Abstract
Background: Although acellular dermal matrix (ADM) is a widely used graft material for abdominal wall repair, differences in processing methods might yield different healing activities. The aim of this study was to compare the healing effects of two human-derived ADM prototypes in abdominal wall reconstruction. Materials and Methods: A standardized 15×50 mm abdominal wall defect was created in 28 Sprague-Dawley rats, which were then implanted with either an EDTA-treated ADM prototype or a salt/solvent-treated ADM prototype. Adhesion formation, tensile strength, tissue ingrowth, neovascularization and inflammatory cell infiltration were then assessed in the two ADM prototypes during the experimental period. Results: In both ADM prototypes, mild adhesion formation with the omentum was observed at autopsy at one and four weeks post-implantation. Tensile strength was higher at four weeks post-implantation than that at one week post-implantation. Good neovascularization was observed in the periphery of the ADM, but not in the ADM core. Muscles facing the ADM and muscle–ADM junctions were thick and long at one week post-implantation and had been replaced by new host collagen at four weeks post-implantation. No mesothelial cells at the margins were observed at one and four weeks post-implantation. The thickness of the remaining implanted ADM at four weeks post-implantation was less than that at one week post-implantation. There were no statistical differences between the two ADM prototypes in terms of adhesion formation, tensile strength, tissue ingrowth, neovascularization and inflammatory cell infiltration during the experimental period. Conclusion: These results indicate that both ADM prototypes are applicable implant materials for repair of abdominal wall defects.
Resection of abdominal wall tumors, ventral hernias, and severe abdominal wall trauma may require abdominal wall reconstruction (AWR) by general surgeons (1). Graft materials for AWR have been developed to satisfy demands for strength and flexibility, scaffolding for good tissue incorporation, and resistance to infection (2). Prosthetic mesh is a popular material for AWR; however, there may be complications associated with the use of prosthetic mesh, including fistula formation, bowel obstruction, and mesh infection (3). Currently, biosynthetic materials composed of an acellular dermal matrix (ADM) to provide tissue incorporation and neovascularization are widely available for use in grafts (2).
Several commercially available ADMs are marketed for AWR. These ADMs are a biological material derived from human cadaveric dermis. All components of the dermis in ADMs are removed by chemical and physical processing, but the extracellular matrix and basement membrane components are preserved. Epidermal and dermal cells are highly immunogenic skin components, and the remaining extracellular matrix is comprised of collagen and proteoglycan (4). Previous evaluations of the efficacy of human-derived ADM for repair in AWR have relied on animal models, including rat, guinea pig, and monkey; all such studies have verified that human ADM is well-tolerated in AWR (2, 5, 6). However, estimates of ADM efficacy in those studies differed due to differences in tissue preparation.
The purpose of this study was to compare adhesion formation, tensile strength, tissue ingrowth, neovascularization and inflammatory cell infiltration in the two ADM prototypes for repair in a rat model of abdominal wall defects.
Materials and Methods
Experimental animals and preparation of ADM. A total of 28 male Sprague-Dawley rats (250±20 g) were supplied from Hanlim Experimental Animal Laboratory Company (Seoul, Korea) and divided into control and test groups. The control group was implanted with salt/solvent-treated ADM prototype for repair of a rat abdominal wall defect model (n=14). This control ADM was derived from human cadaveric skin and obtained commercially (AlloDerm®; LifeCell Co., Branchburg, New Jersey, USA). The tested group was implanted with enzymes and EDTA-treated ADM for repair of an abdominal wall defect (n=14). To strip the epidermis and remove cellular components from the human dermis, the tested ADM was incubated in Tween 20 (Sigma-Aldrich, St. Louis, Missouri, USA) at room temperature for 3 h and subsequently incubated in 5 mM EDTA at room temperature for 32 h, with shaking at regular intervals. The thickness of the ADM was 0.8-1 mm. Thereafter, the material was freeze-dried and stored at 4-8°C. A standardized 15×50 mm abdominal wall defect was created in each of 28 rats and repaired with the ADMs. In each group, seven rats were sacrificed at one week post-implantation and the other seven rats were sacrificed at four weeks post-implantation. The study protocols were approved by the Institutional Animal Care and Use Committee of Chungbuk National University, Korea (CBNU-084-0905-01).
Induction of 15×50 mm abdominal wall defect (A) underlay repair using acellular dermal matrix (B) in a rat model of abdominal wall defects.
External (A, B) and visceral (C, D) aspects at four weeks post-operation in a rat model of abdominal wall defects. Arrows indicate the implantation site and adhesions are shown.
