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Figure 1.
Experimental design.

Experimental design.

Figure 2.
A composite of chondrocytes and scaffold coated onto the skin tube was embedded at the depth of the abdominal muscles.

A composite of chondrocytes and scaffold coated onto the skin tube was embedded at the depth of the abdominal muscles.

Figure 3.
Six weeks after transplantation, the matrix was secreted and there were immature chondrocytes in the graft. The chondrocytes were oval (hematoxylin-eosin, original magnification ×40).

Six weeks after transplantation, the matrix was secreted and there were immature chondrocytes in the graft. The chondrocytes were oval (hematoxylin-eosin, original magnification ×40).

Figure 4.
Twelve weeks after transplantation, chondrocytes were almost mature and the lacunae had formed. The contents of the matrix had obviously increased (hematoxylin-eosin, original magnification ×40).

Twelve weeks after transplantation, chondrocytes were almost mature and the lacunae had formed. The contents of the matrix had obviously increased (hematoxylin-eosin, original magnification ×40).

Figure 5.
Eighteen weeks after transplantation, the neocartilage contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage (toluidine blue, original magnification ×100).

Eighteen weeks after transplantation, the neocartilage contained spherical chondrocytes surrounded by dense extracellular matrix, an appearance similar to that of native cartilage (toluidine blue, original magnification ×100).

Figure 6.
At 18 weeks, the epithelium (arrow) adhered tightly to the inner cartilage (Masson's trichrome, original magnification ×400).

At 18 weeks, the epithelium (arrow) adhered tightly to the inner cartilage (Masson's trichrome, original magnification ×400).

1.
Ochi  M Clinical result of transplantation of tissue-engineered cartilage and future direction of cartilage repair: novel approach with minimally invasive procedure. Yonsei Med J 2004;45 (((suppl)) 74- 79
2.
Huang  JZLi  XSQuan  DP  et al.  In vitro and in vivo study on the degradation and biocompatibility of a poly-DL-lactide (PLA) polymer. Xiandai Kangfu 2001;558- 59
3.
Spriano  GPiantanida  RPellini  R Hypopharyngeal reconstruction using pectoralis major myocutaneous flap and prevertebral fascia. Laryngoscope 2001;111 (3) 544- 547
PubMedArticle
4.
Guang  ZPeng  XRHuang  XM  et al.  Pectoralis major muscle musculo-cutaneous flap repairing tissue defects after surgery of laryngeal pharynx carcinoma. Chin J Microsurg 2004;27300- 301
5.
National Research Council of USA, Guidelines on the Care and Use of Laboratory Animals.  Washington, DC National Academies Press1996;
6.
Cheng  YWang  QPLi  JH  et al.  The modified method for human nasoseptal chondrocyte culture and study on cell's biological character. Zhongguo Erbi Yanhou Toujing Waike 2006;1347- 49
7.
Langer  RVacanti  JP Tissue engineering. Science 1993;260 (5110) 920- 926
PubMedArticle
8.
Britt  JCPark  SS Autogenous tissue-engineered cartilage: evaluation as an implant material. Arch Otolaryngol Head Neck Surg 1998;124 (6) 671- 677Article
9.
Sittinger  MBujia  JMinuth  WW  et al.  Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture. Biomaterials 1994;15 (6) 451- 456Article
10.
Holy  CECheng  CDavis  JE  et al.  Optimizing the sterilization of PLGA scaffolds for use in tissue engineering. Biomaterials 2001;22 (1) 25- 31
PubMedArticle
11.
Cheng  YHuang  JZDu  J  et al.  In vitro and in vivo study on the biodegradation and biocompatibility of chitosan-nonwoven-cloth. Chin J Otorhinolaryngol Skull Base Surg 2005;11229- 231
12.
Cheng  YHuang  JZQuan  DP  et al.  A novel scaffold made by chitosan–nonwoven-cloth coated with poly (dl-lactide-co-glycolide) (PLGA). Chin J Clin Rehabil 2005;960- 61
13.
Vacanti  CAPaige  KTKim  W  et al.  Experimental tracheal replacement using tissue engineered cartilage. J Pediatr Surg 1994;29 (2) 201- 204
PubMedArticle
14.
Vunjak-Novakovic  GObradovic  BMartin  I  et al.  Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 1998;14 (2) 193- 202Article
Original Article
December 15, 2008

Production of Allogenic Cartilage in a Tube Lined With Epithelium and a Novel Scaffold

Author Affiliations

Author Affiliations: Department of Otolaryngology–Head and Neck Surgery, Jinling Hospital, Clinical Medical College of Nanjing University, Nanjing (Drs Cheng, Li, Jiang, and Wang, Ms Zhou, and Mr Wang), and Department of Otolaryngology, Zhujiang Hospital, Southern Medical University, Guangzhou (Dr Huang), China.

