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Mixing collagen with fibrin glue to strengthen the sealing effect for pulmonary air leakage
Mixing Collagen With Fibrin Glue to Strengthen the Sealing Effect for Pulmonary Air Leakage Hiroaki Nomori, MD, Hirotoshi Horio, MD, and Keiichi Suemasu, MD Department of Thoracic Surgery, Saiseikai Central Hospital, Tokyo, Japan
Background. To strengthen the sealing effect of fibrin glue for pulmonary air leakage, atelocollagen was mixed with the glue and the mixing effect was examined. Methods. A mixture of fibrinogen and thrombin with atelocollagen was used as a test sample. The concentrations of atelocollagen were adjusted to levels of 0%, 0.375%, 0.75%, 1.1%, and 1.5%. We next performed air leakage tests on a plastic cap with pin holes and swine lung and also measured the elasticity and the adhesion strength. Results. The pressure required to rupture the sealant on a plastic cap with pin holes increased as the concentration of atelocollagen increased, and the bursting pressures were significantly higher in the glue with 0.75%, 1.1%, and 1.5% of atelocollagen than in the glue without atelocollagen (p < 0.01 and p < 0.001). The air leakage pressure on the swine lung was significantly higher in
the glue with 0.375%, 0.75%, and 1.1% of atelocollagen than in the glue without atelocollagen (p < 0.05 and p < 0.01), and it was the highest with 0.75%. The elasticity of the glue significantly increased as the concentration of atelocollagen increased (p < 0.001). However, the adhesion strength of the glue significantly decreased as the concentration of atelocollagen increased (p < 0.05 to p < 0.001). Conclusions. The mixing of atelocollagen with fibrin glue more effectively sealed pulmonary air leakage due to an increased elasticity of the glue while its adhesion strength decreased. The optimal concentration of atelocollagen in the fibrin glue to obtain the best sealing effect was 0.75%.
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thrombin, which is also freeze-dried; and the calcium chloride solution. Fibrinogen was dissolved using an aprotinin solution according to the original method. Thrombin was dissolved by the calcium chloride solution (11.8 mg/mL), which was twice the concentration and half the volume in comparison with the original method. The thrombin dissolved in the calcium chloride was then mixed with the same volume of either 1.5%, 3.0%, 4.4%, or 6.0% of atelocollagen. The same volume of both fibrinogen solution and the thrombin solution with atelocollagen were then mixed with each other and used as a test sample. The final concentrations of atelocollagen were, therefore, 0.375%, 0.75%, 1.1%, and 1.5%. The original fibrin glue without atelocollagen was used as a control. The sealing effectiveness in vitro was evaluated as the same method reported by Fukunaga and associates [7] (Fig 1). Briefly, five holes, each measuring 1.2 mm in diameter, were made in each of the 50-mL conical tube caps (Falcon 2070; Becton Dickinson Labware, Lincoln Park, NJ). Next, 0.8 mL of the fibrin solution and 0.8 mL of the thrombin solution with each concentration of atelocollagen were applied through a double syringe to cover the holes, as specified by the manufacturer. The caps were replaced and screwed onto the conical tubes. There were two air lines connected to the conical tubes. One was connected to an air supply syringe, and the other was connected to a manometer (POP 760; Okano, Osaka, Japan) to measure the air pressure. Five minutes after applying the glue, the sealing effectiveness was
ibrin glue has been widely used to seal pulmonary air leakage [1–3]. Thoracic surgeons, however, have often found that using fibrin glue for pulmonary parenchymal defects fails to seal air leakage and also causes recurring postoperative leakage. Atelocollagen, which is generally injected into subcutaneous tissue, vocal cords, and urethral sphincter for cosmetic or functional purposes, has also been shown to strengthen the tissue intensity by collagen fiber [4 – 6]. Based on this phenomenon, we mixed atelocollagen with fibrin glue to increase the glue’s sealing effect for pulmonary air leakage and examined the effect of adding atelocollagen by performing air leakage tests in vitro and in vivo, while also measuring the elasticity and testing the adhesion strength.
Material and Methods Atelocollagen is manufactured by Koken Co (Tokyo, Japan). The fibrin glue used in this study is manufactured by Hoechst Marion Roussel Ltd (Beriplast; Frankfurt, Germany). The Beriplast kit includes separate vials containing the individual agents, which together form the glue. They are the fibrinogen, which is freeze-dried; aprotinin solution, which is a fibrinolysis inhibitor; Accepted for publication April 24, 2000. Address reprint requests to Dr Nomori, Department of Surgery, Saiseikai Central Hospital, 1-4-17 Mita, Minato-ku, Tokyo 108-0073, Japan; e-mail: [email protected].
