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Experimental evaluation of photocrosslinkable chitosan as a biologic adhesive with surgical applications
Experimental evaluation of photocrosslinkable chitosan as a biologic adhesive with surgical applications Katsuaki Ono, MD, Masayuki Ishihara, PhD, Yuichi Ozeki, MD, Hiroyuki Deguchi, MD, Mitsuharu Sato, MD, Yoshio Saito, MS, Hirofumi Yura, PhD, Masato Sato, MD, Makoto Kikuchi, PhD, Akira Kurita, MD, and Tadaaki Maehara, MD, Saitama and Kanagawa, Japan
Background. In various surgical cases, effective tissue adhesives are required for both hemostasis (eg, intraoperative bleeding) and air sealing (eg, thoracic surgery). We have designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thereby forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. Methods. The sealing ability of the chitosan hydrogel, determined as a bursting pressure, was assessed with removed thoracic aorta, trachea, and lung of farm pigs and in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle, and the chitosan hydrogel was applied to, respectively, stop the bleeding and the air leakage. In vivo chitosan degradability and biologic responses were histologically assessed in animal models. Results. The bursting pressure of chitosan hydrogel (30 mg/mL) and fibrin glue, respectively, was 225 ± 25 mm Hg (mean ± SD) and 80 ± 20 mm Hg in the thoracic aorta; 77 ± 29 mm Hg and 48 ± 21 mm Hg in the trachea; and in the lung, 51 ± 11 mm Hg (chitosan hydrogel), 62 ± 4 mm Hg (fibrin glue, rubbing method), and 12 ± 2 mm Hg (fibrin glue, layer method). The sealing ability of the chitosan hydrogel was stronger than that of fibrin glue. All rabbits with a carotid artery (n = 8) or lung (n = 8) that was punctured with a needle and then sealed with chitosan hydrogel survived the 1-month observation period without any bleeding or air leakage from the puncture sites. Histologic examinations demonstrated that 30 days after application, a fraction of the chitosan hydrogel was phagocytosed by macrophages, had partially degraded, and had induced the formation of fibrous tissues around the hydrogel. Conclusions. A newly developed photocrosslinkable chitosan has demonstrated strong sealing ability and a great potential for use as an adhesive in surgical operations. (Surgery 2001;130:844-50.) From the Department of Surgery II, Department of Orthopedic Surgery, Department of Medical Engineering, and Division of Biomedical Engineering, Research Institute, National Defense Medical College, Saitama, Japan, and NeTech Inc, Kanagawa, Japan
ALTHOUGH MOST BLEEDING in surgical procedures can be controlled with appropriate sutures, hemostasis is uncontrollable in certain conditions, such as coagulopathy, anticoagulant use, inflammation, infection, and severe adhesion. In addition, intractable air leakage in lung surgery is common, especially in emphysematous lung disease. In many cases of uncontrollable bleeding and intractable air
Accepted for publication May 5, 2001. Reprint requests: Masayuki Ishihara, PhD, Division of Biomedical Engineering, Research Institute, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/56/117197 doi:10.1067/msy.2001.117197
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leakage, a number of adhesives have been used for hemostasis and air sealing (ie, chemically crosslinkable gelatins,1-3 cyanoacrylate polymers,4,5 and fibrin glues).6-9 To be effective, these adhesives must be nonirritating locally and nontoxic systematically, have appropriate flexibility, and be biodegradable. However, cytotoxicity and severe tissue irritability have occurred with resorcinol, formaldehyde, and carbodiimides when use for the crosslink reaction of gelatins or the formation of formaldehyde by degradation of cyanoacrylate.10 Fibrin glue, which contains fibrinogen, thrombin, factor XIII, and a protease inhibitor, uses the blood coagulation system for sealing tissues and currently is the most widely used surgical adhesive. Its effective hemostatic and air-sealing abilities have been reported by many investigators.8,11,12 However, fibrin glue has a disadvantage in its industrial production, since
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human blood is used as its source. Furthermore, when biologic materials are used, fully preventing infectious contaminations is difficult. Chitin is a linear homopolymer of β(1,4)-linked N-acetyl-D-glucosamine, and chitosan is a partially deacetylated chitin. Chitin and chitosan have been proposed as biomaterials with a range of biomedical and industrial applications attributable to their biocompatibility.13,14 Chitosan was observed to accelerate wound healing15-19 by inducing infiltration of inflammatory cells into the wound area,17,18 activation of macrophages,20 and production of cytokines21,22 and by its anti-infection activity.13 Several products that contain chitin and chitosan for wound treatments have already been on the market in the form of a filament, sheet, powder, granule, or sponge.13,19 We have previously reported on the preparation and characterization of a new photocrosslinkable chitosan.23 This material is a viscous solution that is crosslinked on ultraviolet (UV) light irradiation, resulting in an insoluble, soft hydrogel. The purpose of this study was to assess the possible use of chitosan hydrogel as a biologic adhesive and its safety in medical use. The sealing ability of the chitosan hydrogel was evaluated by measuring the bursting pressure in animal tissue models and compared with that of a commercial fibrin glue. In addition, in vivo degradability and toxicity have been studied to assess its biocompatibility. MATERIAL AND METHODS Materials. Photocrosslinkable chitosan molecules (Az-CH-LA) were prepared as previously reported.23 Fig 1 shows the chemical structure of Az-CH-LA. The chitosan used in this study has a molecular weight of 800 to approximately 1000 kDa with a deacetylation ratio of 0.8 (Yaizu Suisankagaku Industry Co, Ltd, Shizuoka, Japan). Azide and lactose moieties were added to the chitosan molecules through a condensation reaction with the amino groups. Addition of lactose resulted in a water-soluble chitosan at neutral hydrogen ion concentration. It was estimated that about 2.5% of the amino groups in the chitosan were substituted by p-azidebenzoic acid and 2% by lactobionic acid.23 A viscous Az-CH-LA aqueous solution was converted into an insoluble hydrogel in 1 to 2 minutes with UV light irradiation at a distance of 2 cm (4-watt, 254-nm UV tube light; Iuchi, Tokyo, Japan) by crosslinking its azide and amino groups.23 Fibrin glue (Beriplast P) was purchased from Hoechst-Marion-Roussel (Tokyo, Japan). New Zealand white rabbits (2 to 3 kg) were purchased from Kitayama Labes, Co, Nagano, Japan, and individually housed in cages.
