Journal of Surgical Research 163, 331–336 (2010)

doi:10.1016/j.jss.2010.03.070

ASSOCIATION FOR ACADEMIC SURGERY

Porous Poly(vinyl alcohol)-Alginate Gel Hybrid Construct for Neocartilage
Formation Using Human Nasoseptal Cells
David A. Bichara, M.D.,* Xing Zhao, M.D.,* Nathaniel S. Hwang, Ph.D.,† Hatice Bodugoz-Senturk, Ph.D.,‡
Michael J. Yaremchuk, M.D.,* Mark A. Randolph, M.A.S.,*,1 and Orhun K. Muratoglu, Ph.D.‡
*Plastic Surgery Research Laboratory, Division of Plastic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston,
Massachusetts; †Koch Institute for Innovative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts;
and ‡Harris Orthopaedic Biomechanics and Biomaterials Laboratory, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts

Submitted for publication January 8, 2010

Conclusion. A novel porous PVA-alginate gel hybrid

Background. Limited options exist for the restora-
tion of craniofacial cartilage. Autologous tissue or po-
rous polyethylene is currently used for nasal and
auricular reconstruction. Both options are associated
with drawbacks, including donor site morbidity and
implant extrusion. Poly(vinyl alcohol) (PVA) is a non-
degradable flexible biocompatible polymer than can
be engineered to mimic the properties of cartilage.
The goal of this study was to engineer a biosynthetic
hybrid construct using a combination of PVA-alginate
hydrogels and human nasal septum chondrocytes.
Materials and Methods. Chondrocytes isolated from
human nasal septum cartilage were expanded and
mixed with 2% sodium alginate hydrogel. The
chondrocyte-alginate mix was injected into a non-
degradable porous PVA hydrogel, creating biosyn-
thetic constructs. A group of these constructs were
implanted into the subcutaneous environment of
nude mice, while the other group was cultured in
a spinner flask bioreactor system for 10 d and then im-
planted. After 6 wk in vivo, the histologic, biochemical,
and biomechanical properties were examined.
Results. Histological analysis demonstrated sul-
fated glycosaminoglycans and deposition of collagen
type II in constructs from both groups. Constructs cul-
tured in the bioreactor system prior in vivo implanta-
tion demonstrated higher levels of DNA,
glycosaminoglycans, and hydroxyproline. An increase
of 22% in the compressive strength of the engineered
constructs exposed to the bioreactor was also ob-
served.
1
To whom correspondence and reprint requests should be ad-
dressed at Massachusetts General Hospital, Room WAC 435, 15 Park-
man Street, Boston, MA 02114. E-mail: marandolph@partners.org.
was used to successfully engineer human cartilage
in vivo. A 10-d period of bioreactor culturing increased
levels of DNA, glycosaminoglycans, hydroxyproline,
and the compressive modulus of the constructs. Ó 2010
Elsevier Inc. All rights reserved.
Key Words: poly(vinyl alcohol); hydrogel; cartilage;
nasoseptal chondrocytes; tissue engineering.
INTRODUCTION
Reconstruction and restoration of the cartilaginous
structures of the craniofacial region continues to be
a challenge for surgeons. The limited availability of bio-
compatible materials with reliable long-term shape re-
tention that are resilient to extrusion or infection has
led to the use of autologous tissue as a preferred source
for repair and restoration of craniofacial cartilage. In
the case of microtia, the gold standard for reconstruc-
tion relies on harvesting costal cartilage and sculpting
an ear-shaped construct [1]. In rhinoplasty procedures,
where a degree of augmentation is required, autologous
cartilage tissue can be utilized. Although biocompatibil-
ity and rejection are not a concern using autologous tis-
sue, harvesting procedures are associated with varying
degrees of donor site morbidities that can lead to long-
term complications. In other settings, limited or lack
of tissue availability in post-traumatic cases leave no
option but for the use of FDA-approved biomaterials.
High density porous polyethylene (HDPPE) has been
extensively used for auricle and nasal reconstruction
[2, 3]. Although satisfactory results from reconstructive
procedures using HDPPE can be achieved for nasal and
auricle reconstruction, this is an inert inflexible