Arch Otolaryngol Head Neck Surg. 2008;134(12):1258-1262. doi:10.1001/archotol.134.12.1258
Abstract

Objective  To study the feasibility of engineering cartilage tissue in a tube lined with epithelium and implanting allogenic chondrocytes in a novel scaffold that is made of chitosan nonwoven cloth coated with poly(DL-lactide-co-glycolide).

Design  Laboratory research.

Subjects  Allogenic chondrocytes were obtained from the auricles of 1-month-old New Zealand white rabbits. After the cells were cultured in vitro for 3 or 4 passages, they were implanted in the scaffolds to form composite grafts and then transplanted into the rabbits. After 6, 12, and 18 weeks, the general histologic characteristics were investigated.

Results  The cobweb-like matrix was observed approximately 1 week after the chondrocytes had been implanted in the scaffolds. At 6 weeks, the matrix was secreted, and there were immature chondrocytes in the grafts. At 12 weeks, the allogenic cartilage in the tube lined with epithelium had been created. Chondrocytes were almost mature and the lacunae had formed. At 18 weeks, the neocartilage was similar to native cartilage.

Conclusion  It is feasible to produce allogenic cartilage in a tube lined with epithelium by implanting allogenic chondrocytes into a novel scaffold made of chitosan nonwoven cloth coated with poly(DL-lactide-co-glycolide).

The key points in repairing laryngeal and tracheal stenosis or defects are to recover the cartilage and its mucosal inner chamber. Because of the finite supply of autologous cartilage, this remains a clinical challenge. Tissue engineering, a relatively new field that enables tissue equivalents to be created from isolated cells in combination with biomaterials and bioreactor culture vessels, can potentially provide a basis for systematic, controlled, in vitro studies of tissue growth and function. Tissue engineering brings the hope of being able to repair cartilage defects of the larynx and trachea.1,2 The function of a large area of mucosal defect in the larynx and trachea can be revitalized by removing a musculocutaneous flap or by performing a free skin graft to allow the area to change to mucosa gradually.3,4 To our knowledge, the simultaneous repair of cartilage and its mucosal integrity has not been reported. In this study, we attempted to construct allogenic cartilage in tubes lined with epithelium in vivo by using a novel scaffold made of chitosan nonwoven cloth coated with poly(DL-lactide-co-glycolide) (PLGA). The focus of the present study is to explore a new way of repairing laryngeal and tracheal defects.

METHODS

Fourteen New Zealand white rabbits from Nanjing Jinling Breeding Rabbit Farm (Jiangsu, China) were used in the study. Animal welfare and experimental procedures were strictly in accordance with the Guidelines on the Care and Use of Laboratory Animals5 and the related ethical regulations of China. The experimental design of the present study is shown in Figure 1.

CHONDROCYTE HARVESTING, ISOLATION, AND CULTURE

The auricles of 1-month-old New Zealand white rabbits were harvested under sterile conditions. After the cartilage was washed 3 times with phosphate-buffered saline (with the addition of penicillin G, 200 U/mL, and streptomycin sulfate, 200 μg/mL; Invitrogen Corporation, Carlsbad, California), it was diced into pieces of approximately 1 mm3 and placed into a centrifuge tube to which double volumes of collagenase II (0.3 mg/mL; Sigma-Aldrich, St Louis, Missouri) were added. After the mixture was digested for 6 hours at 37°C in a homeothermic pulsator (model ZHWY-211C; WitCity Analyzing Instrument Manufactory Co Ltd, Shanghai, China), phosphate-buffered saline was used to terminate the digestion. The material was rinsed 2 times and centrifuged at 2000 rotations per minute for 10 minutes, and the supernatant was discarded.