© 2000 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 2000;70:1666 –70) © 2000 by The Society of Thoracic Surgeons
0003-4975/00/$20.00 PII S0003-4975(00)01936-6
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Fig 1. Apparatus for measuring the bursting pressure.
evaluated by recording the maximum pressure needed to break the fibrin glue (bursting pressure). We also conducted an air leakage test using a swine lung to compare the sealing effect of the fibrin glue mixed with each concentration of atelocollagen, as described previously [8]. Two pigs (33 and 35 kg) were anesthetized, intubated using a Broncho-Cath tracheal tube (Mallinckrodt Med, Athlone, Ireland) for one-lung ventilation, and ventilated with a ventilator (Fancy 80 Monitor Animal; Kimura Med, Tokyo, Japan). After performing a right thoracotomy, a peripheral strip measuring 2 mm deep, 15 mm long, and 10 mm wide was cut from the lateral surface of the lung. The lung was immersed in normal saline solution, and air leakage was quantified by maintaining the pressure at the same level that the leakage first occurred for 15 seconds. The fibrinogen solution (0.5 mL) and the thrombin solution (0.5 mL) with each concentration of atelocollagen were applied through a double syringe to cover the lung wound, as specified by the manufacturer. The glued surface of the lung was kept at rest using one-lung ventilation for 5 minutes. The lung was then again ventilated after immersion in normal saline solution, and the pressure was monitored. The experiment was repeated three times on different wounds for each glue in the two pigs. The elasticity of the glue was examined by a complex modulus of elasticity (E*) using a nonresonant, forced, fixed-frequency oscillation method, which was designated by Japanese Industrial Standard (JIS) [9]. The main parts of the tester are shown in Figure 2. Briefly, a cylindrical-shaped glue measuring 6 mm in diameter and 3 mm in length was prepared using a container. A test piece was set in the tester. The sinusoidal strain was added to the sample by compression from 0.1% to 1.2% of the sample height (3 mm), and the generated stress and strain amount was measured. The complex modulus of elasticity (E*) was calculated by the formula: E* ⫽ L/bt ⫻
Fig 2. Main parts of the tester for tensile oscillation method.
⌬F/⌬L, where E* ⫽ complex modulus of elasticity (Pascal), ⌬F ⫽ dynamic load (N), ⌬L ⫽ dynamic displacement of the length (mm), L ⫽ length of test piece (mm), and bt ⫽ area of section of test piece (mm2). Each measurement was conducted in triplicate, and the mean value was shown as the data. The temperature of the tester was maintained at 23°C. To examine the differences in the adhesion strength of fibrin glue at each concentration of atelocollagen, we performed an adhesion strength test using swine skin strips as described previously [10]. Two swine skin strips, 4 cm long and 2 cm wide, were used (Fig 3). We spread 0.025 mL of fibrinogen solution on the edge of the one strip and 0.025 mL of thrombin solution with each concentration of atelocollagen on the edge of the other strip, measuring 0.5 ⫻ 2 cm in size. Each edge of the two strips
Fig 3. Adhesion strength test. Two swine skin strips were overlapped at the edges after applying fibrinogen solution and thrombin solution with atelocollagen.
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creased as the concentration of atelocollagen increased, and the pressures were significantly higher in the glues with 0.75%, 1.1%, and 1.5% atelocollagen than that in the glue without atelocollagen ( p ⬍ 0.01, p ⬍ 0.001, and p ⬍ 0.001, respectively). The mean pressures required to produce air leakage in the swine lung wounds were 6.8 ⫾ 2.4 cm H2O in the nontreatment, 22.5 ⫾ 5.6 cm H2O in the glue without atelocollagen (0%), 42.5 ⫾ 7.5 cm H2O in the glue with 0.375% of atelocollagen, 58.8 ⫾ 12.4 cm H2O at 0.75%, 35.0 ⫾ 6.1 cm H2O at 1.1%, and 25.1 ⫾ 6.1 cm H2O at 1.5% (Fig 5). The glue with 0.75% of atelocollagen showed the highest pressure tolerance. The mean pressure required to produce air leakage was significantly higher in the glue with 0.375%, 0.75%, and 1.1% atelocollagen than in the glue without atelocollagen (0%) ( p ⬍ 0.05 or p ⬍ 0.01). The pressure required to produce air leakage was significantly higher in the glue with 0.75% of atelocollagen than in the glue with 1.1% atelocollagen ( p ⬍ 0.05). There was no significant difference between the glue without atelocollagen (0%) or with 1.5% atelocollagen.