331 0022-4804/$36.00
Ó 2010 Elsevier Inc. All rights reserved.
332 JOURNAL OF SURGICAL RESEARCH: VOL. 163, NO. 2, OCTOBER 2010
material available in predetermined sizes and shapes
and is predisposed to extrusion if implanted under
a poorly vascularized environment. Furthermore,
implants exposed to traumatic events will invariably
result in construct extrusion due to the limited
potential of the surrounding soft tissues to heal over
the implant. The development of a biomimetic and
flexible scaffold—particularly important in the
auricular and nasal anatomy—could potentially
improve the outcomes of surgical procedures and
decrease post-operative complications.
Poly(vinyl alcohol) (PVA) is a biocompatible synthetic
polymer that possesses great potential for the creation
of synthetic cartilage [4]. In the surgical field, PVA-
based polymers have been used for the prevention of
postsurgical tissue adhesions and embolization of the
uterine artery [5, 6]. Advantages of this material
include the versatile formulations that can be created,
including that of a porous hydrogel, thus creating an
optimal cell-scaffold environment setting for the fabri-
cation of tissue with high water content similar to car-
tilage. Furthermore, PVA can be synthesized to be
flexible and molded into any size or shape, simulating
the mechanics of craniofacial cartilage and allowing
for implant customization (Fig. 1).
The goals of this study were to (1) engineer a biosyn-
thetic construct using a combination of alginate hydro-
gel, a porous non-degradable PVA gel and human nasal
septum chondrocytes, and (2) to quantify and compare
the histologic, biochemical, and biomechanical composi-
tions of engineered constructs cultured under different
conditions.
MATERIALS AND METHODS
PVA Hydrogel Preparation
The synthesis of PVA hydrogel has been previously published [7].
Briefly, hydrogel was prepared by theta gelation by dissolving PVA
(115,000 g/mol) and polyacrylamide-co-acrylic acid (PAAm-co-AAc)
and poly(ethylene glycol) (PEG) in deionized (DI) water at 90 C.
The resulting solution was then molded into sheets measuring
2.1mm in thickness and cooled down to room temperature for gelation
for 24 h. The gel was immersed in DI water for equilibrium and sub-
jected to e-beam sterilization.
Cartilage Harvest, Chondrocyte Isolation, and Expansion
All actions were approved by the Institutional Review Board and
the Institutional Animal Care and Use Committee of the Massachu-
setts General Hospital and followed all of the policies outlined in
the NIH Guide for the Care and Use of Laboratory Animals. Nasal
septum cartilage from a 34-y-old female patient was collected from
the operating room. Cartilage was digested and chondrocytes were
isolated as previously described [8]. Briefly, cartilage was washed
with phosphate buffered saline (PBS) twice and minced into 1 mm3
pieces using razor blades. The minced tissue was placed into 50 mL
polypropylene tubes and 40 mL of HAM’S F-12 solution containing
0.1% collagenase type II was added. The tubes were placed on a rocker
FIG. 1. Porous PVA human shaped auricle. Unlike commercially
available polyethylene implants, the engineered PVA hydrogel is
highly flexible and its porosity allows for seeding of chondrocytes
throughout the construct. (Color version of figure is available online.)
inside an incubator at 37  C for 16 h. The contents were then filtered
through a 100 mm filter and washed twice with PBS. The isolated
chondrocytes were divided into 1.5 3 106 aliquots and placed in 150
cm3 cell culture flasks with HAM’S F-12 media with L-glutamine sup-
plemented with 10% fetal bovine serum, 50 U/mL penicillin, 50 mg/mL
of streptomycin, 50 mg/mL ascorbic acid and 0.1mM nonessential
amino acids mix. Ninety percent confluence was achieved. After
a 14 d expansion period, the non-passaged cells were enzymatically
detached using 0.05% trypsin EDTA and used for the construct prep-
aration.
PVA Constructs Preparation, Spinner Flask Assembly, and
Construct Implantation
For the construct preparation, chondrocytes were mixed with a 2%
sodium alginate (PRONOVA UP LVG) (NovaMatrix, Sandvika, Nor-
way) sterile solution and 40 mL of the alginate-chondrocyte mix was
injected into twelve porous PVA constructs using a 25-gauge needle,
resulting in a final cell concentration of 60 3 106 per mL. Gelation
was achieved by immersion into CaCl2 (100 mg/mL) for 5 s. The final
construct dimensions (n ¼ 12) were 6 mm diameter and 2.1 mm in
thickness. From the 12 constructs, six (group not exposed to a bioreac-
tor system) were washed twice in phosphate buffered saline (PBS) and
implanted into the dorsum of nude mice for 6 wk.
The six remaining constructs were placed in a spinner flask bioreac-
tor system. For the spinner flask assembly, the spinner flask, needle,
silicone stopper, and magnetic stirrer were autoclaved and assembled
as previously described [9]. Three constructs were threaded onto each
13 cm long stylet of a 22-gauge spinal needle. A total of two stylets, each
with three constructs, were then secured to the silicone stopper. A 4 cm
long stir bar was inserted into the spinner flask and 120 mL of cell me-
dia containing the previously described contents was added. The flask
was placed on a magnetic stir plate at 50 rpm at 37  C and 5% CO2 in-
cubator. The side caps of the flask were loosened to permit gas ex-
change and cell media was changed every third d. After a 10
d in vitro period the constructs were implanted into the dorsum of
nude mice. After a 6 wk period in vivo, constructs from both groups
were explanted and grossly examined, subjected to histologic, immu-
nohistochemical, biochemical, and biomechanical analysis.
Cell-alginate solution was also used to create nodules (n ¼ 6). For
the nodule creation, 100 mL of CaCl2 was placed in the bottom of
 BICHARA ET AL.: HYBRID CONSTRUCT FOR NEOCARTILAGE FORMATION 333

