Laparotomy closure using an elastic suture: A promising approach € vel,3 U. P. Neumann,1 A. Lambertz,1* R. R. M. Vogels,1,2* D. Busch,1 P. Schuster,3 S. Jockenho 1 1 U. Klinge, C. D. Klink 1
Department of General, Visceral and Transplantation Surgery, University Hospital of the RWTH Aachen, Aachen, Germany Department of General Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands 3 Institut fuer Textiltechnik at RWTH Aachen University, Aachen, Germany 2
Received 6 January 2014; revised 25 April 2014; accepted 17 May 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33222 Abstract: Background: Midline laparotomy wound failure like burst abdomen remains one of the major complications after abdominal surgery. The use of sutures with a closer resemblance to abdominal wall physiology, like elastic threads, could decrease the risk of these complications occurring. Thus, we evaluated the possibility of using a new elastic thread composed of thermoplastic polyurethane (TPU) as a suture for the closure of midline laparotomies compared to conventionally used polypropylene (PP) in a rabbit model. Methods: The elastic TPU thread was processed and tensile tests were performed. Twenty female chinchilla rabbits underwent midline laparotomy. They were randomized to a TPU and a PP group depending on the suture used for fascia closure. After 7 or 21 days, the abdominal walls were assessed macroscopically for wound healing complications and were explanted for histopathological investigation. Results: Tensile tests showed a mean elastic elongation of 55.5% and a sufficient material strength of the TPU thread. In animal experiments, there was no difference between the
groups at 7 days; however, the TPU suture showed significantly less CD68 positive cells (p < 0.001) and a higher collagen I/III ratio (p 5 0.011) than PP did after 21 days. The amount of apoptotic cells was significantly elevated in the TPU group (p 5 0.007) after 21 days. No differences were found concerning granuloma size and number of Ki67positive cells. Conclusions: The newly developed TPU thread shows promising tensile characteristics. Midline laparotomy closure is feasible and safe in a rabbit model. Immunohistochemistry indicates similar biocompatibility and wound healing after implantation compared to PP after 21 days. To confirm these findings and to proof long-term capability furC 2014 Wiley Periodicals, Inc. ther studies need to be conducted. V J Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: midline laparotomy closure, abdominal wound failure, burst abdomen, elastic thermoplastic polyurethane suture, tensile tests, polypropylene, immunohistochemistry, rabbit model
€ vel S, Neumann UP, Klinge U, Klink CD. 2014. How to cite this article: Lambertz A, Vogels RRM, Busch D, Schuster P, Jockenho Laparotomy closure using an elastic suture: A promising approach. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Burst abdomen is a continuing problem in surgery with an incidence of up to 3% after major laparotomy closure.1,2 It is associated with significant morbidity and mortality rates.3–5 Although various approaches have been made to optimize fascia closure during the last decades, the incidence of burst abdomen has not been reduced significantly.6–9 As the abdominal wall consists of different muscles which require some degree of flexibility,10 the suture after midline laparotomy should aim to reconstruct the physiological conditions.11 The implantation of a nonelastic suturelike polypropylene does not completely resemble the described physiological situation12,13 and could therefore be a predisposing factor for a later fascia burst. Especially when peaks of intraabdominal pressure are reached—for example, during coughing in the early postoperative
period—the rigid polypropylene thread might tear out of the fascia. Furthermore, a rigid thread causes more tissue constriction leading to necrosis in the loops and subsequently causing slacking of the suture and wound failure.14 The application of an elastic suture to close midline laparotomy could thus be a new approach to optimize fascia closure and to prevent the described problems. Polyurethanes are well known and frequently used in surgery for example as vascular grafts because of their excellent mechanical properties and biocompatibility.15,16 Thus, the aim of this study was to show that midline laparotomy closure with a newly developed, elastic nonabsorbable thermoplastic polyurethane (TPU) suture is feasible and safe in a rabbit model. The suture was compared with the conventionally used polypropylene (PP) thread. Our hypothesis is that the elastic nature of the new threads leads to reduced risk of hernia occurrence.
