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Hybrid muscular tissues: Preparation of skeletal muscle cell-incorporated collagen gels
Cell Transplantation, Vol. 6, No. 2, 109-l 18, 1997 Copyright 0 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0963-6897/97 $17.00 + .OO
PI1 SO963-6897(96)00255-2
ELSEVIER
Original Contribution
HYBRID MUSCULAR TISSUES: PREPARATION OF SKELETAL CELL-INCORPORATED COLLAGEN GELS
MUSCLE
TAKAHISA OKANO*? AND TAKEHISA MATSUDA’* *Department of Bioengineering, National Cardiovascular Center Research Institute, Osaka, Japan, and tSecond Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
0 Abstract-We prepared three different types of hybrid muscular tissues in which C2C12 cells (skeletal muscle myoblast cell line) were incorporated in type I collagen gels and then differentiated to myotubes upon culture: a disctype, a polyester mesh-reinforced sheet-type, and a tubular type. A cold mixed solution of the cells and type I collagen was poured into three different types of molds and was kept at 37°C in an incubator to form C2C12 cell-incorporated gels. A polyester mesh was incorporated into a gel to form the sheet-type tissue. The tubular hybrid tissue was prepared by pouring a mixed solution into the interstitial space of a tubular mold consisting of an outer sheath and a mandrel and subsequently culturing after removal of the outer sheath. Hybrid tissues were incubated in a growth medium (20% fetal bovine serum medium) for the first 4 days and then in a differentiation medium (2% horse serum medium) to induce formation of myotubes. Transparent fragile gels shrank with time to form opaque gels, irrespective of type, resulting in the formation of quite dense hybrid tissues. On day 14 of incubation, myoblasts fused and differentiated to form multinucleated myotubes. For a tubular type hybrid tissue, both cells and collagen fiber bundles became circumferentially oriented with incubation time. Periodic mechanical stress loading to a mesh-reinforced hybrid tissue accelerated the cellular orientation along the axis of the stretch. The potential applications for use as living tissue substitutes in damaged and diseased skeletal and cardiac muscle and as vascular grafts are discussed. 0 1997 Elsevier Science Inc. 0 Keywords - Hybrid muscular gel; Stress loading.
tissue; C2C12;
area of medical technology. Living substitutes under development include skin (I), vessels (8,9,15,19,2224,32), corneas (26), and ligaments (11). The essential feature of the key technology common to the hybrid tissues listed above is to immobilize respective viable cells in three-dimensional extracellular matrices such as collagen gels. However, living tissue substitutes for skeletal and cardiac muscle have not yet been developed. Skeletal muscle cells, which have a highly developed intracellular contractile structure, arc present in living tissues in two distinctly different forms: myoblasts (undifferentiated, satellite cells), and muscle fibers (differentiated and multinucleated cells formed through myoblast fusion). The muscle fibers are densely accumulated and highly oriented in one direction. When skeletal muscle is injured, myoblasts proliferate and differentiate to form myotubes and muscle fibers for repair of muscular tissues (3). On the other hand, cardiac muscle cells (cardiomyocytes), which are highly differentiated, do not have the ability to regenerate following injury (29). The regeneration of damaged cardiac muscles has been one of the ultimate goals of cardiovascular medicine. The utilization of electrically responsive contractibility of regenerated skeletal muscle has been considered a choice for replacement or support of cardiac function. In fact, cardiomyoplasty, using skeletal muscle to support ventricular function, has recently been developed (4,5). On the other hand, if a hybrid muscular tissue composed of highly dense and oriented muscle fibers is prepared, such a tissue may contract upon pulsed electrical stimulation, resulting in generation of unidirectional force. Therefore, these hybrid muscular tissues may be useful as transplantation vehicles to repair damaged skel-
Collagen
INTRODUCTION
Tissue engineering to replace diseased tissues with living substitutes has been attracting significant interest in the ACCEPTED
Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-l Fujishirodai, Suita, Osaka 565, Japan.
9110196.
‘Correspondence should be addressed to Takehisa Matsuda, 109
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eta1 and cardiac muscle or as pulsatile-flow-generative vascular prostheses. In the present study, we developed a prototype fabrication technique for hybrid muscular tissues composed of skeletal muscle cells and collagen. Three different types of hybrid muscular tissue were prepared: a disctype, a polyester mesh-reinforced sheet-type, and a tubular type (Fig. 1). Periodic stretching accelerated cellular alignment in the resultant tissues. MATERIALS AND METHODS
Polyester mesh-reinforced sheet-type hybrid tissue. A polyester mesh-reinforced sheet-type hybrid tissue (mesh generously supplied by Teijin Corp., Tokyo, Japan) was prepared as follows. A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into a 24-well plate. The polyester mesh was placed on the gel formed upon incubation at 37°C for 30 min. A similar mixture was then poured over the meshplaced gel in the mold. Similar to the case of the culture of the disc-type tissue, the gel was cultured in DMEM. The medium change from FBS-DMEM to HS-DMEM was performed on day 4 of incubation.
Cell Culture
C2C12 cells (a mouse skeletal muscle myoblast cell line; American Type Culture Collection, Rockville, MD) were used as skeletal muscle cells. The culture mediums used were FBS-DMEM [Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories Inc., Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; CSL Ltd., Victoria, Australia)] as a growth medium and HS-DMEM [DMEM supplemented with 2% horse serum (HS; Gibco Laboratories Inc.)] as a differentiation medium. Cell culture was performed on tissue culture dishes at 37°C in a humidified atmosphere of 95% air and 5% CO1 in an incubator.
