demonstrated significantly smaller aortic diameters than the untreated group, and these groups significantly attenuated a reduction of the elastic fibers and smooth muscle cells in the pathological findings. However, no additional therapeutic effect was noted between the GHSS alone and GHSS + bFGF groups. Immunohistochemical analysis revealed an increase of positive cells for endogenous bFGF in the media and adventitia of both the GHSS alone and GHSS + bFGF groups in comparison to the untreated group. In conclusion, GHSS itself possessed significant therapeutic effects on AAA progression by inducing the production of endogenous bFGF, leading to the preservation of elastic fibers and smooth muscle cells.
Introduction
Abdominal aortic aneurysm (AAA) is a common and potentially life-threatening disorder characterized by an imbalance between the destruction and synthesis of the extracellular matrix in the aortic wall. Aneurysm formation is associated with a chronic transmural inflammation, depletion of the smooth muscle cell (SMC) population, and excessive matrix metalloproteinase (MMP) production causing abnormal degradation of elastins and collagens.1, 2 The standard therapy for AAA is surgical treatment with vascular grafts, including endovascular aneurysm repair, to prevent fatal rupture of the AAA. However, there is still no effective medical treatment to stabilize aneurysmal disease and prevent either further expansion or rupture.
Many researchers have investigated the pathophysiology of AAAs using experimental AAA models, and several studies have shown that new minimally invasive treatments, including medical therapy and gene therapy, inhibited the expansion of experimental AAAs.3, 4, 5, 6, 7, 8, 9, 10 The proliferation of medial SMCs by treatment with gene transfer of basic fibroblast growth factor (bFGF) or SMC seeding is known to inhibit the expansion of experimental AAAs.7, 8, 11 In a recent report, gingival fibroblasts prevented the degradation of rabbit aortic elastin in an ex vivo aortic culture model.12 Therefore, bFGF or fibroblast may provide a novel therapy for human AAA. However, the safety and efficacy of gene transfer or cell seeding has not yet been fully established in clinical settings. On the other hand, the half-life of bFGF in vivo is too short to effectively exert its biological activity when administrated in the free form. We have developed a biodegradable gelatin hydrogel composed of acidic gelatin to enable bFGF to be released at the site of action for an extended time period.13, 14, 15 bFGF tends to bind ionically to the acidic gelatin and is released from the matrix when the gelatin degrades. The release profile is controllable by changing the water content of the hydrogels. A biodegradable gelatin hydrogel is safe in vivo and able to easily prolong the effect of bFGF in comparison to gene transfer of bFGF. Furthermore, biodegradable materials induce the production of endogenous growth factors including bFGF when they degrade.16 Therefore, gelatin hydrogel also may induce it, possibly helping to inhibit the progression of experimental AAAs.
In the present study, we treated experimental AAA rats with a gelatin hydrogel sponge sheet (GHSS) or with GHSS incorporating bFGF to investigate the effects of these treatments on the progression of experimental AAAs.
Materials and Methods
Preparation of the GHSS with or without bFGF
Human recombinant bFGF with an isoelectric point value of 9.6 was supplied from Kaken Pharmaceutical (Tokyo, Japan). GHSSs were prepared as described previously.13 Briefly, GHSS was made by the chemical cross-linking of acidic gelatin with an isoelectric point of 4.9. It was shaped into a sheet (15 × 7 × 2 mm) and freeze-dried. Before placement of the GHSS over the aorta, freeze-dried GHSS was impregnated with an aqueous solution with or without bFGF for 1 hr to obtain a GHSS with or without bFGF, respectively. This gelatin sponge sheet was designed to degrade in 2 weeks.
Experimental Animals
Male Sprague-Dawley rats (250–400 g) were obtained from CLEA (Tokyo, Japan) and used for the experiments. This study was approved by the Animal Care Committee of the Tohoku University Graduate School of Medicine. The animal care complied with the Guide for the Care and Use of Laboratory Animals (National Research Council, Washington DC, 1996).
