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Figure 1.
Cartilage construct grown from cryogenic chondrocytes after 6 weeks in the bioreactor.

Cartilage construct grown from cryogenic chondrocytes after 6 weeks in the bioreactor.

Figure 2.
Cartilage construct grown from fresh chondrocytes after 6 weeks in the bioreactor.

Cartilage construct grown from fresh chondrocytes after 6 weeks in the bioreactor.

Figure 3.
Hematoxylin-eosin–stained histologic section of cartilage constructs grown from cryogenic cells (original magnification ×300).

Hematoxylin-eosin–stained histologic section of cartilage constructs grown from cryogenic cells (original magnification ×300).

Figure 4.
Alcian blue–stained histologic section of the cartilage constructs grown from cryogenic cells (original magnification ×300).

Alcian blue–stained histologic section of the cartilage constructs grown from cryogenic cells (original magnification ×300).

Figure 5.
Dynamic seeding kinetics of cryogenic constructs. This figure shows the seeding rate of chondrocytes onto the scaffold.

Dynamic seeding kinetics of cryogenic constructs. This figure shows the seeding rate of chondrocytes onto the scaffold.

Figure 6.
A transmission electron photomicrograph of the cryogenic cartilage tissue (at 6 weeks) shows procollagen (C), proteoglycan (diamonds), an active Golgi complex (G), a nucleus and nucleolus (N), fat globules (arrows), and endoplasmic reticulum (R) (original magnification ×8250).

A transmission electron photomicrograph of the cryogenic cartilage tissue (at 6 weeks) shows procollagen (C), proteoglycan (diamonds), an active Golgi complex (G), a nucleus and nucleolus (N), fat globules (arrows), and endoplasmic reticulum (R) (original magnification ×8250).

1.
Vetter  UPirsig  WHelbing  GHeit  WHeinze  E Patterns of growth in human septal cartilage: a review of new approaches. Int J Pediatr Otorhinolaryngol.1984;7:63-74.
PubMed
2.
Tomford  WWMenkin  HJ Investigational approaches to articular cartilage preservation. Clin Orthop.1983;174:22-26.
PubMed
3.
Schachar  NSNagao  MMitsuyama  TMcAllister  DIshii  S Metabolic and biochemical status of articular cartilage following cryopreservation and transplantation: a rabbit model. J Orthop Res.1992;10:603-609.
PubMed
4.
Gross  AESilverstein  EAFalk  JFalk  RLanger  F The allo-transplantation of partial joints in the treatment of osteoarthritis of the knee. Clin Orthop.1975;108:7-14.
PubMed
5.
Mankin  HJFogelson  FSThrasher  AZJaffer  F Massive resection and allograft transplantation in the treatment of malignant bone tumors. N Engl J Med.1976;294:1247-1255.
PubMed
6.
Ottolenghi  CEMuscolo  DLMaenza  R Bone defect reconstruction by massive allograft: technique and results of 51 cases followed for 5 to 32 years.  In: Straub  LR, Wilson  PD  Jr, eds. Clinical Trends in Orthopedics. New York, NY: Theime-Stratton; 1982:171-183.
7.
Tomford  WWFredericks  GRMenkin  HJ Studies on cryopreservation of articular cartilage chondrocytes. J Bone Joint Surg Am.1984;66:253-259.
PubMed
8.
Schachar  NNagao  MMatsuyama  TMcAllister  DIshii  S Cryopreserved articular chondrocytes grow in culture, maintain cartilage phenotype, and synthesize matrix components. J Orthop Res.1989;7:344-351.
PubMed
9.
Meryman  HTWilliams  RJDouglas  MS Freezing injury from "solution effects" and its prevention by natural or artificial cryprotection. Cryobiology.1977;14:287-302.
PubMed
10.
Pegg  DE Long-term preservation of cells and tissues: a review. J Clin Pathol.1976;29:271-285.
PubMed
11.
Van Steensel  MHomminga  GNBuma  POlthius  HVandenburg  WB Optimization cryopreservative procedures for human articular cartilage chondrocytes. Arch Orthop Trauma Surg.1994;113:318-321.
PubMed
12.
Falsafi  SKoch  RJ Growth of tissue-engineered human naso-septal cartilage in simulated microgravity. Arch Otolaryngol Head Neck Surg.2000;126:759-765.
PubMed
13.
Vunjak-Novakovic  GMartin  IObradovic  B  et al Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res.1999;17:130-138.
PubMed
14.
Ohlendorf  CTomford  WWMenkin  HJ Chondrocyte survival in cryopreserved osteochondral articular cartilage. J Orthop Res.1996;14:413-416.
PubMed
15.
Dunham  BPKoch  RJ Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Arch Otolaryngol Head Neck Surg.1998;124:1325-1330.
PubMed
Original Article
August 2003

