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Ann Thorac Surg 1999;67:922-926
© 1999 The Society of Thoracic Surgeons

Original Articles

Sealing effect of rapidly curable gelatin-poly (L-glutamic acid) hydrogel glue on lung air leak

Yuto Otani, PhDa, Yasuhiko Tabata, PhDa, Yoshito Ikada, PhDa

a Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

Accepted for publication October 13, 1998.

Address reprint requests to Dr Ikada, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
e-mail: yyikad{at}frontier.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Air leak is a problem commonly occurring in lung and thoracic operations. In this study, a rapidly curable hydrogel glue was prepared as the seal for lung air leak.

Methods. Mixing an aqueous solution of gelatin and poly(L-glutamic acid) with a water-soluble carbodiimide produced a hydrogel. The sealing effect on the air leak wound of rat lung was compared with that of conventional fibrin glue.

Results. The gelatin-poly(L-glutamic acid) hydrogel glue was solidified as rapidly as the fibrin glue, and was significantly more effective in sealing the lung air leak than the fibrin glue. Approximately 80% of the lungs treated with the hydrogel glue exhibited no air leak at the lung pressure of 50 cm H2O. Urea addition could prevent spontaneous gelatination of the mixed solution at room temperature and did not affect the hydrogel sealing effect. The bonding strength of the hydrogel glue both with and without urea to the lung tissue was significantly higher than that of the fibrin glue.

Conclusions. We concluded that this strong lung adhesion of the gelatin-poly(L-glutamic acid) hydrogel glue resulted in its superior sealing effect.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Air leak is a common problem in lung and thoracic operations. Several materials, such as polytetrafluoro-ethylene strips [1, 2] and fibrin-coated collagen fleeces [3], have been used to seal the air leak from the lung. However, they are not always effective in sealing air leak; for example, it is difficult to seal multipoint air leaks produced with suturing and stapling lung tissues. Requirements of an ideal sealant include (1) to be liquid before application, (2) to be rapidly curable even in the presence of water when applied, (3) to be as pliable as the lung tissue when cured, and (4) to be biodegradable in the body. Fibrin glues [4–9], cyanoacrylate adhesives [10], and gelatin adhesives composed of gelatin, resorcinol, and glutaraldehyde or formaldehyde [11] have been originally developed for tissue adhesion and hemostasis [12–16], and later evaluated as sealants for lung air leak. The evaluation studies revealed poor tissue adhesion of the fibrin glue and considerable cytotoxicity with the two other agents [17].

We have been studying a rapidly curable biological glue composed of gelatin and poly(L-glutamic acid) (PLGA) [18–21]. The mixed aqueous solution of gelatin and PLGA set to a hydrogel with the addition of water-soluble carbodiimide (WSC) within several seconds, which is as short as in clinically used fibrin glues. The gelatin aqueous solution spontaneously sets to a physical gel at temperatures lower than about 25°C, but the addition of urea was found to hinder gel formation [22]. The bonding strength of the gelatin-PLGA hydrogel to various soft tissues was higher than that of the fibrin glue [18, 19]. The cured hydrogel was soft, adhered to dog spleen tissue firmly enough to push down the blood oozing from the spleen surface [23], and was gradually degraded in the body without inducing any severe inflammatory response [18].

The objective of the present study is to evaluate feasibility of the WSC-formed gelatin-PLGA hydrogel as a medical sealant. A mixed gelatin and PLGA solution is applied to a lung wound in the presence of WSC to evaluate its sealant effect. We compared the sealing and adhesion capabilities of the hydrogel to the lung tissue with those of fibrin glue.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Material
Alkaline-processed gelatin (molecular weight, 99,000; isoelectric point, 5.0) and PLGA sodium salt (molecular weight, 83,000) were kindly supplied from Nitta Gelatin Co, Ltd, Osaka, and Ajinomoto Co, Ltd, Tokyo, Japan, respectively. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Wako Pure Chemical Industries Ltd, Osaka, Japan) was used as a WSC. Phosphate-buffered saline solution (pH 7.4) and a fibrin glue (BOLHEAL) were purchased from Nissui Pharmaceutical Co, Ltd, Tokyo, Japan, and Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan, respectively. Other chemicals were purchased from Nacalai Tesque, Inc (Kyoto, Japan) and used as received.

