ARTICLE IN PRESS

Biomaterials 25 (2004) 1649–1655

A comparative study of three different biomaterials in the engineering

of skeletal muscle using a rat animal model
F.S. Kamelgera,b,*, R. Marksteinerc, E. Margreiterc, G. Klimad, G. Wechselbergera,b,
S. Heringe, H. Pizaa,b
a
Department of Plastic and Reconstructive Surgery, University Hospital of Innsbruck, Anichstrasse 35, Innsbruck 6020, Austria
b
Ludwig-Boltzmann-Institute for Quality Control in Plastic and Reconstructive Surgery, Anichstrasse 35, Innsbruck 6020, Austria
c
Institute of Biochemical Pharmacology, University of Innsbruck, Innsbruck, Austria
d
Institute of Anatomy, Histology and Embryology, University of Innsbruck, Innsbruck, Austria
e
Institute of Pharmacology and Toxicology, University of Vienna, Austria

Abstract

Defects caused by traumatic or postsurgical loss of muscle mass may result in severe impairments of the functionality of skeletal
muscle. Tissue engineering represents a possible approach to replace the lost or defective muscle. The aim of this study was to
compare the suitability of three different biomaterials as scaffolds for rat myoblasts, using a new animal model. PKH26-fluorescent-
stained cultured rat myoblasts were either seeded onto polyglycolic acid meshes or, alternatively, suspended in alginate or in
hyaluronic acid-hydrogels. In each of the eight Fisher CDF-344 rats, four capsule pouches were induced by subcutaneous
implantation of four silicone sheets. After two weeks the silicone sheets were removed and myoblast-biomaterial-constructs were
implanted in the preformed capsules. Specimens were harvested after four weeks and examined histologically by H&E-staining and
fluorescence microscopy. All capsules were well-vascularized. Implanted myoblasts fused by forming multinucleated myotubes. This
study demonstrates that myoblasts seeded onto different biomaterials can be successfully transplanted into preformed highly
vascularized capsule pouches. Our animal model has paved the way for studies of myoblast-biomaterial transplantations into an
ectopic non-muscular environment.
r 2003 Elsevier Ltd. All rights reserved.

Keywords: Animal model; Muscle; Tissue engineering; Polyglycolic acid; Alginate; Hyaluronic acid

1. Introduction and metabolic disturbances are also responsible for the

Tissue loss and organ failure remain challenging
problems in health care. Loss of voluntary power of
skeletal muscle may result from interruption of the
innervation or, alternatively, may be due to deficiency or
loss of muscle mass. Muscle loss or deficiency may be
primary in congenital anomalies such as Prune-belly
syndrome and high forms of anal atresia, or secondary
after radical resection of malignant tumors, after trauma
or surgical interventions. Intrinsic diseases such as
muscular dystrophies (Duchenne type or Steinert’s
disease), periodic diseases (Myasthenia gravis), develop-
mental defects (hypoplasia or aplasia) and endocrine
*Corresponding author. Department of Plastic and Reconstructive
Surgery, University Hospital of Innsbruck, Anichstrasse 35, Innsbruck
6020, Austria. Tel.: +43-512-504-2731; fax: +43-512-504-2735.
E-mail address: florian.kamelger@uibk.ac.at (F.S. Kamelger).
functional loss of skeletal muscle [1]. The engineering of
three-dimensional skeletal muscle is a new therapeutic
strategy for treatment of diseases of the musculoskeletal
system.
To date myoblast transplantations have been pre-
dominantly performed by injection of myoblast cell
suspensions into mature skeletal muscle. These single
cells have been shown to fuse with the host myofibers
[2]. Saxena et al. were the first to implant successfully
in vitro cultured myoblasts into a non-muscular
environment. Their group used a polyglycolic acid
(PGA) mesh as a scaffold for skeletal muscle cells [1,3].
Different scaffold materials have been used in cell
reimplantation. These materials should fulfill several
preconditions, such as: (i) high levels of biocompatibility
and biodegradability, (ii) low degree of cytotoxicity
and (iii) high affinity to biological surfaces. Further-
more, biodegradable biomaterials should provide

