Surgery Today Jpn. J. Surg. (1992) 22:333-338
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SURGERYTODAY © Springer-Verlag 1992
The Hemostatic Effect of Deacetylated Chitin Membrane on Peritoneal Injury in Rabbit Model MANABU FUKASAWA~1 HIROMASA ABE, 1 TOSHIAKI MASAOKA~1 HIROYUKI ORITA, 1 HIDEO HORIKAWA,2 JOSEPH O. CAMPEAU, 3 and MASAHIKO WASHIO 1
aSecondDepartmentof Surgery,2Departmentof Anesthesiology,YamagataUniversitySchoolof MedicineZao-lida2-2-2, YamagataCity990-23,Japan, and 3LivingstonReproductiveBiologyLaboratory,Universityof SouthernCalifornia,USA
Abstract: In this study, we determined the effect of 80% deacetylated chitin (DAC-80) membrane on postsurgical bleeding after visceral and parietal peritoneal abrasion. Japanese white rabbits underwent a midline laparotomy followed either by a bilateral peritoneal sidewall abrasion (4 × 4cm) or an abrasion of liver surface (3 × 2cm). The injured surface was then covered with a 0.2 mm thick DAC-80 membrane. On postsurgical day 2, the rabbits were sacrificed and the amounts of postsurgical bleeding was determined by quantitating the number of red blood cells recovered in 50 ml peritoneal lavage fluid. The DAC-80 membrane was found to reduce postsurgical bleeding after the abrasion of liver surface (treated with DAC-80 membrane: 2.9 + 0.8; control: 24.6 + 5.9 × l 0 s cells/peritoneal cavity, P < 0.005). This same hemostatic activity was not observed after application in the peritoneal sidewall abrasion model. We also measured plasminogen activator activity (PA) and urokinase inhibitory (PAI) activity in the spent culture media of macrophages recovered from the postsurgical peritoneal exudate. The DAC80 membrane reduced the PA secretion from postsurgical macrophages after liver surface abrasion (treated with DAC8 0 : 2 . 8 _+ 0.7; control: 3.9 + 0.9mPU/ml). The DAC-80 membrane also showed similar effects on PA secretion after peritoneal sidewall abrasion. No significant effects were found in the secretion of PAI by postsurgical macrophages in both surgical models. These findings suggest that the DAC-80 membrane may have hemostatic activity through the modulation of fibrinolytic activity of peritoneal exudative macrophages.
Introduction
Chitin is a large molecular weight polymer of D-1,4acetyl-D-glucosamine. Previous reports have indicated that deacetylated chitin (chitosan) is hemostatic and accelerates wound repair. 1"2 In addition, chitin/ chitosan activates macrophages. 3 Accordingly, this compound was tested for use as a topical agent in tissue repair. Our previous studies addressed the healing of the peritoneum after surgical injury with a focus on adhesion formation. Macrophages are one of the principle cells in peritoneal healing and produce plasminogen activators and protease inhibitors as well as other factors which modulate fibroblast growth. 4-6 Since both hemostasis and wound healing are associated with fibrin polymerization and lysis, a modulation of macrophage fibrinolytic activity may contribute to postsurgical peritoneal repair. In this study, we observed the hemostatic activity of deacetylated chitin membrane after making an abrasion of the parietal peritoneum or visceral peritoneum of the liver surface.
Materials and Methods
Preparation of DA C-80 Membrane Key Words: wound healing, deacetylated chitin
Reprint requests to: M. Fukasawa (Received for publication on Feb. 2, 1991; accepted on May 1, 1992)
Chitin prepared from Queen Crab (Chionoecetes japonica) shells according to the method of Hackman 8 was a gift of Fuji Spinning Co., Ltd., Tokyo, Japan. Partially deacetylated (80%) chitin (DAC-80) was prepared from chitin by deacetylation under alkaline conditions as previously reported) Regeneration of DAC-80 membrane was achieved by the following procedures. The deacetylated chitin was dissolved in acetic acid and then cast into thin membranes (approximately 20 um thick when dried) using ethanolic-NaOH, followed by successive washings of hot and cold water.