Tubular type hybrid tissue. A tubular type hybrid tissue was prepared as follows. A tubular mold, which consisted of an external and an internal glass vessel coaxially fixed at one end with silicone tubes, was prepared. The mold had the following dimensions: inner diameter, 1.5 mm; outer diameter, 7 mm; and length, 7 cm. A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into the space between the sheath and the mandrel of the mold. After subsequent incubation at 37°C for 30 min, the sheath was removed. The resultant gel was cultured in DMEM. The medium change from FBS-DMEM to HS-DMEM was performed on day 4 of incubation. On day 14 of incubation, the mandrel was removed from the tissue.
Fabrication Disc-type hybrid tissue. A disc-type hybrid tissue was prepared as follows. One volume of C2C12 cells suspended in DMEM and one volume of acid-solubilized bovine dermal type I collagen solution (5 mg/mL, CELLGEN, Kohken Corp., Tokyo, Japan) were mixed at 4°C. The cold mixed solution (cell density, 1.0 x lo6 cells/ mL; collagen concentration, 2.5 mg/mL) was poured into a 24-well plate (MICROPLATE; well diameter, 16 mm, Iwaki Glass, Tokyo, Japan) and incubated at 37°C for 30 min. The cell-incorporated collagenous gel was cultured in DMEM. The medium change from FBS-DMEM to HS-DMEM was performed on day 4 of incubation.
Fig. 1. Schema of three different types of hybrid muscular tissues composed of skeletal muscle cells and collagen. (A) Disc-type hybrid tissue. (B) Polyester mesh-reinforced sheettype hybrid tissue. (C) Tubular type hybrid tissue.
Formation of mesh-reinforced hybrid tissue for stress loading. A polyester mesh-reinforced sheet-type tissue for stress loading was prepared as follows. The two shorter sides of a rectangular polyester mesh (1 x 2 cm) were fixed to glass tubes (2 mm in outer diameter). Each glass tube was tied to a loop of silk cord. The polyester mesh thus prepared was placed on a handcrafted rectangular mold (1 x 2.5 cm). A cold mixture of C2C12 cell suspension and type I collagen solution was poured into the mold and incubated at 37°C for 30 min. The meshreinforced hybrid tissue thus prepared was cultured in FBS-DMEM for up to 7 days prior to stress loading. Stress Chamber. The stress chamber was a modified version of the device described in our previous paper (12). A commercially available plastic flask (CORNING, 25 cm2 tissue culture flask) was handcrafted to install the mesh-reinforced hybrid tissue with glass tubes as follows. One loop of silk cord through a glass tube was used to fix the tissues toward the end of the stress chamber. The silk cord from the outer loop was passed through a hole in the cap of the chamber. Thus, two tissues were installed in the chamber. Prior to culture, the stress chambers were sterilized with EOG (ethylene oxide gas). Stress loading. The stretching apparatus used was a custom-designed one that was previously prepared
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(12,13). Briefly, a DC motor drove an eccentric steering disc at 60 RPM, the rotations of which were transferred to a piston rod as directional movement. A stress arm connected to the piston rod periodically moved the glass rod back and forth, which caused periodic stress relaxation of the dynamically stressed hybrid tissues with 5% amplitude of the relaxed tissue length. The periodic movement measured by a position transducer set around the piston rod exhibited a sinelike waveform. The tissues were subjected to cyclic stretching (frequency; 60 RPM, amplitude; 5%) for up to 7 days in HS-DMEM in a stress chamber maintained at 37°C with an atmosphere of humidified 95% air and 5% CO,. A nonstressed tissue, as a control tissue, was floated in HS-DMEM for up to 7 days.
Morphological Evaluation Morphological changes of C2C12 cells cultured on tissue culture dishes and in hybrid tissues were observed with a phase-contrast microscope (DIAPHOT, Nikon, Tokyo, Japan), a light microscope (LM; VANOX-S, Olympus, Tokyo, Japan), and a scanning electron microscope (SEM; S-4000, Hitachi, Tokyo, Japan). For LM study, monolayer tissues on tissue culture dishes were fixed with ethanol for 15 min, and were stained with Papanicolaou stain or were immunohistochemically stained using a muscle-desmin-specific monoclonal antibody (D33; Dako Corp., Glostrup, Denmark). Hybrid
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tissues were fixed with 10% formalin for 1 h, dehydrated with a graded series of ethanol, and embedded in paraffin. For SEM study, specimens were fixed in a fixative (2% glutaraldehyde in a 0.1 M cacodylate buffer, pH 7.4) postfixed with 1% osmium tetroxide in buffer for 15 min, washed with buffer five times, stained with 1% tannic acid for 30 min, and washed with distilled water five times. Subsequently, they were critical point dried, sputter-coated with platinum, and studied under the SEM. Dimensional Change During Hybrid Tissue Formation The diameter of the disc-type hybrid tissue and the length of the tubular type hybrid tissue were macroscopitally measured. The wall thickness of the tubular type hybrid tissue was obtained as the average of the thicknesses measured at four arbitrary points on a micrograph of a transverse section. RESULTS
Differentiation of Myoblasts on Culture Dish C2C 12 is an established cell line from skeletal muscle cells. It has been reported that myoblasts fuse to form myotubes when they are cultured on a dish at high cell density and under very low nutrient conditions, which we demonstrated in the present study as follows. On day 1 of incubation, mononucleated myoblasts spread on the dish exhibited a polygonal shape and random orientation (Fig.
