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Eur J Cardiothorac Surg 2008;34:864-869. doi:10.1016/j.ejcts.2008.05.048
Copyright © 2008, European Association for Cardio-thoracic Surgery. Published by Elsevier. All rights reserved.
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Functional closure of visceral pleural defects by autologous tissue engineered cell sheets

Masato Kanzakia, Masayuki Yamatob, Joseph Yangb, Hidekazu Sekineb, Ryo Takagib, Tamami Isakaa, Teruo Okanob,*, Takamasa Onukia

a The Department of Surgery I, Tokyo Women's Medical University, Tokyo, Japan
b Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Tokyo, Japan

Received 1 January 2008; received in revised form 26 May 2008; accepted 26 May 2008.

* Corresponding author. Address: Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel.: +81 3 3353 8111x66201; fax: +81 3 3359 6046. (Email: tokano{at}abmes.twmu.ac.jp).


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A
 References
 
Objective: The occurrence of intraoperative air leaks is an unavoidable complication during pulmonary surgeries. However, current surgical methods are generally ineffective in closing these visceral pleural defects, resulting in a decreased quality of life for patients. Here, we examined novel tissue engineered cell sheets for the closure of pleural defects in a porcine model. Methods: Skin biopsies were harvested from juvenile swine and tissue sheets composed of dermal fibroblasts were created using ex vivo culture on temperature-responsive dishes. After creating a visceral pleural injury model, the tissue engineered autologous dermal fibroblast sheets were transplanted directly to the defects without the use of sutures or additional adhesive agents, such as fibrin glue. Results: The tissue engineered autologous dermal fibroblast sheets attached directly to the lung surface providing an immediate seal against up to 25 cm H2O of airway pressure. Four weeks after transplantation, the dermal fibroblast sheets remained present on the pleural surface, providing permanent closure. The dermal fibroblast sheets were also responsive to changes in lung volume due to mechanical ventilation. No recurrences of air leaks were observed throughout the follow-up period. Conclusions: This study presents the development of an effective sealant for visceral pleural defects using autologous cells that have the flexibility to respond to expansion and contraction during respiration.

Key Words: Tissue engineering • Visceral pleural defect sealing • Dermal fibroblast


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A
 References
 
Air leaks caused by visceral pleural injury during lung resection are an unavoidable complication and can cause thoracic emphysema leading to increased chest tube durations and prolonged hospital stays [1–4]. In these cases, the prevention of air leaks is a critical objective for patient management after pulmonary surgery. With improved surgical techniques such as manual suturing and electrocautery [5], complications due to intraoperative visceral pleural injuries have been slightly minimized. Nevertheless, difficulty due to surgical constraints of the operations can still result in damage to the surrounding tissues, as well as failure to effectively close the air leaks.

Fibrin-based biological dressings are frequently applied during surgical procedures and have been used as pulmonary sealants for over twenty years [6–9]. In many cases however, the use of fibrin glue to close intra-operative air leaks has yielded inconsistent results [6,7], with similar air leak durations and periods of hospital stay compared to conventional procedures [9]. Similarly, the use of other biological adhesives [10–14] or synthetic materials [15–18] as pulmonary sealants has also resulted in marginal success due either to poor adhesive stability after application, or issues related to biocompatibility. In the development of a successful air leak sealant, the expansion and contraction during normal respiration therefore requires that the material be compliant in withstanding the constant movements of the lungs. Additionally, the sealant material must be easy to apply and remain stably attached to the visceral pleural surface of the surgically operated lung [6].

As an alternative tissue engineering approach, we have developed temperature-responsive culture surfaces to create transplantable sheets composed of living cells, without the use of additional synthetic or biological materials [19]. With this method, cultured cells along with their deposited extracellular matrix [20] can be non-invasively harvested as intact sheets that readily adhere to other surfaces, including host tissues [21] and even damaged tissue wound beds. Additionally, the tissue engineered cell sheets show cytoskeletal reorganization in response to low-temperature cell sheet harvest [22], allowing for the slightly shrunken cell sheets to be expanded by external forces, while remaining functionally and structurally intact. Since these tissue engineered cell sheets can attach to tissue surfaces, owing to their natural adhesive proteins, and are elastic by nature, we investigated the ability of cell sheets to close air leaks in a dynamic function by responding to extensive expansion and contraction during respiration, using a large animal model.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 Appendix A
 References
 
All animals received humane care in compliance with the European Convention on Animal Care and the Guidelines of Tokyo Women's Medical University on Animal Use, and this study was approved by the institutional ethics committee of Tokyo Women's Medical University.