Evaluation of sealing effect
The sealing effect of the gelatin-PLGA hydrogel glue was evaluated according to the method reported by Bellotto and colleagues [11], with some modifications. Lungs were excised carefully from female and male Wistar rats, 10 weeks old (body weight, 210 to 250 g; Shimizu Laboratory Animal Supply Co, Ltd, Kyoto, Japan). A polyurethane tube, outer diameter 2 mm, was inserted into the trachea of the excised lung and fixed with a surgical suture. The other end of the tube had two branches: one was connected to a manometer (EMA-160, Kagaku Kyoeisha Ltd, Osaka, Japan) to measure the lung pressure and the other was connected to a syringe for air inflation. An air leak wound was prepared by pricking the lung with a needle with gauge sizes 20, 23, or 27, at the depth of 2 mm from the lung surface. The needle-pricked lung was placed in phosphate-buffered saline solution at 37°C and inflated with air from the syringe at a constant rate of 1 mL/s, checking for air leaks by observing bubble formation from the pricked wound site. After confirming an air leak, the lung was removed from the phosphate-buffered saline solution and gently wiped with water on the lung surface. A mixed gelatin and PLGA aqueous solution with and without urea preheated at 37°C (50 µL) was applied to the pricked site of the lung. Immediately after application, 17.5 µL of WSC solution in phosphate-buffered saline solution was added to the solution, followed by a 1-minute wait for the hydrogel to form.

Complete sealing was defined as sealing that prevented air leak up to a lung pressure of 50 cm H2O, because normal lung tissue often rips at the higher lung pressures. The concentration of gelatin in the hydrogel preparation was fixed at 100 mg/mL, but the concentrations of PLGA, urea, and WSC varied. As a comparison, 33.8 µL of fibrinogen solution and thrombin solution of the same volume were applied to the lung wound to evaluate the sealing effect. Twelve lungs were used for each experimental group and the percentage of complete sealings to the total wound number (percent sealing) was calculated.

Measurement of bonding strength
Two excised lungs were used for one experiment to measure the bonding strength of hydrogel to lung tissue. An aqueous solution of 100 mg/mL gelatin and 100 mg/mL PLGA with and without 100 mg/mL urea (100 µL) was warmed to 37°C, and applied to the surface of one lung. Immediately after the addition of 35 µL of 9.59 mg/mL WSC solution, the other lung was placed on the glued lung at the adhered area of 1 cm2. The bonding strength of the hydrogel between the two lungs was measured 10 minutes after glue application by use of a tensile machine (Autograph AGS-5D; Shimadzu Ltd, Kyoto, Japan) at 25°C in 60% relative humidity at a separation rate of 10 mm/min. This adhesion test was carried out five times for each sample to obtain the average bonding strength. Similarly, the bonding strength of fibrin glue was measured using a mixture of fibrinogen solution (67.5 µL) and thrombin solution (67.5 µL).

Histologic examination
After 1 minute of application of a gelatin-PLGA solution mixture to the lung wound, the cured hydrogel was pulled off with a forceps to check the adhesion to the lung surface. The concentration of gelatin, PLGA, urea, and WSC used was the same as that used in the lung bonding test. The portion of the lung wound was collected together with the cured hydrogel and fixed in 10% formaldehyde aqueous solution. The fixed specimens were cross-sectioned and stained with hematoxylin and eosin to perform histologic observation at a tissue level.

Statistical analysis
All the data were analyzed with Students’ t test and statistical significance was accepted at a p value less than 0.05. Experimental results were expressed as the means ± the standard error.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
Influence of PLGA, urea, and water-soluble carbodiimide concentrations on sealing effect
Figure 1 shows the sealing effect of the gelatin-PLGA hydrogel glue without urea on the lung air leak as a function of PLGA concentration. The lung pressure required to cause air leak increased with increasing PLGA concentration up to 100 mg/mL. Because phase separation was observed between gelatin and PLGA at PLGA concentrations higher than 125 mg/mL, the PLGA concentration was fixed at 100 mg/mL.



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  		Fig 1. The sealing effect of gelatin-poly(L-glutamic acid) (PLGA) hydrogel glue on the air leak of rat lung wound as a function of poly(L-glutamic acid) concentration. The concentration of gelatin and water-soluble carbodiimide was 100 and 9.59 mg/mL, respectively.

 
Urea was used as an additive to prevent spontaneous physical gelatination of mixed gelatin and PLGA aqueous solution at room temperature. The dependence of the sealing effect on the urea concentration is shown in Figure 2. Air leak pressure was not influenced by the addition of urea up to a concentration of 150 mg/mL, but higher urea concentration remarkably decreased the air leak pressure. Figure 3 shows the influence of WSC concentration on the sealing effect of the gelatin-PLGA hydrogel glue with and without urea. Air leak pressure increased with an increase in WSC concentration up to 10 mg/mL and thereafter leveled off, irrespective of urea addition.



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  		Fig 2. The urea effect on the air leak of rat lung wound sealed with the gelatin-poly(L-glutamic acid) hydrogel glue containing urea. The concentration of gelatin, poly(L-glutamic acid), and water-soluble carbodiimide was 100, 100, and 9.59 mg/mL, respectively.

 


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  		Fig 3. The water-soluble carbodiimide (WSC) effect on the air leak of rat lung wound sealed with the gelatin-poly(L-glutamic acid) hydrogel glue with (open circle) and without urea (open triangle). The concentration of gelatin, poly(L-glutamic acid), and urea was all 100 mg/mL.

