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J Thorac Cardiovasc Surg 2006;132:867-874
© 2006 The American Association for Thoracic Surgery

Evolving Technology

Mesothelium regeneration on acellular bovine pericardia loaded with an angiogenic agent (ginsenoside Rg1) successfully reduces postsurgical pericardial adhesions

Yen Chang, MDa, Po-Hong Lai, MSb, Chung-Chi Wang, MDa, Sung-Ching Chen, PhDb, Wei-Chun Chang, MSb, Hsing-Wen Sung, PhDb,*

a Division of Cardiovascular Surgery, Veterans General Hospital-Taichung and the College of Medicine, National Yang-Ming University, Taipei, Taiwan
b Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.

Received for publication April 26, 2006; revisions received June 5, 2006; accepted for publication June 13, 2006.

* Address for reprints: Hsing-Wen Sung, PhD, Biological Engineering Center, Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan 30013. (Email: hwsung{at}che.nthu.edu.tw).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Objective: Our objective was to reduce postsurgical pericardial adhesions with porous acellular bovine pericardia loaded with ginsenoside Rg1, an angiogenic agent isolated from Panax ginseng (the Acellular/Rg1 patch).

Methods: The acellular/Rg1 patch was used as a substitute to repair a defect created in the pericardium of a rabbit model. A commercially available expanded polytetrafluoroethylene patch, the cellular pericardium (the cellular patch), and the acellular pericardium without loading Rg1 (the acellular patch) were used as controls. The implanted samples were retrieved at 1 and 3 months after surgery (n = 5 per group at each time point).

Results: It was found that each side of the implanted patch could be remesothelialized provided that regeneration of neo–tissue fibrils occurred initially on its surfaces. Because remesothelialization did not take place on the surfaces of the expanded polytetrafluoroethylene and cellular patches, moderate to severe adhesions to the lung and epicardium were clearly observed. As compared with the cellular patch, the acellular patch significantly reduced postsurgical pericardial adhesions, especially on its lung side, as a result of remesothelialization. In the presence of Rg1, a faster remesothelialization was observed on each side of the acellular/Rg1 patch. Therefore, the acellular/Rg1 patch was free of any adhesions to the lung; however, there was still a filmy adhesion to the epicardium observed in 3 of the 5 studied animals at 3 months after surgery, due to incomplete remesothelialization.

Conclusions: The acellular/Rg1 patch effectively repaired pericardial defects in rabbits and successfully reduced the formation of pericardial adhesions.

Abbreviations and Acronyms ECM = extracellular matrix; e-PTFE = expanded polytetrafluoroethylene


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Complete closure of the pericardium after cardiac operations is generally accepted as being advantageous should subsequent cardiac surgery prove to be necessary.1Go Various biologic or synthetic sheets such as bovine pericardia and expanded polytetrafluoroethylene (e-PTFE) have been used as a pericardial substitute.2,3Go However, there have been no adequate substitutes that reduce adhesions.3Go Prevention of adhesions through pericardial substitution has therefore become a matter for investigation.4Go

In our previous study, it was found that acellular bovine pericardial tissues fixed with genipin could provide a natural microenvironment for host cell migration and may be used as a tissue-engineering extracellular matrix (ECM) to accelerate tissue regeneration.5Go Genipin, a naturally occurring cross-linking agent, can be isolated from the fruits of Gardenia jasminoides Ellis.6Go The cytotoxicity of genipin is significantly less than that of glutaraldehyde, a commonly used cross-linking agent.7,8Go

Tissue engineering is aimed at manipulating regeneration of host tissues in an implanted ECM to repair the defects in the human body. In this study, it was hypothesized that tissue regeneration within the implanted substitute might significantly reduce postsurgical pericardial adhesions. The primary challenge for tissue regeneration is to develop a vascular supply that can support the metabolic needs of the engineered tissues.9Go Investigations have incorporated angiogenic factors such as basic fibroblast growth factor and others in ECMs to stimulate angiogenesis.10Go However, the biologicl activity of protein-type growth factors may not last long in vivo because of their poor stability.10Go It was shown in our previous study that ginsenoside Rg1 (Rg1), a natural compound isolated from Panax ginseng, enhanced several human umbilical vein endothelial cell activities in vitro and Rg1-associated induction of angiogenesis enhanced tissue regeneration in vivo.11Go