2.1 Preparation of temperature-responsive cell culture surfaces
The preparation of square-patterned temperature-responsive culture dishes (provided by CellSeed, Inc., Tokyo, Japan) has been previously described [23]. Briefly, N-isopropylacrylamide monomer in 2-propanol solution was spread onto 100 mm diameter tissue culture polystyrene dishes. Dishes were then irradiated by electron beam, resulting in both polymerization and covalent grafting of poly(N-isopropylacrylamide) onto the cell culture surfaces. The temperature-responsive polymer-grafted dishes were rinsed with cold distilled water to remove ungrafted monomer and dried-in nitrogen gas. To prepare square-geometry dishes, the polymer-grafted surfaces were masked with a square glass coverslip (5 cm x 5 cm, Matsunami, Osaka, Japan) and acrylamide monomer (Wako Pure Chemicals, Osaka, Japan) solution in 2-propanol was spread onto the masked dish surface. Then, the dish surfaces were irradiated in the same manner. The resulting culture dishes had center square areas grafted with temperature-responsive polymer with a surrounding border of non cell-adhesive polyacrylamide. All temperature-responsive culture surfaces were finally gas-sterilized by ethylene oxide.

2.2 Culture media
Primary culture and subsequent passages were carried out in culture medium consisting of Dulbecco's modified Eagle's medium (Sigma, St. Louis, Missouri, USA) supplemented with 10% fetal bovine serum (Moregate Biotech, Queensland Australia), 100 units/ml of penicillin and 100 µg/ml of streptomycin (Gibco BRL Life Technologies, Grand Island, New York, USA), 80 µg/ml of gentamicin (Gibco), 2.5 µg/ml of fungizone (Gibco), 5 µg/ml insulin (Gibco), and 5 µg/ml transferrin (Gibco).

2.3 Culture of porcine dermal fibroblasts
Skin specimens, 2 cm x 2 cm, were excised from the abdomen of juvenile specific pathogen-free male pigs (40–45 kg, n = 4) under general anesthesia. The excised tissues were cut into 3 mm x 3 mm pieces and treated with Dulbecco's modified Eagle's medium containing 1500 units/ml dispase I (Godo Shusei, Tokyo, Japan) overnight at 4 °C. After enzymatic treatment, all epithelial layers were removed from the underlying dermis using surgical forceps under a dissecting microscope. The dermis was then finely minced and subjected to dissociation with 0.05% collagenase (Sigma) at 37 °C for 10 min, under gentle shaking. Cell suspensions were then centrifuged, re-suspended in fresh collagenase solution, and treated in the same fashion three additional times. Dermal fibroblasts were then plated at an initial density of 2.0 x 105 cells/cm2 onto commercially available 60 mm diameter tissue culture dishes (BD Biosciences, Franklin Lakes, New Jersey, USA) and cultured for 2 weeks at 37 °C in a humidified atmosphere of 5% CO2. Primary cultured cells were subsequently harvested by treatment with 0.05% trypsin-ethylenediamine tetraacetic acid for 15 min at 37 °C. The expanded cells were seeded on 25 cm2 square-patterned temperature-responsive dishes at a density of 2 x 105 cells/cm2, and cultured for one additional week.

