Tissue regeneration patterns in acellular bovine pericardia implanted in a canine model as a vascular patch

Tissue regeneration patterns in acellular bovine pericardia implanted in a canine model as a vascular patch Yen Chang,1,2 Huang-Chien Liang,3 Hao-Ji Wei,1,2 Chih-Ping Chu,4,5 Hsing-Wen Sung3 1 Division of Cardiovascular Surgery, Veterans General Hospital-Taichung, Taiwan, Republic of China 2 College of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China 3 Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan, Republic of China, 30013 4 Division of Anatomic Pathology, Taipei Institute of Pathology, Taipei, Taiwan, Republic of China 5 Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, Republic of China Received 16 May 2003; revised 8 December 2003; accepted 22 December 2003 Published online 8 March 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30003 Abstract: It was noted in our previous study that acellular tissues can provide a natural microenvironment for host cell migration and proliferation to accelerate tissue regeneration. The purpose of this study was to further investigate the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehydefixed counterpart. At 1 month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde-fixed acellular patch was significantly thicker than its genipin-fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipinfixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaral-

dehyde-fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehydefixed counterpart. At 6 months postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation or a lack of angiogenesis in the regenerated tissue observed at 1 month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 69A: 323–333, 2004

INTRODUCTION

of cells and soluble proteins. They hypothesized that acellular tissue may remove cellular antigens.5,6 Additionally, acellular biological tissues have been proposed to be used as natural biomaterials for tissue repair and tissue engineering.5– 8 Natural biomaterials are composed of extracellular matrix proteins that are conserved among different species and that can serve as scaffolds for cell attachment, migration, and proliferation.8 In addition to inherent cell compatibility, biological tissues possess the desired shape and, primarily, the strength of the tissues from which the materials are derived.8 This can be a large advantage over synthetic materials in terms of materials processing. In our previous study, the biocompatibility of cellular and acellular bovine pericardia fixed with glutaraldehyde or genipin was evaluated subcutaneously in a growing rat model.7 It was noted that acellular

Xenograft pericardia are often used as a biological patch in cardiovascular surgery for pericardial substitute,1 intracardiac defect repair,2 stenosis enlargement,3 and change of blood stream direction.4 Courtman et al.5 and Wilson et al.6 developed a cell extraction process to render bovine pericardium free Correspondence to: H-W Sung; e-mail: hwsung@che. nthu.edu.tw Contract grant sponsor: National Science Council of Taiwan, Republic of China; contract grant number: NSC-912314-B-075A-005 Contract grant sponsor: Veterans General Hospital, TsingHua, Yang-Ming Joint Research Program; contract grant number: VTY-91-P4-23 © 2004 Wiley Periodicals, Inc.

Key words: acellular tissue; genipin; glutaraldehyde; tissue regeneration; metaplasia

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tissues can provide a natural microenvironment for host cell migration and proliferation to accelerate tissue regeneration. The tissue regeneration rate for the genipin-fixed acellular tissue was significantly faster than its glutaraldehyde-fixed counterpart.7 On the contrary, no tissue regeneration was observed in the cellular tissues. The purpose of this study was to further investigate the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined.

MATERIALS AND METHODS Preparation of test samples Bovine pericardia procured from a slaughterhouse were used as raw materials. The procedure used to remove the cellular components from bovine pericardia was as per a method developed by Courtman et al.5 and Wilson et al.6, with slight modifications.7 Acellular tissues were then fixed in a 0.625% aqueous glutaraldehyde solution (Merck KGaA, Darmstadt, Germany) or a 0.625% aqueous genipin solution (Challenge Bioproducts, Taichung, Taiwan) at 37°C for 3 days. The aqueous glutaraldehyde and genipin solutions were buffered with phosphate-buffered saline (0.1M, pH 7.4; Sigma Chemical Co.). The chemical structure of genipin can be found in the literature.9 The degree of crosslinking for each studied group was determined by measuring its fixation index and denaturation temperature (n ⫽ 5). Details of the methods used in the determinations of fixation index and denaturation temperature of test tissues were previously described.10