a sterile Eppendorf tube. Then, 400 mL of the cell-alginate solution Statistical Analysis
was slowly placed on top using a pipette, and another 100 mL of
CaCl2 was placed on top of the cell-alginate mix, creating a CaCl2- Biochemical quantifications are expressed as mean 6 SD. Sta-
cell/alginate-CaCl2 three-layered structure. After 30 s, the constructs tistical significance was determined by ANOVA single factor with
were removed from the CaCl2 layers, washed twice in PBS, and im- P < 0.05.
planted into the dorsum of nude mice for 6 wk. Upon explantation,
the constructs were grossly examined and subjected to histological
and immunohistochemical analysis. RESULTS
For the construct implantation, female nude (nu/nu) mice were ob-
tained at 6 wk of age and allowed to acclimate for 1 wk. Under aseptic Gross Examination
conditions, intraperitoneal anesthesia with tribromoethanol (400 mg/
kg) was achieved, and a 1.5 cm midline incision was made on the dor- Explanted PVA constructs were indistinguishable
sum of each mouse. Two subcutaneous pockets were created through between groups. As expected with a non-degradable hy-
blunt dissection using Stevens tenotomy scissors. A total of two con-
structs was implanted in each mouse. The incision was closed using
drogel, the original dimensions were retained in 12/12
stainless steel Autoclip staples that were removed after 10 d. explants. There were no signs of extrusion, inflamma-
tion, or foreign-body reaction. A fibrous capsule sur-
Histologic and Immunohistochemical Analysis rounded the constructs and was easily removed.
Subsequent dissection allowed for construct palpation.
Briefly, constructs were fixed in 10% phosphate-buffered formalin
Compared with PVA hydrogel alone, described as
overnight, embedded in paraffin, and serial 5 mm sections were ob-
tained. Slides were deparaffinized and stained with safranin O and to- a sponge-like material, the biosynthetic constructs
luidine blue. For the immunohistochemical analysis, deparaffinized were viscoelastic when digitally compressed, similar
sections were treated with 2% bovine testicular hyaluronidase at to auricular cartilage. Implanted cell-alginate nodules
room temperature for 30 min. A blocking reagent consisting of 0.3%
hydrogen peroxide in methanol was added for 30 min. Then, 10%
were explanted as a solid white glossy irregularly
goat serum was added to each slide for 30 min. On separate slides, shaped mass resembling cartilage tissue (Fig. 2). These
the primary antibodies for collagen type I (COL I, Abcam, Cambridge, nodules served as a positive control for neocartilage for-
MA, USA) and II (COL II, Chondrex, Inc., Redmond, WA, USA) were mation.
applied at room temperature for one hour. Both antibodies were di-
luted 1:1000 in 1% bovine serum albumin in PBS. For negative con-
trol, N-Universal Negative Control was applied. The secondary Histologic and Immunohistochemical Analysis
antibody (horseradish peroxidase-labeled polymer) was added to the
slides for 20 min at room temperature. Then 3,3-diaminobenzidine Light microscopy revealed the presence of the non-
was applied to each slide for color development, and cell nuclei were degradable PVA gel infiltrated with neocartilage tissue.
counterstained with hematoxylin.
The main structures identified on the PVA gel were hol-
low spheres and open channels. The alginate-cell mix
Biochemical Evaluations
was injected into the channels that are now occupied
The quantification of deoxyribonucleic acid (DNA) and glycosami- by chondrocytes and extracellular matrix. There was
noglycans was performed using the methodology previously described an absence of an inflammatory infiltrate and the tissue
[10, 11]. The pieces were weighed before and after the 24 h was in close contact with the PVA gel. In all six con-
lyophilization period (n ¼ 3 for each group). The dehydrated
specimens were digested by adding 1 mL solution with 100 mM structs that were cultured in the spinner flask prior to
NaPO4, 10 mM Na ethylenediaminetetraacetic acid/disodium salt/ implantation, intense safranin O and toluidine blue
dihydrate, 10 mM cysteine hydrochloride, 10 mM stains demonstrated abundant glycosaminoglycan de-
ethylenediaminetetraacetic acid, and 125 mg/mL of papain. The
position (Fig. 3A and B). Staining for COL II was also
specimens were incubated in a water bath at 60  C for 16 h. For the
glycosaminoglycan quantification, 30 mL of papain digest were present (Fig. 3C). In the group not exposed to
added to 200 mL of 1,9-dimethylmethylene blue dye and measured
with a spectrophotometer at 525 nm. The DNA contents were
quantified by fluorescence at 360/465 nm after being mixed with
bisbenzimidazole dye. The hydroxyproline content of insoluble
collagen was determined according to Stegemann and Stalder and
normalized to the dry weight [12]. A portion of papain-digested sam-
ple was acid hydrolyzed, neutralized, and reacted with prepare oxidiz-
ing solution of chloramines-T and Ehrlich’s solution. All samples and
standards were analyzed in duplicate. DNA, glycosaminoglycan, and
hydroxyproline contents were expressed as mg/mg of dry weight.