334
M. Fukasawa et al.: Hemostatic Effect of Deacetylated Chitin Membrane on Peritoneal Injury in Rabbit
The resulting membranes were autoclaved and stored in sterilized distilled water. 3
Surgical Procedures Japanese white female rabbits (2 kg) were housed in a light:dark cycle (16:8) controlled vivarium and maintained with water and rabbit chow ad libitum. Rabbit (_->5 rabbits/each time point) underwent a midline laparotomy followed by the indication of either a bilateral peritoneal sidewall abrasion (4 × 4 cm) with a surgical blade or a surface abrasion to the right liver lobe with sterilized sandpaper (3 × 2 cm). In the treatment groups, the area of the injured peritoneum was covered with DAC-80 membrane (membrane size was approximately same as the wound area). The abdominal wall was then closed in two layers with 3-0 nylon sutures (Ethicon, Raritan, N J). In order to minimize any possible contamination by intraoperative bleeding, the peritoneal cavity was protected from the injury site by sterile gauze during the operative procedure. Surgical procedures were performed under the influence of Rompun (25 mg/kg, Bvyet Division Miles Laboratory, KS) and ketamine (130mg/kg, Park-Davis, Morris Plan, N J) anesthesia. All rabbits tolerated the surgical procedures well. The postsurgical time course in the number of red blood cells in peritoneal cavity was initially determined, and then the treatment group which received the DAC-80 membrane for 2 days was compared to the surgical control,
Cell Preparation At a varying number of days after surgery, rabbits were sacrificed with an overdosage of pentobarbiturate (Western Medical Supply, Arcadia, CA). Fifty milliliters of phosphate buffered saline pH 7.4, (PBS) containing 20IU heparin/ml, was injected into the peritoneal cavity of the postsurgical and non-surgical control rabbits. Red- (RBC) and white blood cells (WBC) were recovered by PBS-heparin (50 ml) lavage. RBCs were counted using an improved Neubauer chamber. The lavage fluid was centrifuged at 200 × g for 10 min and WBCs were recovered after hypotonic red cell lysis with a 1:9 PBS:H20 solution as previously described. 9 The cells were washed twice with cold PBS and total cell number was determined by a hemocytometer. A macrophage-enriched population was obtained by Percoll (Pharmacia, Piscataway, N J) discontinuous gradient centrifugation (Percoll 60 per cent, density-l.084). 9 These cells were resuspended in DMEM (Dulbecco's minimum essential medium; Gibco, Gland Island, MA) without serum, plated into 24 well plates (106 cells/ml; 2 ml/well) and incubated for
48h at 37°C in 5% CO~_and 95% air. These peritoneal exudative cells (PEC) maintained over a 90% viability as determined by 1% trypan blue exclusion. 6 Examination after Diff-Quik staining (American Scientific Products, McGaw Park, IL) indicated that at least 90% of the PEC were macrophages after percoll separation. After 48 h incubation, the spent media were harvested and centrifuged at 400 g for 10 rain and the supernatant was stored at -80°C until further assay.