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Fig 1. Chemical structure of the photocrosslinkable chitosan molecule (Az-CH-LA). Azide (p-azidebenzoic acid) and lactose (lactobionic acid) moieties were introduced into 2.5% and 2% of amino groups in the chitosan molecule, respectively.
Bursting pressure of chitosan hydrogels. Farm pigs weighing about 30 kg that were premedicated with an intramuscular injection of 20 mg/kg of ketamine were anesthetized with intravenous injection of 20 mg/kg of sodium pentobarbitone. The pigs were then killed with an overdose of potassium chloride via intravenous injection. A tracheal tube was inserted into a the dead pigs and connected to a mechanical ventilator. The lung was then punctured with a needle (1.2 mm in diameter) about 10mm deep. One drop (about 30 µL) of 30 mg/mL of Az-CH-LA aqueous solution was applied to the puncture site and irradiated with UV light at a distance of 2 cm for 90 seconds. Subsequently, ventilation was gradually started through a linear pulsed-air volume increase. The pressure at which air leakage reoccurred was measured and termed the “bursting pressure” of the chitosan hydrogel. Fibrin glue application according to the manufacturer’s instructions was done by adding the fibrinogen solution first, followed by the thrombin solution (layer method). However, a new, more effective method recently has been reported (the so-called rubbing method), in which a low-viscous thrombin solution is first rubbed into the lung parenchyma, and then the fibrinogen solution is added.24 These 2 methods were performed in this study, and the bursting pressure occurred 5 minutes after application of the fibrin glues. The trachea, thoracic aorta, and small intestine, with diameters of about 20 mm, 10 mm, and 20 mm, respectively, were removed from the dead pigs. One end of the removed artery or small intestine was ligated with suture material, and the other side was intubated with a small catheter held in place by ligatures (Fig 2). The catheter was connected to a syringe and a manometer. The artery and small intestine were punctured with a needle (1.2 mm in diameter), and about 30 µL (1 drop) of Az-CH-LA aqueous solution (30 mg/mL) was applied to the hole and irradiated with UV light for 90 seconds.
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Fig 2. Scheme for the measurement of the bursting pressure with application of chitosan hydrogel on the aorta and trachea.
The artery and small intestine were placed under water, and they were inflated until bubbles could be detected in the water. The pressure required to produce this air leakage was defined as the bursting pressure (millimeters of mercury). The oral side of the trachea was intubated with the catheter, and the bilateral ends of the main bronchus were ligated. Subsequently, a 5-mm long and 5-mm deep transverse incision was made along an annular ligament in the middle section of the trachea. After the chitosan gel (30 µL) was applied, its bursting pressure was determined in the same way as that for the Az-CH-LA aqueous solution (see Fig 2). Similar experiments have been performed with fibrin glue, with bursting pressure measurements starting 5 minutes after application. Sealing effect in vivo and histologic examination. Fourteen male New Zealand white rabbits (2 to 3 kg) were anesthetized with an intramuscular injection of xylazine (12 mg) and ketamine (40 mg). In 7 rabbits, transverse skin incisions were made in the neck, resulting in exposure of the left carotid artery. Bleeding was initiated by pricking the artery with a needle (1.2 mm in diameter) and temporarily stopped by clamping both sides of the bleeding point with vascular clips. About 30 µL (1 drop) of the AzCH-LA aqueous solution was applied to the puncture site and then photocrosslinked by UV irradiation at a distance of 2 cm for 90 seconds. After the vascular clips on both sides of the bleeding point were carefully removed, blood flow and hemostasis were confirmed. Similar experiments have been performed with fibrin glue, with hemostasis confirmations starting 5 minutes after application. In the other 7 rabbits, an anterior lobe of the lung was exposed through a lateral thoracotomy, and a 5 mm-long and 5 mm-deep incision was made on the lung surface. After confirming air leakage as
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a result of the incision of the lung, 3 to 4 drops (total about 100 µL) of the Az-CH-LA aqueous solution were applied to the incision and photocrosslinked by UV irradiation for 90 seconds without stopping the respiration of the rabbits. A small tube was inserted to the thoracic cage, and the skin was then closed by suturing. Intrathoracic air and fluid were aspirated from the thoracic cage through the tube, and the tube was then withdrawn. Similar experiments have been performed with fibrin glue. Fluoroscopic observations (Diagnostic X-ray Apparatus, Model KXO-1000; Tokyo Shibaura Electric Co, Tokyo, Japan) showed that the lungs with punctures sealed by either the chitosan hydrogel or fibrin glue had expanded fully after the operations. The animals were then fed ad libitum (standard feed pellets) and killed after 30 days. The punctured artery and incised lung were removed, fixed in 10% neutral formaldehyde solution, and embedded in paraffin. The samples have been subjected to histologic examination by staining with Congo Red (Wako Pure Chemical Industries, Ltd, Osaka, Japan) (to specifically stain chitin and chitosan). The animal experiments were approved and carried out following the guidelines for animal experimentation at the National Defense Medical College, Tokorozawa, Saitama, Japan. Statistical analysis. All results on the bursting pressures are expressed as mean ± SD or SE. Comparison between multiple group averages were analyzed with one-way analysis of variance and the Scheffé multiple comparisons test. RESULTS Bursting pressure of chitosan hydrogel. The bursting pressure of the chitosan hydrogel in the lung was 51 ± 11 mm Hg (mean ± SD), which is more than that of fibrin glue with the layer method (12 ± 2 mm Hg) (P < .0001). The bursting pressure of the chitosan hydrogel was not statistically different from that of fibrin glue with the rubbing method (P = .138). The bursting pressures of the chitosan hydrogel and fibrin glue (rubbing method) on the trachea were 77 ± 29 mm Hg and 48 ± 21 mm Hg, on the aorta, 225 ± 25 mm Hg and 80 ± 20 mm Hg, and on the small intestine, 65 ± 5 mm Hg and 52 ± 8 mm Hg, respectively. The bursting pressure of the chitosan hydrogel was more than that of the fibrin glue (rubbing method) on the aorta (P < .0001), the trachea (P < .05), and the small intestine (P = .02). The bursting pressure of the fibrin glue with the rubbing method was significantly higher than that with the layer method for the aorta, trachea, and small intestine. These data suggest that the sealing strength of the chitosan