Fig. 2. Micrographs of C2C12 cells on a tissue culture dish. (A, C) On day 1 of incubation and (B, D) on day 11 of incubation, multinucleated cells (myotubes) were observed. Upper: phase-contrast micrographs. Lower: light micrographs stained with Papanicolaou stain.
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2A and C). Upon further culturing, the myoblasts proliferated to form a dense monolayer in the growth medium. On day 4 of incubation the culture medium was substituted with the differentiation medium. Upon further culturing, the myoblasts fused to form multinucleated myotubes. On day 11 of incubation the elongated and spindle-shaped myotubes became dense with local orientation (Fig. 2B and D). Immunohistochemical staining using the muscle-desmin-specific monoclonal antibody revealed the presence of a small quantity of desmin in the myoblasts on day 1 of incubation, but desmin content in the myotubes markedly increased upon long-term culture (11 days) (Fig. 3).
Fabrication of Disc-Type Hybrid Tissue The cold mixture of C2C 12 cells suspended in DMEM and type I collagen solution was poured into a 24-well plate and incubated at 37°C. A C2C12 cell-incorporated gel, which was transparent and fragile when prepared, shrank to form an opaque and round tissue with incubation time. Phase-contrast micrographs (Fig. 4) and scanning electron micrographs (Fig. 5) showed that myoblasts were homogeneously distributed and collagen fiber bundles were randomly oriented in the gel on day 1 of incubation (Figs. 4A and C and 5A). When the growth medium was substituted with the differentiation medium on day 4 of incubation, elongated myoblasts began to fuse, producing multinucleated myotubes. On day 14 of incubation, myotubes and collagen fiber bundles were densely accumulated. Both tended to be randomly oriented in the central region of the tissue and circumferentially oriented at the peripheral region (Figs. 4B and D and 5B). Figure 6 shows time-dependent changes in the diameter of the fabricated disc-type hybrid tissue (relative ratio to initial diameter; 16 mm). The C2C12 cellincorporated gel shrank rapidly for the first 4 days and slowly for the next 10 days of incubation. On day 14 of incubation, the diameter was approximately one-half of the initial diameter (relative ratio; 0.46 + 0.06).
Fabrication of Polyester Mesh-Reinforced Sheet-Type Hybrid Tissue A C2C 12 cell-incorporated gel reinforced with a polyester mesh was comprised of three layers: the polyester mesh was sandwiched between two cell-incorporated collagen gel layers. Similar to the disc-type hybrid tissue, the gel was transparent and fragile immediately following gelation. The gel became opaque with incubation time, but it shrank much less than the disc-type gel did (gel diameter decreased by approximately 10% in contrast with the 46% for the disc-type gel). Scanning electron micrographs showed that polygonal myoblasts were distributed throughout the network of the mesh and collagen fiber bundles were sparsely and randomly oriented on day 1 of incubation (Fig. 7A). When the growth medium was substituted with the differentiation medium on day 4 of incubation, elongated myoblasts began to fuse, producing multinucleated myotubes. On day 12 of incubation, elongated, spindle-shaped myotubes were densely distributed throughout the network of the mesh (Fig. 7B). Figure 7C and D shows phase-contrast micrographs of mesh-reinforced hybrid tissues on day 12 of incubation.
Fabrication of Tubular Type Hybrid Tissue The cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into a cylindrical mold with a mandrel (Fig. 8A). Subsequent incubation for 30 min produced a cylindrical, transparent gel (Fig. 8B). Upon culture, time-dependent shrinkage of the gel occurred and an opaque tissue was formed. On day 4 of incubation, the growth medium was substituted with the differentiation medium. On day 14 of incubation, a considerably shrunk tubular tissue was formed. The resultant tissue became tough. The length and wall thickness of the resultant tissue were approximately one-half of the original length and one-fifth of the original wall thickness, respectively (Fig. SC). Myoblasts were uniformly distributed throughout the gel and collagen fiber bundles were not yet well organized on day 1 of incubation (Fig.
Fig. 3. Micrographs of C2C12 cells on a tissue culture dish stained by antidestnin antibody. (A) On day 1 of incubation, desmin in myoblasts were hardly observed. (B) On day 11 of incubation, a large quantity of desmin in myotubes were observed. Arrow: desmins stained by antidesmin
antibody.
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Fig. 4. Micrographs of a disc-type hybrid tissue. (A, C) On day 1 of incubation, myoblasts were homogeneously distributed in a gel and collagen fiber bundles were randomly oriented. (B, D) On day 14 of incubation, elongated myotubes and collagen fiber bundles tended to become dense and to be randomly oriented in the central region of the tissue and circumferentially oriented at the peripheral region. Upper: phase-contrast micrographs. Lower: light micrographs stained with Masson-trichrome stain (horizontal section).
9A and C). On day 4 of incubation, proliferated myoblasts were induced to fuse upon substitution of the medium. On day 14 of incubation, elongated, spindle-shaped myotubes and collagen fiber bundles became dense and were circumferentially oriented (Fig. 9B and D).
spindle-shaped myotubes and very densely accumulated collagen fiber bundles were oriented parallel to the direction of the stretch (Fig. 10).