Plasminogen Activator Activity and Protease Inhibitor Activity One ml of macrophage conditioned media from each sample well was adiusted to pH 2-3 with 1 N hydrochloric acid and stored overnight at 4°C to inactivate acid-labile protease inhibitor(S) and was then neutralized with 1 N NaOH. 7 The activity of plasminogen activator (PA) in the macrophage conditioned media was determined by using a chromogenic microtiter plate assay (American Diagnostic Inc., New York, NY) as previously described. 1° PA assay incubations were carried out in a 100ul volume in 96-well flat bottomed microtiter plates (Falcon Plastics, Oxnard, CA). Incubation volumes consisted of 25ul of TrisTw buffer (50raM Tris, 0.1M NaC1, 0.01 per cent Tween 80, pH7.5), 25 ul of sample and 25 ul of 1.6mM H-D-norleucyl- hexahydrotyrosyl-lysine-p-nitroaniline (NHLNA) as a chromogenic substrate. Urokinase (Calbiochem, San Diego, CA) was used as the standard plasminogen activator. DMEM (25 ul) was added into the standard well instead of Tris-Tw buffer as a background control. After 6h incubation at 37°C in a humidified incubator, the reaction was stopped with 10ul of 0.25M EACA (epsilon-aminocaproic acid), and the absorbance at 405 nm was measured by using a Beckman spectrophotometer (Beckman DU68, Fullerton, CA). There was no detectable plasminogen independent fibrinolytic activity observed in the macrophage conditioned media. The activity of plasminogen activator inhibitors (PAl) in the macrophage conditioned media on urokinase were determined using a modified indirect solid-phase radiometric assay as previously described. 11 Three hundred microlitters of urokinase standard (25mPU/ml TBS-Tw80) was mixed with 300ul of sample without acid treatment and incubated in a plastic tube for l h at room temperature. Urokinaselike activity was then determined using the radioassay. As a control, fresh DMEM was used instead of conditioned medium. Recoverable urokinase-like activity was initially expressed as mPU/ml ~and then converted to a percent of control activity.7
M. Fukasawa et al.: Hemostatic Effect of Deacetylated Chitin Membrane on Peritoneal Injury in Rabbit
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Fig. 1. Time course study of postsurgical bleeding after peritoneal sidewall abrasion. Rabbits underwent bilateral peritoneal sidewall abrasion (4 × 4 cm each). At various days after surgery, peritoneal lavage with 50ml PBS was performed and the number of red blood cells was counted (× 108 cells/cavity). Data are expressed as mean + SEM
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Fig. 2. Time course study of postsurgical bleeding after liver surface abrasion. Rabbits underwent abrasion of the liver surface (2 × 3 cm). At various days after surgery, peritoneal lavage with 50 ml PBS was performed and the number of red blood cells was counted (× 108 cells/ml). Data are expressed as mean _+ SEM
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@
SURGERYTODAY © Springer-Verlag 1992
The Hemostatic Effect of Deacetylated Chitin Membrane on Peritoneal Injury in Rabbit Model MANABU FUKASAWA~1 HIROMASA ABE, 1 TOSHIAKI MASAOKA~1 HIROYUKI ORITA, 1 HIDEO HORIKAWA,2 JOSEPH O. CAMPEAU, 3 and MASAHIKO WASHIO 1
aSecondDepartmentof Surgery,2Departmentof Anesthesiology,YamagataUniversitySchoolof MedicineZao-lida2-2-2, YamagataCity990-23,Japan, and 3LivingstonReproductiveBiologyLaboratory,Universityof SouthernCalifornia,USA
Abstract: In this study, we determined the effect of 80% deacetylated chitin (DAC-80) membrane on postsurgical bleeding after visceral and parietal peritoneal abrasion. Japanese white rabbits underwent a midline laparotomy followed either by a bilateral peritoneal sidewall abrasion (4 × 4cm) or an abrasion of liver surface (3 × 2cm). The injured surface was then covered with a 0.2 mm thick DAC-80 membrane. On postsurgical day 2, the rabbits were sacrificed and the amounts of postsurgical bleeding was determined by quantitating the number of red blood cells recovered in 50 ml peritoneal lavage fluid. The DAC-80 membrane was found to reduce postsurgical bleeding after the abrasion of liver surface (treated with DAC-80 membrane: 2.9 + 0.8; control: 24.6 + 5.9 × l 0 s cells/peritoneal cavity, P < 0.005). This same hemostatic activity was not observed after application in the peritoneal sidewall abrasion model. We also measured plasminogen activator activity (PA) and urokinase inhibitory (PAI) activity in the spent culture media of macrophages recovered from the postsurgical peritoneal exudate. The DAC80 membrane reduced the PA secretion from postsurgical macrophages after liver surface abrasion (treated with DAC8 0 : 2 . 8 _+ 0.7; control: 3.9 + 0.9mPU/ml). The DAC-80 membrane also showed similar effects on PA secretion after peritoneal sidewall abrasion. No significant effects were found in the secretion of PAI by postsurgical macrophages in both surgical models. These findings suggest that the DAC-80 membrane may have hemostatic activity through the modulation of fibrinolytic activity of peritoneal exudative macrophages.
