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Ann Thorac Surg 2007;84:560-567
© 2007 The Society of Thoracic Surgeons

Original Articles: Cardiovascular

External Application of Rapamycin-Eluting Film at Anastomotic Sites Inhibits Neointimal Hyperplasia in a Canine Model

Satoshi Kawatsu, MDa,*, Katsuhiko Oda, MDa, Yoshikatsu Saiki, MDa, Yasuhiko Tabata, PhD, DMedScib, Koichi Tabayashi, MDa

a Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan
b Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Science, Kyoto University, Sankyo-ku, Kyoto, Japan

Accepted for publication February 15, 2007.

* Address correspondence to Dr Kawatsu, Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Japan 980-8574 (Email: kawa2{at}mail.tains.tohoku.ac.jp).

Presented at the Forty-third Annual Meeting of The Society of Thoracic Surgeons, San Diego, CA, Jan 29–31, 2007.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 
Background: Stenosis at a vascular anastomotic site has been a significant clinical issue. We tested the hypothesis that rapamycin-eluting biodegradable poly L-lactic acid and epsilon-caprolactone copolymer (PLA-CL) film applied externally can inhibit neointimal hyperplasia in a canine vascular anastomosis model.

Methods: Femoral artery graft interposition was performed in 25 beagles. Beagles were divided into five groups (five in each): graft interposition without PLA-CL film (control); with PLA-CL film only; and PLA-CL containing rapamycin 8 µg, 80 µg, and 800 µg. Orthotopic arterial graft interposition was performed on the left side and vein graft from the ipsilateral femoral vein was interposed on the right. Morphometric and immunochemical analyses were performed at four-week intervals.

Results: In arterial graft models, the ratio of intimal area (intimal area divided by the entire vessel area) was significantly reduced in all the three rapamycin-eluting film groups compared with control (0.19, 0.07, 0.05, and 0.38 in 8 µg, 80 µg, 800 µg groups and control, respectively, p < 0.05). In vein graft models, the ratio of intimal area was significantly decreased only in the 800 µg rapamycin group compared with control (0.33 vs 0.54, p < 0.05). Inhibition of neointimal growth was associated with reduced cell proliferation, as evidenced by proliferating cell nuclear antigen immunostaining and diminished alpha-actin positive vascular smooth muscle cells.

Conclusions: Rapamycin-eluting biodegradable PLA-CL film applied externally can inhibit neointimal hyperplasia of arterial and vein grafts in a canine model. The inhibitory effect of rapamycin-eluting film against neointimal growth is more pronounced in the arterial graft than the vein graft.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 
Surgical outcome of coronary artery bypass grafting (CABG) has been improved over the decades [1, 2]. Steady refinement of surgical technique and the use of arterial grafts undoubtedly contributed to the advancement in surgical management of coronary artery disease. Aggressive use of arterial grafts provided excellent clinical outcome, while the liberal use of the saphenous vein grafts is limited by suboptimal graft patency [3–5]. Besides technical failure, graft patency rate is largely affected by the degree of neointimal hyperplasia which develops immediately after graft anastomosis [6, 7].

Restenosis after angioplasty is also induced by neointimal hyperplasia. Mechanism of neointimal overgrowth is mainly attributable to vascular smooth muscle cell migration and proliferation from the media to the intima [8]. Various growth factors, including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and chemotartic factors such as interleukin-6, interleukin-18, and interleukin-1 beta released from inflammatory cells are also involved in the process of vascular remodeling after vascular injury [9–12].