Induction of Experimental AAA
The rats were anesthetized with an i.p. injection of sodium pentobarbital (50–60 mg/kg of body weight: Dainippon Pharmaceutical, Osaka, Japan), and a midline laparotomy was performed under sterile conditions. A 1 cm segment of infrarenal aorta was isolated, and all lumber arteries were ligated. The external aortic diameter before elastase infusion (preinfusion AD) was measured with a digimatic caliper (Mitutoyo, Kanagawa, Japan). A polyethylene tube (Becton Dickinson, Sparks, MD) was inserted into the right external iliac artery and then advanced into the isolated aorta. An atraumatic clamp was placed across the proximal aorta, and a temporary ligation was placed around the distal aorta to secure the tube. The isolated aorta was infused with 2.7 units of type I porcine pancreatic elastase (E-1250, lot 84K7700; Sigma, St. Louis, MO) for 60 min using a syringe pump (TOP-5200; TOP, Tokyo, Japan). After elastase infusion, the ligature and tube were removed and then the right external iliac artery was ligated. The retroperitoneum and abdominal wall were closed. Rats were reexplored on postoperative day 14 (14 POD), and the maximum AD was measured.
Treatment Groups
Experimental rats with elastase infusion were randomly divided into five groups according to the treatments: a group without any treatment (untreated group, n = 10), a group treated with GHSS incorporating distilled water (GHSS alone group, n = 10), three groups treated with GHSS incorporating different amounts of bFGF (GHSS + 100 ng group, GHSS + 1 μg group, and GHSS + 10 μg group; n = 10, 6, and 6, respectively). These GHSSs were placed over the elastase-infused aortas of the rats after elastase infusion, and the retroperitoneum was closed.
Histological Analysis
Three groups of experimental rats, including the untreated group, the GHSS alone group, and the GHSS + 100 ng group, were prepared for histological analysis (n = 6, respectively). After the rats were killed at 14 POD, they were perfused with 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 5 min via the left ventricle, and the harvested aortas were further fixed in 4% paraformaldehyde solution in PBS for 2 hr, followed by 100% ethanol. The specimens were then embedded in paraffin and cut in cross section at 5 μm. These sections were stained with Verhoeff-van Gieson (VVG) for elastic fibers and prepared for immunohistochemistry. The ratio of stained elastic fibers to the media area in VVG staining was calculated using Image-J software (NIH, Bethesda, MD).
Immunohistochemistry was undertaken after deparaffinization and rehydration. The sections were treated for 10 min with H2O2 to block endogenous peroxidase activity. After blocking with 1% bovine serum albumin in PBS for 30 min, sections were incubated with primary antibody overnight at 4°C, including a mouse anti-human α-smooth muscle actin (α-SMA) monoclonal antibody (Sigma) for rat SMC staining and a rabbit anti-rat bFGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Subsequent incubation with biotinylated secondary antibody and an avidin-biotin complex method (Dako Cytomation, Glostrup, Denmark) was performed according to the manufacturer's protocol. The sections were counterstained with hematoxylin. Negative control experiments were performed by replacing the primary antibody with unspecific mouse or rabbit immunoglobulin G. Medial SMC density was determined by the average of α-SMA-positive cells in eight high-power fields chosen from two cross sections.
Statistical Analysis
The data are represented as mean ± standard error (SE). The results were assessed using one-way or two-way analysis of variance (ANOVA) with statistical significance assigned as p < 0.05. When the ANOVA finding was significant, the post hoc Scheffe test was used to compare the individual groups. A statistical analysis was performed using the StatView J-5.0 software program (SAS Institute, Cary, NC).
Results
Alterations of AD in the Five Groups
Alterations of AD in the five groups of experimental AAA rats induced by elastase infusion are demonstrated in Figure 1. The preinfusion ADs were quite similar among the five groups. Elastase infusion for 60 min markedly enlarged the mean AD at 14 POD in the untreated group from 1.66 ± 0.02 to 8.16 ± 0.50 mm. Under the same conditions, all treatment groups (GHSS alone, GHSS + 100 ng, GHSS + 1 μg, and GHSS + 10 μg) demonstrated significantly smaller AD at 14 POD (4.66 ± 0.20, 4.19 ± 0.15, 4.30 ± 0.54, and 4.52 ± 0.64 mm, respectively) than that of the untreated group. No significant difference was noted in the AD among the GHSS alone group and the three groups treated with GHSS incorporating bFGF.
Fig. 1
Alterations of the AD in each group. Preinfusion, before elastase infusion. Data are shown as the mean ± SE. n = 10 in untreated, GHSS alone, and GHSS + 100 ng groups; n = 6 in GHSS + 1 μg and GHSS + 10 μg groups. ∗p < 0.0001 vs. AD at 14 POD in untreated group.