* Both authors contributed equally to this work. Correspondence to: A. Lambertz (e-mail: [email protected]) € r Bildung und Forschung—BMBF, Berlin, Germany Contract grant sponsor: Bundesministerium fu
C 2014 WILEY PERIODICALS, INC. V
1
MATERIALS AND METHODS
The experiments were officially approved by the local Animal Care and Use Review Committee (TVA 8402.04.2012.A320). All animals received humane care in accordance with the requirements of the German Tierschutzgesetz, §8 Abs. 1 and in accordance to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health. Animals Twenty female, 12-week-old chinchilla rabbits (New Zealand white rabbits, Charles River Laboratories International, Erkrath, Germany) with a mean bodyweight of 3000 6 100 g were randomly divided into two groups consisting of a TPU group (n 5 10) and a polypropylene group (n 5 10). The rabbit’s acclimatization period took 1 week. In every group, five rabbits were randomized to be euthanized after 7 days and the remaining five animals were euthanized after 21 days. Abdominal walls were explanted, assessed macroscopically for wound healing complications and immunohistochemically examined to get an insight into the different phases of wound healing and host response. All animals were kept under standardized conditions: temperature between 22 C and 24 C; relative humidity 50–60%; 12 h of light following 12 h of darkness. The animals had free access to food and water. Food was withdrawn 12 h before and after surgery. Materials The experimental suture material was made out of a nonabsorbable monofilament polycarbonate-based thermoplastic urethane (TPU) thread (used polymer: Chronoflex C93A by Advan Source, Wilmington, MA). To process the polymer to monofilaments a single extruder melt spinning plant was used at process temperatures of 193–215 C. The monofilaments were quenched in a water bath and wound without prior drawing at a winding speed of 20 m/min. The polymer was dried under vacuum conditions at a temperature of 80 C for at least 16 h in advance to the spinning process. To avoid a water uptake after drying the polymer reservoir was continuously purged with dry nitrogen. The control thread used was a monofilament polypropylene (ProleneV; Ethicon, Somerville, NJ) of the same size (U.S.P. size 2-0). R
Tensile tests and cyclic tensile tests The tensile tests as well as the cyclic tensile tests were performed on a tensile tester (Z 2.5 by Zwick Roell AG, Ulm, Germany) under standard atmosphere (20 C; 65% humidity). Both, for the tensile tests as well as for the cyclic tensile tests clamping length was chosen to be 100 mm. All samples were tested at a testing speed of 100 mm/min. For the tensile tests an initial load of 0.5 cN/tex has been applied while the actual testing speed was chosen to be 100 mm/min. For the cyclic tensile tests (number of cycles: 10) an initial load of 0.01 cN/tex has been applied and the testing speed was chosen to be 500 mm/min up to an elongation of 60%.
2
LAMBERTZ ET AL.