Fig 4. Sealing test using the apparatus shown in Figure 1. The bursting pressures increased as the concentration of atelocollagen increased, and the pressures in the glue with 0.75%, 1.1%, and 1.5% of atelocollagen were significantly higher than that in the glue without atelocollagen (p ⬍ 0.01 or p ⬍ 0.001).
was then attached firmly to each other. Thirty minutes later, both edges of the strips were pulled horizontally by a spring scale machine (IM-20DX; Intesco, Co, Matsudo, Japan), and the weight at the point when the strips pulled apart was measured. Each experiment was repeated three times.
Statistical Analysis The differences in the data for the air leakage test and the adhesion strength test for each concentration of atelocollagen were analyzed for significance using the two-tailed Student’s t test. The differences in the elasticity for each strain among each concentration of atelocollagen were analyzed using the two-tailed Student’s t test for paired values. Differences of p less than 0.05 were considered to be significant. All the values in the text and tables are the means ⫾ SD.
Results The mean pressures required to rupture the seals on the plastic caps with pin holes were 18.0 ⫾ 2.0 cm H2O in the glue without atelocollagen (0%), 22.0 ⫾ 2.0 cm H2O in the glue with 0.375% of atelocollagen, 26.7 ⫾ 2.3 cm H2O at 0.75%, 51.0 ⫾ 12.1 cm H2O at 1.1%, and 82.0 ⫾ 10.6 cm H2O at 1.5% (Fig 4). The mean bursting pressures in-
Fig 5. Air leakage test in swine lung results showed that the glue with atelocollagen concentrations of 0.375%, 0.75%, and 1.1% had significantly more pressure tolerance than the glue without atelocollagen (p ⬍ 0.05 or p ⬍ 0.01), and the glue with 0.75% of atelocollagen had the highest pressure tolerance.
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Table 1. The Dependence of Elasticity (E⬘) on Each Strain of Fibrin Glue With Each Concentration of Atelocollagen Concentration of Atelocollagen Strain (%)
0%
0.375%
0.75%
1.1%
1.5%
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2
1.42 1.50 1.52 1.55 1.56 1.57 1.58 1.59 1.59 1.60 1.61 1.61
1.80 1.90 2.02 2.05 2.07 2.09 2.10 2.11 2.12 2.12 2.13 2.13
2.40 2.48 2.51 2.54 2.57 2.63 2.65 2.68 2.69 2.70 2.71 2.71
2.63 2.75 2.83 2.87 2.89 2.90 2.91 2.91 2.92 2.92 2.93 2.93
2.81 2.93 3.00 3.04 3.07 3.08 3.09 3.10 3.10 3.11 3.11 3.12
Data are elasticities (⫻105 Pascal).
The mean elasticities on each strain in each glue are summarized in Table 1 and demonstrated in Figure 6. As shown in Figure 6, the elasticity for each strain produced a linear curve, thus suggesting the experimental conditions to be appropriate. The elasticity of the glue increased as the concentration of atelocollagen increased, and there was a significant difference in the elasticity between each concentration of atelocollagen ( p ⬍ 0.001). The glue at all concentrations of atelocollagen showed a significantly higher elasticity than the glue without atelocollagen ( p ⬍ 0.001). The adhesion strength test showed that the adhesive ability of the glue decreased as the concentration of atelocollagen increased (Fig 7). The adhesive strength of the glue without atelocollagen (0%) was 191 ⫾ 22 g, 164 ⫾ 19 g at a concentration of 0.375% atelocollagen, 118 ⫾ 15 g at 0.75%, 92 ⫾ 13 g at 1.1%, and 89 ⫾ 11 g at 1.5%. Fibrin glue with 0.375%, 0.75%, 1.1%, and 1.5% atelocollagen showed a significantly lower adhesive ability than the
Fig 6. Dependence of elasticity (E⬘) on each strain. The elasticity of the glue showed a significant difference between each concentration of atelocollagen (p ⬍ 0.001).
Fig 7. The adhesion strength test results showed a significant decrease in the adhesion ability as the concentration of atelocollagen increased (p ⬍ 0.05 to p ⬍ 0.001).
glue without atelocollagen ( p ⬍ 0.05, p ⬍ 0.001, p ⬍ 0.001, and p ⬍ 0.001, respectively).
Comment Fibrin glue has been experimentally shown to effectively seal pulmonary air leakage [1–3]. Tu¨rk and associates [1]
Fig 8. A schema of the lung with fistula covered by the glue. (A) Air leakage is blocked by elasticity of the glue. (B) Gap between the glue and expanding lung is fixed by adhesion strength of the glue.