Mechanical property The mechanical properties of the glutaraldehyde- and genipin-fixed acellular bovine pericardia were measured and compared with that of the main pulmonary artery of mongrel dogs (similar to those used in the animal study, without crosslinking). Bovine pericardia have been shown to be mechanically and optically anisotropic because of the preferential orientation of collagen fibers in tissue.11 In the study, the preferential orientation of collagen fibers in each test sample was examined with a light placed behind it. Tissue strips in dumbbell shape9 were cut from each studied group parallel to the preferential orientation of collagen fibers for the glutaraldehyde- and genipin-fixed acellular bovine pericardia (n ⫽ 3). For the canine main pulmonary artery, test strips were cut along its longitudinal direction (n ⫽ 3). The procedure used for the mechanical strength measurement was described previously.9

Animal study All test samples were sterilized in a graded series of ethanol solutions with a gradual increase in concentration from 20 to 75% over a period of 4 h. Subsequently, test samples were rinsed in sterilized phosphate-buffered saline and prepared for the animal study. Mongrel dogs, each weighing approximately 20 kg, were used to assess the test samples. After an overnight fast, all animals were anesthetized with intravenous pentothal (10 mg/kg) followed by intubation and maintenance of anesthesia with 1% halothane in oxygen using a volume ventilator. Maintenance intravenous fluid (Ringer’s lactate) was administered at approximately 4 mL/kg per hour. A left lateral thoracotomy at the fourth intercostal space was performed and the pericardium was incised to expose the pulmonary trunk. Two test samples, one from each studied group, were then implanted on the pulmonary trunk (Fig. 1) as described below. A curved vascular clamp was first applied on the main pulmonary artery to partially clamp the pulmonary trunk. A longitudinal defect was then made on the partially clamped pulmonary trunk. Subsequently, one test sample was anchored on the incised defect with continuous 7-0 polypropylene sutures. The smooth (inner) pericardial surface was placed toward the lumen. Finally, the other test sample was anchored on the pulmonary trunk in the same manner. The order of each implanted test sample was rotated from animal to animal. Each implanted test sample was approximately 1.0 cm in width and 1.5 cm in length. The dogs received prophylactic antibiotics for a few days postoperatively (cephalexin, 30 mg/kg orally twice a day). The implanted samples were then retrieved at 1 week (n ⫽ 3), 1 month (n ⫽ 6), or 6 months (n ⫽ 6) postoperatively. Animal care and use was 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. At retrieval, the appearance of each retrieved sample was grossly examined and photographed. Subsequently, the retrieved samples were processed for the light microscopic examination, the denaturation temperature measurement, and the atomic absorption analysis. The atomic absorption analysis was used to determine the calcium content of each retrieved sample. Details of the methods used in the determinations of calcium content of test tissues were previously described. The samples used for light microscopy were fixed in 10% phosphate-buffered formalin for at least 3 days and prepared for histological examination. In the histological examination, the fixed samples were embedded in paraffin and sectioned into a thickness of 5 ␮m and then stained with hematoxylin and eosin (H&E). The stained sections of each test sample were then examined using light microscopy (Nikon Microphoto-FXA) for tissue inflammatory reaction. The number of inflammatory cells observed in each studied case was quantified with a computer-based image analysis system (Image-Pro威 Plus; Media Cybernetics, Silver Spring, MD). Inflammatory cells were visually identified (original magnification ⫻200) and the number was counted for each microscopic field.12 A minimum of five fields was counted