Surgical procedure All operations were performed under general anesthesia, aseptic, and sterile surgical conditions and were performed by two different and independent surgeons. Anesthesia was R 10%, induced with 0.2 mg/kg of 10% Ketamin (KetaminV Fa. Sanofi-Ceva, D€ usseldorf, Germany) and 0.3 mL/kg Domitor (Farmos, Gr€aub). The rabbits were placed in supine position; the abdomen was shaved and disinfected with polyvidone–iodine solution. Midline laparotomy was performed over a length of 15 cm (Figure 1). Afterwards, the fascia was group dependent sutured continuously with a TPU or PP thread (Figure 2). Skin closure was performed intracutaR (U.S.P. size 4-0). neous continuously with MonocrylV After 7 or 21 days, the animals were euthanized using a R, lethal dose of 500 mg Pentobarbital-Natrium (NarcorenV Fa. Rh^ one Merieux, Laupheim). The entire abdominal wall was resected (Figure 3) and immediately fixed in 10% formaldehyde for later histological and immunohistochemical assessment. Fascia defects were documented. Histological assessment and immunohistochemical analysis After formaldehyde fixation, samples were embedded in paraffin and cut in 3 mm sections. All sections were routinely stained with hematoxylin and eosin (H&E) and processed at the same time to reduce internal staining variations. The amount of inflammatory and connective tissue formation was analyzed semiquantitatively by measuring the diameter of the inner and outer foreign body granuloma representing the inflammatory infiltrate and the fibrotic tissue reaction. After capturing four granuloma per sample with a digital camera (Olympus C-3030, Hamburg, Germany) separate measurements were performed with the help of a digital image analyzing software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD). Total granuloma size was calculated by adding the measurements of each granuloma and building averages. Histological investigations were performed by two blinded and independent observers. To analyze the host response, immunohistochemistry was used. After pretreatment of the fixed specimen with microwave and citrate-buffer pH 6 for three times, cells were identified by different monoclonal antibodies from Dako (Glostrup, Denmark). The inflammation was assessed by counting the number of macrophages (CD68 expression) in the foreign body granuloma. Ki67 expression was measured as a marker for cell proliferation. “Terminal deoxynucleotidyl transferase nick end labeling” (TUNEL) histochemistry was used as a staining for DNA fragments indicating apoptosis in the granuloma as previously described by Junge et al.17 Finally, measurements of the quality of collagen were performed using Sirius Red staining. These sections were analyzed for ratio of Collagen I/III by crosspolarization microscopy (CPM). Therefore, 5-mm sections were stained for 1 h in Picrosirius solution (0.1% solution of Sirius Red F3BA in saturated aqueous picric acid, pH 2).18 The sections were washed for 2 min in 0.01N HCl, dehydrated, cleared, and mounted in synthetic resin. In
LAPAROTOMY CLOSURE USING AN ELASTIC SUTURE
ORIGINAL RESEARCH REPORT
All other sections were examined by standard light microscopy (Olympus BX51, Olympus, Hamburg, Germany). The percentage of positive stained cells was assessed in an area of 100 mm 3 100 mm with 400-fold magnification and captured by a digital camera, respectively (Olympus C-3030, Olympus, Hamburg, Germany). Analysis was performed using a digital image analyzing software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD).
FIGURE 1. Midline laparotomy in a rabbit model. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
crosspolarization microscopy, thicker collagen type I fibers were stained in red-orange shades, whereas thinner collagen type III appeared as pale-green shades.
Statistical analysis Statistical analysis was performed using the Statistical PackR , Vers.17.0, Chicago, age for Social Sciences software (SPSSV IL). Differences between study groups were analyzed by Kruskal–Wallis test for non-parametric data and in case of significant differences confirmed by Mann–Whitney test. For numeric data, differences were analyzed by ANOVA and in case of significance confirmed by T-Test. p-Values < 0.05 were considered to be significant. All data are represented as mean 6 standard deviation. RESULTS
Tensile tests and cyclic tensile tests of TPU thread Tensile tests of the newly developed thread showed a tensile strength of 1089 cN and an elongation at break of 177% (Figure 4). In cyclic tensile tests, the mean elastic elongation was 56% after 10 cycles with a mean maximum load of 209 cN (Figure 5). The fiber diameter was 290 mm. Animal experiments Three animals died after anesthesia was induced, possibly due to an overdose of anesthetics. In one rabbit, an operative revision had to be performed directly postoperatively because of gaps between the TPU loops, probably due to a technical error using too little traction suturing the new material. After the operations, all animals recovered quickly. None of the rabbits died during the postoperative observational period. During follow-up there were no signs of wound infection or discomfort due to implanted materials. At time of sacrifice, there was one abdominal wall hernia in the polypropylene group after 21 days. At the day of explantation, animals of both groups had a mean bodyweight of 3400 6 100 g. There were no differences between the two study groups. Size of granuloma Analyzing the sizes of the inner and outer granuloma, no significant difference was found between suture types (Table I). In both groups, TPU and PP, the size of the granuloma was significantly higher after 21 days than after 7 days (Table II).