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reported that fibrin glue sealing of the suture line significantly increased the pressure tolerance compared with conventional lung sutures when tested intraoperatively. Thoracic surgeons, however, have found that for pulmonary parenchymal defects, fibrin without suturing often fails to prevent air leakage during an operation and tends to cause recurring postoperative leakage. In a randomized study, Fleisher and associates [11] found that the routine use of fibrin glue does not effectively reduce the duration of air leakage, chest tube drainage, or hospitalization after a lobectomy. Recently, Tsuda and associates have reported that a human collagen membrane polyglycolic acid sheet using fibrin glue efficiently seals pulmonary air leakage in dogs [12]. Their method, however, necessitates a suture of the sheet at the lung wound, which is not a easy technique under thoracoscopic surgery. Gelatin-resorcinol formaldehyde glutaraldehyde (GRFG) glue has been recently shown to be more effective for sealing pulmonary air leakage and is also useful under thoracoscopic surgery [10]. GRFG glue, however, often induces histotoxicity because it uses formaldehydeglutaraldehyde (FG), which causes tissue necrosis beneath the glue for 2 months after sealing [10]. In contrast, fibrin glue does not induce histotoxicity and does not impair wound healing [13]. We therefore made a fibrin glue stronger by mixing with an atelocollagen. Because atelocollagen generally has been injected in subcutaneous tissue, vocal cords, and urethral sphincter for cosmetic or functional purposes without any complications, the mixing of atelocollagen with fibrin glue presents no problems in its clinical application. Before clinical application, however, we examined an effect of mixing atelocollagen with glue to evaluate its sealing ability and to determine the optimal concentration of atelocollagen in the glue in the present study. The results of a sealing test in vitro showed the pressure tolerance of the glue to increase as the concentration of atelocollagen increased. On the other hand, the results of an air leakage test in swine lung showed the pressure tolerance to be higher in the glue with 0.375%, 0.75%, and 1.1% atelocollagen than in the glue without atelocollagen, while it was the highest with 0.75% atelocollagen, however, a 1.5% concentration of atelocollagen did not increase the pressure tolerance. To examine the cause of this discrepancy between the in vitro and in vivo results and the mechanism of increasing pressure tolerance in those concentrations of atelocollagen, the elasticity and adhesion strength of the glue were both measured. As a result, while the elasticity increased as the concentration of atelocollagen increased, the adhesive ability was decreased. These results could clarify the discrepancy between the in vitro and in vivo sealing effect of the glue, as shown in Figure 8. In the pressure sealing test using a swine lung, the gap between the glue and lung surface occurs when the lung expands with increasing pressure. An adhesion ability of the glue is therefore necessary to seal the expanding lung. On the
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other hand, in the pressure sealing test using a plastic cap, no gap between the glue and cap surface occurs when the pressure increases, as shown in Figure 1, resulting in less effect of adhesive ability of the glue than that observed in the test on swine lung. Therefore, while the results of air leakage test on the swine lung demonstrated both the elasticity and adhesive ability of the glue, the sealing test using the plastic cap demonstrated mainly the elasticity of the glue. These results indicated that the strengthened sealing effect of the glue by mixing with atelocollagen was due to the increased elasticity of the glue, and that the optimal concentration of atelocollagen for sealing the lung wound was determined to be 0.75%. In conclusion, the mixing of atelocollagen made the fibrin glue more effective for sealing pulmonary air leakage, and the optimal concentration of atelocollagen was 0.75%. We are therefore now going to use atelocollagen-mixed fibrin glue in clinical trials.