Rapamycin is an immunosuppressive agent, and inhibits the progression of the cell cycle from the G1 to the S phase of mitotic cycle. The drug forms a complex with FK506 binding protein; the complex inhibits the mammalian target of rapamycin (mTOR). The mTOR is activated by autophosphorylation, by cytokine, or by growth factor-induced signals [13]. The activated mTOR further activates other kinases through phosphorylation, thus intervening in the complex process of cell cycle regulation. Another well-established effect of rapamycin is the inhibition of vascular smooth muscle cell (VSMC) migration and proliferation [14]. With these pharmacologic effects, rapamycin has been successfully applied as a form of drug-eluting stent both in experimental [15] and clinical studies [16]. Endoluminal local delivery of rapamycin dramatically inhibits neointimal formation with a resultant low incidence of restenosis after angioplasty. In an attempt to decrease neointimal hyperplasia at vascular anastomotic sites, we sought to locally apply rapamycin in a sustaining fashion. We have created a rapamycin-eluting biodegradable film composed of poly L-lactic acid and epsilon-caprolactone copolymer (PLA-CL). A PLA-CL is a hydrolysable synthetic polymer plastic and has been used in various experimental studies. In this study, we tested the hypothesis that rapamycin-eluting biodegradable PLA-CL film applied externally can inhibit neointimal hyperplasia in a canine vascular anastomosis model.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Discussion
 Acknowledgments
 References
 
In this experiment, dogs were treated in accordance with the Declaration of Helsinki and the "Guiding Principles in the Care and Use of Animals." The experimental and animal care protocol was also approved by the Animal Care Committee of the Tohoku University School of Medicine.

Material
Rapamycin and other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without any further purification. The PLA-CL is purchased from Corefront Corporation (Tokyo, Japan). The composition of the copolymer was 1:1 of poly-L-lactic acid and caprolactone as a mole ratio.

Preparation of poly L-lactic acid and epsilon caprolactone copolymer film and incorporation of rapamycin into the film
Both rapamycin and PLA-CL are liposoluble agents. We dissolved 50 mg of PLA-CL with 1 mL of 99% chloroform in a laboratory dish. We referred to a clinically available rapamycin-eluting stent in terms of drug concentration. The stent contains 140 µg of rapamycin in 1 cm2 of stent area [17]. To achieve equivalent drug concentration on a round laboratory dish with the diameter of 28 mm, 861 µg of rapamycin is required to spread over it. We tested three different doses of rapamycin to examine a dose-response relationship; 8 µg, 80 µg, and 800 µg of rapamycin per film. One mg of rapamycin was dissolved in 1 mL of 99% chloroform and was titrated to 8 µg, 80 µg, and 800 µg. A previously prepared PLA-CL solution was stirred with these different doses of rapamycin, and dried up at 4°C for 24 hours to eliminate chloroform. The rapamycin-containing film was isolated from a laboratory dish by adding some distilled water to the laboratory dish (Fig 1). The actual size of the rapamycin -containing film was 28 mm in diameter as a round film, and the weight was 60 mg. The weight of the rapamycin in this film was exactly the same as the total components of the prepared film; namely, 8 µg, 80 µg, or 800 µg.


Figure 1
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  		Fig 1. The entire appearance of the rapamycin-containing poly L-lactic acid and epsilon-caprolactone copolymer film. The film is transparent, malleable, and therefore fits compactly onto vascular anastomotic sites.

 
We performed a preliminary test to examine the slow-release rate of rapamycin from the PLA-CL film. We made a 9 mg of PLA-CL film containing 315 µg of rapamycin. Two milligrams of this film containing 70.2 µg of rapamycin was soaked in 1 mL of 0.1% tween20/PBS, which was shaken at 37°C with the speed of 100 rpm at every time point. Rapamycin concentration within the solution increased from 7.09 to 8.23 and to 9.68 when measured 3, 10, and 24 hours after soaking, respectively. This pilot study confirmed gradual release of rapamycin from the PLA-CL film.

Operative Procedures
The experiments were carried out on 25 beagles weighing between 9 and 12 kg. General anesthesia was induced with thiopental injection and maintained with sevoflurane inhalation. We used 30 mg/kg of thiopental sodium for induction of anesthesia and 0.5% sevoflurane to maintain anesthesia. Two different modes of femoral artery reconstruction using arterial and vein grafts constitute our experimental models. Each animal undergoes the right femoral artery reconstruction using an ipsilateral femoral vein graft and simultaneous left femoral artery reconstruction using an in situ femoral artery graft. A schematic diagram is depicted in Figure 2A. The anastomosis was performed by the following procedure. First 100 U/kg heparin was injected as an anticoagulant. We harvested a 1-cm-long femoral vein from the right side as a vein graft. The right femoral artery was clamped and divided. The vein graft was anastomosed to the proximal femoral artery in an end to end fashion using a two-suture technique. Distal anastomosis was done similarly. This anastomotic model was defined as a vein graft model. The left femoral artery was then divided, and a 1-cm-long artery graft was harvested as an autograft. Arterial graft anastomosis was performed similar to the vein grafting. This anastomotic model was defined as an arterial graft model. All anastomoses were performed using 8-0 Prolene (Ethicon, Inc, Somerville, NJ).