FIGURE 2. Fascia closure with TPU (a) and PP (b) suture. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Immunohistochemical observations We characterized the presence of inflammatory cells (CD68), proliferating cells (Ki67), and apoptotic cells in the granuloma surrounding the materials. In expression of Ki67-positive cells, we did not find any significant difference between the two sutures (7 days: p 5 0.732; 21 days: p 5 0.840). After 21 days, the number of Ki67 positive cells
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00
3
FIGURE 5. Cyclic tensile test of TPU thread: elongation under strain. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
groups, the number of apoptotic cells was higher after 21 days than after 7 days (p < 0.001). Collagen type I/III ratio The collagen type I/III ratio reflecting the maturity of the scar was similar for the two sutures without showing any significant differences after 7 days (p 5 0.389). However, after 21 days, the collagen type I/III ratio was significantly elevated for TPU compared to PP (p 5 0.011; Figure 7). In both threads, the collagen type I/III ratio was significantly higher after 21 days compared to the 7 days values (p < 0.001). FIGURE 3. Abdominal wall explantation, TPU group (a) and PP group (b), after 21 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was significantly lower in both sutures than after 7 days (p < 0.001). In the 7 days group, there was no statistical difference in CD68 expression between PP and TPU suture as well (p 5 0.149). Conversely, the polypropylene thread showed significantly more CD68-positive cells after 21 days than the TPU suture did (p < 0.001; Figure 6). Concerning apoptosis, TPU suture showed significantly more apoptotic cells than PP after 21 days (p 5 0.007), whereas there was no difference seen after 7 days (p 5 0.157). In both suture
FIGURE 4. Tensile test of TPU thread: elongation under strain. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
4
LAMBERTZ ET AL.
DISCUSSION
Fascia closure using a polypropylene suture is one of the standard methods in abdominal surgery because of the favorable properties of this material: it is stable, relatively inert, and it only induces a moderate inflammatory host reaction.19,20 Therefore, it is able to remain in the human body for extended periods of time.21 Nevertheless, the development of burst abdomen is a common problem after laparotomy closure.3,4 Considering the biomechanics of the front abdominal wall, a relevant degree of flexibility has been shown.22 The implantation of a non elastic, rather rigid thread might thus be a predisposing factor for a later fascia burst, especially in peaks of intraabdominal pressure like during coughing. In this context, the idea of using an elastic suture for fascia closure to resemble the physiological situation seems to be obvious. Whereas a certain stability of the implanted textile structure may be widely accepted, the demand for elasticity is still under discussion.23 Under strength, a rigid thread might tear out of the fascia, whereas a too elastic thread might lead to unstable wound closure. As other authors could show that high suture tension is a predisposing factor for laparotomy wound failure,24 the tensile characteristics of the TPU thread with a mean elastic elongation of nearly 56% in combination with its sufficient tensile strength might help
LAPAROTOMY CLOSURE USING AN ELASTIC SUTURE
ORIGINAL RESEARCH REPORT
TABLE I. Histological Assessment and Immunohistochemical Analysis: Differences Between TPU and PP Suture After 7 and 21 Days 7 Days
Inner Granuloma size in mm Outer Granuloma size in mm Ki67 % positive cells CD68 % positive cells Apoptosis Collagen I/III quotient
21 Days
TPU (n 5 16)
PP (n 5 20)
p-Value
TPU (n 5 16)
PP (n 5 16)
p-Value
13.7 6 7.4 55.9 6 7.1 22.4 6 6.1 2.2 6 1.8 4.5 6 1.7 6.1 6 1.5
10.5 6 2.5 53.1 6 13.6 23.0 6 6.2 3.4 6 2.1 5.6 6 2.2 5.9 6 1.6
0.594 0.722 0.732 0.149 0.157 0.389
30.0 6 8.8 156.7 6 39.1 12.3 6 5.4 1.3 6 1.3 38.5 6 12.0 9.2 6 2.3
27.6 6 8.3 183.1 6 43.7 12.1 6 4.0 5.1 6 2.9 24.9 6 6.2 8.2 6 1.9
0.565 0.159 0.84
Department of General, Visceral and Transplantation Surgery, University Hospital of the RWTH Aachen, Aachen, Germany Department of General Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands 3 Institut fuer Textiltechnik at RWTH Aachen University, Aachen, Germany 2