0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0142-9612(03)00520-9
 ARTICLE IN PRESS
1650 F.S. Kamelger et al. / Biomaterials 25 (2004) 1649–1655
(iv) mechanical stability to the construct and may
additionally serve as a (v) temporary guide for three-
dimensional tissue regeneration [4–6].
The aim of our study was to design a new animal
model for skeletal muscle tissue engineering applications
and to compare PGA polymers, alginate and hyaluronic
acid hydrogels as delivery vehicles for the implantation
of skeletal muscle cells.
Cultured myoblasts were transplanted into a prefab-
ricated capsule created by a silicone material. Previous
in vivo studies have demonstrated successful transplan-
tations of adipocytes [7], urothelial [8–10] and tracheal
[11] cells to induced capsular tissue. Here we have tested
the suitability of a subcutaneous capsule pouch for
engineering skeletal muscle tissue in an ectopic and
vascularized environment. We hypothesized that with
this approach, migration of cells into surrounding
tissues can be prevented and viability of the implanted
constructs improved.
In this study we managed to successfully develop a
new animal model using capsule induction technique,
which provides a well-vascularized environment. Our
objective was to engineer skeletal muscle outside the
physiological muscle tissue.
2. Material and methods
2.1. Animals
Three 5-day-old Fisher rats CDF-344 (Charles River
Laboratories, Sulzfeld, Germany) served as donors of
myoblasts. Eight adult Fisher CDF-344 rats served as
recipients of implantation. All animals were maintained
and used in conformity with Austrian national regula-
tions and international guidelines of the Council of
Europe of animal welfare.
2.2. Myoblast harvest and isolation
Skeletal muscle tissue was obtained from the latissi-
mus dorsi and the rectus femoris muscles of three
newborn animals and placed in a Petri dish containing
5 ml of plating media (20% fetal calf serum and 5 mg/l
gentamicin (Sigma, Austria) in a solution of DMEM
Dulbecco’s Modified Eagle Medium (Gibco, Austria)).
Connective tissue was removed mechanically and muscle
tissue was minced with small scissors into 1 mm3 pieces
and washed five times in PBS (80 g NaCl, Na2H-
PO4  12H2O, 2 g KCl and 2 g KHPO4 in 1000 ml
distilled water). Skeletal muscle pieces were added
thereafter to 2 ml prewarmed STD (‘Solid Tissue
Digestion’)-solution (Biomedica, Austria) and incubated
at 37 C and 5% CO2 overnight. Cells were mechanically
minced for five minutes, washed with PBS and
centrifuged at 218 rcf (g) for 9 minutes. The cell
suspension was then transferred to two falcon tubes
and centrifuged for a second time. Cells were subse-
quently added to 5 ml of plating media and incubated in
25 cm3 tissue culture flasks (Sarstedt, USA) at 37 C and
5% CO2.
In order to obtain pure myoblast cultures, single cells
were manually collected under microscopic control by
means of a glass micropipette (tip diameter 30 mm)
making use of a Celltram Vario (Eppendorf, Germany).
Single myoblasts were subsequently transferred to 96-
well dishes (Sarstedt, USA) containing 100 ml of plating
media. Desmin antibody staining (Sigma, Austria)
confirmed the purity of the single cell deduced myoblast
cultures.
2.3. Labeling of myoblasts
Prior to seeding the cells onto the different scaffold
materials, the myoblasts were labeled with PKH26
fluorescent staining (Sigma, Austria) according to the
manufacturer’s instructions. In brief: Cells were washed
with PBS and centrifuged and 250 ml of Diluent C
(Sigma) were subsequently added to the cell suspension.
15 ml of the PKH dye were then added to 250 ml of
Diluent C. After a 6 min incubation period at room
temperature fresh culture medium was added to stop
further dye incorporation. The cell suspension was
washed and centrifuged three times.
2.4. Formation of a subcutaneous capsule
Eight adult fisher CDF-344 rats were administered
sevoflurane inhalation anesthesia. Four incisions made
at the backside of each animal were used to enter the
subcutis. Four silicone sheets of 1  1 cm2 with a
thickness of 0.5 mm were then placed subcutaneously
in each rat to stimulate the formation of subcutaneous
and vascularized capsules. Fig. 1 illustrates the positions
of the silicone sheets.
2.5. Tissue constructs
Three different biomaterials were prepared and