2.4 Transplantation of tissue engineered cell sheets to visceral pleural injury models
Three weeks after the harvest of skin specimens, pigs were anesthetized with ketamine hydrochloride (5 mg/kg) and xylazine (1 mg/kg). Animals were intubated with a double-lumen endotracheal tube and anesthesia was maintained using isoflurane inhalation. Animals were placed in the lateral decubitus position and a right-lateral thoracotomy was performed. To create visceral pleural injury models, an incision 15 mm in diameter and 10 mm in depth, was made using scissors and air-leakage from the lung was confirmed by the presence of continuous air bubbles. Dermal fibroblast sheets cultured on temperature-responsive surfaces were placed in an incubator set at 20 °C for 30 min and harvested with the use of a sterile parchment support membrane. An autologous dermal fibroblast sheet was then placed directly on the pleural surface and left undisturbed for 5 min. After attachment to the lung surface, the parchment supporter was carefully removed. To create reinforced air leak sealants, a second autologous cell sheet was transplanted directly over the first, in the same fashion.

2.5 Histological and immunohistochemical analyses
For cross-sectional observations, harvested and transplanted cell sheets were fixed with 10% neutral buffered formalin and routinely processed into 10 µm thick paraffin wax-embedded sections. Hematoxylin and eosin and Azan staining were performed by conventional methods. For immunohistochemistry, de-paraffinized sections were treated with one of the following antibodies: mouse monoclonal anti-elastin (1:500, Novocastra, Newcastle upon Tyne, UK), mouse monoclonal anti-fibronectin (1:200, Santa Cruz Biotechnology, Santa Cruz, California, USA), mouse monoclonal anti-vimentin (1:500, DakoCytomation, Glostrup, Denmark), or mouse monoclonal anti-keratin (1:500, DakoCytomation) antibodies. All sections were peroxidase stained using the LSAB 2 kit (DakoCytomation), according to the manufacturer's suggested protocol.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 Appendix A
 References
 
3.1 Fabrication of transplantable dermal fibroblast sheets
Dermal fibroblasts were isolated from the abdominal skin of juvenile swine (40–45 kg) and expanded ex vivo, prior to seeding on temperature-responsive culture dishes. After 2 weeks in culture, the fibroblasts proliferated to form confluent monolayers (data not shown). To prepare the transplantable dermal fibroblast sheets, primary cultured cells were trypsinized and seeded onto square-patterned temperature-responsive culture dishes. After an additional week in culture, intact 25 cm2 sheets of dermal fibroblasts could be harvested by simple temperature reduction to 20 °C, with the use of a support membrane (Fig. 1a). The harvested dermal fibroblast sheets consisted of one to three stratified cell-dense layers (Fig. 1b) with relatively little extracellular matrix present (Fig. 1c).


Figure 1
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  		Fig. 1. Autologous dermal fibroblast sheets can be harvested by low-temperature treatment. Dermal fibroblasts are harvested as intact sheets from temperature-responsive culture surfaces with the use of a square-shaped supporter (a). Hematoxylin and eosin (b) and Azan (c) staining show that the fibroblast sheets are composed of one to three cell layers with relatively little extracellular matrix. Scale bars indicate 1 cm in (a), 50 µm in (b), and 50 µm in (c).

 
3.2 Transplantation of autologous dermal fibroblast sheets immediately close visceral pleural injuries
After creating a lung puncture 15 mm in diameter and 10 mm in depth (Fig. 2a and b), injury of the visceral pleura with leakage from the pulmonary parenchyma was confirmed by the presence of air bubbles in the thoracic cavity (Fig. 2c). After harvest from a temperature-responsive culture dish (Fig. 2d), an autologous dermal fibroblast sheet was transplanted directly to the pleural defect using a square-shaped support membrane (Fig. 2e). Within 5 min, the dermal fibroblast sheet produced stable attachment to the surrounding lung surface, without the use of sutures, fibrin glue, or staples (Fig. 2f). To create reinforced sealants, a second autologous cell sheet was transplanted directly over the first (Fig. 2g and h) and closure of the air leaks caused by injury to the visceral pleura, was confirmed by the absence of air bubbles from the defect (Fig. 2i).


Figure 2
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  		Fig. 2. Transplantation of autologous dermal fibroblast sheets to seal lung punctures. For transplantation procedures, the thoracic cavity is surgically opened (a) and a 15 mm diameter, 10 mm deep incision is made in the right lung (b). Air leaks are confirmed by the presence of air bubbles on the lung surface (c). The harvested cell sheet along with the supporter (d) is then placed directly on the pleural surface over the defect site (e). After allowing 5 min for the cell sheet to attach to the lung surface, the supporter is carefully removed (f). A second autologous fibroblast sheet is then transplanted directly over the first (g) and the support membrane is removed (h). The transplanted cell sheets immediately act to seal the air leak sites (i). Supplementary Video 1 can be seen as a part of the online supplementary materials.

