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Figure 1.
Bovine chondrocytes in culture. Chondrocytes were isolated and cultured for 28 days. Cultures were processed for hematoxylin-eosin staining (A, original magnification ×165), electron microscopy (B, original magnification ×300), or type II collagen staining (C, original magnification ×83).

Bovine chondrocytes in culture. Chondrocytes were isolated and cultured for 28 days. Cultures were processed for hematoxylin-eosin staining (A, original magnification ×165), electron microscopy (B, original magnification ×300), or type II collagen staining (C, original magnification ×83).

Figure 2.
Scanning electron micrographs of day 14 control (type I collagen) and composite cultures. A and B, Control; confluent epithelium (original magnification: A, ×32; B, ×100). C and D, Respiratory epithelial cells on composite culture. Arrows indicate underlying composite with nonconfluent epithelium (original magnification: C, ×32; D, ×100).

Scanning electron micrographs of day 14 control (type I collagen) and composite cultures. A and B, Control; confluent epithelium (original magnification: A, ×32; B, ×100). C and D, Respiratory epithelial cells on composite culture. Arrows indicate underlying composite with nonconfluent epithelium (original magnification: C, ×32; D, ×100).

Figure 3.
Expression of β-actin, transforming growth factor (TGF)-α and TGF-β in day 14 respiratory epithelial cells from control and composite cultures. M represents RNA ladder; lane 1, β-actin, control; lane 2, β-actin, composite; lane 3, TGF-α, control; lane 4, TGF-α, composite; lane 5, TGF-β control; and lane 6, TGF-β composite.

Expression of β-actin, transforming growth factor (TGF)-α and TGF-β in day 14 respiratory epithelial cells from control and composite cultures. M represents RNA ladder; lane 1, β-actin, control; lane 2, β-actin, composite; lane 3, TGF-α, control; lane 4, TGF-α, composite; lane 5, TGF-β control; and lane 6, TGF-β composite.

1.
Clark  RA The commonality of cutaneous wound repair and lung injury. Chest. 1991;99 (suppl) 57S- 60SArticle
2.
Grillo  HC Tracheal replacement. Ann Thorac Surg. 1990;49864- 865Article
3.
Letang  ESanches-Lloret  JGimferrer  JMRamirez  JVicens  A Experimental reconstruction of the canine trachea with a free revascularized small bowel graft. Ann Thorac Surg. 1990;49955- 958Article
4.
Mulliken  JBGrillo  HC The limits of tracheal resection with primary anastomosis: further anatomical studies in man [abstract]. J Thorac Cardiovasc Surg. 1968;55418
5.
Neville  WEBolanski  PJSoltanzadeh  H Prosthetic reconstruction of the trachea and carina. J Thorac Cardiovasc Surg. 1976;72525- 536
6.
Hirano  MYoshida  TSakaguchi  S Hydroxylapatite for laryngotracheal framework construction. Ann Otol Rhinol Laryngol. 1989;98713- 717
7.
Okumura  NNakamura  TShimizu  YNatsume  TIkada  Y Experimental study of a new tracheal prosthesis made from collagen grafted mesh. Trans Am Soc Artif Organs. 1991;37M317- M319
8.
Langer  RVacanti  JP Tissue engineering. Science. 1993;260920- 926Article
9.
Schultz  GSWhite  MMitchell  R  et al.  Epithelial wound healing enhanced by transforming growth factor-α and vaccinia growth factor. Science. 1987;235350- 352Article
10.
Polk  WH  JrDempsey  PJRussell  WE  et al.  Increased production of transforming growth factor-α following acute gastric injury. Gastroenterology. 1992;1021467- 1474
11.
Madtes  DKBusby  HKStrandjord  TPClark  JG Expression of transforming growth factor-α and epidermal growth factor receptor is increased following bleomycin-induced lung injury in rats. Am J Respir Cell Mol Biol. 1994;11540- 551Article
12.
Massagué  J The transforming growth factor-β family. Ann Rev Cell Biol. 1990;6597- 641Article
13.
Raghow  R Role of transforming growth factor-β in repair and fibrosis. Chest. 1991;99 (suppl) 61S- 65SArticle
14.
Santala  PHeino  J Regulation of integrin-type cell adhesion receptors by cytokines. J Biol Chem. 1991;26623505- 23509
15.
Klagsburn  M Large scale preparation of chondrocytes. Methods Enzymol. 1979;58560- 564
16.
Hicks  WLKuhel  RHall  LLwebuga-Mukasa  JS Rapid isolation of upper respiratory cells [abstract]. Mol Biol Cell. 1994;5 (suppl) 118a
17.
Kawasaki  ES PCR Protocols: A Guide to Methods and Applications.  San Diego, Calif Academic Press1990;
18.
Derynck  R Transforming growth factor-α: structure and biological activities. J Cell Biochem. 1986;32293- 304Article
19.
DeLarco  JETodaro  GJ Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci U S A. 1978;754001- 4005Article
20.
Schreiber  ABWinkler  MEDerynck  R Transforming growth factor-α: a more potent angiogenic mediator than epidermal growth factor. Science. 1986;2321250- 1253Article
21.
Barrandon  YGreen  H Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-α and epidermal growth factor. Cell. 1987;501131- 1137Article
22.
Robinson  CBWu  R Culture of conducting airway epithelial cells in serum-free medium. J Tissue Cult Method. 1991;1395- 102Article
Original Article
November 1999