Orientation and Morphological Response to Stress Loading A mesh-reinforced sheet-type hybrid tissue, which had been incubated for 7 days in FBS-DMEM, was subjected to periodical stretching and recoiling (5% above and below the resting tissue length at 60 RPM frequency) for 7 days in HS-DMEM. In dynamically stressed tissues, compared with nonstressed tissues, both elongated
Skeletal muscles are unique tissues that are composed of bundles of highly oriented and highly dense muscle fibers, each of which is a multinucleated cell derived from myoblasts. The muscle fibers in native skeletal muscle are closely packed together in an extracellular matrix composed mainly of collagen to form an organized tissue with high cell density and cellular orientation. These uniaxially structured cellular assemblages are
DISCUSSION
Fig. 5. Scanning electron micrographs of a disc-type hybrid tissue. (A) On day 1 of incubation, polygonal myoblasts were distributed throughout the network of collagen fibers. (B) On day 14 of incubation, both elongated cylindrical myotubes and collagen fiber bundles tended to be oriented.
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Fig. 6. Time-dependent change in the diameter of disc-type hybrid tissue. (Relative to initial diameter when prepared.) The disc-type hybrid tissue shrank markedly for first 4 days, followed by reduced shrinking rate. On day 14 of incubation, the diameter of the resultant tissue was approximately one-half of the diameter of the initial tissue.
essential of skeletal muscle tissues. A large vector force is generated due to contraction of highly dense and highly oriented muscle fibers when skeletal muscle is subjected to an appropriate stimulus. Muscle fibers can-
not regenerate (29) but during repair of injured muscles, myoblasts (satellite cells) scattered in skeletal muscles (20), which remain in a quiescent and undifferentiated state, can enter the mitotic cycle which induces to proliferate and to fuse to each other to form multinucleated and elongated myotubes, which self-assemble to form a more organized structure, namely a muscle fiber (3). Although there is a similarity in terms of contractibility between skeletal muscle and cardiac muscle, in contrast to skeletal muscle cells, cardiac muscle cells (cardiomyocytes) are incapable of regeneration due to the absence of satellite cells in cardiac muscle (21). Therefore, it has been expected that myoblasts transfer to diseased cardiac muscles may contribute to the restoration of cardiac function. Muscular tissue engineering has been focusing on repair of damaged myocardial tissues upon intramural or transventricular injection of skeletal myoblasts (6,16,18,28). When satellite cells were implanted into injured myocardium, they survived and differentiated to form cardiac-like muscle cells, thus repairing damaged heart muscle in an appropriate environment (‘‘milieu-dependent’ ’ differentiation) (6). The implantation of genetically engineered myoblasts has been utilized as a potential therapy for genetic muscle diseases such as Duchenne muscular dystrophy (2,7,17,27). These findings and stimuli-responsive functional capabilities of skeletal muscle cells could have important clinical implications such as myoblast transfer
Fig. 7. Morphological characteristics of polyester mesh-reinforced sheet-type tissue. (A, B) Scanning electron micrographs. (A) On day 1 of incubation, myoblasts and collagen fibers were sparsely distributed in the mesh. (B) On day 12 of incubation, elongated myotubes and collagen fiber bundles became dense. (C, D) Phase-contrast micrographs on day 12 of incubation. (C) Elongated myotubes were distributed throughout the network of polyester mesh (visualized as black bold lines). (D) At higher density of myotubes was present close to the surface of the tissue than deeper into the tissue. P: polyester mesh. Mb: myoblast. Mt: myotube.
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Fig. 8. Fabrication of a tubular type hybrid tissue. (A) A mold was assembled from an outer and an inner glass tube (Inner diameter, 1.5 mm; outer diameter, 7 mm; length, 7 cm). (B) A transparent and opaque gel formed by 30-min incubation at 37°C after a mixed solution of collagen and C2C12 cells was poured into the interstitial space of the mold. (C) A tubular type hybrid tissue on day 14 of incubation and removal of glass rod.
The length and wall thickness of the resultant tissue were approximately one-half of the original length and one-fifth of the original wall thickness, respectively. or transplantation. On the other hand, studies on hybrid muscular tissues have only recently began. When C2C 12 myoblasts were injected into a fibrous, supple collagen disc, seeded myoblasts exhibited reasonable cellular distribution and myotube formation in the differentiation medium, which was studied to develop autologous seeded/cultured patches for abdominal wall reconstruction (31). Very recently, an attempt to prepare a myoblast-incorporated polyurethane sponge was also reported (25). Our working principle for the preparation of a functional, vital hybrid muscular tissue is that when myoblasts in collagen gels that are cultured at high cell density in the growth medium are induced to fuse to each other in the differentiation medium, multinucleated muscle fiber-incorporated collagen gels are formed, and subsequent continuous external mechanical stress loading may generate reinforced cellular alignment. This may produce a hybrid muscular tissue biomimicking natural ones. When such a well-organized hybrid tissue is subjected to pulsed electrical stimulation, it may contract or
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relax in response to the electrical stimulation. A disctype or a sheet-type hybrid tissue may be useful as a transplantation vehicle to repair damaged and diseased skeletal and cardiac muscle, while a tubular type hybrid tissue may be useful as a vascular prosthesis. The currently available technique of using a collagenase treatment method for harvesting satellite cells from living muscles did not give us a 100% pure preparation of satellite cells, i.e., one not contaminated with cells of other types such as fibroblasts. Also in our experience, more than two or three passages of a culture of harvested satellite cells derived from newborn rats eventually resulted in alteration from a myoblast-rich tissue to a fibroblast-rich tissue. Therefore, a more reliable harvesting method is needed for a 100% pure preparation of satellite cells. In this study, we used C2C 12, which is an established cell line of satellite cells from skeletal muscle of C3H mouse. As reported previously (33), these cells rapidly proliferate to form a