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hydrogel may be sufficient to stop arterial bleeding and air leakage from the lung or trachea in surgical applications. Sealing effects in vivo. Chitosan hydrogel applied to the pinhole on the carotid artery with UV irradiation was able to stop the bleeding (1 ± 1 minutes) faster than fibrin glue (3 ± 1 minutes). However, the bleeding could not be stopped with application of the Az-CH-LA aqueous solution without UV irradiation. Applications of chitosan hydrogel with UV irradiation for 90 seconds (n = 7) or fibrin glue for 5 minutes (n = 2) completely stopped bleeding from the carotid artery of rabbits in vivo. All rabbits were alive 30 days of postoperational observation, with no demonstration of any visual complications. Similarly, the chitosan hydrogel with UV irradiation stopped bleeding from the pinhole on the carotid artery of rabbits (n = 3) that were administered 20 mg/kg of native heparin (185.8 USP U/mg) for 30 minutes (data not shown), indicating that the hemostatic effect of the chitosan hydrogel is independent of the blood coagulation. Although mild fibrous adhesions were noticed 30 days after operation in the thoracic cavity of rabbits treated with chitosan hydrogel, adhesion between the area where chitosan hydrogel was applied and the opposite chest wall was not observed in all cases. The application of chitosan hydrogel with UV irradiation for 90 seconds (n = 7) and the application of fibrin glue (n = 2) also were able to stop the air leakage from the lung, and fluoroscopic observations showed that the sealed lungs expanded fully. All rabbits survived 1 month after the operation and did not demonstrate any visual complications. However, the air leakage from the lung could not be stopped with application of Az-CH-LA aqueous solution without UV irradiation (n = 2) during the operations. Fluoroscopy also showed that the control lungs were deflated during 3 days of postoperational observation, indicating an air leak. The Az-CH-LA aqueous solution is viscous enough to allow application to required sites without any spillage and is easier to apply than the fibrin glue solution. Therefore, the application of Az-CH-LA aqueous solution on the wound site may temporarily stop the air leakage from the lung until gelation of the chitosan occurs with UV irradiation. Microscopic and histologic findings. The area of a removed lung in which chitosan hydrogel was applied was covered with glossy smooth pleura after 30 days, and the chitosan hydrogel was encapsulated by fibrous tissue and covered with regenerated thick pleura (Fig 3, A). Chitosan is orange when Congo Red stain is used, and it was clearly
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shown that a considerable amount of the chitosan gel remained with numerous neutrophils that migrated into the gel (see Fig 3, A). The area in which fibrin glue was applied was replaced with mass of fibrous connective tissue, composed of many fibroblasts and collagen fiber networks; some lymphocytes were observed in the tissue (Fig 3, C). Fig 3, B, and Fig 4, B, show orange granules in many macrophages, indicating that chitosan molecules were phagocytosed by macrophages. Although the chitosan hydrogel is biodegradable, a large fraction of the chitosan gel was encapsulated by thick, fibrous tissue and may remain. Residual chitosan hydrogel was observed 60 days after operation (data not shown). Similarly, the wound of punctured carotid arteries were also closed completely and covered with dense fibrosis and granulation tissue that contained many newly formed capillary vessels and neutrophils 30 days after the operation (Fig 4, A). Although chitosan hydrogels were almost completely degraded, as can be seen on the carotid artery, some residual chitosan hydrogel is still visible (see Fig 4, B). DISCUSSION We previously have reported on a photocrosslinkable chitosan molecule (Az-CH-LA) that contains both lactose moieties and photoreactive azide groups.23 UV irradiation of an Az-CH-LA aqueous solution resulted in an insoluble hydrogel, comparable to a soft rubber, within 90 seconds. This hydrogel could firmly adhere 2 pieces of sliced ham to each other and could effectively seal air leakages (eg, from pinholes in small intestines).23 In this report, animal studies to evaluate the effectiveness and safety of the chitosan hydrogel as a tissue adhesive or sealant were described, with focus on hemostasis of the artery and air-sealing capacity on the lung. Our main conclusion is that the chitosan hydrogel may be a promising new surgical adhesive and sealant. In the animal studies described, chitosan hydrogels exhibited good sealing properties, superior to those of fibrin glue, on a pinhole in the artery and small intestine and an incision on the trachea. On the other hand, the sealing strength of fibrin glue on lung punctures with the rubbing method was almost identical to that of the chitosan hydrogel, although the strength of fibrin glue with the conventional layer method was much lower than that of the chitosan hydrogel. The data about the fibrin glue are identical as previously reported,24 suggesting that an anchoring effect of the fibrin glue on the lung takes place by rubbing on the injured site. However, the high bursting pressure of chitosan
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A
B
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A
B Fig 4. Histologic examination of a rabbit carotid artery 30 days after application of chitosan hydrogel. A, Stained with Congo Red, original magnification ×100. B, Stained with Congo Red, original magnification ×400. The photographs are representative of 7 rabbits in which chitosan hydrogel was applied. Arrows represent residual chitosan hydrogel on application sites.