After filtration of the supernatant through a 100-μm filter, the chondrocytes obtained through incomplete enzyme digestion of the cartilage were seeded with small pieces of cartilage (≈0.4 mm3) in culture flasks that had been coated previously with poly-L-lysine (0.1%). First, small pieces of cartilage that had not undergone filtration were put into vacant flasks, and RPMI 1640 culture medium (fetal bovine serum, 150 g/L; vitamin C, 5 mg/dL; penicillin G, 100 U/mL; streptomycin sulfate, 100 μg/mL; and L-glutamine, 2 mmol/L; HyClone, Thermo Fisher Scientific, Logan, Utah) was added. (To convert vitamin C to micromoles per liter, multiply by 56.78.) The flasks were inverted for 2 hours, and then the chondrocyte suspension was mixed in symbiotic culture with the cartilage and maintained in the incubator at 37°C in a 5% carbon dioxide atmosphere in primary monolayer culture until confluency.6 Culture medium was initially changed after 3 days, and thereafter it was changed every other day. Cells were harvested by treatment with trypsin, 2.5 g/L (Sigma-Aldrich), and counted with a hemocytometer by using trypan blue exclusion and then cultured in monolayer for 3 or 4 passages.

CONSTRUCT PREPARATION

The novel scaffolds made of chitosan nonwoven cloth (degree of deacetylation, ≥90%; molecular weight, 2-5 × 105; Hainan Xinlong Spunlace Materials Co Inc, Laocheng, China) coated with PLGA (DL-lactide and DL-glycolide according to a mole-matching ratio of 75:25; Research Institute of Polymer Chemistry, Sun Yat-sen University) were cut into 2.2 × 2.3– to 4.2-cm sheets and soaked in poly-L-lysine for 6 hours, and then air dried naturally. After being steeped in 75% alcohol for 2 hours and washed 3 times with phosphate-buffered saline, they were placed in culture flasks with RPMI 1640 culture medium at 37°C to incubate for 24 hours. Two hours before chondrocyte implantation, the RPMI 1640 culture medium was removed and the scaffolds remained in the incubator. The chondrocytes were collected, and the density of cell suspension was adjusted to 5 × 107/mL. The cells were then implanted in the scaffolds and cultured at 37°C in the incubator. Fresh medium was added every other day, and the composites were cultured in vitro for 10 days.

CONSTRUCT CULTIVATION IN VIVO

Twelve 4-month-old New Zealand white rabbits (each weighing 2.0-2.5 kg) were anesthetized via venous injection with 3% pentobarbital sodium (30 mg/kg). Under sterile conditions, 2 parallel incisions were made in the abdomen, the subcutaneous tissue was detached between the incisions, and a silica gel tube that sheared approximately 10 pores (2 cm long and 0.6- to 1.2-cm diameter) was placed on the surface of the skin. After the inside margin of each of the 2 parallel incisions was sutured to form a skin tube, with the silica gel tube inside, the composite of chondrocytes and scaffold was convoluted and coated onto the skin tube, which was then stabilized with 4-0 absorbable sutures. Finally, we detached the subcutaneous tissue and the muscles of the abdomen outside of the incisions up to the peritoneum so that they were embedded at the depth of the abdominal muscles (Figure 2); all layers were then sutured. After surgery, the animals received an intramuscular injection of 0.8 million U of penicillin G daily for 3 days.

CONSTRUCT ANALYSIS

Groups of 4 rabbits were humanely killed at 6, 12, and 18 weeks after surgery, and samples of the compounded construct were harvested. After the samples were soaked in 4% neutral formalin, they were embedded in paraffin and stained with hematoxylin-eosin, toluidine blue, and Masson's trichrome.

RESULTS
MORPHOLOGIC CHARACTERISTICS

Chondrocytes adhered to the scaffold fibers individually or in coenobiums, and the cobweb-like matrix was secreted among the fibers after implantation and cultivation for 1 week when viewed under phase contrast microscopy.

Macroscopically, 6 weeks after surgery, the composites of chondrocytes and cytoskeleton retained their approximate primary shape and were covered by a layer of pink connective tissue. When the connective tissue was removed from the samples, they appeared ivory-white and without blood vessel distribution. Their size was approximately equal to that of the originally transplanted size. At 12 weeks, the neocartilage in the tube lined with epithelium had been fabricated. At 18 weeks, the composites had increased in volume and the samples appeared ivory-white and elastic, like cartilage tissue, lined with epithelium. Among the engineered cartilage tubes, the longest was 1.8 cm and the maximum diameter was 0.9 cm.

HISTOLOGIC FINDINGS

After 6 weeks of transplantation, the matrix was secreted and there were immature chondrocytes in the grafts. The chondrocytes were oval (Figure 3). At 12 weeks, the neocartilage in a tube lined with epithelium had been fabricated. Chondrocytes were almost mature and the lacunae had formed. The contents of the matrix had obviously increased (Figure 4). At 18 weeks, the neocartilage was similar to native cartilage. The chondrocytes were spherical and obvious lacunae could be seen. The morphologic features and the matrix contents were almost the same as those of native chondrocytes. The epithelium was well adhered to the inner cartilage tubes (Figure 5 and Figure 6).