This study was to evaluate our hypothesis that using acellular bovine pericardia loaded with Rg1, as a pericardial substitute, may significantly reduce postsurgical pericardial adhesions in a rabbit model. A commercially available e-PTFE patch (Gore-Tex, W. L. Gore & Associates, Inc, Flagstaff, Ariz) together with the cellular bovine pericardium and the acellular pericardium without loading Rg1 were used as controls.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Preparation of Test Samples
The procedures used to remove the cellular components from bovine pericardia were based on a method previously reported by Courtman and colleagues,12Go with slight modifications.13Go To increase the pore size and porosity within test samples, acellular tissues were treated additionally with acetic acid and subsequently with collagenase.14Go Afterward, the cellular (cellular patch) and acellular tissues were fixed in a 0.05% genipin (Challenge Bioproducts, Taichung, Taiwan) aqueous solution (phosphate-buffered saline, pH 7.4) at 37°C for 3 days. The cross-linking degree of each fixed tissue was determined by measuring its fixation index and denaturation temperature (n = 5).8Go Five tissue strips from each studied group were mechanically examined.14Go

To facilitate cell infiltration and repopulation, the dense layer on each side of acellular tissues was sliced off (the Acellular patch) with a cryostat microtome (Leica, Wetzlar, Germany). After preparation of test samples, the cellular and acellular patches were processed for light-microscopic and scanning electron microscopic examinations to investigate their ultrastructures. The pore size of the Acellular patch, stained with hematoxylin and eosin, was determined under a microscope, and its porosity was measured by helium pyknometry.14Go Test samples were sterilized in a graded series of ethanol solutions for the following animal study.

To load Rg1, the sterilized acellular patch was freeze-dried under aseptic conditions and subsequently immersed in an Rg1 aqueous solution (10 mg/mL) for 36 hours (the acellular/Rg1 patch). The amount of Rg1 loaded in the patch was determined by measuring the difference between the initial and residual amounts of Rg1 in the solution by using high-performance liquid chromatography (n = 5). To study the profile of the Rg1 released from the acellular/Rg1 patch, test samples (n = 5) were immersed in phosphate-buffered saline buffer (pH 7.4) and incubated on a rotating incubator at 37°C. The amount of Rg1 in releasing media was determined by high-performance liquid chromatography.

Animal Study
Animal care and use were performed in compliance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (revised 1996). The studied patches (10 x 10 mm) were used to repair pericardial defects surgically created in the pericardium of rabbits (New Zealand White rabbits; 2.5-3.0 kg).2Go The implanted samples were retrieved at 1 or 3 months after surgery (n = 5 per group at each time point).

At retrieval, a grading system was used to describe the degree of pericardial adhesions as follows15Go: no adhesions (grade 0); filmy adhesions easily separable by light blunt dissection (grade 1); more tenacious and cohesive adhesions necessitating division by aggressive blunt or moderate sharp dissection (grade 2, moderate adhesions); and extremely cohesive adhesions that bind the test sample tightly to the lung or epicardium (grade 3, severe adhesions). The adhesion formation was evaluated by 2 independent observers blinded to the animal's treatment group. Subsequently, the retrieved samples were prepared for the histologic examination.

Light Microscopic Examination
In the histologic examination, the fixed samples were embedded in paraffin, sectioned into a thickness of 5 µm, and then stained with hematoxylin and eosin. Also, sections of test samples were stained with Masson trichrome for the detection of collagen fibrils and muscle fibers and stained with safranin O to visualize glycosaminoglycans. Additional sections were stained with a van Gieson solution to visualize mesothelial cells.16Go

A monoclonal antibody against {alpha}-smooth muscle actin (DAKO, Carpinteria, Calif) was used to identify smooth muscle cells. Additional sections were stained for factor VIII with a immunohistological technique with a monoclonal anti–factor VIII antibody (DAKO).17Go The density of neocapillaries in each studied sample was quantified with a computer-based image-analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, Md) and converted to vessels per square millimeter.18Go Five different microscopic fields (400x by ECLIPSE-E800; Nikon, Tokyo, Japan) in each section were randomly selected. Immunohistochemical staining for neocollagen type I and III expression in the rabbit model was performed on paraformaldehyde-fixed slides by using rabbit antibodies (10 µg/mL; Rockland, Gilbertsville, Pa) as the primary antibody and revealed by a peroxidase-antiperoxidase technique. Test samples before implantation were used as a control.

Statistical Analysis
Statistical evaluation was performed by using SAS version 6.08 (SAS Institute, Cary, NC). Results are listed as mean ± SD. The differences between the adhesion scores of groups were statistically analyzed by using the {chi}2 test. One-way analysis of variance was applied to data on fixation index, denaturation temperature, mechanical strength, and cumulative release of Rg1.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Test Samples
Figure E1 presents photomicrographs of the cellular and acellular patches stained with hematoxylin and eosin and their surface morphologies inspected by scanning electron microscope. As shown, the cellular patch showed a number of cells embedded within the connective tissue matrix, whereas the acellular patch revealed large open spaces (pores) with increased interconnectivity. Additionally, the surface of the cellular patch appeared partially sealed in most regions. After the dense layer on each side of the acellular tissue was sliced off, its porous structure beneath was revealed (the acellular patch).


