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Ovid: Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to Produce Tis...
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Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to

Produce Tissue-Engineered Cartilage in the Shape of a Human Ear
Cao, Yilin M.D., Ph.D.; Vacanti, Joseph P. M.D.; ISSN: 0032 -1052
Author(s): Paige, Keith T. M.D.; Upton, Joseph M.D.; Vacanti, Accession: 00006534 -199708000 -00001
Charles A. M.D.
Issue: Volume 100(2), August 1997, pp 297-302
Publication Type: [Articles]
Publisher: © Williams & Wilkins 1997. All Rights Reserved.
From the Department of Surgery at Children's Hospital,
the Department of Plastic and Reconstructive Surgery
at Beth Israel Hospital, and the Department of
Anesthesia at the University of Massachusetts Medical
Center.
Institution(s): Boston, Mass.
Received for publication December 8, 1995; revised
August 16, 1996.
Presented in part at the 39th Annual Meeting of the
Plastic Surgery Research Council, in Ann Arbor,
Michigan, June 4-7, 1994.
Abstract
This study evaluates the feasibility of growing tissue-engineered cartilage in the shape of a human ear using
chondrocytes seeded onto a synthetic biodegradable polymer fashioned in the shape of a 3-year-old child's auricle. A
polymer template was formed in the shape of a human auricle using a nonwoven mesh of polyglycolic acid molded after
being immersed in a 1% solution of polylactic acid. Each polyglycolic acid-polylactic acid template was seeded with
chondrocytes isolated from bovine articular cartilage and then implanted into subcutaneous pockets on the dorsa of 10
athymic mice. The three-dimensional structure was well maintained after removal of an external stent that had been
applied for 4 weeks.
Specimens harvested 12 weeks after implantation and subjected to gross morphologic and histologic analysis
demonstrated new cartilage formation. The overall geometry of the experimental specimens closely resembled the
complex structure of the child's auricle.
These findings demonstrate that polyglycolic acid-polylactic acid constructs can be fabricated in a very intricate
configuration and seeded with chondrocytes to generate new cartilage that would be useful in plastic and
reconstructive surgery.
Total external ear reconstruction with autogenous cartilage still remains one of the most difficult problems in the
field of plastic and reconstructive surgery. The surgeon's goal is to create an ear that is similar in appearance to the
http://www2.lib.purdue.edu:5733/spa/ovidweb.cgi 8/15/2008
Ovid: Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to Produce Tis... Page 2 of 12
contralateral auricle. Such a reconstruction should have a flexible framework in the shape of the complex three-
dimensional structure of the auricle. We report the successful tissue engineering of such a structure.
Two major alternatives are currently available to the plastic and reconstructive surgeon for total external ear
reconstruction: alloplastic implants and sculpted autogenous cartilage grafts. Although sculpted autogenous cartilage
grafts are used more often clinically, alloplastic implants do have some distinct advantages.
Alloplastic implants of silicone or polypropylene are available in limitless supply. Because they are preformed, no
operative time is expended in shaping the implant. They also have a consistent, predetermined shape. However, these
implants suffer from problems associated with any implanted prosthetic device. They have an increased susceptibility to
infection and an uncertain long -term durability. Alloplastic materials have a greater propensity to extrude than
autogenous tissue. Many of these materials, silicone in particular, have an unknown interaction with the host's immune
system.
Pioneering work by Tanzer 1 and then Brent 2 resulted in the use of sculpted autogenous costal cartilage grafts as
an alternative to alloplastic implants. By using autogenous tissue, these grafts overcome the problems associated with
alloplastic materials. Further, Brent has shown that the grafts have good long-term durability and can even grow with
the patient.2 The sculpted graft's shape, although excellent in skilled hands, is less consistent than that of synthetic
implants. Additionally, extensive operative time is required to shape the framework. The harvesting of costal cartilage
results in donor-site morbidity, and occasionally, there is an insufficient supply of usable cartilage.
Tissue engineering offers the potential to grow autogenous cartilage in a precisely predetermined shape and
requires minimal operative time.8 Implanted cell-polymer constructs ultimately result in the formation of a
cartilaginous structure in the shape of the original polymer template. These studies demonstrate that new cartilage of a
complex spatial geometry can be generated on a clinically relevant scale.
Materials and Methods