C Fig 3. Histologic examination of a rabbit lung 30 days after operation. A, Lung with chitosan hydrogel and stained with Congo Red, original magnification ×100. B, Lung with chitosan hydrogel and stained with Congo Red, original magnification ×400. C, Lung with fibrin glue and stained with hematoxylin-eosin, original magnification ×100. The photographs are representative of 7 rabbits in which chitosan hydrogel or fibrin glue was applied. Arrows represent residual chitosan hydrogel on application sites.
hydrogels points to the possibility that the chitosan hydrogels may have the ability to seal the pinholes and incisions in these organs intraoperatively. Thirty days after operation of the lung, the chitosan hydrogel remained and dense fibrous tissue
was observed around the hydrogel in which inflammatory cells were infiltrated (see Fig 3). These responses can be ascribed to originate from foreign body reactions. Chitosan molecules, however, have been reported to induce migration of neutrophils and production of multiple cytokines 21,22 and to accelerate wound healing.15-19 Furthermore, it also has been reported that fibroblasts stimulated by chitosan molecules secrete interleukin-8 (IL-8).25 IL-8 is known to be angiogenic and chemoattractive to endothelial and epidermal cells.23 Fibroblasts that adhere to a chitosan hydrogel may secret IL-8 and other cytokines, which in turn could induce angiogenesis and fibrosis. These reactions may account for the accelerated tissue repair after application of chitosan hydrogels. Chitosan is considered to be a biodegradable molecule, and it has been reported that migrated
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neutrophils degrade chitosan molecules by secretion of lysozyme26 and chitotriosidase,21 in addition to phagocytosis of the molecules. However, a considerable fraction of chitosan hydrogel appeared to be encapsulated by fibrous tissue for longer than several months before its complete degradation (see Fig 3). Another advantage of the chitosan hydrogel is that the viscosity of the Az-CH-LA aqueous solution is higher than that of the fibrin glue and thus can easily be applied without spillage. It should also be noted that components of fibrin glue originate from human blood or bovine lung and hence always contain a certain risk of infectious contamination, such as with hepatitis viruses, human immunodeficiency virus, and unidentified pathogenic organisms. The risk of infection and anaphylaxis cannot be completely ruled out when fibrin glue is used. Since both chitin and chitosan are abundant, Az-CH-LA may be produced at a much lower cost.13 Fibrin glue was effective in controlling bleeding intraoperatively. It was used to control bleeding from anastomoses and aortic bleeding points, which result from needle holes or suture lines, and venous bleeding.7,8,11,12 In addition, fibrin glue also was used in lung procedures to control intractable air leakage from the resected surface or suture and staple lines of the lung, especially in procedures for emphysematous lung disease.24 It may be possible for chitosan hydrogel, having putative advantages over fibrin glue, to be used as a surgical adhesive in a similar situation. Chitosan has been proven to be safe in standard animal toxicologic studies. Chitosan has been used in food and skin wound management. 13,26,27 Azide residues may be toxic and highly reactive to tissue components, such as proteins. However, with a complete crossreaction of azide residues to amino groups, this toxic effect may disappear. Our previous results in mice also indicated that the Az-CH-LA aqueous solution and the chitosan hydrogel were found to be safe. Mice administered about 1200 mg/kg of the Az-CH-LA solution (n = 6) or the chitosan hydrogel (n = 6) intraperitoneally all survived the entire 1 month observation. 23 Cytotoxicity has not been observed with in vitro assays.23 In addition, preliminary toxicity tests of mutagenecity and cytotoxicity have shown the safety of both Az-CH-LA and its hydrogel (data not shown). However, sufficient data are not yet available on the complete toxicity profile of AzCH-LA aqueous solutions and chitosan hydrogels in humans, and standard toxicologic studies
should be performed before this chitosan hydrogel is used as a surgical adhesive.
REFERENCES 1. Braunwald NS, Gay W, Tatooles CJ. Evaluation of crosslinked gelatin as a tissue adhesive and hemostatic agent: an experimental study. Surgery 1966;59:1024-30. 2. Koehnlein HE, Lemperle G. Experimental studies with a new gelatin-resorcine-formaldehyde glue. Surgery 1969;66:377-82. 3. Otani Y, Tabata Y, Ikeda Y. A new biological glue from gelatin and poly (L-glutamic acid). J Biomed Mater Res 1996;31:157-66. 4. Tseng YC, Hyon SH, Idaka Y. Modification of synthesis and investigation of properties for 2-cyanoacrylates. Biomaterials 1990;11:73-9. 5. Vanholder R, Misotten A, Roels H, Matton G. Cyanoacrylate tissue adhesive for closing skin wounds: a double blind randomized comparison with sutures. Biomaterials 1993; 14:737-43. 6. Thetter O. Fibrin adhesive and its application in thoracic surgery. Thorac Cardiovasc Surg 1981;29:290-2. 7. Borst HG, Haverish A, Walterbusch G, Maatz W, Messner B. Fibrin adhesive: an important hemostatic adjunct in cardiovascular operations. J Thorac Cardiovasc Surg 1982;84:54853. 8. Rousou JA, Engelman RM, Breyer RH. Fibrin glue: an effective hemostatic agent for nonsuturable intraoperative bleeding. Ann Thorac Surg 1984;38:409-10. 9. Moy OJ, Peimer CA, Koiuch MP, Howard C, Zeilezny M, Katikaneni PR. Fibrin seal adhesive versus nonabsorbable microsuture in peripheral nerve repair. J Hand Surg [AM] 1988;13A:273-8. 10. Otani Y, Tabata Y, Ikeda Y. Rapidly curable biological glue composed of gelatin and poly (L-glutamic acid). Biomaterials 1996;17:1387-91. 11. Jessen C, Sharma P. Use of fibrin glue in thoracic surgery. Ann Thorac Surg 1985;39:521-4. 12. Stark J, Leval M. Experience with fibrin seal (Tisseel) in operations for congenital heart defects. Ann Thorac Surg 1984;38:411-3. 13. Shigemasa Y, Minami S. Application of chitin and chitosan for biomaterials. Biotechnol Genet Eng Rev 1995;13:383420. 14. Chandy T, Sharma CP. Chitosan as a biomaterial. Biomater Artif Cells Artif Org 1990;18:1-24. 15. Minami S, Okamoto Y, Matsuhashi A, Sashiwa H, Saimoto H, Shigemasa Y, et al. Application of chitin and chitosan in large animal practice. In: Brine CJ, Sandford PA, Zikakis JP, editors. Advances in chitin and chitosan. London: Elsevier Applied Science; 1992. p. 61-9. 16. Pruden JF, Migel P, Hanson P, Friedrich L, Balassa L. The discovery of potent pure chemical wound-healing accelerator. Am J Surg 1970;119:560-4. 17. Okamoto Y, Shibazaki K, Minami S, Matsuhashi A, Tanioka S, Shigemasa Y. Evaluation of chitin and chitosan on open wound healing in dogs. J Vet Med Sci 1995;57:851-4. 18. Ueno H, Yamada H, Tanaka I, Kaba N, Matsuura M, Okumura M, et al. Accelerating effects of chitosan for healing at early phase of experimental open wound in dogs. Biomaterials 1999;20:1407-14. 19. Cho YW, Cho YN, Chung SH, Yoo G, Ko SW. Water-soluble chitin as a wound healing accelerator. Biomaterials 1999;20:2139-45.