Figure 1
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  		Figure E1. Photomicrographs (stained with hematoxylin and eosin [H&E]; original magnification, 200x) and scanning electron micrographs (SEM) of the Cellular and Acellular patches used in the study.

 
The denaturation temperature (74.8°C ± 0.2°C) and fixation index (58.4% ± 4.7%) of the acellular patch were comparable with those (75.2°C ± 0.4°C; 57.8% ± 5.4%) of the cellular patch. However, the fracture tension value (1.1 ± 0.2 kN/m) of the acellular patch was significantly lower than that (6.4 ± 0.5 kN/m) of the cellular patch. The pore size and porosity of the acellular patch were 159.8 ± 26.7 µm and 94.9% ± 1.7%, respectively. The amount of Rg1 loaded in the acellular/Rg1 patch was 500.5 ± 6.7 µg per sample patch. After an initial burst, Rg1 was released gradually in a sustained manner. After 7 days of incubation, the cumulative amount of Rg1 released from the acellular/Rg1 patch was 95.1% ± 3.1%.

Gross Examination
No postoperative complications, such as pneumothorax or respiratory insufficiency, were observed in the study. Table 1 shows the results of the macroscopic inspection of the implanted samples observed at distinct durations after surgery. At 1 month after surgery, filmy to moderate adhesions (25-50% of the patch surface) to the lung and moderate to severe adhesions (>50% of the patch surface) to the epicardium were observed for the e-PTFE and cellular patches, whereas a filmy adhesion (<25% of the patch surface) to the lung and a filmy to moderate adhesion (25-50% of the patch surface) to the epicardium were seen for the acellular patch. In contrast, no adhesions to the lung and a filmy adhesion (<25% of the patch surface) to the epicardium were found for the acellular/Rg1 patch.


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  		TABLE 1. Adhesion scores * of the e-PTFE, cellular, acellular, and acellular/Rg1 patches observed at distinct durations after surgery (n = 5 at each time period)
 
At 3 months after surgery (Figure 1), filmy to moderate adhesions (25-50% of the patch surface) to the lung and moderate to severe adhesions (>75% of the patch surface) to the epicardium were observed for the e-PTFE and cellular patches, whereas a filmy to moderate adhesion (25-50% of the patch surface) to the lung and epicardium was seen for the acellular patch. In contrast, the acellular/Rg1 patch was free of any adhesions to the lung. Whereas 2 of the 5 studied animals in the acellular/Rg1 group were free of any adhesions to the epicardium (Figure 1), the other 3 animals had a filmy adhesion attached to part of the patch surface (< 10%) and along the suture line. No dilation of the implanted patches was seen for all studied groups throughout the entire course of the study.


Figure 1
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  		Figure 1. Photographs of the e-PTFE, cellular, acellular, and acellular/Rg1 patches (marked by * at one of the corners of each implanted patch) retrieved at 3 months after surgery. Arrows point to the sites of pericardial adhesions.

 
Histologic Findings
At 1 month after surgery, a large number of inflammatory cells were found in the vicinity of the e-PTFE and cellular patches, whereas many host cells (mostly inflammatory cells) were infiltrated into the acellular patch (Figure E2). In contrast, host cells (fibroblasts and inflammatory cells) together with neo–tissue fibrils were clearly observed in the inner (the epicardium side) and outer (the lung side) layers of the acellular/Rg1 patch, an indication of tissue regeneration. Additionally, mesothelial-like cells were visible on the outer surface of the acellular/Rg1 patch. However, no such observation was found on the surfaces of the e-PTFE, cellular, or acellular patches. Instead, fibrous tissue was firmly attached to both sides of these studied groups.


Figure 2
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  		Figure E2. Photomicrographs (stained with hematoxylin and eosin; original magnification, 200x) of the e-PTFE, Cellular, Acellular, and Acellular/Rg1 patches retrieved at 1 month after surgery.