Does Cartilage Down-regulate Growth Factor Expression in Tracheal Epithelium?

Author Affiliations

From the Department of Head and Neck Surgery, Roswell Park Cancer Institute (Drs Hicks and Sigurdson and Mr Hall), Departments of Otolaryngology (Dr Gabalski), Anatomy/Cell Biology (Dr Hard), Chemistry (Dr Gardella), and Surgery (Dr Powers), SUNY at Buffalo Medical School, and Department of Pulmonary and Critical Care Medicine, University of Buffalo (Drs Kumar and Lwebuga-Mukasa), Buffalo, NY; and Department of Otolaryngology, Stanford University Medical Center, Stanford, Calif (Dr Gabalski).

Arch Otolaryngol Head Neck Surg. 1999;125(11):1239-1243. doi:10.1001/archotol.125.11.1239
Abstract

Background  Maintaining tracheal integrity and restoring normal physiologic function after injury is complex. Some of the critical events in this process are deposition of a provisional extracellular matrix, tissue remodeling, and angiogenesis. These events are coordinated with epithelial migration and proliferation to restore the mucosal barrier. The ability of respiratory epithelial cells (REC) to migrate and proliferate and restore denuded areas of the large conducting airway after injury is poor.

Objective  To test the hypotheses that (1) the cartilaginous framework, underlying the extracellular matrix (submucosa) and epithelium, decreases the migratory ability of REC when compared with REC on the same provisional extracellular matrix (type I collagen) alone, and (2) this phenomenon is associated with a change in expression of transforming growth factor (TGF)-α and TGF-β, both of which have been demonstrated in cutaneous models to be important in epithelial migration and proliferation.

Design  We developed a culture system that reconstitutes the tracheal lumen in vitro, consisting of dissociated chondrocytes cultured in a manner to form cartilage, submucosa (type I collagen), and REC (termed a "composite culture"). Control cultures consisted of epithelial cells grown on type I collagen alone. Control and composite cultures were evaluated morphologically using scanning electron and light microscopy. Expression of TGF-α and TGF-β was determined in day 14 cultured epithelial cells from control and composite cultures by semiquantitative polymerase chain reaction.

Results  Epithelial cells from composite cultures did not spread and were less squamoid in morphological appearance than epithelial cells on type I collagen alone. Expression of both growth factors was reduced in epithelial cells from composite cultures compared with those on type I collagen.

Conclusions  Cartilage modulates TGF-α and TGF-β expression in REC, and may contribute to regulation of REC proliferation and differentiation.

TRAUMA THAT disrupts intraluminal epithelium, regardless of its cause, often leads to aberrant repair. This process is pathologically manifested by the exuberant proliferation of granulation tissue and replacement of the normal respiratory epithelium with fibroblasts.1,2 This often leads to scar formation, airway stenosis, and eventual physiologic compromise of the host respiratory tract.