Construction of Polymer Devices
Anatomic models of a human ear were created as follows: The ear of a 3 -year -old child was cast using alginate as
the impression material, and then a final cast of plaster was fashioned from the alginate impression. Using the plaster
cast as a mold, synthetic biodegradable polymer constructs were created as described below. A synthetic nonwoven
mesh approximately 100 µm thick, of polyglycolic acid fibers (Davis & Geck, Danbury, Conn.) with a diameter of 15 µm,
having handling characteristics similar to those of a cotton ball and little intrinsic stability, was immersed for
approximately 2 seconds in a 1% (w/v) solution of polylactic acid in methylene chloride. Following immersion, the fabric
was removed and shaped into the form of a human ear using the plaster prosthetic mold. Polymer devices were placed
in 35-mm polystyrene tissue culture dishes (Costar, Cambridge, Mass.).
Isolation of Chondrocytes
Freshly slaughtered (>6 hours) calf forelimbs were obtained from a local slaughterhouse. The forelimbs were
dissected under sterile conditions to expose the articular surfaces of the glenohumeral and humeroulnar joints.
Cartilage fragments were sharply curetted off the articular surface of each joint. Using a method similar to that
described by Klagsbrun,4 the fragments were subjected to collagenase digestion (3 mg/ml) (Worthington Biochemical,
Freehold, N.J.) at 37°C for 12 to 18 hours. The resulting cell suspension was passed through a sterile nylon mesh filter.
The filtrate was centrifuged, and the resulting cell pellet was washed twice with copious amounts of Dulbecco's
phosphate-buffered saline without Ca2+ . Cell number and viability were determined by cell counts using a
hemacytometer and trypan blue vital dye. Chondrocyte preparations having cell viability in excess of 85 percent were
used. Chondrocyte suspensions were concentrated to a cellular density of 5 × 10 7 cells per milliliter.
Seeding and Implantation
Three-milliliter aliquots of the chondrocyte suspension (1.5 × 10 8 cells total) were seeded onto each polymer
matrix in experimental groups I and II and allowed 4 hours at 37°C to attach to the polymer fibers. Tissue culture
medium (Hamm's F-12 medium, Gibco, Grand Island, N.Y.) supplemented with 10% fetal calf serum (Gibco, Grand
Island, N.Y.), 5 µg/ml ascorbic acid, 292 µg/ml L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) was then
added, and the constructs were incubated in vitro at 37°C in 5% CO 2 for 1 week. No external stenting was applied to
either group I or group II during this period. Culture medium was replaced every 3 days (Fig. 1).
Fig. 1. Chondrocytes seeded onto the polymer ear mold in vitro. (Above) Gross appearance of the ear polymer mold
seeded with chondrocytes (15 × 10 7). (Below) Scanning electron micrograph showing adherence of chondrocytes to
polyglycolic acid device before implantation. Note the rounded configuration of the cells and the presence of
extracellular matrix secreted by the cells, indicative of their ability to perform differentiated function. (Scan provided
by David J. Mooney, Ph.D.)
Athymic male mice (nu/nu) (MGH, Boston, Mass.) were purchased at 4 to 6 weeks of age and were acclimated for
no more than 3 weeks before use. Under general anesthesia (methoxyflurane, Pittmann and Moores, Mundelein, Ill.) and
employing sterile surgical technique, subcutaneous pockets were created on the dorsa of 15 athymic mice. Each animal
received one polymer construct according to its random group assignment.
The study was divided into three groups, i.e., two experimental groups (groups I and II) and one control (group III).
Group I (n = 5) consisted of animals with polymer matrices seeded with chondrocytes that were implanted
subcutaneously and then fixed by means of an external stent of the same size and shape on the outside skin (Fig. 2).
Group II (n = 5) consisted of animals with polymer matrices seeded with chondrocytes that were then implanted without
employing an external stent. Group III, the control group (n = 5), was composed of animals with polymer matrices not
seeded with chondrocytes but that were stented externally on implantation.
Fig. 2. An external stent was fixed on the outside skin of the polymer ear implant to maintain polymer mold shape.
Group I consisted of polymer matrices seeded with chondrocytes, implanted subcutaneously, and then fixed by means of
an external stent of the same size and shape. The control group was composed of polymer matrices not seeded with
chondrocytes but stented externally on implantation.
As stated earlier, five mice in each of the two experimental groups (groups I and II) each received implants seeded
with chondrocytes. Five mice (in the control group, group III) had implants not seeded with cells but stented externally.
Implants in groups I and III were stented externally (see Fig. 2). Mice in group II did not have external stents placed.
The animals were sacrificed by an overdose of inhalation anesthesia 12 weeks after implantation. Specimens were
dissected free of surrounding soft tissue and were fixed in 10% phosphate-buffered formalin (Fischer Scientific,
Pittsburgh, Pa.) for gross and histologic analysis.
Histologic Analysis
After fixation with 10% phosphate-buffered formalin for at least 24 hours, specimens were embedded within
paraffin and sectioned. Using standard histochemical techniques, serial sections were stained with hematoxylin and
eosin, fuschian aldehyde-Alcian blue at pH 1.7, or Masson's trichrome solution.
Immunohistochemistry was performed on 4-µm sections of formalin-fixed, paraffinembedded specimens using a