 
Table 1 compares the sealing effect of the gelatin-PLGA hydrogel glue with the fibrin glue. For lungs before glue application, air leak pressure was around 15 cm H2O and no significant effect of needle size on the leak pressure was observed. Both the air leak pressure and percent sealing of the hydrogel glue were significantly higher than those of the fibrin glue, irrespective of urea addition. All the lung wounds pricked with a 27-gauge needle were completely sealed with the hydrogel glue, although the percent sealing increased with the increase in needle gauge size.


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  		Table 1. Sealing Effect of the Gelatin-PLGA Hydrogel With and Without Urea and the Fibrin Glue on the Air Leak of Rat Lung Wounds

 
Bonding strength of gelatin-PLGA hydrogel and fibrin glues to lung tissue
Table 2 shows the bonding strength to the rat lung for the gelatin-PLGA hydrogel and fibrin glues. Bonding strength of the hydrogel glue was significantly higher than that of the fibrin glue, irrespective of urea addition.


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  		Table 2. Bonding Strength Between Rat Lungs by Gelatin-PLGA Hydrogel With or Without Urea and Fibrin Glues

 
Adhesion of gelatin-PLGA hydrogel and fibrin glues to the lung
Irrespective of urea addition, the gelatin-PLGA hydrogel adhered to the lung so firmly that its removal from the lung surface was difficult. On the contrary, the fibrin glue was easily detached from the lung surface in a sheetlike shape (Fig 4 ).



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  		Fig 4. The rat lung wound appearance 1 minute after application of the gelatin-poly(L-glutamic acid) hydrogel glue with (A) and without urea (B), and the fibrin glue (C). Both the hydrogel glues adhered firmly to the lung surface, whereas the fibrin glue did not.

 
Figure 5 shows histologic sections of the gelatin-PLGA hydrogel and fibrin glues adhering to the lung surface. Both hydrogels adhered firmly to the lung surface even at the tissue level, in marked contrast to the fibrin glue. The hydrogel was degraded in the body, disappearing within 12 weeks from the adhesion site without any severe inflammatory response to the hydrogel, irrespective of urea addition (data not shown).



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  		Fig 5. Histologic sections of rat lung tissue after application of the gelatin-poly(L-glutamic acid) (PLGA) hydrogel glue with (A) and without urea (B), the fibrin glue (C), and before glue application (D). (hematoxylin and eosin staining; optical magnification, x70.)

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
Scattered and continuous air leak from multiholes on the lung surface is often observed with suturing and stapling in lung operations. This has been recognized as clinically problematic. In the present study, a needle pricking method was selected to create a lung wound with a constant, steady air leak. As is apparent in Table 1, this method was effective in preparing a pin-hole wound on the lung surface with good reproducibility.

Our previous study revealed that the WSC-formed gelatin-PLGA hydrogel glue adhered firmly to soft tissues with a higher bonding strength as the concentration of PLGA and WSC increased. The adhesion strength of the hydrogel to the soft tissue became maximum at PLGA and WSC concentrations of 100 and 9.59 mg/mL, respectively [18]. A similar concentration profile was seen for the air leak pressure of the gelatin-PLGA hydrogel glue (Figs 1 and 3). This finding indicates that the high bonding strength of the hydrogel to the soft tissue led to its superior sealing effect. However, the bonding strength of 34 gram force/cm2 was not as high as that to the skin, muscle, peritoneum, and small intestine. The reason for this low adhesion of the hydrogel glue to the lung is unclear at present, but the higher bonding strength of the gelatin-PLGA hydrogel glue than the fibrin glue resulted in significantly enhanced sealing effect of the hydrogel on the lung air leak.

The mixed gelatin and PLGA aqueous solution sets in several seconds to a hydrogel at 37°C with the addition of WSC; this is as short as conventional fibrin glue. However, the gelatin and PLGA aqueous solution had to be warmed before applying to the tissue, because this solution undergoes spontaneous gelatination at room temperature probably through intermolecular hydrogen bonding. This solution behavior causes fluctuations in adhesion of the gelatin-PLGA hydrogel in addition to its troublesome warming. As a consequence, it was found that the addition of urea prevented spontaneous gelatination of the mixed solution without affecting the WSC-induced hydrogel formation from the mixed gelatin and PLGA aqueous solution [22]. The present study demonstrated that urea addition did not hinder sealing of the lung wound with the hydrogel glue (Figs 2 and 3, Table 1). In addition, the hydrogel was found to degrade in the body within several weeks without severe inflammatory reaction.

In conclusion, the WSC-formed gelatin-PLGA hydrogel glue was significantly superior to the fibrin glue in terms of lung adhesion and sealing of the air leak. Urea addition could prevent spontaneous gelatination at room temperature, but did not impair the sealing effect of the hydrogel. It was concluded from the maximum air leak pressure and percent sealing that the WSC-formed gelatin-PLGA hydrogel with urea was superior to the fibrin glue as a sealant.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

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