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Figure 1.
Macroscopic view of the different implants used to generate autologous porcine elastic cartilage. A, High-density polyethylene. B, Soft acrylic. C, Polymethylmethacrylate. D, Extrapurified Silastic. E, Conventional Silastic. The implants in (D) and (E) have been removed, and a visible pocket is present.

Macroscopic view of the different implants used to generate autologous porcine elastic cartilage. A, High-density polyethylene. B, Soft acrylic. C, Polymethylmethacrylate. D, Extrapurified Silastic. E, Conventional Silastic. The implants in (D) and (E) have been removed, and a visible pocket is present.

Figure 2.
Histological sections of the engineered cartilage surrounding the different polymers. A, High-density polyethylene. B, Polymethylmethacrylate. C, Soft acrylic. D, Extrapurified Silastic (hematoxylin-eosin, original magnification ×10).

Histological sections of the engineered cartilage surrounding the different polymers. A, High-density polyethylene. B, Polymethylmethacrylate. C, Soft acrylic. D, Extrapurified Silastic (hematoxylin-eosin, original magnification ×10).

Figure 3.
Histological sections of the engineered cartilage surrounding the different polymers. The external perimeter (arrows) and internal interface with polymer (arrowheads) are reminiscent of a perichondrium. A, High-density polyethylene. B, Polymethylmethacrylate. C, Soft acrylic. D, Extrapurified Silastic (Masson trichrome blue, original magnification ×10). Asterisks indicate the location of the internal stent material.

Histological sections of the engineered cartilage surrounding the different polymers. The external perimeter (arrows) and internal interface with polymer (arrowheads) are reminiscent of a perichondrium. A, High-density polyethylene. B, Polymethylmethacrylate. C, Soft acrylic. D, Extrapurified Silastic (Masson trichrome blue, original magnification ×10). Asterisks indicate the location of the internal stent material.

Figure 4.
Elastic fibers are noted in the porcine engineered autologous cartilage covering the high-density polyethylene (Verhoeff, original magnification ×10).

Elastic fibers are noted in the porcine engineered autologous cartilage covering the high-density polyethylene (Verhoeff, original magnification ×10).

Inflammatory Reactions to the Endoskeletal Implants*
Inflammatory Reactions to the Endoskeletal Implants*
1.
Trent  JFKirsner  RS Tissue engineered skin: Apligraf, a bi-layered living skin equivalent. Int J Clin Pract. 1998;52408- 413
2.
Atala  A Tissue engineering in urologic surgery. Urol Clin North Am. 1998;2539- 50Article
3.
Ibarra  CJanetta  CVacanti  CA  et al.  Tissue engineered meniscus: a potential new alternative to allogenic meniscus transplantation. Transplant Proc. 1997;29966- 968Article
4.
Langer  RVacanti  JP Tissue engineering. Science. 1993;260920- 926Article
5.
Cao  YVacanti  JPPaige  KTUpton  JVacanti  CA Transplantation of chondrocytes utilizing a polymer cell construct to produce tissue engineered cartilage in the shape of a human ear. Plast Reconstr Surg. 1997;100297- 302Article
6.
Sakata  JVacanti  CASchloo  BHealy  GBLanger  RVacanti  JP Tracheal composites tissue-engineered from chondrocytes, tracheal epithelial cells and synthetic degradable scaffolding. Transplant Proc. 1994;263309- 3310
7.
Puelacher  WCMooney  DLanger  RUpton  JVacanti  JPVacanti  CA Design of nasoseptal cartilage replacements synthesized from biodegradable polymers and chondrocytes. Biomaterials. 1994;15774- 778Article
8.
Cao  YRodriguez  AVacanti  MIbarra  CArévalo  CAVacanti  CA Comparative study of the use of poly(glycolic acid), calcium alginate and pluronic in the engineering of autologous porcine cartilage. J Biomater Sci Polym Ed. 1998;9475- 487Article
9.
Britt  JCPark  SS Autogenous tissue-engineered cartilage. Arch Otolaryngol Head Neck Surg. 1998;124671- 677Article
10.
BASF Wyandotte Corporation, Central Research and Development, Organic specialties and fine chemicals. PLURONIC Polyols Toxicity and Irritation Data. Wyandotte, Mich BASF Corp1975;
11.
Schmolka  IR Physical basis for poloxamer interactions. Ann N Y Acad Sci. 1994;72092- 97Article
12.
Henry  RLSchmolka  IR Burn wound coverings and the use of poloxamer preparations. Crit Rev Biocompat. 1989;5207- 220
13.
Walker  HLMason  AD  Jr A standard animal burn. J Trauma. 1968;81049- 1051Article
Original Article
December 2000