standard indirect three-step immunoperoxidase technique. The primary antibody was a murine monoclonal antibody
raised against human type II collagen that crossreacts with bovine type II collagen (Chemicon International; Temecula,
Calif.) and that demonstrates no crossreactivity with other collagen subtypes (I, III, V, and VI).
Results
No implants exhibited significant extrusion or infection. Control group specimens consisting of polyglycolic acid
constructs without chondrocytes demonstrated no evidence of cartilage formation after 6 weeks of in vivo incubation.
Once the stent was removed, the skin under it regained its original contour because no tissue had formed and the
polymer was simply absorbed. Experimental groups I and II differed markedly in the overall three-dimensional
appearance of the chondrocyte-polyglycolic acid implants after 12 weeks of in vivo incubation. Specimens from group I,
in which an external stent was applied for 4 weeks, exhibited a morphology nearly identical to that of the initial
implant and mimicked the complex architecture of the original human ear (see Fig. 4, A). In contrast, the specimens in
group II, which were not externally stented, demonstrated only rudimentary three-dimensional structure. These
specimens had decreased in size and became distorted in shape (see Fig. 4, B). The difference in shape was observed
after 2 to 3 weeks of implantation. The complex structure observed in the specimens of group I was well established by
4 weeks and was maintained for an additional 8 weeks after removal of the stent (Fig. 3). Removal of the skin envelope
from group I specimens after sacrifice demonstrated that the neocartilage framework was responsible for the overall
shape seen on the dorsum of the animal (Fig. 4).
Fig. 3. Gross appearance of construct 12 weeks after subcutaneous implantation into a nude mouse. Note the three-
dimensional shape that is almost identical to that of a human ear.
Fig. 4. Photograph comparing the gross appearance of specimens in group I (A) and group II (B). Notice the shrunken and
distorted shape of specimen B. Only a rudimentary three-dimensional structure was maintained. The specimen in group
I was harvested 12 weeks after implantation. Notice the fine contours of the new cartilaginous ear, which are very
similar to those of the original cell-polymer ear construct.
Specimens in both groups I and II manifested neocartilage formation on gross examination. Similarly, histologic
examination using a standard hematoxylin and eosin stain demonstrated cartilage formation in specimens of groups I
and II but not in the control group. Previous data from this laboratory have demonstrated that the chondrocytes survive
the implantation, as documented with BrdU labeling.8 We also demonstrated in another study that the average
internuclear interspaces increased, as evidenced by the lower number of nuclei per high-power field. This indicates
progressive deposition of matrix, suggesting the chondrocytes' viability and function. Fuschian aldehyde-Alcian blue
stains (pH 1.7) of tissue sections from experimental group I and II specimens exhibited purple staining consistent with
the presence of sulfated glycosaminoglycans such as chondroitin sulfate. Masson's trichrome stain demonstrated the
presence of collagen and suggested the presence of type II collagen, specific for cartilage. This finding was confirmed
using a monoclonal antibody raised against human collagen type II that crossreacts with bovine collagen type II.
Discussion
The tissue components of the human auricle are relatively simple, being skin and cartilage. The challenges in total
ear reconstruction are twofold: to have enough viable skin and cartilage available and to arrange these tissues such that