seeded with PKH26-labeled myoblasts:
(1) Non-woven PGA fiber mesh sheets of 1  1 cm2 and
a fiber diameter of approximately 12 mm, specific
gravity of 80.2 mg cm 3, and a porosity of 97%
(Albany International Research Group, Mansfield,
USA) were seeded with myoblasts at a density of 4–
6 million cells/polymer (about 10 million cells/ml,
respectively).
(2) Calcium-alginate-solution (Pronova, Norway) was
brought to gelation with a CaSO4-GDL-system as
described by Kuo and Ma [12] and myoblasts were
added at a density of about 10 million/ml.
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F.S. Kamelger et al. / Biomaterials 25 (2004) 1649–1655
Fig. 1. Position of subcutaneous capsules in rat backsides. Capsule 1
contained the PGA-myoblast-construct, capsule 2 the PGA-alginate-
myoblast-construct, capsule 3 the myoblasts suspended in hyaluronic
acid and capsule 4 the negative controls.
(3) Hyaluronic acid (CollagenAesthetics, Austria) was
mixed with about 10 million myoblasts/ml.
Myoblasts seeded onto the three different biomater-
ials were allowed to attach for 24 h before implantation.
2.6. Implantation
Two weeks after implantation of the silicone sheets,
the rats were anesthetized and the backs of the eight
recipient animals shaved and prepared with betadine.
The four silicone sheets were located and the corre-
sponding capsules were opened for removal of the
implants.
In each animal, the biomaterial-myoblast-constructs
were transplanted by a uniform procedure: In capsule 1,
the PGA-myoblast construct was implanted. In capsule
2, a PGA-mesh was introduced and 0.8 ml of alginate-
myoblast solution was injected. In capsule 3, 0.8 ml of
the hyaluronic acid-myoblast-construct was injected,
and in capsule 4, one of the three biomaterials served as
negative control (Fig. 1). All capsules were sutured with
a monocryl 4/0 and the skin was closed with prolene 3/0
sutures.
2.7. Construct harvest and histological examination
Four weeks after implantation, the recipients were
sacrificed and the constructs harvested. The specimens
were fixed in 4% paraformaldehyde and stored at
70 C. Frozen sections were either stained with H&E
to define general morphology, or used for fluorescence
1651
microscopy to demonstrate the existence of the
implanted myoblasts. Additionally, sections were
labeled with DAPI nuclear staining (Sigma, Austria)
to show the presence of several nuclei in fused
myoblasts.
3. Results
3.1. Capsules
The foreign body reaction against the implanted
silicone sheets resulted in macroscopically well-defined
capsules with sleek walls and new grown blood vessels
(Fig. 2a). Histological analysis showed the capsules to
be embedded within the surrounding tissue. Magnifica-
tion of the capsule-wall revealed a well-developed
vascularization between the inner and the outer layers
of the margin (Fig. 2b).
3.2. Histological analysis of implants
Histological examination under the fluorescence
microscope showed the implanted cells to be located
exclusively within the capsule limits. In other words, no
myoblast migration into the surrounding tissues was
detected (Fig. 2c). The fusion of the PKH26-labeled
implanted myoblasts could be shown with the DAPI
nuclear staining. Figs. 3b and 5b show DAPI-labeled
multinucleated myotubes.
3.2.1. Polyglycolic acid
H&E in PGA-myoblast-constructs demonstrated the
presence of multinucleated myotubes (Fig. 3a). These
structures were revealed by the detection of DAPI-
stained PKH26-labeled cells under the fluorescence
microscope (Fig. 3b).
3.2.2. Alginate
PGA-alginate-myoblast-constructs showed a central
region containing residues of the PGA mesh, alginate
hydrogel and cell-detritus. Myoblasts had fused into
myotubes and showed a concentric growth near the
capsule wall. H&E routine showed multinucleated
myotubes with vascularization in their vicinity (Fig.
4a). Fluorescence microscopy revealed the correspond-
ing structures to consist of the implanted and PKH26-
stained myoblasts (Fig. 4b).
3.2.3. Hyaluronic acid
In hyaluronic acid-myoblast-constructs honeycombed
structures were surrounded by myotubes (Fig. 2c). These
cells were identified as implanted myoblasts by means of
fluorescence microscopy. DAPI-staining shows forma-
tion of multinucleated myotubes (Fig. 5b). The presence
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(a) (b)