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Figure 1. Schematic drawing and photograph of the glutaraldehyde-fixed acellular patch (AGA) and the genipin-fixed acellular patch (AGP) implanted on the pulmonary trunk in a canine model. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] for each retrieved sample. Also, the thickness of intimal thickening observed on each retrieved sample was quantified with the same computer-based image analysis system.13 Additional sections were stained with safranin O to visualize glycosaminoglycans.14 Immunohistological staining of myofibroblasts/smooth muscle cells was performed on deparaffinized sections with ␣-actin antibodies (Dako Co., Carpinteria, CA) and revealed by a peroxidase–antiperoxidase technique.15 Additional sections were stained for factor VIII with immunohistological technique with a monoclonal anti-factor VIII antibody (Dako).16

Statistical analysis Statistical analysis for the determination of differences in the measured properties between groups was accomplished using one-way analysis of variance and determination of confidence intervals, which was performed with a computer statistical program (Statistical Analysis System, version 6.08; SAS Institute Inc., Cary, NC). All data are presented as mean values ⫾ SD.

RESULTS Test samples Figure 2(a,b) shows photomicrographs of bovine pericardia before and after cell extraction stained with H&E. The bovine pericardium before cell extraction showed a number of intact cells embedded within the connective tissue matrix [Fig. 2(a)]. In contrast, the bovine pericardium after cell extraction (acellular tissue) revealed an intact connective tissue matrix with

Figure 2. Photomicrographs of bovine pericardia (a) before and (b) after cell extraction stained with H&E (⫻200 original magnification). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Figure 3. Stress–strain curves for the glutaraldehyde(AGA) and genipin-fixed (AGP) acellular bovine pericardia and the canine main pulmonary artery (without crosslinking).

no evidence of cells [Fig. 2(b)]. Some open spaces within the acellular tissue were apparent. After fixation, it was found that the color of the genipin-fixed acellular tissue became dark-bluish, whereas the glutaraldehyde-fixed acellular tissue turned yellowish (Fig. 1). The fixation indices of the glutaraldehyde(92.2 ⫾ 0.7%) and genipin-fixed (93.3 ⫾ 0.8%) acellular tissues were comparable (p ⬎ 0.05), whereas the denaturation temperature of the glutaraldehyde-fixed acellular tissue (85.1° ⫾ 0.3°C) was significantly greater than its genipin-fixed counterpart (77.8° ⫾ 0.2°C, p ⬍ 0.05). Stress–strain curves for the glutaraldehyde- and genipin-fixed acellular bovine pericardia and the canine pulmonary trunk (without crosslinking) are presented in Figure 3. All stress–strain curves were nonlinear, each material becoming less extensible with increasing stress. The canine pulmonary trunk showed a significantly greater strain than the glutaraldehyde- and genipin-fixed acellular bovine pericardia (p ⬍ 0.05). This indicated that the canine pulmonary trunk was more extensible than the glutaraldehyde- and genipin-fixed acellular bovine pericardia.

Gross examination All test animals survived until retrieval. Figure 4(a–f) shows photographs of the glutaraldehyde- and genipin-fixed acellular patches retrieved at distinct durations postoperatively. At 1 week postoperatively, no apparent tissue adhesion was observed on the outer surface (extravascular side) of the implanted patch for both studied groups [Fig. 4(a)]. The degree of tissue adhesion was graded as per the observation of tissue ingrowth adhered to the extravascular side of

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each retrieved sample. Mild thrombus formation was found on the inner surface (intravascular side) for two of the glutaraldehyde-fixed acellular patches, whereas it was clean for all of the genipin-fixed acellular patches [Fig. 4(b)]. At 1 month postoperatively, moderate tissue adhesion (aggressive dissection required for separation) and intimal thickening were seen on the outer and inner surfaces, respectively, for both studied groups [Fig. 4(c,d)]. At 6 months postoperatively, it was noted that both studied samples became stiff and felt like bony tissue. Moderate to severe (cannot be separated) tissue adhesion was observed on the outer surface of the glutaraldehyde-fixed acellular patch [Fig. 4(e)]. In contrast, mild (gentle dissection required for separation) to moderate tissue adhesion was observed on the outer surface of the genipin-fixed acellular patch. Intimal thickening was observed on the inner surfaces of both studied samples [Fig. 4(f)]. No aneurysmal dilation of the implanted patch was seen for both studied groups throughout the entire course of the study.