References 1. Tu¨rk R, Weidringer W, Hartel W, Blu¨mel G. Closure of lung leaks by fibrin gluing. Experimental investigation and clinical experience. Thorac Cardiovasc Surg 1983;31:185– 6. 2. Bergsland J, Kalmbach T, Balu D, Feldman MJ, Caruana JA, Gage AA. Fibrin seal: an alternative to suture repair in experimental pulmonary surgery. J Surg Res 1986;40:340–5. 3. McCarthy PM, Trastek VF, Bell DG, et al. The effectiveness of fibrin glue sealant for reducing experimental pulmonary air leak. Ann Thorac Surg 1988;45:203–5. 4. Charriere G, Bejot M, Schnitzler L, Ville G, Hartmann DJ. Reactions to bovine collagen implant. Clinical and immunologic study in 705 patients. J Am Acad Dermatol 1989;21: 1203– 8. 5. Ford CN, Martin DW, Warner TF. Injectable collagen in laryngeal rehabilitation. Laryngoscope 1984;94:513– 8. 6. Shortliffe LMD, Freiha FS, Kessler R, Stamey TA, Constantinou CE. Treatment of urinary incontinence by periurethral implantation of glutaraldehyde cross-linked collagen. J Urol 1989;141:538– 41. 7. Fukunaga N, Uchida T, Kaetsu H, Kawakami K, Ishihara Y, Funatsu A. Effective application of fibrin sealant using a spray device with a double-tube structure. Int J Adhesion Adhesives 1998;18:345–9. 8. Nomori H, Horio H, Morinaga S, Suemasu K. Gelatinresorcinol formaldehyde-glutaraldehyde (GRFG) glue for sealing pulmonary air leaks during thoracoscopic operation. Ann Thorac Surg 1999;67:212– 6. 9. Japanese Industrial Standard (JIS). 1991:K 7198. 10. Nomori H, Horio H. Gelatin-resorcinol formaldehydeglutaraldehyde (GRFG) glue-spread stapler prevents air leakage from the lung. Ann Thorac Surg 1997;63:352–5. 11. Fleisher AG, Evans KG, Nelems B, Finley RJ. Effect of routine fibrin glue use on the duration of air leaks after lobectomy. Ann Thorac Surg 1990;49:133– 4. 12. Tsuda T, Nakamura T, Yamamoto Y, et al. Prevention of postoperative air leakage from lungs using a purified human collagen membrane-polyglycolic acid sheet. Ann Thorac Surg 1999;68:339– 42. 13. Romanos GE, Strub JR. Effect of Tissucol® on connective tissue matrix during healing: an immunohistochemical study in rat skin. J Biomed Mater Res 1998;39:462– 8.
Background. To strengthen the sealing effect of fibrin glue for pulmonary air leakage, atelocollagen was mixed with the glue and the mixing effect was examined. Methods. A mixture of fibrinogen and thrombin with atelocollagen was used as a test sample. The concentrations of atelocollagen were adjusted to levels of 0%, 0.375%, 0.75%, 1.1%, and 1.5%. We next performed air leakage tests on a plastic cap with pin holes and swine lung and also measured the elasticity and the adhesion strength. Results. The pressure required to rupture the sealant on a plastic cap with pin holes increased as the concentration of atelocollagen increased, and the bursting pressures were significantly higher in the glue with 0.75%, 1.1%, and 1.5% of atelocollagen than in the glue without atelocollagen (p < 0.01 and p < 0.001). The air leakage pressure on the swine lung was significantly higher in
the glue with 0.375%, 0.75%, and 1.1% of atelocollagen than in the glue without atelocollagen (p < 0.05 and p < 0.01), and it was the highest with 0.75%. The elasticity of the glue significantly increased as the concentration of atelocollagen increased (p < 0.001). However, the adhesion strength of the glue significantly decreased as the concentration of atelocollagen increased (p < 0.05 to p < 0.001). Conclusions. The mixing of atelocollagen with fibrin glue more effectively sealed pulmonary air leakage due to an increased elasticity of the glue while its adhesion strength decreased. The optimal concentration of atelocollagen in the fibrin glue to obtain the best sealing effect was 0.75%.
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thrombin, which is also freeze-dried; and the calcium chloride solution. Fibrinogen was dissolved using an aprotinin solution according to the original method. Thrombin was dissolved by the calcium chloride solution (11.8 mg/mL), which was twice the concentration and half the volume in comparison with the original method. The thrombin dissolved in the calcium chloride was then mixed with the same volume of either 1.5%, 3.0%, 4.4%, or 6.0% of atelocollagen. The same volume of both fibrinogen solution and the thrombin solution with atelocollagen were then mixed with each other and used as a test sample. The final concentrations of atelocollagen were, therefore, 0.375%, 0.75%, 1.1%, and 1.5%. The original fibrin glue without atelocollagen was used as a control. The sealing effectiveness in vitro was evaluated as the same method reported by Fukunaga and associates [7] (Fig 1). Briefly, five holes, each measuring 1.2 mm in diameter, were made in each of the 50-mL conical tube caps (Falcon 2070; Becton Dickinson Labware, Lincoln Park, NJ). Next, 0.8 mL of the fibrin solution and 0.8 mL of the thrombin solution with each concentration of atelocollagen were applied through a double syringe to cover the holes, as specified by the manufacturer. The caps were replaced and screwed onto the conical tubes. There were two air lines connected to the conical tubes. One was connected to an air supply syringe, and the other was connected to a manometer (POP 760; Okano, Osaka, Japan) to measure the air pressure. Five minutes after applying the glue, the sealing effectiveness was
ibrin glue has been widely used to seal pulmonary air leakage [1–3]. Thoracic surgeons, however, have often found that using fibrin glue for pulmonary parenchymal defects fails to seal air leakage and also causes recurring postoperative leakage. Atelocollagen, which is generally injected into subcutaneous tissue, vocal cords, and urethral sphincter for cosmetic or functional purposes, has also been shown to strengthen the tissue intensity by collagen fiber [4 – 6]. Based on this phenomenon, we mixed atelocollagen with fibrin glue to increase the glue’s sealing effect for pulmonary air leakage and examined the effect of adding atelocollagen by performing air leakage tests in vitro and in vivo, while also measuring the elasticity and testing the adhesion strength.