Histological Analysis
We examined elastic fibers and SMCs in the aortic media by VVG staining and immunostaining for α-SMA, respectively. VVG staining revealed a marked reduction of the stained elastic fibers in the media of the untreated group (Fig. 2a, b). On the contrary, staining for elastic fibers was well preserved in both the GHSS alone and GHSS + 100 ng groups (Fig. 2c–f). When the ratio of the stained area for elastic fibers to the media area was calculated by a computerized digital image, it was significantly higher in both the GHSS alone (23.8 ± 2.6%) and GHSS + 100 ng (24.8 ± 2.2%) groups than in the untreated group (16.8 ± 0.9%), although no significant difference was observed in the ratio between the GHSS alone and GHSS + 100 ng groups (Fig. 3). The number of SMCs stained by antibody against α-SMA in the media was significantly larger in both the GHSS alone (81.8 ± 3.6/hpf) and GHSS + 100 ng groups (86.1 ± 2.2/hpf) than in the untreated group (67.6 ± 3.2/hpf), although no significant difference was observed in the number of SMCs between the GHSS alone and GHSS + 100 ng groups (Fig. 4, Fig. 5).
Fig. 2
Photomicrographs of the aortic wall stained with VVG for elastic fibers. Elastic fibers are stained black. Untreated (a, b), GHSS alone (c, d), and GHSS + 100 ng (e, f) groups. M, Media; AD, adventitia. Original magnification ×100 (left panels) and ×400 (right panels).
Fig. 3
Ratio of stained elastic fibers to the media area in VVG staining was calculated using NIH Image-J software. Data are shown as the mean ± SE in each group. n = 6 in all groups. ∗p < 0.05 vs. untreated group.
Fig. 4
Photomicrographs of the aortic wall immunostained with anti-α-SMA for SMCs. The cytoplasms of SMCs in the media are stained brown. Untreated (a), GHSS alone (b), and GHSS + 100 ng (c) groups. M, Media; AD, adventitia. Original magnification ×400.
Fig. 5
Medial SMC density was determined by the average number of α-SMA-positive cells in 8 high-power fields chosen from two cross sections. Data are shown as the mean ± SE. n = 6 in each group. ∗p < 0.01 vs. untreated group.
In addition, immunohistochemical analysis showed an increase of positive cells (cytoplasm and nuclear) for endogenous bFGF in the media and adventitia of both the GHSS alone and GHSS + 100 ng groups in comparison to the untreated group (Fig. 6).
Fig. 6
Photomicrographs of the aortic wall immunostained with anti-bFGF antibody. Untreated (a), GHSS alone (b), and GHSS + 100 ng (c) groups. M, Media; AD, adventitia. These findings reveal an increase of positive cells (cytoplasm and nuclear) for bFGF in the media and adventitia of both the GHSS alone and GHSS + 100 ng groups in comparison to the untreated group. Original magnification ×200.
Discussion
The present study was originally designed to investigate the therapeutic effects of GHSS incorporating bFGF on the progression of experimental AAAs using the rat AAA model. GHSS was used to maintain the local and long-term effects of bFGF. We prepared two groups of AAA rats as control, including a group without any treatment and a group treated with GHSS not incorporating bFGF, because the effect of GHSS on experimental AAAs was unclear. Although the AD at 14 POD of the rats with GHSS + bFGF was significantly smaller than that of the untreated rats, the rats with GHSS alone demonstrated almost the same AD at 14 POD compared to the rats with GHSS + bFGF. Our results also showed no dose-dependant effect of bFGF. These findings suggest that the placement of GHSS over the aorta has significant therapeutic effects on AAA progression, whereas the additional therapeutic effect of bFGF was quite limited.
The induction of experimental AAA by elastase infusion was originally performed by Anidjar et al.,17 and they reported that the pathological findings of this model were similar to those of human AAA. In this study, we evaluated the elastic fibers and SMCs in the media, both of which have been reported to decrease dramatically in both human AAA and experimental AAA.1, 17 Our results demonstrated a marked reduction of both the elastic fibers and SMCs in the untreated rats as reported by earlier studies, whereas the rats treated with GHSS alone significantly attenuated such a reduction of the elastic fibers and SMCs. The treatment with GHSS + bFGF also attenuated such a reduction, but no statistical difference was noted between the rats with GHSS alone and GHSS + bFGF. These results suggest that GHSS itself has an ameliorating effect against the aneurysmal wall by preserving the elastic fibers and SMCs, thus resulting in a smaller AD.
