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Ann Thorac Surg 2007;84:864-870
© 2007 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan
b Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Japan
c Department of Bioenvironmental Medicine, Nara Medical University, Nara, Japan
Accepted for publication April 2, 2007.
* Address correspondence to Dr Yoshioka, Department of Cardiovascular Surgery, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan (Email: iyoshi{at}mail.tains.tohoku.ac.jp).
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Abstract |
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 Top Abstract IntroductionMaterial and MethodsResultsCommentReferences |
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Methods: The mechanical property was assessed by measuring tension of suture pull-out at first break test. Fifteen dogs underwent partial pericardial replacement with the bioabsorbable sheets through a left thoracotomy. Macroscopic assessment for severity of adhesions and microscopic evaluation for histologic changes were made at 2, 4, 12, and 24 weeks postoperatively.
Results: The latticed sheets exhibited tenfold higher tension of disruption at the suturing margin compared to our previously developed gelatin sheets (619 ± 141 versus 62 ± 7 gf, p < 0.001), and demonstrated equivalent strength to that of clinically available expanded polytetrafluoroethylene membrane. During rethoracotomy, adhesions between the epicardium and the pericardial substitutes were moderate at the 4-week interval and resolved completely after 12 weeks postoperatively. Inflammatory reaction scores graded into 4 scales on histologic assessment were 2 ± 0.0, 1.6 ± 0.6, and 0.3 ± 0.5 at 4, 12, and 24 weeks, respectively. Inflammatory reaction significantly decreased from the 4-week interval to the 24-week interval after the pericardial replacement (p < 0.05).
Conclusions: The bioabsorbable gelatin sheets latticed with PGA gained improved mechanical properties compared with the previously reported gelatin sheets without impairing its bioabsorbability. The bioabsorbable sheet could eliminate pericardial adhesions after being replaced with regenerated tissue.
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Introduction |
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Several attempts have been made to resolve the problem related to pericardial adhesions after cardiac surgery by using different types of pericardial substitutes, including expanded polytetrafluoroethylene (ePTFE) patches [3, 4], silicone rubber [5], and glutaraldehyde-treated bovine or porcine pericardium xenografts [6–8]. Recently, many authors reported that bioabsorbable patches effectively prevented the formation of pericardial adhesions in their experimental studies [9–11]. However, none of these materials has yet been routinely applicable in the clinical setting, as solid clinical evidence on their beneficial effects is absent.
Previously, we reported that a bioabsobable gelatin pericardial substitute generates less adhesion and inflammatory reaction compared with an ePTFE sheet [9]. Gelatin sheet is, therefore, a promising alternative as a synthetic pericardium. However, it has two substantial drawbacks in structural properties. First, the sheet is too malleable to be easily handled. Secondly, its tensile strength is not high enough to steadily hold sutures to the native pericardium. If the margin of the sheet is disrupted by chance after chest closure, it may migrate into unintended places with heart beating. To overcome these problems, we have created a new form of gelatin sheet latticed with polyglycolic acid (PGA). We have examined its biomechanical property first, and implanted the sheet as a pericardial substitute in a canine model to assess its bioabsorbability and preventive effect on pericardial adhesion.
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Material and Methods |
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Fifteen dogs weighing from 8 to 27 kg (mean, 12 kg) were used as a pericardial reconstruction model. These animals were observed and sacrificed after 2- to 24-week intervals.
Preparation of Gelatin Sheet Latticed With PGA
Gelatin, type I collagen extracted from porcine skin with the alkaline method, was donated by Nippi (Tokyo, Japan). The latticework was created by multiple PGA threads in perpendicular direction. Each thread was made of four knitted PGA fibers. And each PGA fiber was made of 16 PGA fibrils. The size of each lattice is 2.5 mm by 2.5 mm. After placing the latticed fabric on a polystyrene plate, 5 wt% gelatin aqueous solution was cast and air dried to obtain gelatin and PGA integrated gelatin films of 0.16-mm thick. Cross-linking was introduced by heat treatment at 135°C for 14 hours in a vacuum drier. The sheets were finally sterilized with ethylene oxide gas before use. The sheet was manufactured by Gunze (Kyoto, Japan).