 
Immediately after surgery, all air leaks were completely sealed by the autologous cell sheets and water seal test revealed stable closure of the air leaks against up to 25 cm H2O of airway pressure. While increased airway pressures above 30 cm H2O caused recurrence of air leaks, when the pressure was reduced to 25 cm H2O, no further air leaks were observed (Supplementary Video 1). Therefore when pressures were reduced to values that are used in our hospital to clinically assess air leaks after pulmonary resection, the transplanted cell sheets re-attached and sealed the pleural defects, even after displacement by increased mechanical ventilation.

3.3 Dermal fibroblast sheets provide a long-term, permanent seal of pleural defects
Four weeks after transplantation, the skin fibroblast sheets maintained closure of the pleural defects in all cases (Fig. 3 ), and were flexible to allow for lung expansion and contraction, even under mechanical ventilation (Supplementary Video 2). Histological examination revealed that the transplanted bilayer cell sheets were tightly attached to the pulmonary parenchyma with the pleural defects, and no air spaces were present between cell sheets and the host lung tissues (Fig. 4a and b). Additionally, while the autologous bioengineered sealants were significantly thicker, they resembled the native pleural surface having abundant extracellular matrix (Fig. 4c and d). Immunohistochemistry showed that both fibronectin (Fig. 4f) and elastin (Fig. 4h) were expressed by the skin fibroblast sheets, similar to the native lung pleura (Fig. 4e and g).


Figure 3
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  		Fig. 3. Permanent sealing of lung air leaks with autologous dermal fibroblast sheets. Four weeks after the transplantation of two autologous fibroblast sheets to seal lung punctures, no air leaks were observed. Autologous dermal fibroblast sheets maintained stable closure of the pleural defects without any lung constriction. The black box indicates the location of the sealed lung punctures.

 

Figure 4
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  		Fig. 4. Autologous dermal fibroblast sheets permanently seal lung air leaks. Four weeks after transplantation, the site of the transplanted dermal fibroblast sheets closely resemble the native lung pleura. Hematoxylin and eosin staining (a) and (b) shows the absence of air spaces between the lung pleura and the transplanted cell sheets. Azan staining (c) and (d) demonstrates the presence of abundant extracellular matrix in both the native lung pleura and regions transplanted with the fibroblast sealants. Immunostaining for the extracellular matrix proteins fibronectin (e) and (f), and elastin (g) and (h) show similar expression profiles between the native lung surface and the dermal fibroblast sheets. Left and right panels represent native lung tissues and air leak models transplanted with two autologous dermal fibroblast sheets, respectively. Scale bars represent 50 µm in all panels.

 
The extracellular matrix of the lung interstitium provides a strong and expandable framework for the thin alveolar epithelial–capillary intersection and contains elastic fibers that are responsible for tensile strength, elastic recoil and tissue compliance. Throughout these connective tissues, fibroblasts are responsible for secreting a non-rigid extracellular matrix under normal conditions and during wound healing. Within the cell sheet sealants, positive vimentin staining revealed the presence of extracellular matrix-producing fibroblasts (Fig. 5a). Additionally, keratin expression indicative of epithelial tissues was not observed in the transplanted fibroblast sheets (Fig. 5b). The deposition of an abundant extracellular matrix by the transplanted cell sheet sealants therefore provides their ability to respond in a dynamic function. By demonstrating the flexibility and elasticity that are required to expand and contract in response to normal respiration, the autologous fibroblast sheets were able to maintain stable and permanent closure of the air leaks.