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20. Peluso G, Petillo O, Ranieri M, Santin M, Ambrosio L, Calagro D, et al. Chitosan-mediated stimulation of macrophage function. Biomaterials 1994;15:1215-20. 21. Nishimura K, Ishihara C, Ukei S, Tokura S, Azuma I. Stimulation of cytokine production in mice using deacetylated chitin. Vaccine 1986;4:151-6. 22. Mori T, Okumura M, Matsuura M, Ueno K, Tokura S, Okamoto Y, et al. Effect of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro. Biomaterials 1997;18:947-51. 23. Ono K, Saito Y, Yura H, Ishikawa K, Kurita A, Akaike T, et al. Photocrosslinkable chitosan as a biological adhesive. J Biomed Mater Res 2000;49:289-95.
O
24. Morikawa T, Katoh H. Improved techniques of applying fibrin glue in lung surgery. Eur Surg Res 1999;31:180-6. 25. Koch AE, Polverini PJ, Kunkel SL, Harlow LA, Dipietro LA, Elner VM, et al. Interleukin-8 as a macrophagederived mediator of angiogenesis. Science 1992;258:1798-801. 26. Muzzarelli RA. Biochemical significance of exogenous chitins and chitosans in animals and patients. Carbohydr Polym 1993;20:7-16. 27. Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. J Biomed Mater Res 1997;34:21-8.
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Background. In various surgical cases, effective tissue adhesives are required for both hemostasis (eg, intraoperative bleeding) and air sealing (eg, thoracic surgery). We have designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thereby forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. Methods. The sealing ability of the chitosan hydrogel, determined as a bursting pressure, was assessed with removed thoracic aorta, trachea, and lung of farm pigs and in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle, and the chitosan hydrogel was applied to, respectively, stop the bleeding and the air leakage. In vivo chitosan degradability and biologic responses were histologically assessed in animal models. Results. The bursting pressure of chitosan hydrogel (30 mg/mL) and fibrin glue, respectively, was 225 ± 25 mm Hg (mean ± SD) and 80 ± 20 mm Hg in the thoracic aorta; 77 ± 29 mm Hg and 48 ± 21 mm Hg in the trachea; and in the lung, 51 ± 11 mm Hg (chitosan hydrogel), 62 ± 4 mm Hg (fibrin glue, rubbing method), and 12 ± 2 mm Hg (fibrin glue, layer method). The sealing ability of the chitosan hydrogel was stronger than that of fibrin glue. All rabbits with a carotid artery (n = 8) or lung (n = 8) that was punctured with a needle and then sealed with chitosan hydrogel survived the 1-month observation period without any bleeding or air leakage from the puncture sites. Histologic examinations demonstrated that 30 days after application, a fraction of the chitosan hydrogel was phagocytosed by macrophages, had partially degraded, and had induced the formation of fibrous tissues around the hydrogel. Conclusions. A newly developed photocrosslinkable chitosan has demonstrated strong sealing ability and a great potential for use as an adhesive in surgical operations. (Surgery 2001;130:844-50.) From the Department of Surgery II, Department of Orthopedic Surgery, Department of Medical Engineering, and Division of Biomedical Engineering, Research Institute, National Defense Medical College, Saitama, Japan, and NeTech Inc, Kanagawa, Japan
ALTHOUGH MOST BLEEDING in surgical procedures can be controlled with appropriate sutures, hemostasis is uncontrollable in certain conditions, such as coagulopathy, anticoagulant use, inflammation, infection, and severe adhesion. In addition, intractable air leakage in lung surgery is common, especially in emphysematous lung disease. In many cases of uncontrollable bleeding and intractable air
Accepted for publication May 5, 2001. Reprint requests: Masayuki Ishihara, PhD, Division of Biomedical Engineering, Research Institute, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. Copyright © 2001 by Mosby, Inc. 0039-6060/2001/$35.00 + 0 11/56/117197 doi:10.1067/msy.2001.117197
844 SURGERY
leakage, a number of adhesives have been used for hemostasis and air sealing (ie, chemically crosslinkable gelatins,1-3 cyanoacrylate polymers,4,5 and fibrin glues).6-9 To be effective, these adhesives must be nonirritating locally and nontoxic systematically, have appropriate flexibility, and be biodegradable. However, cytotoxicity and severe tissue irritability have occurred with resorcinol, formaldehyde, and carbodiimides when use for the crosslink reaction of gelatins or the formation of formaldehyde by degradation of cyanoacrylate.10 Fibrin glue, which contains fibrinogen, thrombin, factor XIII, and a protease inhibitor, uses the blood coagulation system for sealing tissues and currently is the most widely used surgical adhesive. Its effective hemostatic and air-sealing abilities have been reported by many investigators.8,11,12 However, fibrin glue has a disadvantage in its industrial production, since
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human blood is used as its source. Furthermore, when biologic materials are used, fully preventing infectious contaminations is difficult. Chitin is a linear homopolymer of β(1,4)-linked N-acetyl-D-glucosamine, and chitosan is a partially deacetylated chitin. Chitin and chitosan have been proposed as biomaterials with a range of biomedical and industrial applications attributable to their biocompatibility.13,14 Chitosan was observed to accelerate wound healing15-19 by inducing infiltration of inflammatory cells into the wound area,17,18 activation of macrophages,20 and production of cytokines21,22 and by its anti-infection activity.13 Several products that contain chitin and chitosan for wound treatments have already been on the market in the form of a filament, sheet, powder, granule, or sponge.13,19 We have previously reported on the preparation and characterization of a new photocrosslinkable chitosan.23 This material is a viscous solution that is crosslinked on ultraviolet (UV) light irradiation, resulting in an insoluble, soft hydrogel. The purpose of this study was to assess the possible use of chitosan hydrogel as a biologic adhesive and its safety in medical use. The sealing ability of the chitosan hydrogel was evaluated by measuring the bursting pressure in animal tissue models and compared with that of a commercial fibrin glue. In addition, in vivo degradability and toxicity have been studied to assess its biocompatibility. MATERIAL AND METHODS Materials. Photocrosslinkable chitosan molecules (Az-CH-LA) were prepared as previously reported.23 Fig 1 shows the chemical structure of Az-CH-LA. The chitosan used in this study has a molecular weight of 800 to approximately 1000 kDa with a deacetylation ratio of 0.8 (Yaizu Suisankagaku Industry Co, Ltd, Shizuoka, Japan). Azide and lactose moieties were added to the chitosan molecules through a condensation reaction with the amino groups. Addition of lactose resulted in a water-soluble chitosan at neutral hydrogen ion concentration. It was estimated that about 2.5% of the amino groups in the chitosan were substituted by p-azidebenzoic acid and 2% by lactobionic acid.23 A viscous Az-CH-LA aqueous solution was converted into an insoluble hydrogel in 1 to 2 minutes with UV light irradiation at a distance of 2 cm (4-watt, 254-nm UV tube light; Iuchi, Tokyo, Japan) by crosslinking its azide and amino groups.23 Fibrin glue (Beriplast P) was purchased from Hoechst-Marion-Roussel (Tokyo, Japan). New Zealand white rabbits (2 to 3 kg) were purchased from Kitayama Labes, Co, Nagano, Japan, and individually housed in cages.