Cartilage Tissue Engineering Using Cryogenic Chondrocytes

Author Affiliations

From the Wound Healing and Tissue Engineering Laboratory, Division of Otolaryngology–Head and Neck Surgery (Drs Gorti, Lo, Falsafi, and Koch, Ms Quan, and Mr Khuu), and the Department of Pathology (Dr Kosek), Stanford University Medical Center, Stanford, Calif. The authors have no relevant financial interest in this article.

Arch Otolaryngol Head Neck Surg. 2003;129(8):889-893. doi:10.1001/archotol.129.8.889
Abstract

Objective  To generate in vitro hyaline cartilage from cryogenically preserved human septal chondrocytes in a simulated microgravity environment on a 3-dimensional biodegradable scaffolding material.

Methods  In this experiment, cryogenically frozen chondrocytes were thawed and cultured in a monolayer in serum-based chondrocyte media. They were seeded onto 3-dimensional biopolymer scaffolds in a spinner flask. The seeded constructs were then transferred to a bioreactor (an environment of solid-body rotation) for 6 weeks. Chondrocyte growth and extracellular matrix production in the constructs were confirmed by cell count, cell viability, and histologic analysis and by electron microscopy.

Results  Histologic sections stained with hematoxylin-eosin and Alcian blue (for acidic proteoglycans) confirmed the presence of hyaline cartilage in the cartilage constructs. Ultrastructural examination using transmission electron microscopy demonstrated matrix formation and chondrocyte viability.

Conclusions  This study proves that chondrocytes that are cryogenically stored for extended periods can be used to grow cartilage in vitro. Cryogenically preserved chondrocytes retain their ability to grow in tissue culture, redifferentiate, and produce extracellular matrix.

IT IS GENERALLY AGREED among surgeons performing head and neck reconstructive surgery that septal cartilage is the best implant to use when repairing cartilaginous defects. It has a firm, nonbendable quality that gives it superior supportive properties. It is usually preferable to the cartilage borrowed from the patient's rib or ears and to allografts, which can be rejected by the body. Moreover, unlike articular chondrocytes, septal chondrocytes isolated from the core and anterior of the nasal septum have remarkable regenerative ability.1 The optimal reconstructive situation usually entails taking some of the patient's own septal cartilage and moving it to a region where it can lend more support. Unfortunately, there is only a finite amount of septal cartilage available in a person's nose. A potential answer to this dilemma is the in vitro growth of cartilage from a patient's own chondrocytes obtained during elective septoplasty.

Investigators from the late 19th century to the present have attempted to preserve functional chondrocytes, both isolated and in a matrix, in an attempt to transplant viable tissue that is capable of functioning like cartilage.2 Cryopreservation, freezing to ultralow temperatures after exposure to cryoprotective agents, is one method of long-term preservation of transplant tissues like cartilage.3 For cryopreservation to succeed as a viable alternative banking method, the individual chondrocytes must be able to survive the freezing process, and the cartilage grown from these cells must be able to maintain the metabolic and biomechanical integrity of normal cartilage.

Many authors have suggested that long-term failure of cartilage grafts is mainly due to cartilage degeneration.46 To deal with such a problem, these authors suggest that the preservation of cartilage in the form of individual chondrocytes, with the ability to store and transplant viable chondrocytes, might improve the functional survival of these cartilage grafts. Tomford et al,7 in 1984, studied isolated chondrocytes and concluded that viability and function can be retained after freeze preservation. Up to 90% of frozen isolated cells will survive, and these cells were capable of producing proteoglycans in culture. In 1989, Shachar et al8 showed that cryogenically frozen chondrocytes could be successfully cultured in monolayer in vitro.