(c)
Fig. 2. Capsule induction technique leads to the formation of vascularized capsule pouches with the neoformation of supplying vessels (arrows (a))
(X=capsule pouch; bar=5 mm). Histologically, the capsule wall appears to be highly vascularized. Formation of a capillary bed was observed
between the inner and the outer layers of the wall (stars in (b)) (arrowheads highlight the inner layer of the capsule wall; bar=75 mm). Fluorescence
microscopy of a hyaluronic-myoblast-construct shows the PKH26-labeled cells to be located exclusively within the capsule walls (arrowheads in
Fig. 2c; bar=2 mm). Honeycomb structures defined by PKH26-stained fluorescent fused muscle cells.

(a) (b) (c)

Fig. 3. (a) H&E of PGA-construct. Black arrowhead highlights a multinucleated (stars marking nuclei) myoblast along a partially degraded polymer
fiber (P), bar=100 mm. (b) Overlay of PKH26- and DAPI-staining. Fluorescence microscopy of a PGA-construct. Arrowheads show the two
multinucleated myoblasts. Nuclei are labeled by DAPI blue, red PKH26-staining suggests fusion of myoblasts, bar=100 mm. (c) Fluorescence of
PGA-construct. White arrows indicating the PKH26-marked implanted myoblasts fused myotubes, bar=200 mm.

of fibrovascular tissue indicates a well-developed blood myotubes was observed in all biomaterials tested. In
supply to the construct (Fig. 5a). constructs containing PGA fibers, a light form of
Vascularization was found in all specimens and the fibrovascular growth without, however, any signs of
tendency of the myoblasts to fuse into multinucleated inflammation could be detected.
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F.S. Kamelger et al. / Biomaterials 25 (2004) 1649–1655 1653

(a) (b)
Fig. 4. (a) H&E of PGA-alginate-construct. Arrowheads show the capsule wall. Arrows (black) indicate the myotubes. C=capillary, bar=200 mm.
(b) Fluorescence of PGA-alginate-construct (corresponding slide). Arrowheads highlight the capsule wall. Arrows indicating the myotubes with
typical fluorescent flecked appearance of the PKH26-staining, bar=200 mm.

(a) (b)

Fig. 5. (a) H&E of hyaluronic acid-constructs. Arrows show the fused muscle cells which are situated at the edge of honeycomb structures.
C=capillary, bar=100 mm. (b) Overlay of DAPI- and PKH26-staining. Fluorescence microscopy of a hyaluronic acid-construct. Arrowheads show a
well-defined multinucleated myotube with DAPI-stained cell nuclei, bar=100 mm.