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Fig 1. Chemical structure of the photocrosslinkable chitosan molecule (Az-CH-LA). Azide (p-azidebenzoic acid) and lactose (lactobionic acid) moieties were introduced into 2.5% and 2% of amino groups in the chitosan molecule, respectively.
Bursting pressure of chitosan hydrogels. Farm pigs weighing about 30 kg that were premedicated with an intramuscular injection of 20 mg/kg of ketamine were anesthetized with intravenous injection of 20 mg/kg of sodium pentobarbitone. The pigs were then killed with an overdose of potassium chloride via intravenous injection. A tracheal tube was inserted into a the dead pigs and connected to a mechanical ventilator. The lung was then punctured with a needle (1.2 mm in diameter) about 10mm deep. One drop (about 30 µL) of 30 mg/mL of Az-CH-LA aqueous solution was applied to the puncture site and irradiated with UV light at a distance of 2 cm for 90 seconds. Subsequently, ventilation was gradually started through a linear pulsed-air volume increase. The pressure at which air leakage reoccurred was measured and termed the “bursting pressure” of the chitosan hydrogel. Fibrin glue application according to the manufacturer’s instructions was done by adding the fibrinogen solution first, followed by the thrombin solution (layer method). However, a new, more effective method recently has been reported (the so-called rubbing method), in which a low-viscous thrombin solution is first rubbed into the lung parenchyma, and then the fibrinogen solution is added.24 These 2 methods were performed in this study, and the bursting pressure occurred 5 minutes after application of the fibrin glues. The trachea, thoracic aorta, and small intestine, with diameters of about 20 mm, 10 mm, and 20 mm, respectively, were removed from the dead pigs. One end of the removed artery or small intestine was ligated with suture material, and the other side was intubated with a small catheter held in place by ligatures (Fig 2). The catheter was connected to a syringe and a manometer. The artery and small intestine were punctured with a needle (1.2 mm in diameter), and about 30 µL (1 drop) of Az-CH-LA aqueous solution (30 mg/mL) was applied to the hole and irradiated with UV light for 90 seconds.
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Fig 2. Scheme for the measurement of the bursting pressure with application of chitosan hydrogel on the aorta and trachea.
The artery and small intestine were placed under water, and they were inflated until bubbles could be detected in the water. The pressure required to produce this air leakage was defined as the bursting pressure (millimeters of mercury). The oral side of the trachea was intubated with the catheter, and the bilateral ends of the main bronchus were ligated. Subsequently, a 5-mm long and 5-mm deep transverse incision was made along an annular ligament in the middle section of the trachea. After the chitosan gel (30 µL) was applied, its bursting pressure was determined in the same way as that for the Az-CH-LA aqueous solution (see Fig 2). Similar experiments have been performed with fibrin glue, with bursting pressure measurements starting 5 minutes after application. Sealing effect in vivo and histologic examination. Fourteen male New Zealand white rabbits (2 to 3 kg) were anesthetized with an intramuscular injection of xylazine (12 mg) and ketamine (40 mg). In 7 rabbits, transverse skin incisions were made in the neck, resulting in exposure of the left carotid artery. Bleeding was initiated by pricking the artery with a needle (1.2 mm in diameter) and temporarily stopped by clamping both sides of the bleeding point with vascular clips. About 30 µL (1 drop) of the AzCH-LA aqueous solution was applied to the puncture site and then photocrosslinked by UV irradiation at a distance of 2 cm for 90 seconds. After the vascular clips on both sides of the bleeding point were carefully removed, blood flow and hemostasis were confirmed. Similar experiments have been performed with fibrin glue, with hemostasis confirmations starting 5 minutes after application. In the other 7 rabbits, an anterior lobe of the lung was exposed through a lateral thoracotomy, and a 5 mm-long and 5 mm-deep incision was made on the lung surface. After confirming air leakage as
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a result of the incision of the lung, 3 to 4 drops (total about 100 µL) of the Az-CH-LA aqueous solution were applied to the incision and photocrosslinked by UV irradiation for 90 seconds without stopping the respiration of the rabbits. A small tube was inserted to the thoracic cage, and the skin was then closed by suturing. Intrathoracic air and fluid were aspirated from the thoracic cage through the tube, and the tube was then withdrawn. Similar experiments have been performed with fibrin glue. Fluoroscopic observations (Diagnostic X-ray Apparatus, Model KXO-1000; Tokyo Shibaura Electric Co, Tokyo, Japan) showed that the lungs with punctures sealed by either the chitosan hydrogel or fibrin glue had expanded fully after the operations. The animals were then fed ad libitum (standard feed pellets) and killed after 30 days. The punctured artery and incised lung were removed, fixed in 10% neutral formaldehyde solution, and embedded in paraffin. The samples have been subjected to histologic examination by staining with Congo Red (Wako Pure Chemical Industries, Ltd, Osaka, Japan) (to specifically stain chitin and chitosan). The animal experiments were approved and carried out following the guidelines for animal experimentation at the National Defense Medical College, Tokorozawa, Saitama, Japan. Statistical analysis. All results on the bursting pressures are expressed as mean ± SD or SE. Comparison between multiple group averages were analyzed with one-way analysis of variance and the Scheffé multiple comparisons test. RESULTS Bursting pressure of chitosan hydrogel. The bursting pressure of the chitosan hydrogel in the lung was 51 ± 11 mm Hg (mean ± SD), which is more than that of fibrin glue with the layer method (12 ± 2 mm Hg) (P < .0001). The bursting pressure of the chitosan hydrogel was not statistically different from that of fibrin glue with the rubbing method (P = .138). The bursting pressures of the chitosan hydrogel and fibrin glue (rubbing method) on the trachea were 77 ± 29 mm Hg and 48 ± 21 mm Hg, on the aorta, 225 ± 25 mm Hg and 80 ± 20 mm Hg, and on the small intestine, 65 ± 5 mm Hg and 52 ± 8 mm Hg, respectively. The bursting pressure of the chitosan hydrogel was more than that of the fibrin glue (rubbing method) on the aorta (P < .0001), the trachea (P < .05), and the small intestine (P = .02). The bursting pressure of the fibrin glue with the rubbing method was significantly higher than that with the layer method for the aorta, trachea, and small intestine. These data suggest that the sealing strength of the chitosan