monolayer tissue on a tissue culture dish in the growth medium at containing a high concentration of FBS, and subsequently differentiate to form multinucleated myotubes in the differentiation medium at containing a low concentration of HS (Fig. 2). An increased intracellular content of desmin was observed upon differentiation, which was verified by immunohistochemical staining of muscle-desmin-specific monoclonal antibody. A basic feature of the hybrid skeletal muscle tissues which we designed was C2C12 cell-incorporated collagen gel. The preparation procedure was very similar to those used for living tissue substitutes for skin (l), vessels (8,9,15,19,22-24,32), corneas (26), and ligaments (11). We prepared three different types of hybrid muscular tissues as prototype hybrid muscular tissues (Fig. 1): a disc-type, a tubular type and a polyester meshreinforced sheet-type. The former two were free of a synthetic scaffold, and the latter was structurally reinforced with a polyester mesh. C2C12 cell-incorporated collagen gels were prepared by thermal gelation of a cell-mixed collagenous solution in respective vessels upon incubation at 37°C. Irrespective of tissue type, C2C12 cells differentiated with time to form myotubes in the collagen gels in the differentiation medium. Transparent and fragile gels when prepared shrank time dependently to form opaque and dense tissues. C2C12 cells and collagen fiber bundles in the hybrid muscular tissues tended to become denser and to be highly oriented with time. Such a self-organized tissue formation process is driven by contraction of cells interacting with collagen fibers. The rapid reduction of the size of the hybrid tissues incorporated with mesenchymal cells has been generally observed. This phenomena is common to mesenchymal cells such as fibroblast and smooth muscle cell (1,13,14,30). However, little study on immobilization of
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Fig. 9. Micrographs of a tubular type hybrid tissue. (A, C) On day 1 of incubation, myoblasts were uniformly distributed throughout the tissue and collagen fiber bundles were not yet well organized. Top shows luminal surface. (B, D) On day 14 of incubation, elongated, spindle-shaped myotubes and collagen fiber bundles tended to become dense and to be circumferentially oriented. Upper: light micrographs stained with Masson-trichrome stain (cross-section). Lower: scanning electron micrographs. Arrow: circumferential direction. Mb_ myoblast. Mt: myotube. myoblasts
into
dependent
dimensional
A polyester designed
collagen
or other change
mesh-reinforced
to improve
matrix
and
its time-
have not been reported. sheet-type
the mechanical
hybrid
strength
tissue,
of the hy-
brid tissue, shrank markedly less than the mesh-free disctype tissue. Polyester mesh significantly improved mechanical strength but produced a minimal degree of gel shrinkage. Some difference of the state of C2C12 in the mesh-reinforced and mesh-free tissue was observed. Myotubes in mesh reinforced-tissue were more stretched than in the mesh-free disc-type tissue. In the tubular type hybrid tissue, circumferential orientation of cells and collagen fibers proceeded with time particularly at the luminal surface, similar to observations in our previous study of SMC-incorporated hybrid vascular medial tissue (8,9,13,14). If contraction and relaxation of the tubular type hybrid tissue occur upon pulsed electrical stimulation, this may cause narrowing and enlargement of the tube, resulting in the generation of pulsatile flow. To accelerate the orientation of skeletal muscle cells, the mesh-reinforced hybrid tissue was subjected to periodic stress loading. The C2C12 cells in the dynamically stressed tissue became elongated and spindle-shaped, collagen fiber bundles became densely accumulated with time, and both were aligned to the direction of the stretch in contrast to those in the nonstressed tissue (Fig. 10). This is in good agreement with our previous finding (13,14) in which accelerated accumulation and orienta-
tion of SMCs in collagenous tissues occurred with time under dynamic stress loading. The periodic stretching of the hybrid tissues extended the collagen fiber bundles in the direction of the stretch, which increased the orientation of the collagen fibers, resulting in more prominent elongation and orientation of skeletal muscle cells in the direction by the contact guidance mechanism, as was previously proposed (14). Although myoblasts differentiated to form myotubes in the three-dimensional collagen gels, cellular density and orientation of cells and collagen fibers in the hybrid muscular tissues were still much lower than those of native skeletal muscle. In addition, individual muscle fibers in a native tissue are surrounded by rich capillary beds, via which oxygen and nutritive elements are supplied to maintain metabolism. Therefore, a much higher density of oriented muscle fibers as well as incorporation of capillary networks (or microvascularization) into a hybrid tissue is essential for a transplantation vehicle for repair of damaged and diseased muscular tissues. To this end, the following factors should be taken into account when preparing of hybrid muscular tissue: 1) high cell density and high degree of orientation of myotubes and 2) angiogenesis. Use of an optimal design incorporating biomechanical and tissue engineering principles may yield a transplantation vehicle for repair of damaged and diseased skeletal and cardiac muscle and a muscularpowered vascular prosthesis for tubular type tissues.
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t
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IOOpm
stress
Fig. 10. Morphological characteristics of periodically stretched mesh-reinforced sheet-type hybrid tissues. (A-C): Masson-trichrome staining of cross-section parallel to the direction of the stretch. (A) On day 1 of incubation (prior to stress loading), myoblasts were homogeneously distributed throughout the tissue. (B) On day 14 of incubation in the nonstressed tissue, spindle-shaped myotubes were distributed in the tissues. (C) On day 14 of incubation (7 days after stress loading) in the dynamically stressed tissues, elongated and spindle-shaped myotubes were closely accumulated and oriented in the direction of the stretch. (D-F): Scanning electron micrographs of hybrid tissue surfaces. (D) On day 1 of incubation, collagen fibers were coarse and randomly oriented. (E) On day 14 of incubation, in the nonstressed tissues, collagen fibers remained coarse and randomly oriented. (F) On day 14 of incubation, in the dynamically stressed tissues, densely collagen fibers were oriented parallel to the direction of the stretch.