Compression Strain

Compression strain was performed at the same day of explantation.

Mechanical characterization of the biosynthetic constructs was per-
formed using an Instron Universal Testing Apparatus (Norwood,
MA, USA) (n ¼ 3 for each group). Compressive stress and strain
were calculated and plotted as a stress-strain diagram for both
PVA-alginate-cell construct groups and for the PVA hydrogel alone.
FIG. 2. Explanted alginate-chondrocyte nodule. Mass grossly re-
sembled cartilage tissue after a 6-wk in vivo period. Note the irregu-
larly formed shaped due in part by the gelatation kinetics of
alginate hydrogel. (Color version of figure is available online.)
334 JOURNAL OF SURGICAL RESEARCH: VOL. 163, NO. 2, OCTOBER 2010

FIG. 3. Histologic and immunohistochemical analysis. Top three images: two structures are visualized including open channels infiltrated
with neocartilage tissue and non-collapsible hollow spheres. Bottom three images: neocartilage formation from the alginate-chondrocyte nod-
ules. Stains: A, D ¼ safranin O; B, E ¼ toluidine blue; C, F: collagen type II. (Color version of figure is available online.)

a bioreactor, glycosaminoglycan deposition was also
identified by a less intense stain. For the cartilage nod-
ules, safranin O and toluidine blue stains revealed gly-
cosaminoglycan deposition, identical to the tissue
described before that was housed by the PVA hydrogel.
Staining for COL II was abundant and revealed a simi-
lar intensity compared with the tissue throughout the
PVA (Fig. 3D, E, and F).
(12.31 6 2.03 mg/mg versus 10.67 6 2.28 mg/mg), and hy-
droxyproline (20.67 6 4.5 mg/mg versus 15.49 6 2.3 mg/
mg). The increased levels in the group exposed to the
bioreactor, expressed as a percent, were the following:
22% more DNA, 15% more glycosaminoglycans, and
33% more hydroxyproline. Hydroxyproline, an indica-
tion of total collagen content in constructs, was found
to be statistically significant (P < 0.05).