AWR model. Anesthesia was provided by intraperitoneal injection of zolazepam+tiletamine (20 mg/kg) and xylazine (5 mg/kg). The abdominal median skin was incised for separation of the subcutaneous tissue and exposure of the right abdominal wall, and a 15×50 mm abdominal wall defect was created (Figure 1A). The basement membrane side of the ADM prototype was positioned facing the peritoneal cavity, and a 20×60 mm-sized ADM was implanted and sutured in an underlay fashion using 4-0 nylon (Figure 1B). The same suture material was used in a subcuticular suture pattern. Cefazolin (50 mg/kg) and tramadol (10 mg/kg) were injected subcutaneously twice a day for three days post-implantation.
Peritoneal adhesion scores at one and four weeks post-operation in a rat model of abdominal wall defects. No statistical differences in the amount of adhesion formation between acellular dermal matrix (ADM) prototypes were evident. Data are the mean±S.D. (n=7).
Evaluation of peritoneal adhesions. Adhesion of abdominal contents to the ADM was graded according to Hooker's adhesion scoring scale (9); on the 4-point scale: no adhesion=0; gentle blunt dissection required to free an adhesion=1; aggressive blunt dissection required to free an adhesion=2; and sharp dissection required to free an adhesion=3.
Tensile strength test. At one and four weeks post-implantation, all suture materials were removed and abdominal wall specimens were collected and divided into four equal strips. The middle two strips of the abdominal wall were tested for tensile strength and the other strips were used for histology. For tensile strength testing, each strip was placed vertically between two clamps of a MultiTest 1-I tensiometer (Mecmesin Limited, Slinfold, West Sussex, UK). Force was then applied with a 100 N load cell at a constant speed of 50 mm/min and displacement of 30 mm until rupture occurred. The maximal force for disruption was recorded for each sample.
Histological analysis of wound healing. The tissues to be examined were embedded in paraffin block and 4-μm thick sections were obtained. The sections were stained with Masson's trichrome. Tissue ingrowth and neovascularization and infiltration of inflammatory cells were ascertained by light microscopy examination. Additionally, the sections were stained with cellular marker specific for activated rat macrophages (ED1, AbD Serotec, Oxford, UK) as a macrophage marker or cluster of differentiation –34 (CD34) antibody (Lab Vision Corp., Fremont, CA, USA) as an endothelial cell marker. Each CD34-stained slide or ED1-stained slide was examined by two independent observers under a high-power field (×200). The three most vascularized areas were identified and the vessels were then calculated and recorded. The thickness of the remaining ADM was measured using a digital image analyzer (Image Partner Software, Anyang, Korea).
Tensile strength at one and four weeks post-operation in a rat model of abdominal wall defects. Data are the mean±S.D. (n=7). *p<0.05.
Statistical analysis. The results are expressed as the mean ± SD. Statistical differences were analyzed using Student's t-test, and differences were considered significant when the p-value was less than 0.05.
Results
Peritoneal adhesion assessment. All rats displayed peritoneal adhesion to a greater or lesser extent. Mild adhesion of the abdominal contents (largely omentum) to the ADM margin or to suture holes was evident, while less adhesion to the ADM surface was apparent (Figure 2C, D). No intraperitoneal organ adhesion was apparent in any rat. At autopsy at one and four weeks post-implantation, there were no statistical differences in the abdominal content adhesion scores for the two groups (Figure 3).
Tensile strength scores. Tensile strengths in both groups were greater at four weeks post-implantation than those at one week post-implantation (Figure 4, p<0.05). There were no statistical differences in tensile strength between the two groups during the experimental period.
Histological findings. Collagen fibers before implantation in the two ADM prototypes were thin and short. Good integration of the ADMs with the abdominal wall was observed in all rats four weeks post-implantation compared to those observed one week post-implantation. At one week post-implantation, many inflammatory cells had infiltrated the muscles facing the ADM and the muscle–ADM junction area (Figure 5A); those areas were observed to be thick and long at one week post-implantation and had been replaced by new host collagen at four weeks post-implantation. However, few inflammatory cells had infiltrated the peritoneal cavity facing the ADM. Good neovascularization was observed in the periphery of the ADM, but not in the ADM core at one week (Figure 5B). There were many inflammatory cells in muscles facing the ADM and in the muscle–ADM junctions at one week post-implantation. At four weeks post-implantation, there were few inflammatory cell infiltrations in the peritoneal cavity facing the ADM and the remaining implanted ADM (Figure 6). No mesothelial cells at the margins were observed at one and four weeks post-implantation. The thickness of the remaining implanted ADM at four weeks post-implantation was less than that at one week post-implantation (Figure 7, p<0.05), and there were no differences between the two groups in this regard.