Figure 5
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  		Fig. 5. Characterization of autologous dermal fibroblast air leak sealants. Four weeks after transplantation, immunostaining for vimentin demonstrates the presence of extracellular matrix producing fibroblasts within the autologous bilayer tissue sealants (a). Negative staining for keratins demonstrates the lack of epithelial cells in the transplanted dermal fibroblast sheets (b). Scale bars represent 50 µm in (a) and (b).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 Appendix A
 References
 
Due to the various limitations of currently available tissue adhesives, new alternatives have been aggressively pursued. In particular, because of the high possibility of intraoperative air leaks due to visceral pleural injuries, as well as the inherent difficulty in closing visceral pleural defects during pulmonary surgery, novel materials to effectively seal lung air leaks are of particular importance. Using temperature-responsive cell culture surfaces, we have previously developed and evaluated lung fibroblast sheets as an air leak sealant using small animals [24]. Our results showed that the tissue engineered cell sheets could readily close visceral pleural defects, followed by the deposition of an extracellular matrix-enriched tissue on the lung surface with excellent biocompatibility. Based on our previous results, we have now evaluated the use of autologous dermal fibroblasts as a living biomaterial that acts to immediately and permanently stop intraoperative air leaks that occur due to visceral pleural defects, in a clinically relevant large animal model. Our present results therefore indicate that autologous dermal fibroblast sheets can likely be readily translated into clinical practice due to their relative ease of use and promising effectiveness.

The development of an effective surgical sealant material requires that it be biocompatible and applicable in a safe manner. The use of autologous cell transplantation therefore provides several key advantages for use in future clinical applications. Since the indicated surgical operations would involve non-emergent cases, autologous cell sheet transplantation could be applied during these procedures that are generally scheduled approximately four weeks in advance. Prior to surgery, autologous blood is often obtained for potential transfusions during the operations. Similarly, autologous dermal fibroblast sheets could also be fabricated during this period. In addition, the use of allogeneic cell sheets from the frozen stocks should not be excluded in cases of emergency surgery.

Since dermal fibroblasts have high proliferative potential, they can be easily obtained from small, non-invasive biopsies and simply expanded using ex vivo culture. Prior to high-risk lung resections such as in immunosuppressed individuals, elderly patients or those suffering from bullous disease, diabetes, or malnutrition, tissue specimens could therefore be acquired at preoperative examination, allowing for cell sheet fabrication for subsequent surgical application. Finally, the use of autologous cells also eliminates the risk of both host rejection, as well as inflammatory responses to foreign materials.

With cases of chronic emphysema, surgical procedures such as decortication (the removal of superficial tissues to relieve lung constriction) can result in the development of air leaks. During the surgical removal of thicker surface layers, suturing can become difficult and therefore visceral pleural injury and damage to healthy lung tissues can result from repeated attempts to close the air leaks. Since dermal fibroblast sheets can be transplanted without influencing pulmonary function, the application of cell sheet sealants allows for resolution of air leaks by simply covering the affected regions of the visceral pleural surface to create airtight closures.

Postoperatively, the tissue engineered dermal fibroblast sheets undergo active tissue remodeling with the transplanted cells depositing large amounts of extracellular matrix on the visceral pleural surface. While the bilayer cell sheet sealants were significantly thicker than the normal lung pleura, the tissue surface showed similar structural and functional properties when compared to the native lung. This remodeling process leading to tissue compliance may be a key factor in the permanent and stable closure of the air leaks.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
Appendix A
 References
 
Tissue engineered skin fibroblast sheets are easy to handle and apply during surgical procedures. Due to their ability to contour and adhere to both the lung and visceral pleura surface immediately after transplantation, the cell sheets can provide an air-tight seal to visceral pleural defects, while at the same time allowing for lung expansion and contraction in response to both normal and mechanical ventilation. In contrast to currently available sealant materials, which are generally only moderately effective, the use of autologous fibroblast sheets demonstrates, for the first time, a quick and reliable method to permanently close air leaks using a readily available autologous cell source, and can be expected to reduce surgical complications during high risk pulmonary resections.


   Appendix A
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A
References
 
Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejcts.2008.05.048.


   Footnotes
 
{star} This work was supported by the Center of Excellence (COE) Program for the 21st Century, the High-Tech Research Center Program, and the Formation of Innovation Center for Fusion of Advanced Technologies in the Special Coordination Funds for Promoting Science and Technology, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Appendix A
 References
 

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