Material and Methods Atelocollagen is manufactured by Koken Co (Tokyo, Japan). The fibrin glue used in this study is manufactured by Hoechst Marion Roussel Ltd (Beriplast; Frankfurt, Germany). The Beriplast kit includes separate vials containing the individual agents, which together form the glue. They are the fibrinogen, which is freeze-dried; aprotinin solution, which is a fibrinolysis inhibitor; Accepted for publication April 24, 2000. Address reprint requests to Dr Nomori, Department of Surgery, Saiseikai Central Hospital, 1-4-17 Mita, Minato-ku, Tokyo 108-0073, Japan; e-mail: [email protected].
© 2000 by The Society of Thoracic Surgeons Published by Elsevier Science Inc
(Ann Thorac Surg 2000;70:1666 –70) © 2000 by The Society of Thoracic Surgeons
0003-4975/00/$20.00 PII S0003-4975(00)01936-6
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Fig 1. Apparatus for measuring the bursting pressure.
evaluated by recording the maximum pressure needed to break the fibrin glue (bursting pressure). We also conducted an air leakage test using a swine lung to compare the sealing effect of the fibrin glue mixed with each concentration of atelocollagen, as described previously [8]. Two pigs (33 and 35 kg) were anesthetized, intubated using a Broncho-Cath tracheal tube (Mallinckrodt Med, Athlone, Ireland) for one-lung ventilation, and ventilated with a ventilator (Fancy 80 Monitor Animal; Kimura Med, Tokyo, Japan). After performing a right thoracotomy, a peripheral strip measuring 2 mm deep, 15 mm long, and 10 mm wide was cut from the lateral surface of the lung. The lung was immersed in normal saline solution, and air leakage was quantified by maintaining the pressure at the same level that the leakage first occurred for 15 seconds. The fibrinogen solution (0.5 mL) and the thrombin solution (0.5 mL) with each concentration of atelocollagen were applied through a double syringe to cover the lung wound, as specified by the manufacturer. The glued surface of the lung was kept at rest using one-lung ventilation for 5 minutes. The lung was then again ventilated after immersion in normal saline solution, and the pressure was monitored. The experiment was repeated three times on different wounds for each glue in the two pigs. The elasticity of the glue was examined by a complex modulus of elasticity (E*) using a nonresonant, forced, fixed-frequency oscillation method, which was designated by Japanese Industrial Standard (JIS) [9]. The main parts of the tester are shown in Figure 2. Briefly, a cylindrical-shaped glue measuring 6 mm in diameter and 3 mm in length was prepared using a container. A test piece was set in the tester. The sinusoidal strain was added to the sample by compression from 0.1% to 1.2% of the sample height (3 mm), and the generated stress and strain amount was measured. The complex modulus of elasticity (E*) was calculated by the formula: E* ⫽ L/bt ⫻
Fig 2. Main parts of the tester for tensile oscillation method.
⌬F/⌬L, where E* ⫽ complex modulus of elasticity (Pascal), ⌬F ⫽ dynamic load (N), ⌬L ⫽ dynamic displacement of the length (mm), L ⫽ length of test piece (mm), and bt ⫽ area of section of test piece (mm2). Each measurement was conducted in triplicate, and the mean value was shown as the data. The temperature of the tester was maintained at 23°C. To examine the differences in the adhesion strength of fibrin glue at each concentration of atelocollagen, we performed an adhesion strength test using swine skin strips as described previously [10]. Two swine skin strips, 4 cm long and 2 cm wide, were used (Fig 3). We spread 0.025 mL of fibrinogen solution on the edge of the one strip and 0.025 mL of thrombin solution with each concentration of atelocollagen on the edge of the other strip, measuring 0.5 ⫻ 2 cm in size. Each edge of the two strips
Fig 3. Adhesion strength test. Two swine skin strips were overlapped at the edges after applying fibrinogen solution and thrombin solution with atelocollagen.