Based on the pilot experiments, we confirmed that it takes 6 weeks for the latticed gelatin sheet to biodegrade (Fig 1).
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Fifteen samples (5 in each material) were cut to the size of 3 x 3 cm square, and immersed in phosphate-buffered saline at 25°C for 1 hour. Once all the samples were stabilized, a Nescosuture 5-0 Nylon (Alfresa Pharma Corporation, Osaka, Japan) was passed through a sample at 2 mm distance from the margin. They are fixed on the analyzer, EZ-graph (Shimadzu, Kyoto, Japan). The edge of the suture was pulled at 100 mm/min speed until the sample sheet tears at the suturing margin. Maximal tension at disruption was defined as failure force.
Surgical Procedures
An intravenous line and electrocardiographic monitoring were established. Anesthesia was induced with 2.5% sodium thiopental injection at a dose of 25 mg/kg, and maintained with inhaled halothane in oxygen. Mechanical ventilation was instituted with an approximate tidal volume set at 10 mL/kg body weight, with 100% oxygen, at a rate of 8 to 14 cycles per minute. Under sterile conditions, a left lateral thoracotomy incision was made through the fourth or fifth intercostal space. After pericardial fat and feeding vessels were carefully separated and the surface of the pericardium was exposed, a 3 x 3 cm segment of pericardium was excised about 1 cm anterior to the phrenic nerve. After scattering 5 mL of autologous blood into the pericardial defect in an attempt to promote the formation of adhesions [9], a sheet was anchored to the surrounding pericardium with 5-0 Prolene (Ethicon, Somerville, New Jersey) mattress sutures at each corner of the sheet. Prophylactic antibiotics (cefazolin sodium 40 mg/kg) were given intravenously immediately before the operation.
The dogs were sacrificed at 2 to 24 weeks after operation, and the en bloc hearts, including the entire pericardium and left lung, were removed through a median sternotomy incision.
Evaluations
The implanted membranes were retrieved at 2 weeks (n = 1), 4 weeks (n = 3), 12 weeks (n = 5), and 24 weeks (n = 6) after implantation. Adhesion formation was evaluated by macroscopic findings. The degree of adhesion was quantitatively classified from 0 to 3, according to the adhesion score used by Heydorn and colleagues [12]: 0 = no adhesion; 1 = adhesion that could be readily separated by finger dissection; 2 = adhesion of intermediate strength; and 3 = adhesion necessitated sharp dissection.
In each animal, the specimens of the myocardium, epicardium, and pericardium were taken for light microscopic studies. These were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned at 5 µm. Then, sections were stained with hematoxylin-eosin and elastica-Masson to assess inflammatory reaction along with changes in the pericardial substitutes per se.
Escalating severity of the inflammatory reaction was histopathologically defined by increasing numbers of infiltrating inflammatory cells and inflammatory foci. The inflammatory reaction of each sheet was quantitatively classified from 0 to 3, using the inflammatory reaction score reported by Lu and colleagues [13]: 0 = no cell infiltration; 1 = sparse, focal infiltration of neutrophils, lymphocytes, and plasma cells; 2 = focal infiltration of neutrophils, plasma cells, and lymphocytes; 3 = diffuse infiltration of neutrophils, plasma cells, and lymphocytes. Inflammatory reactions were evaluated by the independent two pathologists, who were masked to the treatment. Quoted scores were the mean of the two scores classified by the pathologists.
Finally, the fibrous membranes regenerated from the gelatin sheet latticed with PGA were harvested 24 weeks after implantation. Immunostaining for cytokeratin and HBME-1 was performed using these samples. Cytokeratin, an intermediate filament, is a constituent of epithelial and mesodermal cells; and HBME-1 has developed as a mesothelial cell marker. The antibodies we used were Pan-keratin (Ventana, Kanagawa, Japan) for cytokeratin and anti HBME-1 antibody (Dako, Kyoto, Japan). The immunostaining process we followed has been reported elsewhere [14, 15].