Internal Support of Tissue-Engineered Cartilage

Author Affiliations

From the Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, and the Department of Otology and Laryngology, Harvard Medical School, Boston (Drs Arévalo-Silva and Eavey); and the Department of Anesthesia, Laboratory for Tissue Engineering (Drs Arévalo-Silva, Cao, M. Vacanti, Weng, and C. A. Vacanti), and the Department of Pathology (Dr M. Vacanti), University of Massachusetts Medical Center, Worcester.

Arch Otolaryngol Head Neck Surg. 2000;126(12):1448-1452. doi:10.1001/archotol.126.12.1448
Abstract

Background  Auricles previously created by tissue engineering in nude mice used a biodegradable internal scaffold to maintain the desired shape of an ear. However, the biodegradable scaffold incited a compromising inflammatory response in subsequent experiments in immunocompetent animals.

Objective  To test the hypothesis that tissue-engineered autologous cartilage can be bioincorporated with a nonreactive, permanent endoskeletal scaffold.

Materials and Methods  Auricular elastic cartilage was harvested from Yorkshire swine. The chondrocytes were isolated and suspended into a hydrogel (Pluronic F-127) at a cell concentration of 5 × 107 cells/mL. Nonbiodegradable endoskeletal scaffolds were formed with 1 of 5 polymers: (1) high-density polyethylene, (2) soft acrylic, (3) polymethylmethacrylate, (4) extrapurified Silastic, and (5) conventional Silastic. Three groups were studied: (1) a control group using only the 5 polymers, (2) the 5 polymers enveloped by Pluronic F-127 only, and (3) the implants coated with Pluronic F-127 seeded with chondrocytes. All constructs were implanted subdermally; implants containing cells were implanted into the same animal from which the cells had been islolated. The implants were harvested after 8 weeks of in vivo culture and histologically analyzed.

Results  Only implants coated by hydrogel plus cells generated healthy new cartilage. With 3 polymers (high-density polyethylene, acrylic, and extrapurified Silastic), the coverage was nearly complete by elastic cartilage, with minimal fibrocartilage and minimal to no inflammatory reaction. The Food and Drug Administration–approved conventional Silastic implants resulted in fragments of fibrous tissue mixed with elastic cartilage plus evidence of chronic inflammation. The polymethylmethacrylate implant was intermediate in the amount of cartilage formed and degree of inflammation.

Conclusions  This pilot technique combining tissue-engineered autologous elastic cartilage with a permanent biocompatible endoskeleton demonstrated success in limiting the inflammatory response to the scaffold, especially to high-density polyethylene, acrylic, and extrapurified Silastic. This model facilitates the potential to generate tissue of intricate shape, such as the human ear, by internal support.

TISSUE ENGINEERING already provides tissue supply for specific clinical applications, such as burned skin,1 ureterovesicular reflux,2 and cellular cartilage repair for the knee.3 However, a main objective of this science is to generate an autologous tissue structure of predetermined shape.4 Previous laboratory generation of cartilage in predetermined shapes, such as the ear on the back of the mouse,5 the trachea,6 and the nasal septum,7 was accomplished using polyglycolic acid/polylactic acid (PGA/PLA) copolymer as the scaffold material; PGA/PLA is nonreactive in immunotolerant nude mice. Autologous experiments8,9 in immunocompetent animal models have shown that PGA/PLA generates an inflammatory reaction that interferes with the viability and shape of the tissue constructs.