the rich three-dimensional structure of the ear is approximated.
Although skin coverage is a critical element of any ear reconstruction, the techniques reported in this paper are
designed to address the formation of a cartilage scaffold. In most patients, an autogenous rib cartilage framework is
feasible. However, in a subset of patients, the rib cartilage needed to perform the reconstruction either already has
Ovid: Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to Produce ... Page 10 of 12
been used or is of such poor quality as to be unusable.
The field of tissue engineering has developed to address the problem of donor-tissue scarcity either as whole organs
or as reconstructive grafts, such as cartilage frameworks for total ear reconstruction. Tissue-engineering techniques are
used to combine isolated parenchymal cells with biocompatible and biodegradable polymer scaffolds to produce a
cellpolymer implant. Earlier work with chondrocytes and polyglycolic acid fiber meshes has demonstrated that
chondrocyte-polyglycolic acid constructs can be fashioned and, further, that when they are implanted into an animal
host, neocartilage is generated.7 Studies have demonstrated that the final morphology of the neocartilage is
determined by the shape of the polymer template.8 The current studies demonstrate that the technology can be
applied on a clinically useful scale. Fig. 5
Fig. 5. Histology of chondrocyte-polymer ear constructs stained with hematoxylin and eosin (× 320). A section of tissue
from a 6-week specimen showing a region of mature cartilage with the cells organized in nodules surrounded by
basophilic ground substance and internodular strands.
An earlier report concerning the creation of an ear using tissue engineering was less than perfect.10 The
neocartilage ear lacked much of the rich three-dimensional structure that one associates with the human auricle. In this
current report, a 3-year-old child's ear was used as the model for creation of the polymer scaffold. The scaffold was
fabricated as a full-scale replica, seeded with chondrocytes in appropriate concentrations determined following the
original report, and then implanted. Although neocartilage was produced in all cases, only those implants which were
stented externally for 4 weeks precisely replicated the complex convolutions of the auricle. This finding suggests that
during the initial formation of the neocartilage, its structure is not rigid enough to resist the contraction forces of
healing. When the structure of the neocartilage matures, it ultimately is able to counteract the contracting forces of
the skin envelope. The evidence of this transformation is the maintenance of three-dimensional structure for up to 8
weeks after the external stent is removed. In this way, the mature neocartilage seems to function as the native
auricular cartilage.
These studies are preliminary and leave many avenues of investigation open. Future studies will investigate the
physical properties in a controlled manner using rigorous biomechanical testing protocols. The choice for using articular
chondrocytes versus auricular chondrocytes stems not only from our experience with the latter but also from the fact
that the auricular cartilage containing fibroblasts has the propensity for overgrowth of fibroblasts in tissue culture, with
the possibility of decreased cartilage production. In contrast, articular cartilage contains only one cell type, the
chondrocytes, and avoids this problem. Future studies should examine the use of other chondrocyte sources. The
framework that was created was of a clinically applicable size; however, a framework appropriate for implantation into
a patient would be shaped with accentuated prominences and depressions if a cutaneous or fasciocutaneous flap were