Histological findings Figure 5(a– d) shows the inner and outer layers of the glutaraldehyde- and genipin-fixed acellular patches stained with H&E retrieved at 1 week postoperatively. The solid lines in the figure represent the boundaries of the inner or outer surfaces of the implanted acellular patches. The degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart (Fig. 6, p ⬍ 0.05). Additionally, it was found that inflammatory cells were able to infiltrate into the inner and outer layers of the acellular patches for both studied groups. A thin intimal thickening with some thrombus was observed on two of the glutaraldehyde-fixed acellular patches [Fig. 5(a)], whereas it was clean for the genipin-fixed acellular patches [Fig. 5(b)]. No endothelial cells were visible on the inner surfaces of both studied samples. Figure 7(a– d) presents the inner and outer layers of the glutaraldehyde- and genipin-fixed acellular patches stained with H&E retrieved at 1 month postoperatively. Intimal thickening was observed on the inner surfaces of both studied samples [Fig. 7(a,b)]. The thickness of intimal thickening seen on the glutaraldehyde-fixed acellular patch (1.0 ⫾ 0.2 mm) was significantly greater than that on the genipin-fixed acellular patch (0.4 ⫾0.1 mm, p ⬍ 0.05). Fibroblasts migrated from the host tissue together with neocollagen fibrils were observed in the inner and outer layers of both studied groups. The neocollagen fibrils regenerated in the acellular patches can be further confirmed by the denaturation temperature measure-

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Figure 4. Photographs of the glutaraldehyde- (AGA) and genipin-fixed (AGP) acellular patches retrieved at distinct durations postoperatively. One week postoperatively: (a) and (b); 1 month postoperatively: (c) and (d); 6 months postoperatively: (e) and (f). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ments. Figure 8 shows a thermogram of the genipinfixed acellular patch retrieved at 1 month postoperatively measured by differential scanning calorimetry (DSC). As shown, there were two denaturation-temperature peaks (59.2° and 74.7°C) observed in the DSC thermogram. The amounts of fibroblasts and neocollagen fibrils observed in the genipin-fixed acellular patch appeared to be greater than its glutaraldehyde-fixed counterpart. The degree of inflammatory reaction observed for the genipin-fixed acellular patch was still significantly less than its glutaraldehyde-fixed counterpart (Fig. 6, p ⬍ 0.05). Figure 9(a– d) shows the inner and middle layers of the glutaraldehyde- and genipin-fixed acellular patches stained with factor VIII or ␣-actin retrieved at 1 month postoperatively. As shown, an intact layer of endothelial cells was identified on the intimal thickening generated on the genipin-fixed acellular patch. In contrast, endothelial cells did not universally and totally cover the entire inner surface of the glutaraldehyde-fixed acellular patch [Fig. 9(a,b)]. The middle layers of the glutaraldehyde- and genipin-fixed acel-

lular patches were positively stained with ␣-actin antibodies [Fig. 9(c,d)]. This indicated that myofibroblasts or smooth muscle cells were observed in both the glutaraldehyde- and genipin-fixed acellular patches. However, the amount of cells observed in the genipin-fixed acellular patch appeared to be greater than its glutaraldehyde-fixed counterpart. At 6 months postoperatively, the intimal thickening observed on the glutaraldehyde-fixed acellular patch (1.3 ⫾ 0.2 mm) was significantly thicker than its genipin-fixed counterpart (0.8 ⫾ 0.1 mm, p ⬍ 0.05). Figure 10(a– d) shows the middle layers of the glutaraldehyde- and genipin-fixed acellular patches stained with H&E or safranin O retrieved at 6 months postoperatively. It was found that all implanted test samples had developed areas of chondroid and/or bony metaplasia in their middle layers for both studied groups. Those fibroblasts and myofibroblasts/smooth muscle cells observed at 1 month postoperatively had all disappeared. Instead, chondrocytes, osteoclasts, lamellate bone trabecula, and bone marrow were observed in the acellular patches in four of the test animals, an