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hydrogel may be sufficient to stop arterial bleeding and air leakage from the lung or trachea in surgical applications. Sealing effects in vivo. Chitosan hydrogel applied to the pinhole on the carotid artery with UV irradiation was able to stop the bleeding (1 ± 1 minutes) faster than fibrin glue (3 ± 1 minutes). However, the bleeding could not be stopped with application of the Az-CH-LA aqueous solution without UV irradiation. Applications of chitosan hydrogel with UV irradiation for 90 seconds (n = 7) or fibrin glue for 5 minutes (n = 2) completely stopped bleeding from the carotid artery of rabbits in vivo. All rabbits were alive 30 days of postoperational observation, with no demonstration of any visual complications. Similarly, the chitosan hydrogel with UV irradiation stopped bleeding from the pinhole on the carotid artery of rabbits (n = 3) that were administered 20 mg/kg of native heparin (185.8 USP U/mg) for 30 minutes (data not shown), indicating that the hemostatic effect of the chitosan hydrogel is independent of the blood coagulation. Although mild fibrous adhesions were noticed 30 days after operation in the thoracic cavity of rabbits treated with chitosan hydrogel, adhesion between the area where chitosan hydrogel was applied and the opposite chest wall was not observed in all cases. The application of chitosan hydrogel with UV irradiation for 90 seconds (n = 7) and the application of fibrin glue (n = 2) also were able to stop the air leakage from the lung, and fluoroscopic observations showed that the sealed lungs expanded fully. All rabbits survived 1 month after the operation and did not demonstrate any visual complications. However, the air leakage from the lung could not be stopped with application of Az-CH-LA aqueous solution without UV irradiation (n = 2) during the operations. Fluoroscopy also showed that the control lungs were deflated during 3 days of postoperational observation, indicating an air leak. The Az-CH-LA aqueous solution is viscous enough to allow application to required sites without any spillage and is easier to apply than the fibrin glue solution. Therefore, the application of Az-CH-LA aqueous solution on the wound site may temporarily stop the air leakage from the lung until gelation of the chitosan occurs with UV irradiation. Microscopic and histologic findings. The area of a removed lung in which chitosan hydrogel was applied was covered with glossy smooth pleura after 30 days, and the chitosan hydrogel was encapsulated by fibrous tissue and covered with regenerated thick pleura (Fig 3, A). Chitosan is orange when Congo Red stain is used, and it was clearly
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shown that a considerable amount of the chitosan gel remained with numerous neutrophils that migrated into the gel (see Fig 3, A). The area in which fibrin glue was applied was replaced with mass of fibrous connective tissue, composed of many fibroblasts and collagen fiber networks; some lymphocytes were observed in the tissue (Fig 3, C). Fig 3, B, and Fig 4, B, show orange granules in many macrophages, indicating that chitosan molecules were phagocytosed by macrophages. Although the chitosan hydrogel is biodegradable, a large fraction of the chitosan gel was encapsulated by thick, fibrous tissue and may remain. Residual chitosan hydrogel was observed 60 days after operation (data not shown). Similarly, the wound of punctured carotid arteries were also closed completely and covered with dense fibrosis and granulation tissue that contained many newly formed capillary vessels and neutrophils 30 days after the operation (Fig 4, A). Although chitosan hydrogels were almost completely degraded, as can be seen on the carotid artery, some residual chitosan hydrogel is still visible (see Fig 4, B). DISCUSSION We previously have reported on a photocrosslinkable chitosan molecule (Az-CH-LA) that contains both lactose moieties and photoreactive azide groups.23 UV irradiation of an Az-CH-LA aqueous solution resulted in an insoluble hydrogel, comparable to a soft rubber, within 90 seconds. This hydrogel could firmly adhere 2 pieces of sliced ham to each other and could effectively seal air leakages (eg, from pinholes in small intestines).23 In this report, animal studies to evaluate the effectiveness and safety of the chitosan hydrogel as a tissue adhesive or sealant were described, with focus on hemostasis of the artery and air-sealing capacity on the lung. Our main conclusion is that the chitosan hydrogel may be a promising new surgical adhesive and sealant. In the animal studies described, chitosan hydrogels exhibited good sealing properties, superior to those of fibrin glue, on a pinhole in the artery and small intestine and an incision on the trachea. On the other hand, the sealing strength of fibrin glue on lung punctures with the rubbing method was almost identical to that of the chitosan hydrogel, although the strength of fibrin glue with the conventional layer method was much lower than that of the chitosan hydrogel. The data about the fibrin glue are identical as previously reported,24 suggesting that an anchoring effect of the fibrin glue on the lung takes place by rubbing on the injured site. However, the high bursting pressure of chitosan
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A
B
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A
B Fig 4. Histologic examination of a rabbit carotid artery 30 days after application of chitosan hydrogel. A, Stained with Congo Red, original magnification ×100. B, Stained with Congo Red, original magnification ×400. The photographs are representative of 7 rabbits in which chitosan hydrogel was applied. Arrows represent residual chitosan hydrogel on application sites.