Figure 2
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  		Fig 2. (A) Schematic drawings of arterial and vein graft models; the right femoral vein was used as a vein graft. End to end anastomosis was performed with a running polypropylene suture. The left femoral artery was used for an arterial graft model. A 1-cm-long transected femoral artery graft was sutured back in place as an autograft. (B) A schematic drawing of the cross-section of a graft; the cross-sectional area of a graft was classified into three components. The most inner area was intimal area (IA). The IA was often thickened with neointimal hyperplasia. The medial area (MA) was delineated with both intimal and external lamina elastica and was mostly composed of smooth muscle cells and extracellular matrix. The outermost area was the adventitial area (AdA). We defined the vessel area (VA) as the summation of IA and MA.

 
The 25 beagles were divided into five experimental groups (five in each group) according to the encircling materials at anastomotic sites on the femoral arteries: the control group, with no material; the PLA-CL group, with a PLA-CL film without rapamycin; and three rapamycin-eluting groups containing 8, 80, and 800 µg of rapamycin incorporated into PLA-CL films. The beagles were sacrificed four weeks later.

Histologic analysis
The grafts were resected beyond the suture lines including the native femoral arteries and fixed with 4% phosphate-buffered formaldehyde, and were embedded in paraffin. Two portions were cut out from each specimen, and then the transmural sections were stained with elastica-Masson (EM) and hematoxylin-eosin (HE) for morphometric analysis.

The areas of the neointima, media, and adventitia at the anastomotic site were measured using the sections stained with EM. We defined the vessel area (VA) as the summation of the neointimal area (IA) and the medial area (MA). The adventitial area (AdA) was difficult to delineate due to adhesion to the surrounding tissue. Thereafter, the area of the IA and MA were calculated (Fig 2B).

To characterize neointimal hyperplasia, IA divided by VA (IA/VA) was calculated. This morphometric analysis was performed using Image-Pro Plus version 4.0 for Windows (MediaCybernetics, Silver Spring, MD).

Immunohistochemical studies
Immunohistochemical staining was carried out on paraffin-embedded sections to detect cellular proliferative activity and to identify migrating vascular smooth muscle cells. Mouse antiproliferating cell nuclear antigen (PCNA) antibody (NeoMarkers, CA) and mouse anti-alpha-actin antibody (DAKO, CA) were used for those purposes. The number of PCNA positive cells per field was counted in each vascular component. Regarding the delineation of the outer margin of the adventitia was not sufficient enough; the demarcation of the outermost adventitial layer is not obvious in the histologic specimens in contrast to the clearly identifiable external elastic laminae. The areas for PCNA positive cell counting were selected on the external elastic laminae side in the adventitia. Eight fields were randomly selected from each specimen under medium power field (x100). The average number per 1 mm2 was calculated for comparison. Alpha-actin positive cells were evaluated to localize the distribution of migrating smooth muscle cells.

Statistical Analysis
Statistical analysis was performed using Excel for Windows (Microsoft, Redmond, WA) with the add-in software StatMate III (ATMS Company, Ltd., Tokyo, Japan). The data were analyzed by one-way analysis of variance (ANOVA). If the ANOVA finding was significant, then the least significant difference multiple comparison test was used as a post-hoc test. Experimental results were expressed as mean ± standard error of the mean. A difference with a p value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Discussion
 Acknowledgments
 References
 
All the animals survived to the end of our protocol. There were no occlusions of the graft or necrosis in the limbs as visualized at autopsy. No infection was detected.