Received 6 January 2014; revised 25 April 2014; accepted 17 May 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33222 Abstract: Background: Midline laparotomy wound failure like burst abdomen remains one of the major complications after abdominal surgery. The use of sutures with a closer resemblance to abdominal wall physiology, like elastic threads, could decrease the risk of these complications occurring. Thus, we evaluated the possibility of using a new elastic thread composed of thermoplastic polyurethane (TPU) as a suture for the closure of midline laparotomies compared to conventionally used polypropylene (PP) in a rabbit model. Methods: The elastic TPU thread was processed and tensile tests were performed. Twenty female chinchilla rabbits underwent midline laparotomy. They were randomized to a TPU and a PP group depending on the suture used for fascia closure. After 7 or 21 days, the abdominal walls were assessed macroscopically for wound healing complications and were explanted for histopathological investigation. Results: Tensile tests showed a mean elastic elongation of 55.5% and a sufficient material strength of the TPU thread. In animal experiments, there was no difference between the
groups at 7 days; however, the TPU suture showed significantly less CD68 positive cells (p < 0.001) and a higher collagen I/III ratio (p 5 0.011) than PP did after 21 days. The amount of apoptotic cells was significantly elevated in the TPU group (p 5 0.007) after 21 days. No differences were found concerning granuloma size and number of Ki67positive cells. Conclusions: The newly developed TPU thread shows promising tensile characteristics. Midline laparotomy closure is feasible and safe in a rabbit model. Immunohistochemistry indicates similar biocompatibility and wound healing after implantation compared to PP after 21 days. To confirm these findings and to proof long-term capability furC 2014 Wiley Periodicals, Inc. ther studies need to be conducted. V J Biomed Mater Res Part B: Appl Biomater 00B: 000–000, 2014.
Key Words: midline laparotomy closure, abdominal wound failure, burst abdomen, elastic thermoplastic polyurethane suture, tensile tests, polypropylene, immunohistochemistry, rabbit model
€ vel S, Neumann UP, Klinge U, Klink CD. 2014. How to cite this article: Lambertz A, Vogels RRM, Busch D, Schuster P, Jockenho Laparotomy closure using an elastic suture: A promising approach. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Burst abdomen is a continuing problem in surgery with an incidence of up to 3% after major laparotomy closure.1,2 It is associated with significant morbidity and mortality rates.3–5 Although various approaches have been made to optimize fascia closure during the last decades, the incidence of burst abdomen has not been reduced significantly.6–9 As the abdominal wall consists of different muscles which require some degree of flexibility,10 the suture after midline laparotomy should aim to reconstruct the physiological conditions.11 The implantation of a nonelastic suturelike polypropylene does not completely resemble the described physiological situation12,13 and could therefore be a predisposing factor for a later fascia burst. Especially when peaks of intraabdominal pressure are reached—for example, during coughing in the early postoperative
period—the rigid polypropylene thread might tear out of the fascia. Furthermore, a rigid thread causes more tissue constriction leading to necrosis in the loops and subsequently causing slacking of the suture and wound failure.14 The application of an elastic suture to close midline laparotomy could thus be a new approach to optimize fascia closure and to prevent the described problems. Polyurethanes are well known and frequently used in surgery for example as vascular grafts because of their excellent mechanical properties and biocompatibility.15,16 Thus, the aim of this study was to show that midline laparotomy closure with a newly developed, elastic nonabsorbable thermoplastic polyurethane (TPU) suture is feasible and safe in a rabbit model. The suture was compared with the conventionally used polypropylene (PP) thread. Our hypothesis is that the elastic nature of the new threads leads to reduced risk of hernia occurrence.