Biochemical Analysis Compression Strain

All biochemical quantifications normalized to the dry
weight of the engineered tissues can be visualized in
Fig. 4. The biochemical composition of the constructs
that were implanted in vivo immediately after assem-
bly was compared with those cultured in a bioreactor
prior implantation. The constructs exposed to a bioreac-
tor demonstrated increased levels of DNA (5.63 6 0.71
mg/mg versus 4.616 0.43 mg/mg), glycosaminoglycans
The synthesized PVA hydrogel had a compressive
equilibrium modulus of 25 kPa. Constructs exposed to
a bioreactor demonstrated a 22% higher compressive
modulus compared with those that were implanted
immediately after assembly (P < 0.05) (Fig. 5). Addition
of the cell-alginate component to the PVA hydrogel sig-
nificantly increased the compressive modulus after the
in vivo period.

FIG. 4. DNA, glycosaminoglycan and hydroxyproline quantification between groups: Control ¼ PVA gel only (acellular). In vivo ¼ group
implanted without bioreactor exposure. Bioreactor þ in vivo ¼ group with a 10 d bioreactor culture period prior implantation. Data presented
as mean þ SEM.
 BICHARA ET AL.: HYBRID CONSTRUCT FOR NEOCARTILAGE FORMATION
FIG. 5. Stress-strain curves. Control ¼ porous PVA hydrogel. In vivo ¼ group implanted without bioreactor exposure. Bioreactor þ in vivo ¼
group with a 10 d bioreactor culture period prior implantation. (Color version of figure is available online.)
DISCUSSION
PVA is a versatile material that has been extensively
researched for engineering synthetic articular carti-
lage. Our group has developed flexible PVA hydrogels
formulations with the potential for replacing defective
cartilage with a synthetic cartilage-like material
[7, 13]. However, the subcutaneous use of these inert
acellular gels could pose similar complications
experienced with HDPPE, including extrusion if
placed under a poorly vascularized environment, and
the limited capacity of tissues to heal over the implant
after direct trauma. In this preliminary study, we
decided to combine a natural and a novel porous, non-
degradable synthetic hydrogel—engineering a biosyn-
thetic construct—to evaluate the neocartilage-forming
potential of human chondrocytes derived from the nasal
septum. Furthermore, we evaluated if a 10 d period in
a bioreactor would allow ECM deposition and in turn
improve the histologic, immunohistochemical, biochem-
ical, and biomechanical properties of the constructs.
These data demonstrate that cartilage can be engi-
neered in a subcutaneous environment using a human
tissue and a porous PVA-alginate hybrid gel. Due to
lower costs and easier access, a vast majority of pub-
lished studies regarding engineering of cartilage utilize
young healthy animal chondrocytes rather than human
tissue. Research reproducibility and utilizing a cell
source that is consistent in both specie and age are ad-
vantages of using animal versus human chondrocytes.
However, studies using human cell lines are equally
or more important for the field of tissue engineering.
In this study, we have used a limited amount of human
tissue to engineer cartilage under different conditions.
In the constructs exposed to a bioreactor, the shear
force created inside the spinner flask acted against
the constructs prior in vivo implantation, and increased
the amounts of DNA and glycosaminoglycans, as well
as the compressive properties. Although subjective,
335
the intensity of the safranin O, toluidine blue, and
COL II stains correlate with the biochemical and biome-
chanical data presented. Unaware of the nutrient diffu-
sion properties throughout the PVA gel, the engineered
cartilage nodules served as a positive control for neocar-
tilage formation, achieving results similar to native
tissue.
Numerous investigators have researched alginate
hydrogel, a biocompatible gel derived from seaweed
that has been previously used to engineer cartilage. Do-
bratz et al. injected human nasal chondrocytes sus-