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Figure 5. Photomicrographs of the inner [(a) and (b)] and outer [(c) and (d)] layers of the glutaraldehyde- (AGA) and genipin-fixed (AGP) acellular patches stained with H&E retrieved at 1 week postoperatively (⫻200 original magnification). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

indication of bony metaplasia [Fig. 10(a,b)]. In the safranin O stain, significant amounts of glycosaminoglycans were observed in both studied groups [Fig. 10(c,d)]. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart. That is, the area of chondroid and/or bony metaplasia and the amount of glycosaminoglycans observed in the genipin-fixed acellular patch were greater than those in the glutaraldehydefixed acellular patch.

Denaturation temperature and calcium content Results of the denaturation temperature and calcium content for each studied group before implantation and those retrieved at distinct durations postoperatively are presented in Tables I and II. Generally, the denaturation temperature of the glutaraldehydefixed acellular patch declined slightly as the duration of implantation increased (p ⬍ 0.05), whereas that of the genipin-fixed acellular patch stayed approximately the same (p ⬎ 0.05, Table I). However, the

calcium contents for both studied groups increased significantly at 6 months postoperatively (p ⬍ 0.05, Table II).

DISCUSSION In the study, the cell extraction process developed by Courtman et al.5 and Wilson et al.6 was adopted to remove the cellular components of bovine pericardia as an extracellular matrix. Light microscopic examination of the extracted tissue stained with H&E revealed an intact connective tissue matrix with no evidence of cells [Fig. 2(a,b)]. After cell extraction, it left open spaces in the acellular tissue. Our previous transmission electron microscopic examination indicated that all cellular constituents were removed from the bovine pericardium.17 Additionally, the biochemical analyses confirmed that the acellular bovine pericardium consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans.17 Furthermore, the thermal stability (denaturation temperature), mechan-

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Figure 6. Densities of inflammatory cells observed for the glutaraldehyde-fixed acellular patch (AGA) and the genipin-fixed acellular patch (AGP) retrieved at distinct durations postoperatively.

ical property, and capability against enzymatic degradation of the bovine pericardial tissue remained unaltered after cell extraction. Because cells bear classes I and II histocompatibility antigens that are involved in rejection, cell removal may reduce tissue antigenicity and rejection.15 Nevertheless, even with complete extraction of cellular proteins, a cross-species response directed toward the structural proteins if acellular tissue matrices were used as a xenograft would still be anticipated. In an evaluation of a cell-free arterial extracellular matrix in an animal study, Allaire et al.15 found a cross-species graft antigenicity. This cross-species response due to the structural proteins may be further reduced by modifying acellular tissues with a crosslinking agent. Glutaraldehyde has been used extensively as a crosslinking agent for the fixation of biological tissues.18 It was found in our previous study that genipin can react with free amino groups such as lysine, hydroxylysine, or arginine residues in biological tissues.19 The reaction mechanism of genipin with biological tissues was previously proposed.20 It was found in the study that the fixation indices of the glutaraldehyde- and genipin-fixed acellular bovine pericardia were comparable. This indicated that the percentages of free amino groups in test tissues reacted with glutaraldehyde or genipin were approximately equivalent. It is known that reduction of free amino groups in biological tissue diminishes its antigenicity.21 The denaturation temperature of the glu-