Statistical Methods
All the results were expressed as mean ± SD. Intergroup comparisons for failure force with tension of suture pull-out at first break test were performed using analysis of variance, and the Scheffe test was conducted as the post-hoc test. Intragroup comparisons for adhesion and inflammatory scores were performed using the corresponding nonparametric test (Mann-Whitney test). A p value of less than 0.05 was considered to denote significance. Analyses were performed with StatMate software (ATMS, Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Macroscopic Findings
After 2 weeks, the gelatin sheet latticed with PGA retained the implanted form and had minimal adhesions between the epicardium and the sheet. This adhesion was easily separated by finger blunt dissection. The surface of the heart was smooth, and the coronary vessels were clearly identifiable (Fig 2A).
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After 12 weeks, the adhesions between the epicardium and the pericardial substitute were none in all five animals. The sheets had been completely absorbed and regenerated with a thin fibrous membrane in 2 animals and in the remaining 3 animals, approximately one fifth of the sheets remained in the center of the replaced area and the remaining peripheral area was regenerated with a thin fibrous membrane. In those 2 animals with diminished sheets, the changes in macroscopic appearance at the surface of the heart were minimal and the underlying structures were identifiable, whereas in those 3 animals with partial absorption of the sheets, the epicardial reaction was moderate and recognition of the underlying structures was impeded by fibrous tissue on the epicardium (Fig 2C).
After 24 weeks, all of the pericardial sheets were absorbed completely and regenerated with a thin fibrous membrane. The adhesions between the pericardial substitute and the epicardium were none or minimal. The fibrous changes at th minimal, and underlying structures was identifiable (Fig 2D).
The adhesion scores are summarized in Table 1 (numbers represent mean ± SD of the adhesion scores). Adhesion formation on the latticed gelatin sheet was significantly less than that on the e-PTFE sheet, at 12 weeks after operation (p < 0.05), when the actual numbers reported in our previous paper were directly compared [9]. In the latticed gelatin group, adhesion formation tended to be less as postoperative time progressed (Table 1).
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After 12 weeks, the pericardial substitute turned out to be the bilayered membrane. The most of gelatin and PGA had been absorbed. The inflammatory reaction resolved practically, but PGA was slower than gelatin to be degradated, therefore, some amount of granulous tissue and chronic inflammatory reaction were observed at the site of the remaining PGA. The surface layer of fibrous connective tissue at the membrane seemed to be regenerative autologus pericardium mimicking the native pericardium (Fig 3C).
After 24 weeks, almost no inflammatory reaction was observed. The pericardial substitute had been completely absorbed and regenerated with the thin membrane. The membrane of fibrous connective tissue became thinner (Fig 3D).
The microscopic scores of inflammatory reaction are summarized in Table 1 (numbers represent mean ± SD of inflammatory scores). Inflammatory reaction scores were high at 4 weeks and decreased after 12 weeks. The actual inflammatory reaction scores in the latticed gelatin sheets were equivalent to those in the gelatin sheets without PGA latticework, when compared with the numbers reported in our previous paper [9].
Immunostaining for Cytokeratin and HBME-1
In the regenerated membrane 24 weeks after pericardial replacement with the latticed gelatin sheet, cytokeratin was positive on both epicardial and pleural sides (Fig 4A); HBME-1 was positive on the epicardial side only (Fig 4B). These findings imply that the regenerated membrane bears the characteristics of mesothelium.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentReferences |
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Prosthetic membranes include silicone rubber, ePTFE membrane, polyethylene film, and Dacron mesh [3–5, 16, 17]. Among them, the efficacy of ePTFE membrane is widely recognized; however, its clinical use has been limited by concerns over the permanence of this sheet and extensive inflammatory reaction resulting in formation of a fibrous capsule. This poses a potential problem for pediatric patients as the heart grows, and leaving a foreign body in place per se predisposes patients to infection over time [18]. Furthermore, ePTFE is not transparent and may interfere with visualization of the cardiac architecture covered with fibrous capsule underneath an ePTFE sheet during reoperations. In contrast, the gelatin sheet latticed with PGA is transparent, and did not generate fibrous capsule underneath the sheet during 24 weeks of follow-up. Therefore, anatomy of the heart, specifically location of the coronary artery was distinctively identifiable at reoperation. More to the point, unlike ePTFE sheet, our latticed gelatin sheet did not leave any foreign materials by being replaced with autologus tissue, which is the virtue of bioabsorbable material.