When cells are frozen to between 0°C and –40°C, there is usually damage to the cell as the intracellular and extracellular water is converted to ice.9,10 The 2 factors important in determining cell death due to freezing are (1) the concentration of solutes and (2) the intracellular growth of large ice crystals. These 2 properties are interrelated and may act synergistically to destroy the cells while freezing.3,11 However, the use of cryopreservatives such as dimethyl sulfoxide (DMSO) to some extent prevents these adverse processes. The mechanism of cell protection is unclear, but Meryman et al9 suggest that colligative properties of these cryopreservatives may be the cryoprotective elements.

In our laboratory, we have successfully grown ex vivo hyaline cartilage from human nasal septal chondrocytes using serum and serum-free media.12 We have investigated the formation of cell scaffolding constructs using a magnetically stirred spinner flask and bioreactor. The magnetically stirred spinner flask allows for optimal cellular attachment to the polymer surface during the seeding phase, and the bioreactor maintains a shear-attenuated environment of simulated microgravity, which enhances induction of matrix formation. We have been able to show evidence of hyaline cartilage formation by this method and have placed emphasis on histologic examination to determine the cellularity as well as the phenotypic matrix formation. The focus of the present study is to evaluate the feasibility of using cryogenic cells for the in vitro growth of human septal cartilage.

METHODS
CHONDROCYTE HARVESTING AND ISOLATION

The chondrocytes were harvested from cartilage specimens that were obtained after elective septoplasty. These specimens would normally be discarded, and their use was approved by the human subjects committee of the Stanford University Medical Center. The chondrocytes were harvested and isolated via the same method that was developed in our laboratory years ago.12 Two separate and distinct chondrocyte cell lines were used in the present experiment: a cryopreserved cell line where the cells were frozen for 3 years (SC1999) and a fresh cell line where the cells were established from a fresh cartilage specimen (SC0002).

CRYOPRESERVATION

The media from the culture flasks of cell line SC1999 were aspirated out. The flasks were washed 3 times with phosphate-buffered saline solution and then trypsinized using 1 mL of trypsin. Five milliliters of medium was added to the flasks after 1 minute to deactivate the trypsin. Cell viability was determined using the trypan blue dye exclusion method, and cells were counted using a hemocytometer and phase microscopy. The cell suspension was then centrifuged at 1200 rpm for 10 minutes. While the cells were spinning, solutions were made of 20% fetal bovine serum medium (1 mL per flask being frozen), and 10% DMSO in 20% fetal bovine serum medium (0.5 mL per flask being frozen). These solutions were refrigerated to ensure coldness.

The supernatant was removed, and the cells were resuspended in 0.5 mL of 20% fetal bovine serum medium and refrigerated. One-half milliliter of 10% DMSO solution was added in a dropwise fashion to the cell suspension and mixed. One milliliter of cell suspension was then transferred to each freezer vial and labeled with the cell line, date, initials, and passage number. The freezer vials were placed in a low-temperature freezer (−80°C) overnight. These vials were then transferred to liquid nitrogen for cryostorage for 3 years at −196°C.

THAWING

After 3 years, this sample line of frozen cultures was thawed by gentle agitation in a 37°C water bath. Five milliliters of 20% fetal calf serum medium was added to each vial, and the vials were centrifuged at 1200 rpm for 10 minutes. The supernatant was removed, and the cell pellet resuspended 3 times. The suspended cells were then transferred to culture flasks. Cell viability was confirmed using trypan blue and the hemocytometer.

AMPLIFICATION AND 3-DIMENSIONAL CELL CULTURE

These cells were cultured in a monolayer and amplified through 3 passages to a population of 130 million cells in 120 mL of growth medium. The polymer scaffold, knitted polygalactin 910 woven mesh (Vicryl; Ethicon Inc, Somerville, NJ), was weighed, cut into 10 × 10-mm pieces in a sterile fashion, and bathed in medium prior to being suspended in magnetically stirred spinner flasks. The spinner flasks with the scaffolds were incubated at 37°C and 5% carbon dioxide in humidified air for 18 to 24 hours. The scaffolds were fixated in the spinner flasks as described by Vunjak-Novakovic et al.13 The cell solution (1.3 × 108 cells/scaffold seeding density) was then added to the spinner flask via the side ports and incubated at 37°C in the presence of 5% carbon dioxide in humidified air. The spinner flask was set at 50 rpm, and the seeding kinetics were estimated by performing periodic cell counts on the bulk medium using the hemocytometer during the first 24 hours.