To avoid this inflammatory response, a different polymer has been used for immunocompetent animals. Excellent results have been obtained using the hydrogel Pluronic F-127, a copolymer formed with 70% polyethylene oxide and 30% polypropylene oxide.8,10 In a solution with Ham F-12 culture media, Pluronic F-127 forms a reversible thermosensitive hydrogel that at cold temperatures exists in a liquid state and at warm physiologic temperatures transforms into a thick gel.11 With greater viscosity, Pluronic F-127 becomes a 3-dimensional environment that allows the chondrocytes to multiply and to produce matrix, which replaces the gel that is gradually bioabsorbed. The end product is engineered cartilage tissue without residual foreign material. Pluronic F-127 is nontoxic, biocompatible, and bioabsorbable12 and has been used for human benefit as a biodressing.13 However, despite the useful characteristics that allow Pluronic F-127 to generate excellent-quality cartilage, this hydrogel polymer does not maintain a specific shape.

Nonabsorbable materials with permanent shape, such as high-density polyethylene, Silastic, and various polymers for intraocular lens implants (polymethylmethacrylate [PMMA], extrapurified Silastic, and acrylic), have demonstrated clinical success as nonreactive implants in human patients. The objective of this preliminary study was to evaluate the combination of Pluronic F-127 hydrogel as a coating for several nonbiodegradable polymers already commonly used in surgical practice to generate cartilage around a permanent endoskeleton.

MATERIALS AND METHODS

This study was conducted in compliance with animal research procedures at the University of Massachusetts Medical Center, Worcester.

CHONDROCYTE ISOLATION

Under sterile conditions and after sedation with intramuscular injection of xylazine hydrochloride (2 mg/kg) and ketamine hydrochloride (20 mg/kg) and inhaled anesthesia by tracheal intubation with isoflurane at 2%, auricular cartilage was harvested from male Yorkshire swine (aged 8-10 weeks) weighing 16 to 18 kg. The isolated cartilage was minced into small fragments; washed with phosphate-buffered saline solution containing penicillin potassium (100 U/L), streptomycin sulfate (100 mg/L), and amphotericin B (0.25 mg/L) (GIBCO, Grand Island, NY); and digested with 0.3% collagenase II (Worthington Biochemical Corp, Freehold, NJ) at 37°C for 12 hours. The chondrocyte suspensions were filtered using a sterile 250-mm polypropylene mesh filter (Spectra/Mesh 146-426; Spectrum Medical Industries Inc, Laguna Hills, Calif) and centrifuged at 6000 rpm for 10 minutes. The resulting pellets of cells were washed twice with phosphate-buffered saline solution (GIBCO) and then resuspended in Ham F-12 culture media with levoglutamide, L-ascorbic acid (50 mg/L), penicillin (100 U/L), streptomycin sulfate (100 mg/L), and amphotericin B (0.25 mg/L) (GIBCO) and 10% fetal bovine serum (Sigma-Aldrich Corp, St Louis, Mo). Cell number was quantitated using a hemocytometer, and cell viability was determined using trypan blue vital dye (Sigma-Aldrich, Irvine, Calif).

PLURONIC F-127 PREPARATION

The powder of Pluronic F-127 was weighed and slowly added (over approximately 6 hours) in a cold room into an autoclavable glass bottle that contained culture media. The mixture was prepared at 30% (wt/vol). The mixture was autoclaved for 20 minutes and, after cooling, was stored at less than 4°C until use.

CHONDROCYTE-POLYMER SUSPENSION

After chondrocyte count at 4°C, the cells were suspended in a 30%(wt/vol) solution of copolymer gel of ethylene and propylene oxide, Pluronic F-127, and Ham F-12 (BASF, Mount Olive, NJ) at a cellular density of 5 × 107 cells/mL and stored on ice until implantation.