Such
a study
reported
is now underway,
and the results
will be
in the near future. 7.
Acknowledgments - The authors thank Dr. Shigeko Takaichi of the Department of Etiology and Pathophysiology of the National Cardiovascular Center, who kindly instructed us in electron microscopic techniques. The first author (T.O.) is grateful for the continuous encouragement of Professor Takahiro Oka of the Second Department of Surgery, Kyoto Prefectural University of Medicine, from which T.O. was on leave.
8.
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15. Kanda, K.; Oka, T.; Matsuda, T. Ultrasmall diameter vascular prosthesis composed of highly oriented dense collagen fiber bundles induced by the traction force of embedded arterial smooth muscle cells. Jpn. J. Artif. Organs 23: 585-589; 1994. 16. Koh, G.Y.; Klug, M.G.; Soonpaa, M.H.; Field, L.J. Differentiation and long-term survival of C2Cl2 myoblast grafts in heart. J. Clin. Invest. 92:1548-1554; 1993. 17. Law, P.K.; Goodwin, T.G.; Fang, Q.; Deering, M.B.; Duggirala, V.; Larkin, C.; Florendo, J.A.; Kirby, D.S.; Li, H.J.; Chen, M.; Cornet& J.; Li, L.M.; Shirzad, A.; Quinley, T.; Yoo, T.J.; Holcomb, R. Cell transplantation as an experimental treatment for Duchenne muscular dystrophy. Cell Transplant. 2:485-505; 1993. 18. Marelli, D.; Desrosiers, C.; El-Alfy, M.; Kao, R.L.; Chiu, R.C.-J. Cell transplantation for myocardial repair: An experimental approach. Cell Transplant. 1:383-390; 1992. 19. Matsuda, T.; Miwa, H. A hybrid vascular model biomimicking the hierarchic structure of arterial wall: Neointimal stability and neoarterial regeneration process under arterial circulation. J. Thorac. Cardiovasc. Surg. 110:988-997; 1995. 20. Mauro, A. Satellite cell of skeletal muscle libres. J. Biophys. Biochem. Cytol. 9:493-495; 1961. 21. Midsukami, M. The structure and distribution of satellite cells of cardiac muscles in decapod crustaceans. Cell Tissue Res. 219:69-83; 1981. 22. Miwa, H.; Matsuda, T. An integrated approach to the design and engineering of hybrid arterial prostheses. J. Vast. Surg. 19:658-667; 1994. 23. Miwa, H.; Matsuda, T.; Iida, F. Development of a hierarchically structured hybrid vascular graft biomimicking natural arteries. J. Am. Sot. Artif. Intern. Organs 39: M273-277; 1993.
24. Miwa, H.; Matsuda, T.; Tani, N.; Kondo, K.; Iida, F. An in vitro endothelialized compliant vascular graft minimizes anastomotic hyperplasia. J. Am. Sot. Artif. Intern. Organs 39:M501-M505; 1993. 25. Mulder, M.M.; McElwain, J.F.; Tresco, P.A. Three dimensional culture system for myocyte growth and differentiation. J. Am. Sot. Artif. Intern. Organs 41:90; 1995. 26. Nakao, H.; Matsuda, T.; Nakayama, Y.; Hara, Y.; Saishin, M. Design concept and construction of a hybrid lamellar keratoprosthesis. J. Am. Sot. Artif. Intern. Organs 39:M257-M260; 1993. 27. Neumeyer, A.N.; DiGregorio, D.M.; Brown, R.H., Jr. Arterial delivery of myoblasts to skeletal muscle. Neurology 42:2258-2262; 1992. 28. Robinson, S.W.; Cho, P.W.; Levitsky, H.I.; Olson, J.L.; Hruban, R.H.; Acker, M.A.; Kessler, P.D. Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: Long-term survival and phenotypic modification of implanted myoblasts. Cell Transplant. 5:77-91; 1996. 29. Rumyanstev, P.P. Some comparative aspects of myocardial regeneration. In: Mauro, A., ed. Muscle regeneration. New York: Raven Press; 1979:335-355. 30. Stopak, D.; Harris, A.K. Connective tissue morphogenesis by fibroblast traction. Dev. Biol. 90:383-398; 1982. 31. van Wachen, P.B.; van Luyn, M.J.A.; Ponte da Costa, M.L. Myoblast seeding in a collagen matrix elevated in vitro. J. Biomed. Mater. Res. 30:353-360; 1996. 32. Weinberg, C.E.; Bell, E. A blood vessel model constructed from collagen and cultured vascular cells. Science. 231: 397-400; 1986. 33. Yaffe, D.; Saxal, 0. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270:725-727; 1977.