 
At 3 months after surgery, many inflammatory cells were still observed adjacent to the e-PTFE and cellular patches, with no signs of tissue regeneration (fibroblasts and neo–tissue fibrils; Figure 2). For the acellular patch, inflammatory cells had almost disappeared; instead, fibroblasts and neo–tissue fibrils, as well as neocapillaries (ie, endothelial cells stained with factor VIII; Figure E3), were observed. In contrast, more fibroblasts, neo–tissue fibrils, and neocapillaries were seen in the acellular/Rg1 patch as compared with its counterparts observed at 1 month after surgery. It was found that the density of neocapillaries infiltrated into the acellular/Rg1 patch (260 ± 19 vessels per square millimeter) was significantly greater than that for the acellular patch (164 ± 27 vessels per square millimeter).


Figure 2
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  		Figure 2. Photomicrographs (stained with hematoxylin and eosin; original magnification, 200x) of the e-PTFE, Cellular, Acellular, and Acellular/Rg1 patches retrieved at 3 months after surgery.

 

Figure 3
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  		Figure E3. Photomicrographs of the Acellular and Acellular/Rg1 patches retrieved at 3 months after surgery: stained with Factor VIII (original magnification, 800x), Masson trichrome (original magnification, 200x), a monoclonal antibody against {alpha}-smooth muscle actin ({alpha}-SMA; brown staining; original magnification, 800x), and safranin O ( red staining; original magnification, 200x).

 
The neo–tissue fibrils regenerated in the acellular and acellular/Rg1 patches were identified to be neocollagen fibrils (stained blue) by the Masson trichrome stain (Figure E3). The neocollagenous tissues were further shown by the immunohistochemical stains to contain collagen type I and III fibrils (Figure 3). Additionally, neomuscle fibers (stained red by the Masson trichrome stain) together with {alpha}-smooth muscle actin positively stained cells were observed in the acellular/Rg1 patch (Figure E3). Furthermore, there were neoglycosaminoglycans regenerated within the pores of the acellular and acellular/Rg1 patches recognized by the safranin O stain (stained pink).


Figure 3
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  		Figure 3. Photomicrographs of the acellular and acellular/Rg1 patches retrieved at 3 months after surgery: immunohistochemical stains identified neocollagen type III (brown staining; original magnification, 800x); van Gieson stain identified mesothelial cells (original magnification, 800x).

 
Neomesothelial cells, identified by the van Gieson stain, lying on the regenerated tissue fibrils were clearly observed on the outer surface (the lung side) and the inner surface (the epicardium side) of the acellular/Rg1 patch (Figure 3). For that not covered with neomesothelial cells on the inner surface of the acellular/Rg1 patch, fibrous tissue adhesion was found in 3 of the 5 studied animals. In contrast, neomesothelial cells were observed only on the outer surface of the acellular patch.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
Inflammatory cells typical of a foreign-body response were mostly present adjacent to the e-PTFE and cellular patches, and no tissue regeneration was observed at 1 and 3 months after surgery (Figures E2 and 2). In contrast, host cells were able to infiltrate into the acellular patch. This may be attributed to the fact that the acellular patch, with a porous structure, can provide a larger open space for cell infiltration and repopulation (Figure E1) as compared with the e-PTFE and cellular patches.

At 1 month after surgery, fibroblasts and neo–tissue fibrils, an indication of tissue regeneration, were clearly observed to fill the pores within the acellular/Rg1 patch, whereas there were merely a large number of inflammatory cells in the acellular patch (Figure E2). This indicated that in the presence of Rg1, tissue regeneration in the acellular patch can be significantly promoted because of the greater number of neocapillaries infiltrated. Vascularization in ECMs to support the metabolic needs of the engineered tissues is generally a prerequisite for achieving appropriate tissue regeneration and function.19Go

Rg1 is one of the active components of saponin in Panax ginseng.20Go Panax ginseng has long been used in herbal medicine in the repair of intractable skin ulcers of patients with diabetes mellitus.21Go Angiogenesis is known to play an important role in the repair of ulcers. Sengupta and colleagues22Go found that Rg1 promoted the proliferation of, chemoinvasion of, and tubulogenesis by endothelial cells in vitro. Their results suggested that in addition to promoting the synthesis of nitric oxide synthase enzyme, Rg1 can activate through the phosphatidylinositol-3-kinase -> phospho-Akt -> nitric oxide synthase pathway.