IMPLANTS AND SURGICAL IMPLANTATION

The day after cartilage harvest, using general anesthesia and sterile technique, the autologous chondrocyte and Pluronic F-127 suspensions were collected into sterile 5-mL syringes using a 16-gauge needle. Small squares of the 5 biocompatible materials were cut to approximately 1 × 1 × 0.2 cm and sterilized using ethylene oxide. The materials used were (1) high-density polyethylene (Medpor; Porex Surgical Inc, College Park, Ga); (2) soft acrylic (Acrysoft; Alcon Laboratories, Fort Worth, Tex); (3) PMMA (polymethacrylate; Ioptex Research Inc, Irwindale, Calif); (4) extrapurified Silastic (Allergan Medical Optics, Irvine); and (5) conventional Silastic (Food and Drug Administration [FDA]–approved for body implantation). These 5 materials were studied in 3 groups of animals. Group 1 was the control, using only the 5 materials described. Group 2 consisted of the same 5 materials coated only with Pluronic F-127. Group 3 consisted of the same 5 materials coated with Pluronic F-127 that had been seeded with autologous chondrocytes. In groups 2 and 3, each material was slowly covered with the Pluronic F-127 gel and the chondrocyte-Pluronic F-127 gel suspension, respectively, resurfacing the materials with 2 thin (approximately 1 mm per layer) layers using the syringe and needle and waiting until the previous layer had solidified; the suspension viscosity at that time was similar to hair gel. Once the materials were totally covered by 2 layers of the gel or the chondrocyte and gel suspension, the constructs were implanted subcutaneously through small incisions on the ventral surface (inguinal and axillary regions) of the 2 donor animals. Before wound closure, the materials in groups 2 and 3 were resurfaced with additional gel (at least 3 mm total) to ensure that they were completely covered. The incisions were closed using a running suture. Groups 1 and 2 had 2 samples of the 5 different materials implanted and group 3 had 4 samples of each material implanted, for a total of 40 implants. The implants were monitored weekly by external inspection until harvest.

HARVEST OF THE IMPLANTS

Implants were harvested after 8 weeks in vivo under general anesthesia. Implants were carefully dissected, examined, and analyzed, comparing their gross appearance. All implants were fixed in 10% phosphate-buffered formalin (Fisher Scientific, Fair Lawn, NJ) for histological analysis.

HISTOLOGICAL ANALYSIS

After being fixed for 24 hours, implants were embedded in paraffin and transversely sectioned. Slide sections were stained with hematoxylin-eosin, safranin O, Masson trichrome blue, and Verhoeff solution.

RESULTS
EXTERNAL INSPECTION ANALYSIS

Minimal to no evidence of inflammation was noted in all groups for implants of high-density polyethylene, acrylic, and extrapurified Silastic. For implants of PMMA and conventional FDA-approved Silastic, seromas that required drainage were detected in all specimens in groups 1 and 2 and an inflammatory reaction of erythema and edema was present in all specimens in group 3.

HISTOLOGICAL ANALYSIS

Analysis using hematoxylin-eosin stains for groups 1 and 2 demonstrated fibrous tissue that created a pocket around the scaffolds. All specimens in these 2 groups demonstrated infiltration with chronic inflammatory cells (Table 1).

In group 3, specimens of high-density polyethylene were approximately 95% covered by an elastic cartilage layer approximately 0.5 cm thick, and fibrocartilage covered the remaining 5% (Figure 1A). Histological sections with hematoxylin-eosin stain showed lobules of cartilage surrounded but not invaded by vascularized fibrous tissue containing a rare sprinkling of chronic inflammatory cells. The cartilage contained somewhat evenly spaced, round to triangular lacunae with single chondrocytes. Occasional bicellular groups were seen, which is characteristic of elastic cartilage. The matrix was heterochromic, indicating maturity (Figure 2A). Safranin O stain was strongly and evenly positive in the matrix, indicating proteoglycans production. Trichrome stain highlighted the peripheral rim of fibrous tissue surrounding the lobules of cartilage, reminiscent of a perichondrium. Also noted was fibrous tissue enveloping the high-density polyethylene material and connecting it to the cartilage, resembling an internal layer of perichondrium (Figure 3A). Verhoeff elastic stain showed coiled fibers (Figure 4). Use of polarized light defined a central core of foreign body material. No foreign body giant cell reaction was seen. No significant inflammation or cellular atypia was seen. Histological sections were consistent with mature elastic cartilage.