* Both authors contributed equally to this work. Correspondence to: A. Lambertz (e-mail: [email protected]) € r Bildung und Forschung—BMBF, Berlin, Germany Contract grant sponsor: Bundesministerium fu
C 2014 WILEY PERIODICALS, INC. V
1
MATERIALS AND METHODS
The experiments were officially approved by the local Animal Care and Use Review Committee (TVA 8402.04.2012.A320). All animals received humane care in accordance with the requirements of the German Tierschutzgesetz, §8 Abs. 1 and in accordance to the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health. Animals Twenty female, 12-week-old chinchilla rabbits (New Zealand white rabbits, Charles River Laboratories International, Erkrath, Germany) with a mean bodyweight of 3000 6 100 g were randomly divided into two groups consisting of a TPU group (n 5 10) and a polypropylene group (n 5 10). The rabbit’s acclimatization period took 1 week. In every group, five rabbits were randomized to be euthanized after 7 days and the remaining five animals were euthanized after 21 days. Abdominal walls were explanted, assessed macroscopically for wound healing complications and immunohistochemically examined to get an insight into the different phases of wound healing and host response. All animals were kept under standardized conditions: temperature between 22 C and 24 C; relative humidity 50–60%; 12 h of light following 12 h of darkness. The animals had free access to food and water. Food was withdrawn 12 h before and after surgery. Materials The experimental suture material was made out of a nonabsorbable monofilament polycarbonate-based thermoplastic urethane (TPU) thread (used polymer: Chronoflex C93A by Advan Source, Wilmington, MA). To process the polymer to monofilaments a single extruder melt spinning plant was used at process temperatures of 193–215 C. The monofilaments were quenched in a water bath and wound without prior drawing at a winding speed of 20 m/min. The polymer was dried under vacuum conditions at a temperature of 80 C for at least 16 h in advance to the spinning process. To avoid a water uptake after drying the polymer reservoir was continuously purged with dry nitrogen. The control thread used was a monofilament polypropylene (ProleneV; Ethicon, Somerville, NJ) of the same size (U.S.P. size 2-0). R
Tensile tests and cyclic tensile tests The tensile tests as well as the cyclic tensile tests were performed on a tensile tester (Z 2.5 by Zwick Roell AG, Ulm, Germany) under standard atmosphere (20 C; 65% humidity). Both, for the tensile tests as well as for the cyclic tensile tests clamping length was chosen to be 100 mm. All samples were tested at a testing speed of 100 mm/min. For the tensile tests an initial load of 0.5 cN/tex has been applied while the actual testing speed was chosen to be 100 mm/min. For the cyclic tensile tests (number of cycles: 10) an initial load of 0.01 cN/tex has been applied and the testing speed was chosen to be 500 mm/min up to an elongation of 60%.
2
LAMBERTZ ET AL.
Surgical procedure All operations were performed under general anesthesia, aseptic, and sterile surgical conditions and were performed by two different and independent surgeons. Anesthesia was R 10%, induced with 0.2 mg/kg of 10% Ketamin (KetaminV Fa. Sanofi-Ceva, D€ usseldorf, Germany) and 0.3 mL/kg Domitor (Farmos, Gr€aub). The rabbits were placed in supine position; the abdomen was shaved and disinfected with polyvidone–iodine solution. Midline laparotomy was performed over a length of 15 cm (Figure 1). Afterwards, the fascia was group dependent sutured continuously with a TPU or PP thread (Figure 2). Skin closure was performed intracutaR (U.S.P. size 4-0). neous continuously with MonocrylV After 7 or 21 days, the animals were euthanized using a R, lethal dose of 500 mg Pentobarbital-Natrium (NarcorenV Fa. Rh^ one Merieux, Laupheim). The entire abdominal wall was resected (Figure 3) and immediately fixed in 10% formaldehyde for later histological and immunohistochemical assessment. Fascia defects were documented. Histological assessment and immunohistochemical analysis After formaldehyde fixation, samples were embedded in paraffin and cut in 3 mm sections. All sections were routinely stained with hematoxylin and eosin (H&E) and processed at the same time to reduce internal staining variations. The amount of inflammatory and connective