Introduction
Chitin is a large molecular weight polymer of D-1,4acetyl-D-glucosamine. Previous reports have indicated that deacetylated chitin (chitosan) is hemostatic and accelerates wound repair. 1"2 In addition, chitin/ chitosan activates macrophages. 3 Accordingly, this compound was tested for use as a topical agent in tissue repair. Our previous studies addressed the healing of the peritoneum after surgical injury with a focus on adhesion formation. Macrophages are one of the principle cells in peritoneal healing and produce plasminogen activators and protease inhibitors as well as other factors which modulate fibroblast growth. 4-6 Since both hemostasis and wound healing are associated with fibrin polymerization and lysis, a modulation of macrophage fibrinolytic activity may contribute to postsurgical peritoneal repair. In this study, we observed the hemostatic activity of deacetylated chitin membrane after making an abrasion of the parietal peritoneum or visceral peritoneum of the liver surface.
Materials and Methods
Preparation of DA C-80 Membrane Key Words: wound healing, deacetylated chitin
Reprint requests to: M. Fukasawa (Received for publication on Feb. 2, 1991; accepted on May 1, 1992)
Chitin prepared from Queen Crab (Chionoecetes japonica) shells according to the method of Hackman 8 was a gift of Fuji Spinning Co., Ltd., Tokyo, Japan. Partially deacetylated (80%) chitin (DAC-80) was prepared from chitin by deacetylation under alkaline conditions as previously reported) Regeneration of DAC-80 membrane was achieved by the following procedures. The deacetylated chitin was dissolved in acetic acid and then cast into thin membranes (approximately 20 um thick when dried) using ethanolic-NaOH, followed by successive washings of hot and cold water.
334
M. Fukasawa et al.: Hemostatic Effect of Deacetylated Chitin Membrane on Peritoneal Injury in Rabbit
The resulting membranes were autoclaved and stored in sterilized distilled water. 3
Surgical Procedures Japanese white female rabbits (2 kg) were housed in a light:dark cycle (16:8) controlled vivarium and maintained with water and rabbit chow ad libitum. Rabbit (_->5 rabbits/each time point) underwent a midline laparotomy followed by the indication of either a bilateral peritoneal sidewall abrasion (4 × 4 cm) with a surgical blade or a surface abrasion to the right liver lobe with sterilized sandpaper (3 × 2 cm). In the treatment groups, the area of the injured peritoneum was covered with DAC-80 membrane (membrane size was approximately same as the wound area). The abdominal wall was then closed in two layers with 3-0 nylon sutures (Ethicon, Raritan, N J). In order to minimize any possible contamination by intraoperative bleeding, the peritoneal cavity was protected from the injury site by sterile gauze during the operative procedure. Surgical procedures were performed under the influence of Rompun (25 mg/kg, Bvyet Division Miles Laboratory, KS) and ketamine (130mg/kg, Park-Davis, Morris Plan, N J) anesthesia. All rabbits tolerated the surgical procedures well. The postsurgical time course in the number of red blood cells in peritoneal cavity was initially determined, and then the treatment group which received the DAC-80 membrane for 2 days was compared to the surgical control,
Cell Preparation At a varying number of days after surgery, rabbits were sacrificed with an overdosage of pentobarbiturate (Western Medical Supply, Arcadia, CA). Fifty milliliters of phosphate buffered saline pH 7.4, (PBS) containing 20IU heparin/ml, was injected into the peritoneal cavity of the postsurgical and non-surgical control rabbits. Red- (RBC) and white blood cells (WBC) were recovered by PBS-heparin (50 ml) lavage. RBCs were counted using an improved Neubauer chamber. The lavage fluid was centrifuged at 200 × g for 10 min and WBCs were recovered after hypotonic red cell lysis with a 1:9 PBS:H20 solution as previously described. 9 The cells were washed twice with cold PBS and total cell number was determined by a hemocytometer. A macrophage-enriched population was obtained by Percoll (Pharmacia, Piscataway, N J) discontinuous gradient centrifugation (Percoll 60 per cent, density-l.084). 9 These cells were resuspended in DMEM (Dulbecco's minimum essential medium; Gibco, Gland Island, MA) without serum, plated into 24 well plates (106 cells/ml; 2 ml/well) and incubated for
48h at 37°C in 5% CO~_and 95% air. These peritoneal exudative cells (PEC) maintained over a 90% viability as determined by 1% trypan blue exclusion. 6 Examination after Diff-Quik staining (American Scientific Products, McGaw Park, IL) indicated that at least 90% of the PEC were macrophages after percoll separation. After 48 h incubation, the spent media were harvested and centrifuged at 400 g for 10 rain and the supernatant was stored at -80°C until further assay.