taraldehyde-fixed acellular tissue was significantly greater than its genipin-fixed counterpart, due to the differences in their crosslinking structures.9 The mechanical study found that the canine pulmonary trunk (without crosslinking) was significantly more extensible than the glutaraldehyde- and genipinfixed acellular bovine pericardia (Fig. 3). This is because of the very small elastin content in the bovine pericardial tissue (⬃4% by dry weight).5 It is known that elastin can account for ⬎50% by dry weight of an artery.22 Additionally, tissue fixation with a crosslinking agent can limit its extensibility. Consequently, this could cause a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation. In the animal study, it was found that the inflammatory reaction of the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart (Fig. 6). This may be because the cytotoxicity of genipin is significantly lower than glutaraldehyde.7 Inflammatory cells were able to infiltrate into the inner and outer layers of the glutaraldehyde- and genipin-fixed acellular patches [Figs. 5(a– d) and 7(a– d)]. Penetration of cells into the acellular tissue may be caused by the extraction of soluble proteins, lipids, nucleic acids, salts, and carbohydrates, making the tissue more permeable to cellular infiltrates. At 1 month postoperatively, intimal thickening covered with endothelial cells was found on the inner surfaces of both studied groups [Fig. 9(a,b)]. It is known that the pathogenesis of intimal thickening is complex and

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Figure 7. Photomicrographs of the inner [(a) and (b)] and outer [(c) and (d)] layers of the glutaraldehyde- (AGA) and genipin-fixed (AGP) acellular patches stained with H&E retrieved at 1 month postoperatively (⫻200 original magnification). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

may be propagated in part by suboptimal compliance characteristics.23 The intimal thickening observed on the glutaraldehyde-fixed acellular patch was signifi-

Figure 8. Thermogram of the genipin-fixed acellular patch retrieved at 1 month postoperatively measured by a differential scanning calorimeter.

cantly thicker than that on the genipin-fixed acellular patch. An intact layer of endothelial cells was found on the intimal thickening of the genipin-fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehydefixed acellular patch [Fig. 9(a,b)]. It is anticipated that host cells cannot migrate and proliferate on the cytotoxic area. In an in vitro study, we found that the 3T3 fibroblasts cultured on the glutaraldehyde-fixed tissue were not able to survive, whereas those cultured on the genipin-fixed tissue could significantly proliferate and secrete neocollagen fibrils.24 Accordingly, it is speculated that the migration and proliferation of host endothelial cells on the genipin-fixed acellular patch is more rapid, because of a lower cytotoxicity, than its glutaraldehyde-fixed counterpart. As a result, it takes a thicker intimal thickening for the glutaraldehydefixed acellular patch than its genipin-fixed counterpart to block the possible cytotoxic residues released from the implanted samples before host endothelial cells can migrate and proliferate on their surfaces. In the same way, fibroblasts with neocollagen fibrils

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Figure 9. Photomicrographs of the inner and middle layers of the glutaraldehyde- (AGA) and genipin-fixed (AGP) acellular patches stained with factor VIII [⫻800 original magnification, (a) and (b)] or ␣-actin [⫻400 original magnification, (c) and (d)] retrieved at 1 month postoperatively. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

and myofibroblasts/smooth muscle cells were observed in the acellular patches for both studied groups. This indicated that tissue was being regenerated in the acellular patch. The neocollagen fibrils regenerated in the acellular patches can be further confirmed by our denaturation temperature measurements. As shown in Figure 8, two peaks were observed in the DSC thermogram for the genipin-fixed acellular patch retrieved at 1 month postoperatively: one was the denaturation temperature of the original bovine collagen fixed with genipin (74.7°C), and the other was that of the neocollagen fibrils regenerated from the host (i.e., the canine tissue, 59.2°C). Again, this phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart. Tissue regeneration in the implanted acellular patch may prevent it from dilation. It was reported that one of the major pathologic consequences of arterial graft rejection is graft dilation.15 In the study, no aneurysmal dilation was observed for both studied groups throughout the entire course of the study [Fig. 4(a–f)]. At 6 months postoperatively, foci of chondroid