tissue formation was analyzed semiquantitatively by measuring the diameter of the inner and outer foreign body granuloma representing the inflammatory infiltrate and the fibrotic tissue reaction. After capturing four granuloma per sample with a digital camera (Olympus C-3030, Hamburg, Germany) separate measurements were performed with the help of a digital image analyzing software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD). Total granuloma size was calculated by adding the measurements of each granuloma and building averages. Histological investigations were performed by two blinded and independent observers. To analyze the host response, immunohistochemistry was used. After pretreatment of the fixed specimen with microwave and citrate-buffer pH 6 for three times, cells were identified by different monoclonal antibodies from Dako (Glostrup, Denmark). The inflammation was assessed by counting the number of macrophages (CD68 expression) in the foreign body granuloma. Ki67 expression was measured as a marker for cell proliferation. “Terminal deoxynucleotidyl transferase nick end labeling” (TUNEL) histochemistry was used as a staining for DNA fragments indicating apoptosis in the granuloma as previously described by Junge et al.17 Finally, measurements of the quality of collagen were performed using Sirius Red staining. These sections were analyzed for ratio of Collagen I/III by crosspolarization microscopy (CPM). Therefore, 5-mm sections were stained for 1 h in Picrosirius solution (0.1% solution of Sirius Red F3BA in saturated aqueous picric acid, pH 2).18 The sections were washed for 2 min in 0.01N HCl, dehydrated, cleared, and mounted in synthetic resin. In
LAPAROTOMY CLOSURE USING AN ELASTIC SUTURE
ORIGINAL RESEARCH REPORT
All other sections were examined by standard light microscopy (Olympus BX51, Olympus, Hamburg, Germany). The percentage of positive stained cells was assessed in an area of 100 mm 3 100 mm with 400-fold magnification and captured by a digital camera, respectively (Olympus C-3030, Olympus, Hamburg, Germany). Analysis was performed using a digital image analyzing software (Image-Pro Plus, Media Cybernetics, Silver Spring, MD).
FIGURE 1. Midline laparotomy in a rabbit model. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
crosspolarization microscopy, thicker collagen type I fibers were stained in red-orange shades, whereas thinner collagen type III appeared as pale-green shades.
Statistical analysis Statistical analysis was performed using the Statistical PackR , Vers.17.0, Chicago, age for Social Sciences software (SPSSV IL). Differences between study groups were analyzed by Kruskal–Wallis test for non-parametric data and in case of significant differences confirmed by Mann–Whitney test. For numeric data, differences were analyzed by ANOVA and in case of significance confirmed by T-Test. p-Values < 0.05 were considered to be significant. All data are represented as mean 6 standard deviation. RESULTS
Tensile tests and cyclic tensile tests of TPU thread Tensile tests of the newly developed thread showed a tensile strength of 1089 cN and an elongation at break of 177% (Figure 4). In cyclic tensile tests, the mean elastic elongation was 56% after 10 cycles with a mean maximum load of 209 cN (Figure 5). The fiber diameter was 290 mm. Animal experiments Three animals died after anesthesia was induced, possibly due to an overdose of anesthetics. In one rabbit, an operative revision had to be performed directly postoperatively because of gaps between the TPU loops, probably due to a technical error using too little traction suturing the new material. After the operations, all animals recovered quickly. None of the rabbits died during the postoperative observational period. During follow-up there were no signs of wound infection or discomfort due to implanted materials. At time of sacrifice, there was one abdominal wall hernia in the polypropylene group after 21 days. At the day of explantation, animals of both groups had a mean bodyweight of 3400 6 100 g. There were no differences between the two study groups. Size of granuloma Analyzing the sizes of the inner and outer granuloma, no significant difference was found between suture types (Table I). In both groups, TPU and PP, the size of the granuloma was significantly higher after 21 days than after 7 days (Table II).