There is currently no effective way to study intraluminal events of reepithelialization after injury. Present approaches to tracheal repair include resection and reanastomosing the injured airway, replacement of the damaged portion by synthetic material, and use of autologous tissue for reconstruction of the tracheal defect.35 Recently, tissue engineering approaches have been taken, including forming an in vivo tracheal cartilaginous scaffolding by injecting dissociated chondrocytes into a preformed synthetic construct.68 Such devices were of limited success owing to lack of reepithelialization. In the case of synthetic replacement, migration of the prosthesis can occur and may result in chronic ulceration, and even fatal hemorrhage.2

We have developed a 3-dimensional in vitro model system that incorporates both cartilaginous and epithelial elements, a feature unique among currently available models of upper airway injury and repair. Using this model, we hypothesized that communication between epithelial cells and underlying stroma would lead to modulation of epithelial cell growth and differentiation through the release of growth factors. To test this hypothesis, we investigated expression of transforming growth factors α and β (TGF-α and TGF-β) in respiratory epithelial cells (REC) cultured on type I collagen or composite cultures of chondrocytes and type I collagen.

Transforming growth factor α is a member of the epidermal growth factor family and plays an important role in wound healing.911 τransforming growth factor β1 is a multifunctional polypeptide with differing cell-specific effects, including stimulation or inhibition of proliferation, and regulation of extracellular matrix production and remodeling.1214

Respiratory epithelial cells in a composite culture system with cartilage represents a 3-dimensional model of the tracheal luminal surface, which facilitates studies of the interactions between epithelium and its underlying stroma. Using this model, we have shown that cartilage may modulate the migration of REC, and changes in expression of TGF-α and TGF-β, when compared with REC grown on type I collagen alone.

MATERIALS AND METHODS
CELL ISOLATION AND CULTURE

Chondrocytes were harvested from bovine articulator cartilage under clean conditions, minced finely, and digested for 12 to 16 hours at 37°C in phosphate-buffered saline with antibiotics and collagenase II (Worthington, Freehold, NJ) and DNAse I (Sigma-Aldrich Corporation, St Louis, Mo), as described elsewhere.15 Cell viability was determined by trypan blue staining, and cell type was confirmed by staining with hematoxylin-eosin and antibody to extracellular type II collagen. Chondrocytes were plated on collagen inserts (Costar Transwell; VWR, Rochester, NY) at 20 to 40×106 cells per well in Dulbecco modified Eagle medium (DMEM/F-12) (Gibco, Grand Island, NY), with antibiotics, 10% fetal calf serum, and 50-µg/mL ascorbic acid. After the chondrocytes formed a layer of extracellular matrix, 0.5 mL of type I collagen (Vitrogen; Collagen Biomaterials, Palo Alto, Calif) was added.

Human tissue specimens were obtained through Manhattan Eye, Ear, Nose and Throat Hospital, New York, NY, under a human institutional review board–approved protocol. Samples were healthy tissues from surgical procedures performed on adults. Specimens were removed aseptically, rinsed in isotonic sodium chloride solution to remove debris, and shipped at 4°C within 24 hours of procurement. Upper respiratory tract epithelium from bronchus, nasal polyps, or turbinates was dissociated as described elsewhere16 and REC were added to type I collagen–coated chondrocytes at 2×105/cm2, in DMEM/F-12, containing antibiotics, epidermal growth factor (10 ng/mL), hydrocortisone (1 µmol/L), insulin (5 µg/mL), L-isoproterenol (1 µmol/L), and 5% fetal calf serum. Cultures were maintained for 2 weeks and then processed for scanning electron microscopy, histochemical analysis, or RNA isolation. Epithelial cells were also seeded at the same density onto type I collagen–coated culture dishes and kept for the same length of time as the composite cultures.

ELECTRON MICROSCOPY

Fourteen-day-old type I collagen REC cultures, and REC from composite cultures were processed for scanning electron micrcoscopy and examined with a scanning electron microscope (model 35CF; JEOL, Japan) at 10 to 12 kV.

IMMUNOHISTOCHEMISTRY

Twenty-eight-day-old chondrocyte cultures were formalin fixed and processed for immunoperoxidase histochemical analysis. Rabbit polyclonal antibody to type II collagen (DAKO, Carpinteria, Calif) was applied, washed, and followed by a biotinylated universal secondary antibody and streptavidin peroxidase (Immunon/Shandon-Lipshaw, Pittsburgh, Pa).