PI1 SO963-6897(96)00255-2
ELSEVIER
Original Contribution
HYBRID MUSCULAR TISSUES: PREPARATION OF SKELETAL CELL-INCORPORATED COLLAGEN GELS
MUSCLE
TAKAHISA OKANO*? AND TAKEHISA MATSUDA’* *Department of Bioengineering, National Cardiovascular Center Research Institute, Osaka, Japan, and tSecond Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
0 Abstract-We prepared three different types of hybrid muscular tissues in which C2C12 cells (skeletal muscle myoblast cell line) were incorporated in type I collagen gels and then differentiated to myotubes upon culture: a disctype, a polyester mesh-reinforced sheet-type, and a tubular type. A cold mixed solution of the cells and type I collagen was poured into three different types of molds and was kept at 37°C in an incubator to form C2C12 cell-incorporated gels. A polyester mesh was incorporated into a gel to form the sheet-type tissue. The tubular hybrid tissue was prepared by pouring a mixed solution into the interstitial space of a tubular mold consisting of an outer sheath and a mandrel and subsequently culturing after removal of the outer sheath. Hybrid tissues were incubated in a growth medium (20% fetal bovine serum medium) for the first 4 days and then in a differentiation medium (2% horse serum medium) to induce formation of myotubes. Transparent fragile gels shrank with time to form opaque gels, irrespective of type, resulting in the formation of quite dense hybrid tissues. On day 14 of incubation, myoblasts fused and differentiated to form multinucleated myotubes. For a tubular type hybrid tissue, both cells and collagen fiber bundles became circumferentially oriented with incubation time. Periodic mechanical stress loading to a mesh-reinforced hybrid tissue accelerated the cellular orientation along the axis of the stretch. The potential applications for use as living tissue substitutes in damaged and diseased skeletal and cardiac muscle and as vascular grafts are discussed. 0 1997 Elsevier Science Inc. 0 Keywords - Hybrid muscular gel; Stress loading.
tissue; C2C12;
area of medical technology. Living substitutes under development include skin (I), vessels (8,9,15,19,2224,32), corneas (26), and ligaments (11). The essential feature of the key technology common to the hybrid tissues listed above is to immobilize respective viable cells in three-dimensional extracellular matrices such as collagen gels. However, living tissue substitutes for skeletal and cardiac muscle have not yet been developed. Skeletal muscle cells, which have a highly developed intracellular contractile structure, arc present in living tissues in two distinctly different forms: myoblasts (undifferentiated, satellite cells), and muscle fibers (differentiated and multinucleated cells formed through myoblast fusion). The muscle fibers are densely accumulated and highly oriented in one direction. When skeletal muscle is injured, myoblasts proliferate and differentiate to form myotubes and muscle fibers for repair of muscular tissues (3). On the other hand, cardiac muscle cells (cardiomyocytes), which are highly differentiated, do not have the ability to regenerate following injury (29). The regeneration of damaged cardiac muscles has been one of the ultimate goals of cardiovascular medicine. The utilization of electrically responsive contractibility of regenerated skeletal muscle has been considered a choice for replacement or support of cardiac function. In fact, cardiomyoplasty, using skeletal muscle to support ventricular function, has recently been developed (4,5). On the other hand, if a hybrid muscular tissue composed of highly dense and oriented muscle fibers is prepared, such a tissue may contract upon pulsed electrical stimulation, resulting in generation of unidirectional force. Therefore, these hybrid muscular tissues may be useful as transplantation vehicles to repair damaged skel-
Collagen
INTRODUCTION
Tissue engineering to replace diseased tissues with living substitutes has been attracting significant interest in the ACCEPTED
Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-l Fujishirodai, Suita, Osaka 565, Japan.
9110196.
‘Correspondence should be addressed to Takehisa Matsuda, 109
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eta1 and cardiac muscle or as pulsatile-flow-generative vascular prostheses. In the present study, we developed a prototype fabrication technique for hybrid muscular tissues composed of skeletal muscle cells and collagen. Three different types of hybrid muscular tissue were prepared: a disctype, a polyester mesh-reinforced sheet-type, and a tubular type (Fig. 1). Periodic stretching accelerated cellular alignment in the resultant tissues. MATERIALS AND METHODS
Polyester mesh-reinforced sheet-type hybrid tissue. A polyester mesh-reinforced sheet-type hybrid tissue (mesh generously supplied by Teijin Corp., Tokyo, Japan) was prepared as follows. A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into a 24-well plate. The polyester mesh was placed on the gel formed upon incubation at 37°C for 30 min. A similar mixture was then poured over the meshplaced gel in the mold. Similar to the case of the culture of the disc-type tissue, the gel was cultured in DMEM. The medium change from FBS-DMEM to HS-DMEM was performed on day 4 of incubation.
Cell Culture
C2C12 cells (a mouse skeletal muscle myoblast cell line; American Type Culture Collection, Rockville, MD) were used as skeletal muscle cells. The culture mediums used were FBS-DMEM [Dulbecco’s modified Eagle’s medium (DMEM; Gibco Laboratories Inc., Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; CSL Ltd., Victoria, Australia)] as a growth medium and HS-DMEM [DMEM supplemented with 2% horse serum (HS; Gibco Laboratories Inc.)] as a differentiation medium. Cell culture was performed on tissue culture dishes at 37°C in a humidified atmosphere of 95% air and 5% CO1 in an incubator.
Tubular type hybrid tissue. A tubular type hybrid tissue was prepared as follows. A tubular mold, which consisted of an external and an internal glass vessel coaxially fixed at one end with silicone tubes, was prepared. The mold had the following dimensions: inner diameter, 1.5 mm; outer diameter, 7 mm; and length, 7 cm. A cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into the space between the sheath and the mandrel of the mold. After subsequent incubation at 37°C for 30 min, the sheath was removed. The resultant gel was cultured in DMEM. The medium change from FBS-DMEM to HS-DMEM was performed on day 4 of incubation. On day 14 of incubation, the mandrel was removed from the tissue.