Immunostaining of cellular marker specific for activated rat macrophages (ED1) and cluster of differentiation–34 (CD34) at one week post-implantation in the EDTA-treated group. Photomicrographs show many macrophages (A; arrows) in muscles facing the ADM (▴) and blood vessels (B; arrows) in the periphery of the ADM (p). Original magnification, ×200; bar=100 μm.
Photomicrographs of implanted acellular dermal matrix (ADM) prototypes in a rat model of abdominal wall defects. Many inflammatory cells (i) are present in the muscle (m) facing the ADM at one week post-operation. Fewer inflammatory cells are present in the part of the peritoneal cavity facing the ADM at four weeks post-operation. Note the low amount of ADM graft (g) left in the peritoneal cavity area facing the ADM and the thick new collagen deposition (n) in the muscle facing the ADM at four weeks post-operation. Original magnification, ×100, Masson-trichrome stain; bar=100 μm.
Discussion
The present study was designed to compare the ability of two ADM prototypes to close an abdominal wall defect in a rat model. Both prototypes of human-derived ADM induced good integration with the abdominal wall, low adhesion formation, good tensile strength, and remodeling of host collagen. In this rat model of abdominal wall defect, good integration between the two ADM prototypes and the abdominal wall was evident at four weeks post-implantation. Mild adhesion formation with the omentum was observed at one and four weeks post-implantation in both groups. Tensile strength was higher at four weeks post-implantation than that at one week post-implantation in both groups. At four weeks post-implantation, a small amount of the ADM facing the peritoneal cavity remained in both groups. It is likely that, with more time, the remaining implanted ADM would be replaced by new host collagen, further increasing the tensile strength.
The degradation rate of ADM prototypes should not persistently outpace new collagen deposition in order to prevent weakness, bulging, or failure of ADM grafts (10). The fast degradation rate of salt-/solvent-treated ADM is suitable for cleft palate repair or tympanoplasty (7), while a slower degrading ADM is more suitable for correction of post-rhinoplasty dorsal nasal irregularities or for facial augmentation (11, 12). In our study, both implanted ADMs were mostly replaced by new host collagen at four weeks post-implantation; thus, these degradation rates appear to be suitable to prevent weakness and hernia in this rat model of abdominal wall defects.
In the present study, collagen fibers in the two groups were thinner and shorter than the new host collagen fibers. Macrophages infiltrated the muscles facing the ADM and the junction between ADM and muscles at one week post-implantation, but such infiltration in those regions was replaced by new host collagen at four weeks post-implantation in both groups. A small amount of implanted ADM (less than approximately 25%) remained in the peritoneal cavity facing the ADM at four weeks post-implantation. It is assumed that the remaining implanted ADM would be replaced by new host collagen in both groups.
The thickness of the remaining implanted acellular dermal matrix (ADM) at one and four weeks post-operation in a rat model of abdominal wall defects. Data are the mean±S.D. (n=7). *p<0.05.
According to a previous report, human-derived ADM showed abundant neovascularization in a rabbit subcutaneous implant model (13), and porcine-derived ADM showed limited evidence of vascular ingrowth in a rat subcutaneous implant model (14). In a rat AWR model, the use of a porcine-derived acellular cross-linked dermal matrix revealed neovascularization at the periphery of the implanted ADM, due to the acellular cross-linked architecture of the implant (2). Furthermore, a porcine-derived small intestinal submucosa produced neovascularization in the ADM core (2) due to the presence of multiple factors including vascular endothelial growth factor, transforming growth factor-β, and connective tissue growth factor in the collagen of small intestinal submucosa (15). In the present study, both ADM prototypes displayed neovascularization in the periphery of the ADM, but not in the ADM core. Further animal studies of neovascularization of acellular collagen produced by different species, sources, and processes will be required.
Our results support the belief that ADM prototypes implanted into the human body induce fibroblast incorporation, collagen deposition and collagen maturation (16). However, it has been reported that acellular collagen matrix from human fascia lata was replaced by rodent muscle fiber in a rat AWR model at eight weeks post-implantation (17). It is difficult to compare two different collagen sources, dermis and fascia; however, if implanted substitutes are replaced by muscle tissue for reconstruction of abdominal wall defects, the fascia-derived matrix may be the more suitable product.
In conclusion, both ADM prototypes were replaced by new host collagen in a rat model of abdominal wall defects. Moreover, there was no neoperitonealisation at four weeks post-implantation. These results indicate that the EDTA-treated ADM prototype is thus applicable as an implant material for reconstruction of abdominal wall defects.
Acknowledgements
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009744), Rural Development Administration, Republic of Korea.
- Received July 22, 2013.
- Revision received September 26, 2013.
- Accepted September 30, 2013.
- Copyright © 2013 The Author(s). Published by the International Institute of Anticancer Research.
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