The acrylic implants were 95% enveloped by elastic cartilage and 5% by fibrocartilage (Figure 1B). Minimal to no chronic inflammatory changes were noted. Hematoxylin-eosin stain demonstrated lobules of mature cartilage. The cartilage had round to triangular lacunae containing single chondrocytes. The matrix was heterochromic, indicating maturity (Figure 2B). The proteoglycans production noted by safranin O stain was strongly and evenly positive. Trichrome stain showed a peripheral rim and internal coating of fibrous tissue reminiscent of a perichondrium. The fibrous tissue did not invade the elastic cartilage (Figure 3B). Verhoeff elastic stain was strongly positive. Use of polarized light defined a central core of foreign body material. The findings were consistent with mature elastic cartilage.

The PMMA implants were 85% covered by cartilage and 15% by fibrocartilage (Figure 1C). The cartilage contained small lacunae with poor organization. The cartilage was surrounded by vascularized fibrous tissue containing some chronic inflammatory cells. The matrix was heterochromic (Figure 2C). The trichrome stain was densely positive surrounding the cartilage and interwoven in the matrix, suggestive of fibrocartilage (Figure 3C). Safranin O stain was strongly and evenly positive in the matrix, indicating proteoglycans production. The Verhoeff stain for elastic fibers was not uniform, with areas that stained strongly alternating with areas that stained lightly. Use of polarized light defined a central core of foreign body material. The PMMA implants generated a tissue with features of fibrocartilage and elastic cartilage.

Implants made with extrapurified Silastic were 95% covered by elastic cartilage and 5% by fibrocartilage (Figure 1D). The hematoxylin-eosin stain showed a uniform layer of cartilage and a vascularized fibrous tissue perimeter with focal chronic inflammatory cells external to the cartilage. The cartilage contained evenly spaced, round to triangular lacunae with single chondrocytes. Characteristic bicellular groups were present. The matrix was heterochromic, indicating maturity (Figure 2D). Safranin O stain was strongly positive in the matrix, indicating proteoglycans production. The peripheral rim of fibrous tissue was highlighted by the trichrome stain, suggestive of a perichondrium, and an internal layer was also present (Figure 3D). Verhoeff elastic stain was mildly positive for coiled fibers. Use of polarized light defined a central core of foreign body material. No significant inflammation or cellular abnormalities were seen. Histological sections were consistent with mature elastic cartilage.

The conventional FDA-approved Silastic implants resulted in fragments of mostly fibrous tissue mixed with elastic cartilage. Hematoxylin-eosin stain demonstrated areas of cartilage with focal areas of cell disorganization, with some surrounding fibrous tissue containing chronic inflammatory cells. No cellular atypia or evidence of malignancy was seen. The tissue contained round to triangular lacunae containing single chondrocytes. The matrix was heterochromic, indicating maturity. The proteoglycans production was noted by safranin O stain to be strongly and evenly positive. Trichrome stain highlighted a rim of fibrous tissue reminiscent of a perichondrium that in a few areas showed fibrous tissue mixed with elastic cartilage. Verhoeff elastic stain was strongly positive. Polarized light did not demonstrate any foreign body because the Silastic had been removed. Overall, the histological features indicated a mixture of elastic and approximately 25% fibrocartilage.

COMMENT

The engineering of autologous tissue is a promising technology with potential for tissue and organ replacement. Initial results using an immunocompromised xenograft model did not suggest that maintenance of shape using scaffolds of FDA-approved PGA/PLA would be an issue, other than requiring temporary placement of an external stent. However, subsequent autologous models using PGA/PLA molds have not provided satisfactory long-term results because of an inflammatory reaction against the scaffold that deforms the shape and impairs the quality of the tissue.8,9 In our experience, the best results to reproduce cartilage in an autologous model have been obtained using a hydrogel as a scaffold.8 Because this gel does not have the capacity to maintain a specific shape, use of a permanent, nonreactive endoskeleton seemed to be a possible option.