The efficacy of other types of bioabsorbable films made from polylactide [19] and polyethylene glycol mixed with polylactic acid [20] [21] in the prevention of adhesion formation has been reported. As discussed by Okuyama and colleagues [21], a bioabsorbable film may only be required during the initial postoperative period to limit adhesion formation. Similarly, Iliopoulos and coworkers [19] sought to reduce the retrosternal adhesions using polylactide film with different absorption rates. Their examination demonstrated that the film with slower absorption rate tended to be more effective in reducing adhesion formation. The fact may be explained by the presumption that the polylactide film acts as scaffold for tissue regeneration which ultimately eliminate adhesions. Malm and coworkers [22] studied the course of tissue regeneration after implantation of patches made from polyhydroxybutyrate in a sheep model, and found that these patches were effective in reducing the formation of adhesions and preservation of the coronary anatomy. These materials have also functioned as supporting scaffolds for regeneration of the pericardium. However, some polyhydroxybutyrate still resided in the regenerated pericardium, and phagocytosis by macrophage was histologically ongoing in their experiments. Prolonged chronic inflammatory reaction around the pericardial substitute might in return cause adhesions. Hence, with regard to bioabsorbability of the various pericardial substitutes, it is still controversial to define the optimal absorption rate.
We have previously compared gelatin films with L-lactic acid-alpha-caprolactone copolymer (L-C copolymer) films in a canine model, and demonstrated that gelatin is superior to the L-C copolymer sheet in terms of prevention of pericardial adhesion with efficient absorption process and less evoked inflammation [9]. In an attempt to improve mechanical strength of the film to achieve a function as a sufficient barrier without loosing excellent characteristics of the gelatin sheet, we created a new form of bioabsorbable gelatin sheet by adding PGA as a latticework.
In this study, the histologic examination demonstrated that the inflammatory reaction to the latticed gelatin sheet was heterogeneously present and more remarkable around the PGA filaments than the area of remaining gelatin. Hence, the PGA filaments as a latticework evoked inflammatory response on the biodegradable sheet. This augmented inflammatory reaction during early postoperative period could have resulted in more intense adhesion formation. On the contrary, almost no inflammatory reaction was observed at 24 weeks after implantation. This reduced inflammatory reaction at late term might closely be related to diminished adhesion around the latticed gelatin sheet.
We found that the gelatin sheet latticed with PGA had been replaced with autologus membrane by 12 weeks after implantation. The membrane was composed of the bilayered structure mimicking the native pericardium as evidenced by immunostaining for cytokeratin and HBME-1 positve methothelium-like cells. This finding imply that latticed gelatin sheet remained in place for appropriate period of time to act as a scaffold for tissue regeneration. The placement of a biodegradable film between the beating heart and the sternum during the actual human cardiac surgery will probably impose a multiaxial loading condition to the film. Under such condition, degradation or breakdown of the sheet may be accelerated. The optimal absorption time, therefore, has yet to be determined in a clinically relevant setting.
In summary, we have created a new form of bioabsorbable gelatin sheet latticed with PGA. Its mechanical property and characteristics for manipulation have remarkably improved compared with our previously developed gelatin sheet. We confirmed the usefulness of the sheet in a canine pericardial replacement model. The sheet exerted the benefits of bioabsorbale material, elimination of adhesions, and regeneration of autologus tissue, without a side effect.
Limitations
Limitation of this study should be noted. The experimental findings might not be completely reproducible clinically because the epicardial damage in this study was limited, cardiopulmonary bypass was not performed, and bleeding was minimal. In addition, unlike in the human chest, where the heart is fixed in its place by the surrounding pleura, creation of retrosternal adhesions in a canine model is difficult because of the large space between the sternum and the pericardium. In this study, therefore, formation of adhesions between the posterior aspect of the sternum and the pericardium, seen in humans, was not observed.
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  J. B. Lopes, L. A.O. Dallan, S. P. Campana-Filho, L. A.F. Lisboa, P. S. Gutierrez, L. F. P. Moreira, S. A. Oliveira, and N. A.G. Stolf Keratinocyte growth factor: a new mesothelial targeted therapy to reduce postoperative pericardial adhesions Eur. J. Cardiothorac. Surg., February 1, 2009; 35(2): 313 - 318. [Abstract] [Full Text] [PDF]
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