RNA ISOLATION AND REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION

Total cellular RNA was isolated from day 14 REC composite cultures, and REC cultured on type I collagen, using a reagent (TriReagent; Molecular Research Center, Cincinnati, Ohio), as described by the manufacturer. Under a dissecting microscope, the REC layer was removed from composite cultures with a sterile, RNAse-free spatula into TriReagent. One to five micrograms of total RNA was reverse transcribed using murine Moloney leukemia virus reverse transcriptase.17 Polymerase chain reaction amplification was performed using primer sets for human TGF-α, TGF-β1, and β-actin (Clontech, Palo Alto, Calif), following manufacturer's instructions. One-tenth volumes of polymerase chain reaction products were run on 2.5% or 3% agarose gels and visualized by ethidium bromide staining. The polymerase chain reaction products obtained were of expected sizes (human β-actin, 838 base pairs [bp]; human TGF-β, 161 bp; and human TGF-α, 297 bp).

RESULTS
CHONDROCYTE AND REC CULTURES

Bovine chondrocytes established in primary culture were morphologically similar to in vivo bovine cartilage (Figure 1, A and B). Cartilage cultured for less than 2 months did not always form lacunae, but always produced an abundant extracellular matrix of type II collagen (Figure 1, C).

Respiratory epithelial cells grown on type I collagen formed a continuous sheet (Figure 2, A and B). In contrast, REC grown on composite cultures did not spread to confluence, but rather grew as patches of epithelium (Figure 2, C and D). The epithelial cell layer in both the composite cultures and on type I collagen was undifferentiated.

TGF-α AND TGF-β GENE EXPRESSION ON TYPE I COLLAGEN AND IN COMPOSITE CULTURES

In REC from day 14 composite cultures, expression of both TGF-α and TGF-β was reduced (Figure 3, lane 4, TGF-α; lane 6, TGF-β) compared with REC on type I collagen (Figure 3, lane 3, TGF-α; lane 5, TGF-β). Relative expression of β-actin was equal (Figure 3, lanes 1 and 2).

Paraffin sections of REC grown on type I collagen and on composites were immunostained for TGF-α and TGF-β. Both growth factors were expressed in chondrocytes, and to a lesser extent, in epithelial cells (data not shown).

COMMENT

A frequent problem seen in tracheal repair with synthetic or autologous materials is the failure of luminal surface reepithelialization. Failure of reepithelialization to reestablish luminal integrity is an important reason why no acceptable surgical procedure exists for the repair of extended segments of trachea compromised by inhalation injury, congenital anomalies, or neoplastic disease.

Why the rate of reepithelialization in the large conducting airway is different from that seen within other epithelial-lined or -covered surfaces is unclear. The phenomenon of "slowed" reepithelialization is seen after both ablative surgical reconstruction and denudation injury, where the epithelium and basement membrane are removed with an intact cartilaginous superstructure (eg, inhalation injury).

One of the difficulties in understanding the relationship between respiratory epithelium and its underlying substructure (cartilage and submucosa) is the inaccessibility of the tissue for direct observation. Our model facilitates the examination the tissue interactions and mechanisms resulting in REC migration.

Both TGF-α and TGF-β play crucial roles in new tissue formation and remodeling. Transforming growth factor α stimulates proliferation in cultured epithelial cells,18 fibroblasts,19 and endothelial cells.20 It is chemotactic for epithelial cells in vitro21 and enhances epithelial wound healing when applied topically.9 Transforming growth factor β is mitogenic for cells of mesenchymal origin and plays a role in repair through its ability to modulate extracellular matrix formation and tissue remodeling.

When isolated human REC were cultured on type I collagen, the cells spread to form a confluent layer as has been previously reported by other authors.22 When plated onto composite cultures with a layer of type I collagen on top of cartilage, the cells did not spread efficiently but formed epithelial nests. Complete reepithelialization of the surface did not occur, even after 3 weeks.

We examined TGF-α and TGF-β expression in REC from composite cultures and found that both of these growth factors were reduced in epithelial cells from 14-day composite cultures when compared with the expression of these factors in REC cultured on type I collagen alone. This suggests that the cartilage modulates the behavior of epithelial cells. One hypothesis for the observed diminished expression of TGF-α and TGF-β is the secretion of soluble factors from the cartilage. Transforming growth factor α was expressed in cartilage (not shown), where it may have acted on the epithelium in a paracrine manner to decrease its expression.