Ratio of the Neointimal Area
The representative photographs of graft cross-sections stained with EM are shown in Figures 3 and 4. Go In the arterial graft models, the ratios of neointimal areas in the control, PLA-CL, 8 µg, 80 µg, and 800 µg were 0.38 ± 0.19, 0.28 ± 0.10, 0.19 ± 0.10, 0.07 ± 0.06, and 0.05 ± 0.02, respectively (Fig 5A). The rapamycin-eluting films significantly inhibited neointimal hyperplasia in a dose-responsive manner. The biodegradable film with 800 µg of rapamycin, a theoretically equivalent dose to the clinically available stent, reduced neointimal hyperplasia to one fifth of that in the control. In the vein graft models, development of neointimal hyperplasia was more remarkable than that in the arterial graft models (Fig 5B). The ratios of neointimal areas in the control, PLA-CL, 8 µg, 80 µg, and 800 µg were 0.54 ± 0.03, 0.58 ± 0.06, 0.50 ± 0.21, 0.47 ± 0.14, and 0.33 ± 0.11, respectively. The rapamycin-eluting films tended to suppress the neointimal growth with increasing doses. The film with 800 µg of rapamycin only reduced the neointimal hyperplasia significantly.


Figure 3
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  		Fig 3. Histologic section of the arterial grafts at the anastomotic site after four week interval. (A) Control; (B) the poly L-lactic acid and epsilon-caprolactone film group; (C) the 8 µg rapamycin-eluting film group; (D) the 80 µg rapamycin-eluting film group; (E) the 800 µg rapamycin-eluting film group. The neointimal growth was suppressed in the rapamycin groups. (Original magnification: x40.)

 

Figure 4
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  		Fig 4. Histologic sections of the vein grafts at the anastomotic site after four week interval. (A) Control; (B) the PLA-CL film group; (C) the 8 µg rapamycin-eluting film group; (D) the 80 µg rapamycin-eluting film group; (E) the 800 µg rapamycin-eluting film group. The neointimal growth was suppressed in the rapamycin groups. The neointimal growth was more remarkable than that in the arterial graft model, yet still less in the higher doses of rapamycin groups. (Original magnification: x20.)

 

Figure 5
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  		Fig 5. (A) The ratio of neointimal area in the arterial graft models. The neointimal hyperplasia was inhibited by rapamycin in a dose-response manner. (B) The ratio of neointimal area in vein graft models. The IA/VA in the 800 µg rapamycin-eluting film group was significantly lower compared with control and the PLA-CL group. (IA/VA = neointimal area divided by vessel area; *p < 0.05; **p < 0.01; PLA-CL = poly L-lactic acid and epsilon-caprolactone copolymer.)

 
Immunohistochemical Studies for PCNA and Alpha-Actin
The number of PCNA positive cells are summarized in Table 1. In the arterial graft models, the PCNA positive cell number in the intimal layer was significantly less in the 800 µg group compared with control. The numbers at the adventitial layer tended to decrease in the higher doses of rapamycin groups, but the difference did not reach statistical significance, whereas the numbers of the PCNA positive cells in the media remained essentially unchanged. These findings in the arterial graft models were also reproducible in the vein graft models (Table 1). Immunohistochemical staining with anti-alpha-actin antibody confirmed that the positive cells were predominantly localized in the media. Furthermore, alpha-actin positive cells were detected in the neointima; those were diminished in the higher doses of rapamycin groups (Figs 6, 7). Go


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  		Table 1 Number of PCNA Positive Cells per Square Millimeter, Quantitative Analysis of Immunochemical Staining for Proliferating Cell Nuclear Antigen Positive Cells in Arterial and Vein Grafts at Four Weeks Postoperatively b
 

Figure 6
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  		Fig 6. (A) to (E) Immunohistochemical staining of the arterial graft sections at the anastomotic site after a four-week interval using anti-alpha-actin antibody. Micrographs (original magnification, x100) are the representative photographs from each group: (A) Control group; (B) PLA-CL film group; (C) 8 µg rapamycin-eluting film group; (D) 80 µg rapamycin-eluting film group; and (E) 800 µg rapamycin-eluting film group. The proliferation and migration of the vascular smooth muscle cells are present in the neointimal layer (arrows), and are diminished with increasing doses of rapamycin.