C Fig 3. Histologic examination of a rabbit lung 30 days after operation. A, Lung with chitosan hydrogel and stained with Congo Red, original magnification ×100. B, Lung with chitosan hydrogel and stained with Congo Red, original magnification ×400. C, Lung with fibrin glue and stained with hematoxylin-eosin, original magnification ×100. The photographs are representative of 7 rabbits in which chitosan hydrogel or fibrin glue was applied. Arrows represent residual chitosan hydrogel on application sites.
hydrogels points to the possibility that the chitosan hydrogels may have the ability to seal the pinholes and incisions in these organs intraoperatively. Thirty days after operation of the lung, the chitosan hydrogel remained and dense fibrous tissue
was observed around the hydrogel in which inflammatory cells were infiltrated (see Fig 3). These responses can be ascribed to originate from foreign body reactions. Chitosan molecules, however, have been reported to induce migration of neutrophils and production of multiple cytokines 21,22 and to accelerate wound healing.15-19 Furthermore, it also has been reported that fibroblasts stimulated by chitosan molecules secrete interleukin-8 (IL-8).25 IL-8 is known to be angiogenic and chemoattractive to endothelial and epidermal cells.23 Fibroblasts that adhere to a chitosan hydrogel may secret IL-8 and other cytokines, which in turn could induce angiogenesis and fibrosis. These reactions may account for the accelerated tissue repair after application of chitosan hydrogels. Chitosan is considered to be a biodegradable molecule, and it has been reported that migrated
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neutrophils degrade chitosan molecules by secretion of lysozyme26 and chitotriosidase,21 in addition to phagocytosis of the molecules. However, a considerable fraction of chitosan hydrogel appeared to be encapsulated by fibrous tissue for longer than several months before its complete degradation (see Fig 3). Another advantage of the chitosan hydrogel is that the viscosity of the Az-CH-LA aqueous solution is higher than that of the fibrin glue and thus can easily be applied without spillage. It should also be noted that components of fibrin glue originate from human blood or bovine lung and hence always contain a certain risk of infectious contamination, such as with hepatitis viruses, human immunodeficiency virus, and unidentified pathogenic organisms. The risk of infection and anaphylaxis cannot be completely ruled out when fibrin glue is used. Since both chitin and chitosan are abundant, Az-CH-LA may be produced at a much lower cost.13 Fibrin glue was effective in controlling bleeding intraoperatively. It was used to control bleeding from anastomoses and aortic bleeding points, which result from needle holes or suture lines, and venous bleeding.7,8,11,12 In addition, fibrin glue also was used in lung procedures to control intractable air leakage from the resected surface or suture and staple lines of the lung, especially in procedures for emphysematous lung disease.24 It may be possible for chitosan hydrogel, having putative advantages over fibrin glue, to be used as a surgical adhesive in a similar situation. Chitosan has been proven to be safe in standard animal toxicologic studies. Chitosan has been used in food and skin wound management. 13,26,27 Azide residues may be toxic and highly reactive to tissue components, such as proteins. However, with a complete crossreaction of azide residues to amino groups, this toxic effect may disappear. Our previous results in mice also indicated that the Az-CH-LA aqueous solution and the chitosan hydrogel were found to be safe. Mice administered about 1200 mg/kg of the Az-CH-LA solution (n = 6) or the chitosan hydrogel (n = 6) intraperitoneally all survived the entire 1 month observation. 23 Cytotoxicity has not been observed with in vitro assays.23 In addition, preliminary toxicity tests of mutagenecity and cytotoxicity have shown the safety of both Az-CH-LA and its hydrogel (data not shown). However, sufficient data are not yet available on the complete toxicity profile of AzCH-LA aqueous solutions and chitosan hydrogels in humans, and standard toxicologic studies
should be performed before this chitosan hydrogel is used as a surgical adhesive.
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20. Peluso G, Petillo O, Ranieri M, Santin M, Ambrosio L, Calagro D, et al. Chitosan-mediated stimulation of macrophage function. Biomaterials 1994;15:1215-20. 21. Nishimura K, Ishihara C, Ukei S, Tokura S, Azuma I. Stimulation of cytokine production in mice using deacetylated chitin. Vaccine 1986;4:151-6. 22. Mori T, Okumura M, Matsuura M, Ueno K, Tokura S, Okamoto Y, et al. Effect of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro. Biomaterials 1997;18:947-51. 23. Ono K, Saito Y, Yura H, Ishikawa K, Kurita A, Akaike T, et al. Photocrosslinkable chitosan as a biological adhesive. J Biomed Mater Res 2000;49:289-95.
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24. Morikawa T, Katoh H. Improved techniques of applying fibrin glue in lung surgery. Eur Surg Res 1999;31:180-6. 25. Koch AE, Polverini PJ, Kunkel SL, Harlow LA, Dipietro LA, Elner VM, et al. Interleukin-8 as a macrophagederived mediator of angiogenesis. Science 1992;258:1798-801. 26. Muzzarelli RA. Biochemical significance of exogenous chitins and chitosans in animals and patients. Carbohydr Polym 1993;20:7-16. 27. Rao SB, Sharma CP. Use of chitosan as a biomaterial: studies on its safety and hemostatic potential. J Biomed Mater Res 1997;34:21-8.
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