We hypothesized that GHSS itself induced production of endogenous bFGF when it degraded. When we performed immunostaining for bFGF, our findings revealed an increase in the number of positive cells for endogenous bFGF in the media and adventitia of the rats treated with GHSS alone in comparison to untreated rats. Furthermore, mild fibrosis was observed around the aorta of rats with GHSS + bFGF and of those with GHSS alone when we performed a relaparotomy in several rats at 7 POD (data not shown), suggesting that GHSS induced the same growth factors around the aorta. These results are compatible with those which reported that placement of a biomaterial polymer in vivo induces the production of endogenous growth factors.16 Hoshina et al.7 reported that a gene transfer of bFGF limited the aneurysm enlargement due to medial SMC proliferation. Gogly et al.12 also reported that fibroblasts had protective effects against the degradation of elastic fibers. Our results also showed preservation of the SMCs and elastic fibers in rats treated with GHSS alone. Therefore, we believe that GHSS itself induces production of endogenous bFGF, which proliferates the SMCs and fibroblasts, which may preserve the degradation of the elastic fibers; and these ameliorations in the aortic wall prevent the expansion of experimental AAAs. Another possible mechanism is the effect of external support of aorta by GHSS itself. The wrapping of an intracranial aneurysm is an effective treatment for inhibiting the progression or rupture of aneurysms due to reinforcement of the aneurysmal wall.18 Haraguchi et al.19 also demonstrated that wrapping vein grafts in gelatin hydrogel sheet without bFGF significantly inhibited the graft dilatation due to the mechanical support and endothelial cell protection in association with some autocrine growth factors. However, GHSS with an aqueous solution is a soft hydrogel, and it largely degrades by 14 POD. Furthermore, we placed it over the aorta and did not wrap it. Therefore, the dynamic effect of GHSS to reinforce the aneurysmal wall may have helped to inhibit the expansion of experimental AAAs in this study, but it is thought that this was not so strong. GHSS may actually have several other effects on experimental AAAs. Further study is needed to clarify the effects of GHSS on the progression of experimental AAAs.
The addition of bFGF had little therapeutic effect in the present study. Furthermore, no dose-dependant effect of bFGF was observed on the progression of experimental AAA. Although bFGF is known to proliferate the various mesenchymal cells, several studies have reported that an excessive dose of bFGF inhibits the proliferation of them and that its efficacy is not dose-dependent.20, 21 Therefore, our results suggest that GHSS alone induces a sufficient amount of bFGF to inhibit the progression of experimental AAAs, and this is why the addition of bFGF had no significant effect against the aneurysmal wall.
The therapeutic value of a gelatin hydrogel incorporating bFGF has been widely studied experimentally regarding angiogenesis22 and the regeneration of bone or skin,15, 23 and some clinical applications have been also started.24 For the application of treatment using GHSS to human AAA, methodological improvements are required to place GHSS by a less invasive method, such as by a minilaparotomy or a laparoscopy. Since it is actually difficult to isolate the whole abdominal aorta and wrap it by GHSS in humans, we placed the GHSS over the abdominal aorta in this study, not wrapping it. In fact, the placement of GHSS over the aorta induced a sufficient effect. Therefore, this study suggests that it is enough to apply GHSS to the anterolateral side of the aorta as a treatment of human AAA. Furthermore, gelatin hydrogel can change into an injectable microsphere,24, 25 and it may be possible to inject such a gelatin microsphere around an AAA. If a better method is established, then this minimally invasive therapy may be applicable for either small AAAs or AAAs in high-risk patients who are unable to withstand an invasive operation.
In conclusion, our present study clearly demonstrated the therapeutic effects of GHSS on the progression of experimental AAAs. Treatment with GHSS induced the production of endogenous bFGF, which increased SMCs and fibroblasts, which might preserve the degradation of elastic fibers. The amelioration in the histological findings resulted in the inhibition of experimental AAA expansion. Our present findings suggest a potential clinical application of biodegradable GHSS to treat human AAA using a minimally invasive method.
We are grateful to Dr. Shojiro Matsuda, Gunze Limited Research and Development Center, for providing the GHSS, and Kaken Pharmaceutical for providing bFGF. This work was supported by a grant-in-aid for scientific research (JSPS-18591402) from the Japanese Society for Promotion of Science.
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Part of this work was initially presented at the Twenty-second World Congress of the International Union of Angiology, Lisbon, Portugal, June 24–28, 2006.
PII: S0890-5096(08)00246-X
doi:10.1016/j.avsg.2008.06.009
© 2009 Annals of Vascular Surgery Inc. Published by Elsevier Inc All rights reserved.