 

Figure 7
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  		Fig 7. (A) to (E) Immunohistochemical staining of the vein graft sections at the anastomotic site after four-week interval using anti-alpha-actin antibody. Micrographs (original magnification, x100) are the representative photographs from each group: (A) Control group; (B) PLA-CL film group; (C) 8 µg rapamycin-eluting film group; (D) 80 µg rapamycin-eluting film group; and (E) 800 µg rapamycin-eluting film group. The proliferation and migration of the vascular smooth muscle cells are present in the neointimal layer (arrows). The positive cells in the 800 µg rapamycin-eluting film group were less than the other groups.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Discussion
 Acknowledgments
 References
 
We have demonstrated that a rapamycin-eluting biodegradable PLA-CL film applied externally at an anastomotic site can inhibit neointimal hyperplasia of arterial and vein grafts in a canine femoral artery graft interposition model. Inhibition of neointimal growth was associated with reduced mitotic activity and diminished alpha-actin positive smooth muscle cells in the intimal layer. These data indicate that rapamycin-eluting film can prevent stenosis at a vascular anastomotic site, and implicate that the biodegradable film may improve long-term patency of the vascular graft after surgical revascularization. In this viewpoint the drug-eluting biodegradable film is a promising and clinically applicable mode of treatment.

Our drug-eluting film exhibited its inhibitory effect of neointimal hyperplasia on the arterial graft in a dose-response manner. A similar effect was observed in the vein graft model, but the higher dose was required to suppress the neointimal growth to a significant level. This may be related to the fact that graft disease is, in general, more advanced in a vein graft than in an arterial graft. The vein graft bears the potential risk by nature to develop progressive vasculopathy, when used as a bypass conduit [4, 5], which is related to accelerated cellular proliferation and migration in vein grafts. It is challenging to suppress such vein graft disease and to attain long-term patency. Our novel strategy, which can be performed at the time of surgical intervention, should have a significant impact on the management of graft disease.

We proved our hypothesis that rapamycin-eluting biodegradable PLA-CL film can exert a preventive effect of neointimal hyperplasia from the adventitial side. Lately the mechanism of neointimal formation has been investigated extensively, and an important function of adventitia in the process has been reported [18, 19]. The mechanical injury to the vessel per se induces an adventitial angiogenic response. The augmentation of this response leads to an exaggerated increased neointimal formation [20, 21]. It is suggested that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors and possibly migrating into the neointima. We have focused on this pathologic process at the adventitia and developed an externally applicable biodegradable film as a new form of drug delivery system. Schachner and colleagues [22, 23] used rapamycin-containing gel to a perivascular space to observe reduced intimal lesion in a mouse carotid artery vein graft model. Compared with gel, our film has an advantage to be able to localize the target area for drug administration in a sustaining fashion. The film can be applied in various clinical settings, including coronary artery bypass grafting, where the drug-containing gel would spread easily to the pericardial space due to contractile movement of the beating heart, whereas a rapamycin-containing film is placed encircling the end to side anastomotic site in a skirt-like fashion. Our film is very malleable and can be accommodated to variable anastomotic suture sites.

In the present study we constructed arterial and vein anastomotic models by using an end to end fashion, while the anastomotic site of CABG is constructed by using an end to side fashion. End to side anastomosis is supposed to generate turbulence immediately below the anastomosis compared with the end to end anastomosis. The drive toward neointimal hyperplasia is potentially stronger than that being developed at end to end anastomosis. This different mode of anastomosis may influence the outcome in anastomotic graft lesions after application of the rapamycin-eluting film. Although rapamycin was delivered from the adventitial side in this experiment, the origin of the alpha-actin positive cells could not be determined and might be derived from circulating progenitor cells.

In conclusion, we have created a rapamycin-eluting biodegradable film. The film inhibited neointimal hyperplasia of arterial and vein grafts in a canine model. External application of rapamycin is effective to prevent anastomotic stenosis. External application of rapamycin may be effective to prevent anastomotic stenosis, although further studies and longer follow-up are warranted before its clinical use.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
Acknowledgments
 References
 
DR MARC RUEL (Ottawa, Ontario, Canada): Congratulations for your talk and a very interesting work. I have two questions.