The medium was exchanged every 24 hours. After 14 days of incubation in the spinner flask, the cell-polymer constructs were transferred to a 110-mL slowly turning lateral vessel (STLV; Synthecon, Houston, Tex) and incubated at 37°C in the presence of 5% carbon dioxide in humidified air for 6 weeks. In the initial stage, the STLV was set at 15 rotations/min, and 50% of the medium was replaced every week. The medium exchange rate was based on qualitative colorimetric assessment. As metabolic by-products accumulated and the polymer degraded, the color of the medium changed from clear ruby red to turbid orange, at which point the medium was exchanged. The rotation rate of the STLV was periodically varied to keep the constructs in a freely suspended mode. After 6 weeks, the constructs were removed for analysis. The methods for amplification, 3-dimensional culture, and construct analysis were similar for cryogenic and fresh cell lines.

CONSTRUCT ANALYSIS

Four representative samples of the constructs from each line, fresh and cryogenic, were fixed in 10% formalin and paraffin embedded. They were sectioned and stained for histologic analysis; hematoxylin-eosin stained for general morphologic observations; and stained with Alcian blue (at pH 1.5) to detect the presense of chondroitin sulfate and glycosaminoglycans. Portions of the constructs were fixed in 1.5% glutaraldehyde in 0.1M cacodylate buffer at a pH of 7.2 for at least 12 hours. These portions were then fixed in 1.5% osmium tetroxide, dehydrated with ethanol, and embedded in epoxy resin. One-micrometer-thick scout sections were stained with toluidine blue. Thin sections were serially stained with uranyl acetate and lead citrate before being studied under the Philips 201 Transmission Electron Microscope (Philips Electronics NV, Eindhoven, the Netherlands).

RESULTS
GROSS MORPHOLOGIC CHARACTERISTICS

The cryogenic and fresh cell constructs were grown from 130 × 106 cell seedings per scaffold. After 14 days in the spinner flask, they did not show any evidence of cartilage formation. At the end of 6 weeks of the STLV phase, 1 of the cryogenic cell constructs had a smooth, glistening, gray-surface and rounded corners. The other 3 had buckled and completely disintegrated in the media after 21 days in the STLV. The surviving construct was very friable and was not mechanically robust. Figure 1 shows the surviving construct in the bioreactor after 6 weeks in the STLV.

The fresh cell constructs were grown from similar concentrations of seeded cells as the cryogenic cell constructs. They also did not show any evidence of cartilage formation after 14 days in the spinner flask. However, at the end of the STLV phase, all 4 fresh cell constructs were intact, had glistening surfaces, and were robust in appearance; they did not disintegrate (Figure 2). The cryogenic and fresh cell constructs taken from the STLV were sent for histologic and ultrastructural analysis.

HISTOLOGIC FINDINGS

The hematoxylin-eosin–stained sections from the core region of the cryogenic cell construct were obtained after 6 weeks of growth in the STLV. The stained sections showed the homogeneous matrix and lacunae with mature chondrocytes (Figure 3). The matrix reflects the presence of extracellular substance, suggesting hyaline cartilage formation. The polyaniline Alcian blue (pH 1.5) characteristics of chondroitin sulfate were used to verify the presence of cartilage-specific matrix, which was clearly shown as blue (Figure 4).

DYNAMIC SEEDING KINETICS

Dynamic seeding kinetics refers to the cell concentration over the time during which cells separate from solution and adhere to the scaffold. Figure 5 shows the dynamic seeding data recorded by a hemocytometer (accurate to within 105 cells) from concentrations of cryogenic chondrocytes during the first 12 hours of seeding as a function of time. At the 12-hour mark, no more viable cells were visible, and seeding was complete. The undetectable cutoff point was presumed to be 105 cells. The cryogenic cells showed a decaying seeding kinetic comparable with that of fresh chondrocytes.12

TRANSMISSION ELECTRON MICROSCOPY

Figure 6 shows a transmission electron micrograph of a 6-week-old cryogenic cell construct. Prominent nucleoli, abundant rough endoplasmic reticulum, and small budding matrix vesicles all suggest active matrix biosynthesis. Flocculent material containing Golgi vacuoles as well as mitochondria, lipovesicles, and dense filamentous material are present. There are ample amounts of proteoglycan-containing matrix and a small amount of procollagen surrounding the chondrocytes.