COMMENT

The larynx and trachea are hollow tubes, so an ideal graft should have the same sustainability, permanent viability, and integrity as the epithelium inner chamber. The native columnar epithelium with cilia is crucial to prevent secretion storing and cicatrization, but it is very difficult to achieve such a graft. At present, often a musculocutaneous flap is removed or a free skin graft is performed to allow the area to change to mucosa gradually and to implement its function. In our experiments, we used the technology of tissue engineering to construct neocartilage in tubes and we created coating with an epithelium inner chamber by using a tubular skin flap. These experiments may offer a clinical basis and reference for the simultaneous repair of laryngeal and tracheal defects in the future.

Three-dimensional cytoskeleton and its role in the temporary extracellular matrix are vital in the process of cartilage engineering.7 The cytoskeleton provides a locus for adhesion, differentiation, metabolism, and proliferation of chondrocytes.8,9 An ideal biomaterial carrier should have satisfactory biocompatibility, biodegradation, absorption, cell interface, permeability, intensity, and 3-dimensional structure.2 For the hollow tubes of the larynx and trachea, sufficient mechanical intensity and agile plasticity are the requirements for cytoskeleton; however, these qualities remain deficient in the currently used scaffolds. The PLGA that we used was a copolymer of DL-lactide and DL-glycolide based on a mole-matching ratio of 75:25. It possessed satisfactory tensile strength, biocompatibility, and biodegradation and has been widely used in cartilage tissue engineering10; it was friable and insufficient in plasticity. The chitosan nonwoven cloth is a novel biomaterial. Findings from our previous experiments in animals confirmed that chitosan nonwoven cloth is not toxic and does not produce an inflammatory reaction. It had appropriate degradation properties in vitro or in vivo and showed ideal biocompatibility in general histologic analysis.11 It was plastic but insufficient in tensile strength. Integrating the superiority and peculiarity of the 2 materials, we reported our method of producing the novel scaffold made by chitosan nonwoven cloth coated with PLGA.12 The novel scaffolds made in this way had a stable 3-dimensional microstructure with controllable variables and reliable biomechanical performance and plasticity. The porosity of the scaffolds was 82% to 86%, and the pore size ranged from 100 to 300 μm. The chondrocytes implanted in the scaffolds adhered to the fibers and grew well. Thus, we deduced that the specifications of the scaffolds met the requirements of engineered cartilage for the hollow larynx and trachea. This would be promising in the research of repairing cartilage defects of the larynx and trachea through the use of tissue engineering.

Vacanti et al13 first produced engineered cartilage tubes in vivo in athymic nude mice by using polyglycolic acid scaffolds. We constructed engineered cartilage in tubes lined with epithelium by implanting allogenic chondrocytes in the novel scaffolds in vivo for use in immune animals.

The use of allogenic chondrocytes as seeding cells for cartilage tissue engineering is receiving great attention. Our research demonstrated that the composites of allogenic chondrocytes and the novel scaffold could produce cartilage tissue after being transplanted in vivo without immunologic rejection. This may be because cartilage has inferior immunity and its surface antigen is weakened or is destroyed by digestion, isolation, culture in vitro, and carrier implantation. Therefore, the immunogenicity of allogenic chondrocytes is weakened.14

The idea in using tissue engineering technology to treat laryngeal and tracheal stenosis or defects is to combine engineered cartilage, engineered mucosa and submucosa, and engineered intrinsic laryngeal muscle to simultaneously fabricate larynx and trachea organs in situ or ectopia. Our research represented merely a pilot study. The maximum diameter of harvested engineered cartilage was 0.9 cm. Experiments with larger-caliber tubing demonstrated that the neocartilage was not unsatisfactorily formed, which could be related to the rabbits' small somatotype, their lack of local muscles and soft tissues, and their frequent activity. Perhaps sufficient nourishment was not available. The specific reasons remain to be clarified. Furthermore, the shearing intensity, ductility, energy absorption efficiency, and degree of stress of the harvested neocartilage were perhaps insufficient to achieve or approach the demand of practical application. More appropriate biomaterials and methods for clinical application are required for further research.