and/or bony metaplasia were found in each retrieved sample [Fig. 10(a,b)]. Metaplasia is a reversible change in which one cell type replaces another cell type.25 The phenomenon of chondroid and/or bony metaplasia may involve fibroblasts and myofibroblasts/smooth muscle cells, observed at 1 month postoperatively, which differentiate along a new pathway into cartilage cells. However, it is known that mature smooth muscle cells cannot be differentiated into chondrocytes.26 Therefore, the cells positively stained by ␣-actin antibodies observed in the middle layers of the glutaraldehyde- and genipin-fixed acellular patches retrieved at 1 month postoperatively may be myofibroblasts [Fig. 9(c,d)]. The finding of myofibroblasts in healing anastomosis has been reported in the literature.23 The observations of chondroid metaplasia in arterial grafts were also reported by other research groups.15,27 Pardo-Mindan et al.27 reported their observations of chondroid metaplasia in the tunica media of aorta, an incidental finding after bypass experiments with saphenous vein grafts between the aorta and the superior vena cava in a canine model. Three dogs also had bony metaplasia. Allaire et al.15 re-

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Figure 10. Photomicrographs of the middle layers of the glutaraldehyde- (AGA) and genipin-fixed (AGP) acellular patches stained with H&E [(a) and (b)] or safranin O [(c) and (d)] retrieved at 6 months postoperatively (⫻200 original magnification). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

ported that there were areas of chondroid metaplasia observed in a cell-free arterial xenograft implanted in a rat model. It is known that tissue may adapt to environmental stimuli by a change in cell differentiation. Pardo-Mindan et al.27 suggested that the compliance mismatch of the bypass over the area of suture could be the reason for chordoid metaplasia. Additionally, they proposed that chronic ischemia due to rupture of vasa vasorum TABLE I Results of the Denaturation Temperature for Each Studied Group Before Implantation and Those Retrieved at Distinct Durations Postoperatively Denaturation Temperature (°C)

in the aorta during an operation may cause metaplasia. It is recognized that ischemia is a usual cause of metaplasia.28 Accordingly, the adverse response of chondroid metaplasia observed in our study may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation or a lack of angiogenesis in the regenerated tissue observed at 1 month postoperatively. Bony metaplasia may then develop as in other chondroid tissues.27 This may explain the observation of significant increases in TABLE II Results of the Calcium Content for Each Studied Group Before Implantation and Those Retrieved at Distinct Durations Postoperatively Calcium Content (␮g/mg)

Implantation Duration

AGA

AGP

Implantation Duration

AGA

AGP

Before implantation One week postoperatively One month postoperatively Six months postoperatively

85.1 ⫾ 0.3 84.0 ⫾ 0.4 82.5 ⫾ 0.2 81.6 ⫾ 0.1

77.8 ⫾ 0.2 75.4 ⫾ 0.2 75.5 ⫾ 0.8 76.2 ⫾ 0.5

Before implantation One week postoperatively One month postoperatively Six month postoperatively

0.4 ⫾ 0.1 6.7 ⫾ 1.2 9.5 ⫾ 1.3 24.4 ⫾ 1.7

0.4 ⫾ 0.1 4.0 ⫾ 1.9 2.3 ⫾ 0.2 32.8 ⫾ 4.8

AGA, the glutaraldehyde-fixed acellular patch; AGP, the genipin-fixed acellular patch.

AGA, the glutaraldehyde-fixed acellular patch; AGP, the genipin-fixed acellular patch.

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the calcium contents for both studied groups at 6 months postoperatively (Table II).

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CONCLUSIONS The results observed at 1 month postoperatively indicated that host fibroblasts, myofibroblasts, and endothelial cells were able to migrate and proliferate in the acellular patches fixed with glutaraldehyde or genipin, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart, perhaps because of the lower cytotoxicity of genipin. However, foci of chondroid and/or bony metaplasia were observed in the acellular patches for both studied groups at 6 months postoperatively.

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