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creased as the concentration of atelocollagen increased, and the pressures were significantly higher in the glues with 0.75%, 1.1%, and 1.5% atelocollagen than that in the glue without atelocollagen ( p ⬍ 0.01, p ⬍ 0.001, and p ⬍ 0.001, respectively). The mean pressures required to produce air leakage in the swine lung wounds were 6.8 ⫾ 2.4 cm H2O in the nontreatment, 22.5 ⫾ 5.6 cm H2O in the glue without atelocollagen (0%), 42.5 ⫾ 7.5 cm H2O in the glue with 0.375% of atelocollagen, 58.8 ⫾ 12.4 cm H2O at 0.75%, 35.0 ⫾ 6.1 cm H2O at 1.1%, and 25.1 ⫾ 6.1 cm H2O at 1.5% (Fig 5). The glue with 0.75% of atelocollagen showed the highest pressure tolerance. The mean pressure required to produce air leakage was significantly higher in the glue with 0.375%, 0.75%, and 1.1% atelocollagen than in the glue without atelocollagen (0%) ( p ⬍ 0.05 or p ⬍ 0.01). The pressure required to produce air leakage was significantly higher in the glue with 0.75% of atelocollagen than in the glue with 1.1% atelocollagen ( p ⬍ 0.05). There was no significant difference between the glue without atelocollagen (0%) or with 1.5% atelocollagen.
Fig 4. Sealing test using the apparatus shown in Figure 1. The bursting pressures increased as the concentration of atelocollagen increased, and the pressures in the glue with 0.75%, 1.1%, and 1.5% of atelocollagen were significantly higher than that in the glue without atelocollagen (p ⬍ 0.01 or p ⬍ 0.001).
was then attached firmly to each other. Thirty minutes later, both edges of the strips were pulled horizontally by a spring scale machine (IM-20DX; Intesco, Co, Matsudo, Japan), and the weight at the point when the strips pulled apart was measured. Each experiment was repeated three times.
Statistical Analysis The differences in the data for the air leakage test and the adhesion strength test for each concentration of atelocollagen were analyzed for significance using the two-tailed Student’s t test. The differences in the elasticity for each strain among each concentration of atelocollagen were analyzed using the two-tailed Student’s t test for paired values. Differences of p less than 0.05 were considered to be significant. All the values in the text and tables are the means ⫾ SD.
Results The mean pressures required to rupture the seals on the plastic caps with pin holes were 18.0 ⫾ 2.0 cm H2O in the glue without atelocollagen (0%), 22.0 ⫾ 2.0 cm H2O in the glue with 0.375% of atelocollagen, 26.7 ⫾ 2.3 cm H2O at 0.75%, 51.0 ⫾ 12.1 cm H2O at 1.1%, and 82.0 ⫾ 10.6 cm H2O at 1.5% (Fig 4). The mean bursting pressures in-
Fig 5. Air leakage test in swine lung results showed that the glue with atelocollagen concentrations of 0.375%, 0.75%, and 1.1% had significantly more pressure tolerance than the glue without atelocollagen (p ⬍ 0.05 or p ⬍ 0.01), and the glue with 0.75% of atelocollagen had the highest pressure tolerance.
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Table 1. The Dependence of Elasticity (E⬘) on Each Strain of Fibrin Glue With Each Concentration of Atelocollagen Concentration of Atelocollagen Strain (%)
0%
0.375%
0.75%
1.1%
1.5%
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2
1.42 1.50 1.52 1.55 1.56 1.57 1.58 1.59 1.59 1.60 1.61 1.61
1.80 1.90 2.02 2.05 2.07 2.09 2.10 2.11 2.12 2.12 2.13 2.13
2.40 2.48 2.51 2.54 2.57 2.63 2.65 2.68 2.69 2.70 2.71 2.71
2.63 2.75 2.83 2.87 2.89 2.90 2.91 2.91 2.92 2.92 2.93 2.93
2.81 2.93 3.00 3.04 3.07 3.08 3.09 3.10 3.10 3.11 3.11 3.12
Data are elasticities (⫻105 Pascal).
The mean elasticities on each strain in each glue are summarized in Table 1 and demonstrated in Figure 6. As shown in Figure 6, the elasticity for each strain produced a linear curve, thus suggesting the experimental conditions to be appropriate. The elasticity of the glue increased as the concentration of atelocollagen increased, and there was a significant difference in the elasticity between each concentration of atelocollagen ( p ⬍ 0.001). The glue at all concentrations of atelocollagen showed a significantly higher elasticity than the glue without atelocollagen ( p ⬍ 0.001). The adhesion strength test showed that the adhesive ability of the glue decreased as the concentration of atelocollagen increased (Fig 7). The adhesive strength of the glue without atelocollagen (0%) was 191 ⫾ 22 g, 164 ⫾ 19 g at a concentration of 0.375% atelocollagen, 118 ⫾ 15 g at 0.75%, 92 ⫾ 13 g at 1.1%, and 89 ⫾ 11 g at 1.5%. Fibrin glue with 0.375%, 0.75%, 1.1%, and 1.5% atelocollagen showed a significantly lower adhesive ability than the
Fig 6. Dependence of elasticity (E⬘) on each strain. The elasticity of the glue showed a significant difference between each concentration of atelocollagen (p ⬍ 0.001).