pended in 2% alginate gel in a subcutaneous
immunodeficient environment for up to 38 wk [14].
The authors wanted to achieve a shape-specific con-
struct by using an external mold that would shape the
cell-alginate-CaCl2 mix as it was being injected. Al-
though neocartilage formation was achieved, the ex-
plants were irregularly shaped and did not retain
their original dimensions over time. Previously, Chang
et al. also used alginate gel to suspend chondrocytes and
engineer auricular cartilage in an ovine model using
autologous cells [15]. In their study, the cell-alginate-
CaSO4 mix was injected into a mold shaped like
a nose bridge and chin implant. The constructs were im-
planted for 30 wk and surprisingly maintained their
original dimensions at the time of explantation. How-
ever, it is possible that the alginate gel has not fully de-
graded contributing to the retention in size and shape.
Described by Kuo and Ma, the gelation kinetics of algi-
nate are difficult to control, largely due to the fast cross-
linking that occurs when in contact with calcium
cations [16]. This results in the inability to form intri-
cate shapes that require a defined structure upon im-
plantation. For this reason, our group decided to
synthesize a non-degradable porous PVA gel with a pre-
defined shape that could subsequently be infiltrated
with alginate gel and chondrocytes.
In tissue engineering approaches where an intricate
formed shape is desired, such as a human shaped
336 JOURNAL OF SURGICAL RESEARCH: VOL. 163, NO. 2, OCTOBER 2010
auricle, the use of a non-degradable PVA hydrogel pos-
sesses numerous advantages over other degradable hy-
drogels or polymers. Previously utilized biomaterials
used to engineer human shaped auricles range from fi-
brin gel [17, 18] to polylactic acid/polyglycolic acid [19]
to poly-l-lactide/poly-3-caprolactone copolymer blends
[20, 21]. Although the degradation rate of these
materials can be controlled and neocartilage
engineering can be achieved, shape retention,
specifically of an intricately formed shape under an
environment with overlying tensile forces, is unlikely.
The use of a non-degradable material with adequate
mechanical properties would be more adequate in this
scenario. Our group has extensive experience using hy-
drogels, including collagen [22], fibrin [8], poly(ethylene
glycol) [23], and hyaluronic acid [24]. However, this is
our first experience using alginate in combination
with a non-degradable hydrogel. To our knowledge,
this is the first report describing the use of a flexible po-
rous biocompatible PVA hydrogel in combination with
a natural alginate hydrogel and chondrocytes derived
from human nasal septum.
Unaware of the effects of shear force acting against
and throughout the PVA gel, a set of constructs were
placed in a simple bioreactor system. If future clinical
applications include placing the hybrid gel under an
area of tension, such as scarred or scarring tissue, im-
planting a construct with ECM-producing chondrocytes
can potentially be beneficial over time, preventing con-
struct deformation from the overlying contractile forces
by the skin. In this study, compression stress testing
varied among groups. Biosynthetic constructs exposed
to shear force exhibited a 22% higher (P < 0.05) com-
pressive modulus compared with the unexposed group
that was implanted immediately after assembly.
In conclusion, successful engineering of cartilage has
been demonstrated using a novel flexible PVA-alginate
hybrid gel scaffold using a limited amount of human tis-
sue. The engineered neocartilage histologically resem-
bles native tissue, and exposing constructs to shear
force for 10 d in a spinner flask increases the DNA, gly-
cosaminoglycans, and hydroxyproline contents, as well
as the mechanical integrity.
ACKNOWLEDGMENTS
This study was supported in part by the American Society of Max-
illofacial Surgeons Research Grant and the Department of Orthopae-
dic Surgery Academic Enrichment Fund of the Massachusetts