COMMENT

Tissue engineering opens the door for an unending supply of graft material for reconstructive purposes. In reconstructive surgery, morphometric and structural constraints that are imposed by the tissue being replaced preclude the use of traditional cell culture techniques.

The results obtained from our experiment indicate that chondrocyte growth from cryogenic cells has a lag period of 14 days during culture compared with fresh cells and that the cartilage constructs are weaker and more friable than those grown from fresh cells. This could be because of the type of scaffolding material or the toxic effects of DMSO. The lag period in growth of the cryogenic cells observed during culture may be explained by the effect of the freezing process. However, after the lag period of 14 days, cryogenic chondrocyte proliferation was similar to that of fresh cells.

The growth of the cartilage constructs from fresh cells validates our proposed optimal experimental method for in vitro growth of hyaline cartilage. Ohlendorf et al14 have shown that cell survival is confined to the superficial layer of the cartilaginous matrix if frozen cells are used. They also reported no evidence of cell survival in the middle or deeper layers of the cartilage formed by frozen cells with or without using DMSO. They used 8% DMSO and phosphate-buffered saline solution for freezing cells. The slide sections they used were 1.5 mm and 100 µm in thickness. In contrast, we have seen viable chondrocytes from the core of the construct analyzed for histologic characteristics.

In the present study, we could not quantify the amount of proteoglycans in the cryogenic cartilage owing to the lack of adequate cartilage material for analysis. The proteoglycan analysis was strictly qualitative. We did not perform any quantitative analysis and so cannot definitively say that the proteoglycans in the cartilage grown from cryogenic cells is similar to or different in content from the proteoglycans in the cartilage grown from fresh cells. Much research remains before we can prove that cartilage grown from cryogenically stored cells is suitable as a graft material for transplantation. As realized in our study, there are some problems to solve, including the slower growth rate of the cryogenic cells in culture, the friable nature of the grown cartilage, and the identification of the ideal 3-dimensional scaffold. The lower growth rate may be improved by supplementing the growth medium with growth factors such as insulinlike growth factor 1, basic fibroblast growth factor, and transforming growth factor β1.15

We have shown evidence that cryogenic cells frozen for 24 months retain growth characteristics similar to those of fresh cells. Their ability to proliferate, differentiate, and form extracellular matrix does not change with time. Histologically, they look similar to cartilage grown from fresh cells,12 and they have a lag period of 14 days in monolayer culture. The rate of dynamic seeding is similar in cryogenic and fresh cells. Morphologically, the cartilage grown from cryogenic cells is friable and lacks the integrity of the cartilage constructs grown from fresh chondrocytes. This might be attributed (though it is unlikely) to the use of different scaffolding materials (Vicryl for cryogenic cartilage constructs and polyglycolic acid [PGA] for fresh cartilage constructs) or to the cell characteristics of cryogenically frozen cells. While it would have been optimal to use the same scaffolding material in test and control cultures, the use of PGA and Vicryl should not have contributed any significant difference. Vicryl is a copolymer of 90% PGA and 10% polyactic acid. It is possible in a future study to grow cryopreserved cells on PGA scaffolds and draw comparisons using the same type of scaffold, but it is preferable to grow test and control cultures on more advanced and recently developed scaffolding material.

We will evaluate other biomaterials to find the optimal scaffolding material for the growth of cryogenic cells. These morphologic data, however, do not demonstrate that cryopreserved cultures are not of sufficient quality to make useful cartilage. Other factors must be considered, such as the use of different and better scaffolding materials and the effect of supplementing the growth medium with growth factors. If later consideration of these factors shows that cartilage made from cryopreserved cultures remains more friable than that of cartilage from fresh cultures, we may then conclude that there is a difference in quality. In addition, we will need to perform a construct analysis in comparison with fresh cells.