Could you first tell us what 800 micrograms of rapamycin represents from a clinical perspective? In comparison, how much rapamycin is found in drug-coated stents? This would give us a better idea of the translational relevance of your findings. Are we talking about a dose that we eventually could apply on a film, put it on human bypass grafts, and have no toxic effect from it?

My second question is about the localization of the effects. Where did you see most neointimal hyperplasia? Was it at the suture line? Was it at the mid segment of the graft? And, compared to controls, where was the rapamycin film having most effect?

DR KAWATSU: Thank you for your question. Is your first question about concentration of rapamycin? In terms of drug concentration, we referred to the clinically available rapamycin-eluting stent. The stent contains 140 micrograms of rapamycin in one centimeter square of stent area. To achieve equivalent drug concentration on a round laboratory dish with the diameter of 28 mm, 861 micrograms of rapamycin is required to spread over it. So we tested 800 micrograms of rapamycin. And we tested 8 and 80 micrograms of rapamycin to examine the dose-response relationship.

And your second question is?

DR RUEL: Where did you see most neointimal hyperplasia and where did you see most of the effect, at the anastomotic suture lines or at the mid segments?

DR KAWATSU: We observed neointimal hyperplasia at anastomotic sites. At graft mid portion, neointimal hyperplasia might be occurred. But in our experiments, we did not observe a microscopic section at mid portion. Sorry, my coauthor will answer your question.

DR SAIKI: I am one of the coauthors of this paper. I appreciate the comment and questions from Dr Ruel. With regard to the rapamycin dose, we referred to the concentration of rapamycin in a commercially available drug-eluting stent as the presenter mentioned. As for the second question, this experiment was designed as an acute model; we therefore specifically looked at the anastomotic sites on the graft side right adjacent to the suture line, since the area is the place where various histological changes occur. It would be very intriguing to see what will happen to the mid portion of the graft. However, the venovasculopathy is rather a chronic lesion. Therefore, we could not assess histologic changes at the mid portion of the graft in our acute model. If we were allowed to mention our impression through the experiment, there was no significant difference in the neointimal lesion occurring at the mid portion of the vein graft in this setting of experiment.

DR FRANK W. SELLKE (Boston, MA): The effect on the arterial anastomosis seemed to be greater than on the venous anastomosis. Do you have a scientific basis for this?

DR SAIKI: I’m sorry, could you repeat the question.

DR SELLKE: The rapamycin seemed to be more effective on the arterial anastomosis compared to the venous anastomosis. Do you have a scientific basis for this?

DR SAIKI: Right. Generally speaking, vasculopathy develops more extensively in the vein graft. But, your point is that what is the underlying mechanism on the altered reactions to rapamycin in the arterial and venous graft in this short-term observation as a separate matter from chronic venous vasculopathy. We speculate that turbulence generated at the anastomotic site was, at least in part, attributable to the exaggerated neointimal growth in the vein graft. As one could appreciate in the slide presentation, the vein graft is larger in diameter than the artery. The difference in the calibers would reasonably cause turbulence. That might be one of the mechanisms we can speculate on the fact that we required larger doses of rapamycin to suppress neointimal growth in the vein graft; namely, rapamycin appeared to be more effective on the artery graft. Furthermore, there should be inherent histological differences between the vein and the artery graft.

DR RUEL: Let me ask you one more question about your film. What are the release kinetics of the rapamycin in the film? And, is the film completely biodegradable? And if so, how long does it take?

DR SAIKI: That’s a very important question. By the end of four week interval, the film had been completely absorbed. We tested other films with various component ratios, for instance, 85 to 15 and 75 to 15 of PLA and CL. These films remained in place, preserving its contour at the end of four weeks of follow-up. For our four weeks of studies, we selected 1 to 1 mole ratio of this PLA and CL copolymer. If you intend to assess a long-term effect of a certain agent incorporated into such a film, you can modulate its component ratio of the bioabsorbable polymer according to the targeted absorption period.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
References
 
The authors wish to express their appreciation to Hideki Fujiwara, MD, Ichiro Yoshioka, MD, and Shinya Masuda, MD, for their superb technical assistance during the operative procedure. This study was supported by a grant-in-aid scientific research from the Japan Society for the Promotion of Science.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Discussion
 Acknowledgments
 References
 

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