General Hospital.
REFERENCES
1. Tollefson TT. Advances in the treatment of microtia. Curr Opin-
ion Otolaryngol Head Neck Surg 2006;14:412.
2. Romo T, Reitzen SD. Aesthetic microtia reconstruction with
Medpor. Facial Plast Surg FPS 2008;24:120.
3. Peled ZM, Warren AG, Johnston P, et al. The use of alloplastic
materials in rhinoplasty surgery: A meta-analysis. Plast Recon-
struct Surg 2008;121:85e.
4. Oka M, Ushio K, Kumar P, et al. Development of artificial artic-
ular cartilage. Proceedings of the Institution of Mechanical En-
gineers. Part H. J Eng Med 2000;214:59.
5. Metwally M, Cheong Y, Li TC. A review of techniques for adhe-
sion prevention after gynecological surgery. Curr Opinion
Obstet Gynecol 2008;20:345.
6. Spies JB, Allison S, Flick P, et al. Polyvinyl alcohol particles and
tris-acryl gelatin microspheres for uterine artery embolization
for leiomyomas: Results of a randomized comparative study. J
Vasc Intervent Radiol 2004;15:793.
7. Bodugoz-Senturk H, Macias CE, Kung JH, et al. Poly (vinyl alco-
hol) acrylamide hydrogels as load-bearing cartilage substitute.
Biomaterials 2009;30:589.
8. Mesa JM, Zaporojan V, Weinand C, et al. Tissue engineering car-
tilage with aged articular chondrocytes in vivo. Plast Recon-
struct Surg 2006;118:41. discussion 50.
9. Vunjak-Novakovic G, Obradovic B, Martin I, et al. Dynamic cell
seeding of polymer scaffolds for cartilage tissue engineering. Bi-
otechnol Prog 1998;14:193.
10. Kim YJ, Sah RL, Doong JY, et al. Fluorometric assay of DNA in
cartilage explants using Hoechst 33258. Anal Biochem 1988;
174:168.
11. Enobakhare BO, Bader DL, Lee DA. Quantification of sulfated
glycosaminoglycans in chondrocyte/alginate cultures, by use of
1,9-dimethylmethylene blue. Anal Biochem 1996;243:189.
12. Stegemann H, Stalder K. Determination of hydroxyproline.
Clinica Chimica Acta 1967;18:267.
13. Bodugoz-Senturk H, Choi J, Oral E, et al. The effect of polyeth-
ylene glycol on the stability of pores in polyvinyl alcohol hydro-
gels during annealing. Biomaterials 2008;29:141.
14. Dobratz EJ, Kim SW, Voglewede A, et al. Injectable cartilage:
Using alginate and human chondrocytes. Arch Facial Plast
Surg 1995;11:40.
15. Chang SC, Tobias G, Roy AK, et al. Tissue engineering of autol-
ogous cartilage for craniofacial reconstruction by injection mold-
ing. Plast Reconstr Surg 2003;112:793. discussion 800.
16. Kuo CK, Ma PX. Ionically crosslinked alginate hydrogels as scaf-
folds for tissue engineering: Part 1. Structure, gelation rate and
mechanical properties. Biomaterials 2001;22:511.
17. Ting V, Sims CD, Brecht LE, et al. In vitro prefabrication of hu-
man cartilage shapes using fibrin glue and human chondrocytes.
Ann Plast Surg 1998;40:413. discussion 420.
18. Xu J, Johnson TS, Motarjem PM, et al. Tissue-engineered
flexible ear-shaped cartilage. Plast Reconstr Surg 2005;
115:1633.
19. Liu Y, Zhang L, Zhou G, et al. In vitro engineering of human ear-
shaped cartilage assisted with CAD/CAM technology. Biomate-
rials 2010;31:2176.
20. Isogai N, Morotomi T, Hayakawa S, et al. Combined
chondrocyte-copolymer implantation with slow release of basic
fibroblast growth factor for tissue engineering an auricular car-
tilage construct. J Biomed Mat Res Part A 2005;74:408.
21. Kusuhara H, Isogai N, Enjo M, et al. Tissue engineering a model
for the human ear: Assessment of size, shape, morphology, and
gene expression following seeding of different chondrocytes.
Wound Repair Regeneration 2008;17:136.
22. Ibusuki S, Papadopoulos A, Ranka M, et al. Engineering carti-
lage in a photochemically crosslinked collagen gel. J Knee
Surg 2009;22:72.
23. Elisseeff J, Anseth K, Sims D, et al. Transdermal photopolyme-
rization of poly(ethylene oxide)-based injectable hydrogels for
tissue-engineered cartilage. Plast Reconstr Surg 1999;
104:1014.
24. Chung C, Mesa J, Miller GJ, et al. Effects of auricular chondro-
cyte expansion on neocartilage formation in photocrosslinked
hyaluronic acid networks. Tissue Eng 2006;12:2665.