3.2.4. Controls enabling muscle regeneration [13]. Enzymatically iso-

The control implants did not contain structures
similar to those in the constructs described above.
Neither single myoblasts nor fluorescent spots were
detected. In some specimens we observed degraded PGA
fibers or honeycomb structures that did not, however,
contain myoblasts.
4. Discussion
Mature skeletal muscle cells are multinucleated
syncytia arising from the fusion of mononucleated
precursors. Some of the precursors (so-called satellite
cells) persist in mature muscle. Upon muscle injury they
start proliferating and fuse to adjacent myofibers,
lated and in vitro cultured myoblasts provide an almost
unlimited source for muscle reconstruction. Experimen-
tal myoblast transplantation has been performed for the
treatment of various pathophysiologically very different
diseases such as: (i) neurodegenerative diseases; (ii)
Duchenne’s muscular dystrophy. Besides, this technique
may also be suitable for treating coagulation disorders
or hormone deficiencies via gene transfer using myo-
blasts as a shuttle vehicle [14–17].
Biomaterials provide mechanical stability to the
construct in the short term and serve as a template for
the three-dimensional organization for the developing
tissue. The effective repair and regeneration of injured
tissues and organs depend on early reestablishment of
the blood flow needed for metabolic support. An
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1654 F.S. Kamelger et al. / Biomaterials 25 (2004) 1649–1655
implantable biomaterial should be three-dimensional
and highly porous for cell growth and flow transport of
nutrients and metabolic waste. It should be biodegrad-
able and have a suitable surface chemistry for cell
attachment and proliferation. Furthermore, it should be
capable of being easily processed to form a variety of
shapes and sizes [5].
The biomaterials tested in this study have been used in
various previous investigations. PGA meshes served as
scaffolds in the engineering of cartilage [18,19], bone
[20], skin [21] and skeletal muscle [1,3]. Alginate
hydrogels were used as delivery vehicles of engineered
cartilage [18,22] and in the treatment of vesicoureteral
reflux [23]. Hyaluronic acid, a naturally occurring
glycosaminoglycan derived from rooster combs, was
used as a biomaterial in the engineering of autologous
fibroblasts and keratinocytes [24] and in the transplan-
tation of chondrocytes [25].
Our study clearly demonstrates that myoblasts seeded
onto different biomaterials can be successfully trans-
planted into a preformed capsule. PKH26-staining has
been established as an effective identification technique
for implanted cells [26–29]. As expected, the implanted
myoblasts mixed with the different scaffold materials
(PGA, alginate- and hyaluronic acid hydrogels) re-
mained within the preformed capsules and did not
migrate into the surrounding tissue. The formation of
multinucleated myotubes demonstrates the suitability of
PGA and alginate- and hyaluronic acid hydrogels and
suggests that this technique may be suitable for future
skeletal muscle engineering applications. Cell growth
and fusion within the capsule limits suggests that this
technique might be suitable for filling volume defects in
ectopic locations outside skeletal muscles.
In summary, our results show that a macroscopically
and microscopically well-vascularized compartment can
be induced in which biomaterial-myoblast-constructs
can be successfully implanted. We found, particularly in
specimens containing the polymer scaffold, a fibrovas-
cular growth seemingly reflecting also a reaction against
the biomaterial. Our data suggest that the technique of
preforming vascularized capsules might represent a
helpful tool for achieving adequate and rapid sprouting
of new vessels that provide an optimal environment for
the biomaterial-myoblast-constructs.
There remain additional issues regarding the engi-
neering of skeletal muscle that future research must
address such as motor innervation and orientation of
skeletal muscle fibers.
Acknowledgements
This study was supported by the Ludwig Boltzmann
Institute for Quality Control in Plastic and Reconstruc-
tive Surgery Innsbruck, Austria and grants P12828 and
P15527 of the FWF Austrian Science Fund. The authors
are indebted to Ms. Karin Gutleben for technical help
and to Ms. Rajam Csordas for critical reading of the
manuscript and editorial assistance.
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