Plasminogen Activator Activity and Protease Inhibitor Activity One ml of macrophage conditioned media from each sample well was adiusted to pH 2-3 with 1 N hydrochloric acid and stored overnight at 4°C to inactivate acid-labile protease inhibitor(S) and was then neutralized with 1 N NaOH. 7 The activity of plasminogen activator (PA) in the macrophage conditioned media was determined by using a chromogenic microtiter plate assay (American Diagnostic Inc., New York, NY) as previously described. 1° PA assay incubations were carried out in a 100ul volume in 96-well flat bottomed microtiter plates (Falcon Plastics, Oxnard, CA). Incubation volumes consisted of 25ul of TrisTw buffer (50raM Tris, 0.1M NaC1, 0.01 per cent Tween 80, pH7.5), 25 ul of sample and 25 ul of 1.6mM H-D-norleucyl- hexahydrotyrosyl-lysine-p-nitroaniline (NHLNA) as a chromogenic substrate. Urokinase (Calbiochem, San Diego, CA) was used as the standard plasminogen activator. DMEM (25 ul) was added into the standard well instead of Tris-Tw buffer as a background control. After 6h incubation at 37°C in a humidified incubator, the reaction was stopped with 10ul of 0.25M EACA (epsilon-aminocaproic acid), and the absorbance at 405 nm was measured by using a Beckman spectrophotometer (Beckman DU68, Fullerton, CA). There was no detectable plasminogen independent fibrinolytic activity observed in the macrophage conditioned media. The activity of plasminogen activator inhibitors (PAl) in the macrophage conditioned media on urokinase were determined using a modified indirect solid-phase radiometric assay as previously described. 11 Three hundred microlitters of urokinase standard (25mPU/ml TBS-Tw80) was mixed with 300ul of sample without acid treatment and incubated in a plastic tube for l h at room temperature. Urokinaselike activity was then determined using the radioassay. As a control, fresh DMEM was used instead of conditioned medium. Recoverable urokinase-like activity was initially expressed as mPU/ml ~and then converted to a percent of control activity.7
M. Fukasawa et al.: Hemostatic Effect of Deacetylated Chitin Membrane on Peritoneal Injury in Rabbit
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Fig. 1. Time course study of postsurgical bleeding after peritoneal sidewall abrasion. Rabbits underwent bilateral peritoneal sidewall abrasion (4 × 4 cm each). At various days after surgery, peritoneal lavage with 50ml PBS was performed and the number of red blood cells was counted (× 108 cells/cavity). Data are expressed as mean + SEM
>
Statistics D a t a were determine groups was The results
Fig. 2. Time course study of postsurgical bleeding after liver surface abrasion. Rabbits underwent abrasion of the liver surface (2 × 3 cm). At various days after surgery, peritoneal lavage with 50 ml PBS was performed and the number of red blood cells was counted (× 108 cells/ml). Data are expressed as mean _+ SEM
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