to be used for coverage, but it may be possible that the framework shape may not have to be modified to compensate
for skin coverage. A pseudoperichondrium has been observed on neocartilage specimens, the presence of which raises
the possibility of using a full-thickness skin graft to cover the constructs, which would produce a better aesthetic result,
being thinner and more conformable than a flap.
In summary, these studies demonstrate that a neocartilage implant of clinically applicable size can be fabricated
and made to produce new cartilage. Further, the early period of neocartilage formation in constructs of complex
architecture such as the human ear may require the application of a stent to resist the contraction forces of wound
healing; however, once formed, the neocartilage seems able to resist contraction forces and maintain its shape. The
studies only examined the early period of neocartilage formation in an earshaped construct. The question of long-term
results is open and under investigation. The current model is a xenograft model, and its extension to an autograft
system is reasonable and currently being developed.
Yilin Cao, M.D., Ph.D.
Laboratory for Tissue Engineering; Department of Anesthesia; University of Massachusetts Medical Center; 55 Lake
Avenue North; Worcester, Mass. 01655
REFERENCES
1. Tanzer, R. C. Total reconstruction of the external ear. Plast. Reconstr. Surg. 23: 1, 1959. SFX Bibliographic Links
Library Holdings [Context Link]
2. Brent, B. Auricular repair with autogenous rib cartilage grafts: Two decades of experience with 600 cases. Plast.
Reconstr. Surg. 90: 355, 1992. SFX Bibliographic Links Library Holdings [Context Link]
3. Paige, K. T., and Vacanti, C. A. Tissue-Engineered Cartilage and Bone. In C. Ricordi (Ed.), Methods in Cell
Transplantation. Austin, Texas: R. G. Landes, 1993.
4. Klagsbrun, M. Large-scale preparation of chondrocytes. Methods Enzymol. 58: 560, 1979. SFX Bibliographic Links
Library Holdings [Context Link]
5. Wilkes, G. H., and Wolfaardt, J. F. Osseointegrafted alloplastic versus autogenous ear reconstruction: Criteria for
treatment selection. Plast. Reconstr. Surg. 93: 967, 1994.
6. Wellisz, T. Reconstruction of the burned external ear using a Medpor porous polyethylene pivoting helix framework.
Plast. Reconstr. Surg. 91: 811, 1993. SFX Bibliographic Links Library Holdings
7. Vacanti, C. A., Langer, R., Schloo, B., and Vacanti, J. P. Synthetic biodegradable polymers seeded with chondrocytes
provide a template for new cartilage formation. Plast. Reconstr. Surg. 88: 753, 1991. [Context Link]
8. Kim, W. S., Vacanti, J. P., Cima, L., et al. Cartilage engineered in predetermined shapes employing cell
transplantation on synthetic biodegradable polymers. Plast. Reconstr. Surg. 94: 233, 1994. [Context Link]
9. Vacanti, J. P., Morse, M. A., Saltzman, W. M., and Domb, A. J. Selective cell transplantation using bioabsorbable
artificial polymers as matrices. J. Pediatr. Surg. 23: 3, 1988. SFX Bibliographic Links Library Holdings
10. Vacanti, C. A., Cima, L., Ratkowski, D., et al. Tissue engineering of new cartilage in the shape of a human ear
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Ovid: Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to Produce Tis...

Transplantation of Chondrocytes Utilizing a Polymer-Cell Construct to

Materials and Methods

Seeding and Implantation

Immunohistochemistry was performed on 4-µm sections of formalin-fixed, paraffinembedded specimens using a

been used or is of such poor quality as to be unusable.

Yilin Cao, M.D., Ph.D.

Copyright (c) 2000 -2008 Ovid Technologies, Inc.

Version: OvidSP_UI01.02.00, SourceID 36376

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