The results of our experiment have important implications in cryostorage or cryobanking of cells. They suggest that chondrocytes will survive freezing and thawing for long periods and retain matrix memory much like unfrozen chondrocytes.

This preliminary study proves that septal chondrocytes that have been cryogenically stored for extended periods can be used to grow cartilage ex vivo. Cryogenically preserved chondrocytes retain their ability to grow in tissue culture, redifferentiate, and produce extracellular matrix. The ability to store septal chondrocytes for later hyaline cartilage graft generation should prove to be extremely valuable in reconstructive surgery. We plan to pursue refinements in our cryogenic techniques and to perform biomechanical analysis of the cartilage constructs. It is the degree of optimization of the in vitro phase that determines the quality of the engineered cartilage at implantation.

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Article Information

Corresponding author: R. James Koch, MD, MS, Division of Otolaryngology–Head and Neck Surgery, Edwards Bldg, R-135, Stanford University Medical Center, Stanford, CA 94305-5328 (e-mail: RJK@stanford.edu).

Submitted for publication April 30, 2002; final revision received October 22, 2002; accepted December 3, 2002.

References
1.
Vetter  UPirsig  WHelbing  GHeit  WHeinze  E Patterns of growth in human septal cartilage: a review of new approaches. Int J Pediatr Otorhinolaryngol.1984;7:63-74.
PubMed
2.
Tomford  WWMenkin  HJ Investigational approaches to articular cartilage preservation. Clin Orthop.1983;174:22-26.
PubMed
3.
Schachar  NSNagao  MMitsuyama  TMcAllister  DIshii  S Metabolic and biochemical status of articular cartilage following cryopreservation and transplantation: a rabbit model. J Orthop Res.1992;10:603-609.
PubMed
4.
Gross  AESilverstein  EAFalk  JFalk  RLanger  F The allo-transplantation of partial joints in the treatment of osteoarthritis of the knee. Clin Orthop.1975;108:7-14.
PubMed
5.
Mankin  HJFogelson  FSThrasher  AZJaffer  F Massive resection and allograft transplantation in the treatment of malignant bone tumors. N Engl J Med.1976;294:1247-1255.
PubMed
6.
Ottolenghi  CEMuscolo  DLMaenza  R Bone defect reconstruction by massive allograft: technique and results of 51 cases followed for 5 to 32 years.  In: Straub  LR, Wilson  PD  Jr, eds. Clinical Trends in Orthopedics. New York, NY: Theime-Stratton; 1982:171-183.
7.
Tomford  WWFredericks  GRMenkin  HJ Studies on cryopreservation of articular cartilage chondrocytes. J Bone Joint Surg Am.1984;66:253-259.
PubMed
8.
Schachar  NNagao  MMatsuyama  TMcAllister  DIshii  S Cryopreserved articular chondrocytes grow in culture, maintain cartilage phenotype, and synthesize matrix components. J Orthop Res.1989;7:344-351.
PubMed
9.
Meryman  HTWilliams  RJDouglas  MS Freezing injury from "solution effects" and its prevention by natural or artificial cryprotection. Cryobiology.1977;14:287-302.
PubMed
10.
Pegg  DE Long-term preservation of cells and tissues: a review. J Clin Pathol.1976;29:271-285.
PubMed
11.
Van Steensel  MHomminga  GNBuma  POlthius  HVandenburg  WB Optimization cryopreservative procedures for human articular cartilage chondrocytes. Arch Orthop Trauma Surg.1994;113:318-321.
PubMed
12.
Falsafi  SKoch  RJ Growth of tissue-engineered human naso-septal cartilage in simulated microgravity. Arch Otolaryngol Head Neck Surg.2000;126:759-765.
PubMed
13.
Vunjak-Novakovic  GMartin  IObradovic  B  et al Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J Orthop Res.1999;17:130-138.
PubMed
14.
Ohlendorf  CTomford  WWMenkin  HJ Chondrocyte survival in cryopreserved osteochondral articular cartilage. J Orthop Res.1996;14:413-416.
PubMed
15.
Dunham  BPKoch  RJ Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Arch Otolaryngol Head Neck Surg.1998;124:1325-1330.
PubMed
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