The results demonstrate a difference in outcome depending on the material used as an internal scaffold. Moreover, the differences between materials were more evident when comparing the control group of materials alone with the other 2 groups coated with Pluronic F-127 with or without chondrocytes. The delay of the inflammatory reaction by external inspection in the group 2 implants suggests that the gel temporarily (approximately 8 days after implantation) protected the foreign material from the immune system. The results of the Pluronic F-127 and chondrocyte group revealed that the combination of cell multiplication and matrix continued to protect the material from the inflammatory reaction. The quality of the tissue varied depending on the polymer, the most ideal being tissue generated around high-density polyethylene, acrylic, or, to a slightly less ideal degree, extrapurified Silastic. Conversely, specimens of PMMA and conventional FDA-approved Silastic resulted in a more intense inflammatory reaction that produced fibrous tissue around and in the elastic cartilage.

Future studies should focus on the materials that resulted in the least reactive constructs. Increasing the porosity of such materials may allow the Pluronic F-127 and chondrocyte suspension to permeate the polymer to provide enhanced anchorage of the cartilage to the endoskeleton. Using life-sized and shaped auricular scaffolds will permit evaluation of how realistic this internal skeleton concept of chondrocyte engineering might be for patient use.

In conclusion, this pilot technique combining tissue-engineered autologous elastic cartilage with a permanent biocompatible endoskeleton demonstrated success for the generation of healthy cartilage and in limiting the inflammatory response to the scaffold, especially to high-density polyethylene, acrylic, and extrapurified Silastic.

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Article Information

Accepted for publication June 28, 2000.

We thank Betty Treanor for providing word processing services.

Corresponding author and reprints: Roland D. Eavey, MD, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA 02114.

References
1.
Trent  JFKirsner  RS Tissue engineered skin: Apligraf, a bi-layered living skin equivalent. Int J Clin Pract. 1998;52408- 413
2.
Atala  A Tissue engineering in urologic surgery. Urol Clin North Am. 1998;2539- 50Article
3.
Ibarra  CJanetta  CVacanti  CA  et al.  Tissue engineered meniscus: a potential new alternative to allogenic meniscus transplantation. Transplant Proc. 1997;29966- 968Article
4.
Langer  RVacanti  JP Tissue engineering. Science. 1993;260920- 926Article
5.
Cao  YVacanti  JPPaige  KTUpton  JVacanti  CA Transplantation of chondrocytes utilizing a polymer cell construct to produce tissue engineered cartilage in the shape of a human ear. Plast Reconstr Surg. 1997;100297- 302Article
6.
Sakata  JVacanti  CASchloo  BHealy  GBLanger  RVacanti  JP Tracheal composites tissue-engineered from chondrocytes, tracheal epithelial cells and synthetic degradable scaffolding. Transplant Proc. 1994;263309- 3310
7.
Puelacher  WCMooney  DLanger  RUpton  JVacanti  JPVacanti  CA Design of nasoseptal cartilage replacements synthesized from biodegradable polymers and chondrocytes. Biomaterials. 1994;15774- 778Article
8.
Cao  YRodriguez  AVacanti  MIbarra  CArévalo  CAVacanti  CA Comparative study of the use of poly(glycolic acid), calcium alginate and pluronic in the engineering of autologous porcine cartilage. J Biomater Sci Polym Ed. 1998;9475- 487Article
9.
Britt  JCPark  SS Autogenous tissue-engineered cartilage. Arch Otolaryngol Head Neck Surg. 1998;124671- 677Article
10.
BASF Wyandotte Corporation, Central Research and Development, Organic specialties and fine chemicals. PLURONIC Polyols Toxicity and Irritation Data. Wyandotte, Mich BASF Corp1975;
11.
Schmolka  IR Physical basis for poloxamer interactions. Ann N Y Acad Sci. 1994;72092- 97Article
12.
Henry  RLSchmolka  IR Burn wound coverings and the use of poloxamer preparations. Crit Rev Biocompat. 1989;5207- 220
13.
Walker  HLMason  AD  Jr A standard animal burn. J Trauma. 1968;81049- 1051Article
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