Patients with critical large conducting airway injury requiring medical intervention often succumb to their condition because no reliable means of restoring the large conducting airway exists. Lack of REC migration and proliferation remains the key physiologic and biologic issue underlying failure of surgical repair. Understanding the factors that influence epithelial migration and regeneration in the large conducting airway remains a central issue in solving this clinical problem. The model presented demonstrates one potential mechanism to explain the phenomenon of impaired reepithelialization seen in the large conducting airway after injury.

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Article Information

Accepted for publication March 31, 1999.

Reprints: Wesley L. Hicks, Jr, MD, Head and Neck Surgery, Roswell Park Cancer Institute, Elm and Carlton streets, Buffalo, NY 14263.

References
1.
Clark  RA The commonality of cutaneous wound repair and lung injury. Chest. 1991;99 (suppl) 57S- 60SArticle
2.
Grillo  HC Tracheal replacement. Ann Thorac Surg. 1990;49864- 865Article
3.
Letang  ESanches-Lloret  JGimferrer  JMRamirez  JVicens  A Experimental reconstruction of the canine trachea with a free revascularized small bowel graft. Ann Thorac Surg. 1990;49955- 958Article
4.
Mulliken  JBGrillo  HC The limits of tracheal resection with primary anastomosis: further anatomical studies in man [abstract]. J Thorac Cardiovasc Surg. 1968;55418
5.
Neville  WEBolanski  PJSoltanzadeh  H Prosthetic reconstruction of the trachea and carina. J Thorac Cardiovasc Surg. 1976;72525- 536
6.
Hirano  MYoshida  TSakaguchi  S Hydroxylapatite for laryngotracheal framework construction. Ann Otol Rhinol Laryngol. 1989;98713- 717
7.
Okumura  NNakamura  TShimizu  YNatsume  TIkada  Y Experimental study of a new tracheal prosthesis made from collagen grafted mesh. Trans Am Soc Artif Organs. 1991;37M317- M319
8.
Langer  RVacanti  JP Tissue engineering. Science. 1993;260920- 926Article
9.
Schultz  GSWhite  MMitchell  R  et al.  Epithelial wound healing enhanced by transforming growth factor-α and vaccinia growth factor. Science. 1987;235350- 352Article
10.
Polk  WH  JrDempsey  PJRussell  WE  et al.  Increased production of transforming growth factor-α following acute gastric injury. Gastroenterology. 1992;1021467- 1474
11.
Madtes  DKBusby  HKStrandjord  TPClark  JG Expression of transforming growth factor-α and epidermal growth factor receptor is increased following bleomycin-induced lung injury in rats. Am J Respir Cell Mol Biol. 1994;11540- 551Article
12.
Massagué  J The transforming growth factor-β family. Ann Rev Cell Biol. 1990;6597- 641Article
13.
Raghow  R Role of transforming growth factor-β in repair and fibrosis. Chest. 1991;99 (suppl) 61S- 65SArticle
14.
Santala  PHeino  J Regulation of integrin-type cell adhesion receptors by cytokines. J Biol Chem. 1991;26623505- 23509
15.
Klagsburn  M Large scale preparation of chondrocytes. Methods Enzymol. 1979;58560- 564
16.
Hicks  WLKuhel  RHall  LLwebuga-Mukasa  JS Rapid isolation of upper respiratory cells [abstract]. Mol Biol Cell. 1994;5 (suppl) 118a
17.
Kawasaki  ES PCR Protocols: A Guide to Methods and Applications.  San Diego, Calif Academic Press1990;
18.
Derynck  R Transforming growth factor-α: structure and biological activities. J Cell Biochem. 1986;32293- 304Article
19.
DeLarco  JETodaro  GJ Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci U S A. 1978;754001- 4005Article
20.
Schreiber  ABWinkler  MEDerynck  R Transforming growth factor-α: a more potent angiogenic mediator than epidermal growth factor. Science. 1986;2321250- 1253Article
21.
Barrandon  YGreen  H Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-α and epidermal growth factor. Cell. 1987;501131- 1137Article
22.
Robinson  CBWu  R Culture of conducting airway epithelial cells in serum-free medium. J Tissue Cult Method. 1991;1395- 102Article
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