FIGURE 2. Fascia closure with TPU (a) and PP (b) suture. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Immunohistochemical observations We characterized the presence of inflammatory cells (CD68), proliferating cells (Ki67), and apoptotic cells in the granuloma surrounding the materials. In expression of Ki67-positive cells, we did not find any significant difference between the two sutures (7 days: p 5 0.732; 21 days: p 5 0.840). After 21 days, the number of Ki67 positive cells
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | MONTH 2014 VOL 00B, ISSUE 00
3
FIGURE 5. Cyclic tensile test of TPU thread: elongation under strain. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
groups, the number of apoptotic cells was higher after 21 days than after 7 days (p < 0.001). Collagen type I/III ratio The collagen type I/III ratio reflecting the maturity of the scar was similar for the two sutures without showing any significant differences after 7 days (p 5 0.389). However, after 21 days, the collagen type I/III ratio was significantly elevated for TPU compared to PP (p 5 0.011; Figure 7). In both threads, the collagen type I/III ratio was significantly higher after 21 days compared to the 7 days values (p < 0.001). FIGURE 3. Abdominal wall explantation, TPU group (a) and PP group (b), after 21 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was significantly lower in both sutures than after 7 days (p < 0.001). In the 7 days group, there was no statistical difference in CD68 expression between PP and TPU suture as well (p 5 0.149). Conversely, the polypropylene thread showed significantly more CD68-positive cells after 21 days than the TPU suture did (p < 0.001; Figure 6). Concerning apoptosis, TPU suture showed significantly more apoptotic cells than PP after 21 days (p 5 0.007), whereas there was no difference seen after 7 days (p 5 0.157). In both suture
FIGURE 4. Tensile test of TPU thread: elongation under strain. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
4
LAMBERTZ ET AL.
DISCUSSION
Fascia closure using a polypropylene suture is one of the standard methods in abdominal surgery because of the favorable properties of this material: it is stable, relatively inert, and it only induces a moderate inflammatory host reaction.19,20 Therefore, it is able to remain in the human body for extended periods of time.21 Nevertheless, the development of burst abdomen is a common problem after laparotomy closure.3,4 Considering the biomechanics of the front abdominal wall, a relevant degree of flexibility has been shown.22 The implantation of a non elastic, rather rigid thread might thus be a predisposing factor for a later fascia burst, especially in peaks of intraabdominal pressure like during coughing. In this context, the idea of using an elastic suture for fascia closure to resemble the physiological situation seems to be obvious. Whereas a certain stability of the implanted textile structure may be widely accepted, the demand for elasticity is still under discussion.23 Under strength, a rigid thread might tear out of the fascia, whereas a too elastic thread might lead to unstable wound closure. As other authors could show that high suture tension is a predisposing factor for laparotomy wound failure,24 the tensile characteristics of the TPU thread with a mean elastic elongation of nearly 56% in combination with its sufficient tensile strength might help
LAPAROTOMY CLOSURE USING AN ELASTIC SUTURE
ORIGINAL RESEARCH REPORT
TABLE I. Histological Assessment and Immunohistochemical Analysis: Differences Between TPU and PP Suture After 7 and 21 Days 7 Days
Inner Granuloma size in mm Outer Granuloma size in mm Ki67 % positive cells CD68 % positive cells Apoptosis Collagen I/III quotient
21 Days
TPU (n 5 16)
PP (n 5 20)
p-Value
TPU (n 5 16)
PP (n 5 16)
p-Value
13.7 6 7.4 55.9 6 7.1 22.4 6 6.1 2.2 6 1.8 4.5 6 1.7 6.1 6 1.5
10.5 6 2.5 53.1 6 13.6 23.0 6 6.2 3.4 6 2.1 5.6 6 2.2 5.9 6 1.6
0.594 0.722 0.732 0.149 0.157 0.389
30.0 6 8.8 156.7 6 39.1 12.3 6 5.4 1.3 6 1.3 38.5 6 12.0 9.2 6 2.3
27.6 6 8.3 183.1 6 43.7 12.1 6 4.0 5.1 6 2.9 24.9 6 6.2 8.2 6 1.9
0.565 0.159 0.84