In conclusion, our experiments demonstrated that the composite of allogenic chondrocytes and the novel scaffold, made with chitosan nonwoven cloth coated with PLGA (75:25 mole-matching ratio), could produce engineered cartilage after being transplanted in vivo in rabbits. It was feasible to fabricate neocartilage in a tube lined with epithelium in vivo in immune animals without immunologic rejection occurring. We found a new way of simultaneously repairing the cartilage and its epithelial integrity in the larynx and trachea. Therefore, we believe that, in the future, with the progress and perfection of tissue engineering technology and other relevant research areas, and using our experimental design, human allogenic cartilage can be fabricated in a tube lined with epithelium to allow for the simultaneous clinical repair of laryngeal and tracheal defects.

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Article Information

Correspondence: Qiu-ping Wang, MD, Department of Otolaryngology–Head and Neck Surgery, Jinling Hospital, 305 E Zhongshan Rd, Nanjing 210002, China (qpwang1016@vip.sina.com).

Submitted for Publication: January 4, 2008; final revision received April 28, 2008; accepted May 12, 2008.

Author Contributions: All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Cheng, Huang, Li, Zhou, T. Wang, Jiang, and Q. Wang. Drafting of the manuscript: Cheng, Huang, Li, and Q. Wang. Critical revision of the manuscript for important intellectual content: Zhou and T. Wang. Obtained funding: Cheng, Huang, and Q. Wang. Administrative, technical, and material support: Cheng, Huang, and Q. Wang. Study supervision: Cheng, Li, and Q. Wang.

Financial Disclosure: None reported.

Funding/Support: This research is supported by grant 990418 from the Natural Science Foundation of Guangdong Province.

Additional Contributions: Daping Quan, ScD, Research Institute of Polymer Chemistry, Sun Yat-sen University, Guangzhou, provided the PLGA scaffold material.

References
1.
Ochi  M Clinical result of transplantation of tissue-engineered cartilage and future direction of cartilage repair: novel approach with minimally invasive procedure. Yonsei Med J 2004;45 (((suppl)) 74- 79
2.
Huang  JZLi  XSQuan  DP  et al.  In vitro and in vivo study on the degradation and biocompatibility of a poly-DL-lactide (PLA) polymer. Xiandai Kangfu 2001;558- 59
3.
Spriano  GPiantanida  RPellini  R Hypopharyngeal reconstruction using pectoralis major myocutaneous flap and prevertebral fascia. Laryngoscope 2001;111 (3) 544- 547
PubMedArticle
4.
Guang  ZPeng  XRHuang  XM  et al.  Pectoralis major muscle musculo-cutaneous flap repairing tissue defects after surgery of laryngeal pharynx carcinoma. Chin J Microsurg 2004;27300- 301
5.
National Research Council of USA, Guidelines on the Care and Use of Laboratory Animals.  Washington, DC National Academies Press1996;
6.
Cheng  YWang  QPLi  JH  et al.  The modified method for human nasoseptal chondrocyte culture and study on cell's biological character. Zhongguo Erbi Yanhou Toujing Waike 2006;1347- 49
7.
Langer  RVacanti  JP Tissue engineering. Science 1993;260 (5110) 920- 926
PubMedArticle
8.
Britt  JCPark  SS Autogenous tissue-engineered cartilage: evaluation as an implant material. Arch Otolaryngol Head Neck Surg 1998;124 (6) 671- 677Article
9.
Sittinger  MBujia  JMinuth  WW  et al.  Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture. Biomaterials 1994;15 (6) 451- 456Article
10.
Holy  CECheng  CDavis  JE  et al.  Optimizing the sterilization of PLGA scaffolds for use in tissue engineering. Biomaterials 2001;22 (1) 25- 31
PubMedArticle
11.
Cheng  YHuang  JZDu  J  et al.  In vitro and in vivo study on the biodegradation and biocompatibility of chitosan-nonwoven-cloth. Chin J Otorhinolaryngol Skull Base Surg 2005;11229- 231
12.
Cheng  YHuang  JZQuan  DP  et al.  A novel scaffold made by chitosan–nonwoven-cloth coated with poly (dl-lactide-co-glycolide) (PLGA). Chin J Clin Rehabil 2005;960- 61
13.
Vacanti  CAPaige  KTKim  W  et al.  Experimental tracheal replacement using tissue engineered cartilage. J Pediatr Surg 1994;29 (2) 201- 204
PubMedArticle
14.
Vunjak-Novakovic  GObradovic  BMartin  I  et al.  Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol Prog 1998;14 (2) 193- 202Article
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