At 3 months after surgery, the neotissues regenerated in the acellular/Rg1 patch seemed to be more compact and organized than those in the acellular patch (Figures E3 and 3). Additionally, the density of neocapillaries infiltrated into the acellular/Rg1 patch was significantly greater than that with the acellular patch. It is interesting to note that the acellular/Rg1 patch was positively stained with a monoclonal antibody against {alpha}-smooth muscle actin (Figure E3), thus indicating that smooth muscle cells were present. Smooth muscle cells permit formation of a muscular tissue (observed in the acellular/Rg1 patch, stained red by Massson trichrome; Figure E3) in addition to collagen formation (stained blue by Massson trichrome).23Go This finding suggested that multipotential cells from the systemic circulation (ie, via the neocapillaries) or from the surrounding tissues may be relevant to the presence of smooth muscle cells in the acellular/Rg1 patch.24Go Such an observation was not found in the acellular patch up to 3 months after implantation, possibly as a result of a lower density of neocapillaries infiltrated into the acellular patch than the acellular/Rg1 patch.

At 1 month after surgery, an intact layer of neomesothelial cells (ie, completed remesothelialization), identified by the van Gieson stain, was already present on top of the neo–tissue fibrils regenerated in the outer layer (the lung side) of the acellular/Rg1 patch. This finding was not seen on the other side (the epicardium side) of the acellular/Rg1 patch until 3 months after implantation. At 3 months after surgery, an intact layer of neomesothelial cells was resting on the regenerated tissues in the inner layer of the acellular/Rg1 patch in 2 of the 5 studied animals (Figure 3), whereas remesothelialization was still not completed for the rest of the studied animals. No neomesothelial cells were present on the surfaces of the acellular patch until 3 months after implantation, and these cells were observed only on its outer surface (the lung side). In contrast, no neomesothelial cells were seen on either sides of the e-PTFE and cellular patches throughout the entire course of the study.

The above-mentioned results suggest that each side of the implanted patch can be remesothelialized provided that regeneration of neo–tissue fibrils occurred initially on its surfaces. Several authors have suggested that multipotential cells present within the collagen matrix can differentiate into mesothelial cells and contribute to surface re-epithelialization.25Go The process of remesothelialization on the inner surface (the epicardium side) of the implanted patch seemed to be slower than on the outer surface (the lung side). It is speculated that the rubbing between the host epicardium and the inner surface of the implanted patch during each heartbeat may interfere with the attachment of multipotential cells on the regenerated neo–tissue fibrils.

The native pericardium consists of a single layer of flattened mesothelial cells resting on each side of loose connective tissues.26Go Remesothelialization on the surfaces of the implanted patch is assumed to play an important role in the prevention of postsurgical pericardial adhesions. It is well documented that mesothelial cells prevent adhesions.27Go Whitaker and associates28Go reported that a pure culture of mesothelial cells was able to induce fibrinolysis. Another study suggested that the mesothelial fibrinolytic properties are associated with the secretion of tissue plasminogen activator.27,29Go These results likely explained the observation that once the surface of the implanted patch was populated with mesothelial cells, it remained resistant to adhesion formation.

Because remesothelialization did not take place, as a result of a lack of regeneration of neo–tissue fibrils, on the surfaces of the e-PTFE and cellular patches, pericardial adhesions to the lung and epicardium were clearly observed. The pericardial adhesions for both studied groups became more remarkable with time (Table 1). The e-PTFE patch is the most widely used synthetic pericardial substitute. Unfortunately, the e-PTFE patch has a number of significant disadvantages; it remains in situ as a permanent foreign body and causes an extensive inflammatory reaction.30Go

As compared with the cellular patch, the acellular patch significantly reduced postsurgical pericardial adhesions, especially on its lung side (Figure 1). As discussed previously, the reduction of formation of pericardial adhesions on the lung side of the acellular patch can be attributed to remesothelialization on its outer surface. In the presence of Rg1, angiogenesis and tissue regeneration were further promoted in the acellular patch, and, thus, a faster remesothelialization was observed on each side of the acellular/Rg1 patch. Therefore, the acellular/Rg1 patch was free of any adhesions to the lung throughout the entire course of the study (Figure 1). However, there was still a filmy adhesion to its epicardium side in 3 of the 5 studied animals at 3 months after surgery, because of incomplete remesothelialization on the outer surface of the acellular/Rg1 patch.

On the basis of the aforementioned results, it is expected that a greater amount of Rg1 loaded in the acellular patch may further speed up angiogenesis and tissue regeneration in the implanted substitute. This may accelerate remesothelialization on its inner surface and eventually prevent its adhesions to the epicardium.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
The results obtained in the study indicate that the acellular/Rg1 patch effectively repaired pericardial defects in rabbits and successfully reduced the formation of pericardial adhesions. The neomesothelium formed on each side of the acellular/Rg1 patch physically and functionally replaced the normal pericardium.


   Footnotes
 
This work was supported by grants from the Ministry of Economic Affairs (94-EC-17A-17S1-0009) and the National Health Research Institute (NHRI-EX95-9518EI), Taiwan, ROC.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

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