Fig 7. The adhesion strength test results showed a significant decrease in the adhesion ability as the concentration of atelocollagen increased (p ⬍ 0.05 to p ⬍ 0.001).
glue without atelocollagen ( p ⬍ 0.05, p ⬍ 0.001, p ⬍ 0.001, and p ⬍ 0.001, respectively).
Comment Fibrin glue has been experimentally shown to effectively seal pulmonary air leakage [1–3]. Tu¨rk and associates [1]
Fig 8. A schema of the lung with fistula covered by the glue. (A) Air leakage is blocked by elasticity of the glue. (B) Gap between the glue and expanding lung is fixed by adhesion strength of the glue.
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NOMORI ET AL FIBRIN GLUE MIXED WITH COLLAGEN
reported that fibrin glue sealing of the suture line significantly increased the pressure tolerance compared with conventional lung sutures when tested intraoperatively. Thoracic surgeons, however, have found that for pulmonary parenchymal defects, fibrin without suturing often fails to prevent air leakage during an operation and tends to cause recurring postoperative leakage. In a randomized study, Fleisher and associates [11] found that the routine use of fibrin glue does not effectively reduce the duration of air leakage, chest tube drainage, or hospitalization after a lobectomy. Recently, Tsuda and associates have reported that a human collagen membrane polyglycolic acid sheet using fibrin glue efficiently seals pulmonary air leakage in dogs [12]. Their method, however, necessitates a suture of the sheet at the lung wound, which is not a easy technique under thoracoscopic surgery. Gelatin-resorcinol formaldehyde glutaraldehyde (GRFG) glue has been recently shown to be more effective for sealing pulmonary air leakage and is also useful under thoracoscopic surgery [10]. GRFG glue, however, often induces histotoxicity because it uses formaldehydeglutaraldehyde (FG), which causes tissue necrosis beneath the glue for 2 months after sealing [10]. In contrast, fibrin glue does not induce histotoxicity and does not impair wound healing [13]. We therefore made a fibrin glue stronger by mixing with an atelocollagen. Because atelocollagen generally has been injected in subcutaneous tissue, vocal cords, and urethral sphincter for cosmetic or functional purposes without any complications, the mixing of atelocollagen with fibrin glue presents no problems in its clinical application. Before clinical application, however, we examined an effect of mixing atelocollagen with glue to evaluate its sealing ability and to determine the optimal concentration of atelocollagen in the glue in the present study. The results of a sealing test in vitro showed the pressure tolerance of the glue to increase as the concentration of atelocollagen increased. On the other hand, the results of an air leakage test in swine lung showed the pressure tolerance to be higher in the glue with 0.375%, 0.75%, and 1.1% atelocollagen than in the glue without atelocollagen, while it was the highest with 0.75% atelocollagen, however, a 1.5% concentration of atelocollagen did not increase the pressure tolerance. To examine the cause of this discrepancy between the in vitro and in vivo results and the mechanism of increasing pressure tolerance in those concentrations of atelocollagen, the elasticity and adhesion strength of the glue were both measured. As a result, while the elasticity increased as the concentration of atelocollagen increased, the adhesive ability was decreased. These results could clarify the discrepancy between the in vitro and in vivo sealing effect of the glue, as shown in Figure 8. In the pressure sealing test using a swine lung, the gap between the glue and lung surface occurs when the lung expands with increasing pressure. An adhesion ability of the glue is therefore necessary to seal the expanding lung. On the
Ann Thorac Surg 2000;70:1666 –70
other hand, in the pressure sealing test using a plastic cap, no gap between the glue and cap surface occurs when the pressure increases, as shown in Figure 1, resulting in less effect of adhesive ability of the glue than that observed in the test on swine lung. Therefore, while the results of air leakage test on the swine lung demonstrated both the elasticity and adhesive ability of the glue, the sealing test using the plastic cap demonstrated mainly the elasticity of the glue. These results indicated that the strengthened sealing effect of the glue by mixing with atelocollagen was due to the increased elasticity of the glue, and that the optimal concentration of atelocollagen for sealing the lung wound was determined to be 0.75%. In conclusion, the mixing of atelocollagen made the fibrin glue more effective for sealing pulmonary air leakage, and the optimal concentration of atelocollagen was 0.75%. We are therefore now going to use atelocollagen-mixed fibrin glue in clinical trials.
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