Fabrication Disc-type hybrid tissue. A disc-type hybrid tissue was prepared as follows. One volume of C2C12 cells suspended in DMEM and one volume of acid-solubilized bovine dermal type I collagen solution (5 mg/mL, CELLGEN, Kohken Corp., Tokyo, Japan) were mixed at 4°C. The cold mixed solution (cell density, 1.0 x lo6 cells/ mL; collagen concentration, 2.5 mg/mL) was poured into a 24-well plate (MICROPLATE; well diameter, 16 mm, Iwaki Glass, Tokyo, Japan) and incubated at 37°C for 30 min. The cell-incorporated collagenous gel was cultured in DMEM. The medium change from FBS-DMEM to HS-DMEM was performed on day 4 of incubation.
Fig. 1. Schema of three different types of hybrid muscular tissues composed of skeletal muscle cells and collagen. (A) Disc-type hybrid tissue. (B) Polyester mesh-reinforced sheettype hybrid tissue. (C) Tubular type hybrid tissue.
Formation of mesh-reinforced hybrid tissue for stress loading. A polyester mesh-reinforced sheet-type tissue for stress loading was prepared as follows. The two shorter sides of a rectangular polyester mesh (1 x 2 cm) were fixed to glass tubes (2 mm in outer diameter). Each glass tube was tied to a loop of silk cord. The polyester mesh thus prepared was placed on a handcrafted rectangular mold (1 x 2.5 cm). A cold mixture of C2C12 cell suspension and type I collagen solution was poured into the mold and incubated at 37°C for 30 min. The meshreinforced hybrid tissue thus prepared was cultured in FBS-DMEM for up to 7 days prior to stress loading. Stress Chamber. The stress chamber was a modified version of the device described in our previous paper (12). A commercially available plastic flask (CORNING, 25 cm2 tissue culture flask) was handcrafted to install the mesh-reinforced hybrid tissue with glass tubes as follows. One loop of silk cord through a glass tube was used to fix the tissues toward the end of the stress chamber. The silk cord from the outer loop was passed through a hole in the cap of the chamber. Thus, two tissues were installed in the chamber. Prior to culture, the stress chambers were sterilized with EOG (ethylene oxide gas). Stress loading. The stretching apparatus used was a custom-designed one that was previously prepared
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(12,13). Briefly, a DC motor drove an eccentric steering disc at 60 RPM, the rotations of which were transferred to a piston rod as directional movement. A stress arm connected to the piston rod periodically moved the glass rod back and forth, which caused periodic stress relaxation of the dynamically stressed hybrid tissues with 5% amplitude of the relaxed tissue length. The periodic movement measured by a position transducer set around the piston rod exhibited a sinelike waveform. The tissues were subjected to cyclic stretching (frequency; 60 RPM, amplitude; 5%) for up to 7 days in HS-DMEM in a stress chamber maintained at 37°C with an atmosphere of humidified 95% air and 5% CO,. A nonstressed tissue, as a control tissue, was floated in HS-DMEM for up to 7 days.
Morphological Evaluation Morphological changes of C2C12 cells cultured on tissue culture dishes and in hybrid tissues were observed with a phase-contrast microscope (DIAPHOT, Nikon, Tokyo, Japan), a light microscope (LM; VANOX-S, Olympus, Tokyo, Japan), and a scanning electron microscope (SEM; S-4000, Hitachi, Tokyo, Japan). For LM study, monolayer tissues on tissue culture dishes were fixed with ethanol for 15 min, and were stained with Papanicolaou stain or were immunohistochemically stained using a muscle-desmin-specific monoclonal antibody (D33; Dako Corp., Glostrup, Denmark). Hybrid
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tissues were fixed with 10% formalin for 1 h, dehydrated with a graded series of ethanol, and embedded in paraffin. For SEM study, specimens were fixed in a fixative (2% glutaraldehyde in a 0.1 M cacodylate buffer, pH 7.4) postfixed with 1% osmium tetroxide in buffer for 15 min, washed with buffer five times, stained with 1% tannic acid for 30 min, and washed with distilled water five times. Subsequently, they were critical point dried, sputter-coated with platinum, and studied under the SEM. Dimensional Change During Hybrid Tissue Formation The diameter of the disc-type hybrid tissue and the length of the tubular type hybrid tissue were macroscopitally measured. The wall thickness of the tubular type hybrid tissue was obtained as the average of the thicknesses measured at four arbitrary points on a micrograph of a transverse section. RESULTS
Differentiation of Myoblasts on Culture Dish C2C 12 is an established cell line from skeletal muscle cells. It has been reported that myoblasts fuse to form myotubes when they are cultured on a dish at high cell density and under very low nutrient conditions, which we demonstrated in the present study as follows. On day 1 of incubation, mononucleated myoblasts spread on the dish exhibited a polygonal shape and random orientation (Fig.
Fig. 2. Micrographs of C2C12 cells on a tissue culture dish. (A, C) On day 1 of incubation and (B, D) on day 11 of incubation, multinucleated cells (myotubes) were observed. Upper: phase-contrast micrographs. Lower: light micrographs stained with Papanicolaou stain.
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2A and C). Upon further culturing, the myoblasts proliferated to form a dense monolayer in the growth medium. On day 4 of incubation the culture medium was substituted with the differentiation medium. Upon further culturing, the myoblasts fused to form multinucleated myotubes. On day 11 of incubation the elongated and spindle-shaped myotubes became dense with local orientation (Fig. 2B and D). Immunohistochemical staining using the muscle-desmin-specific monoclonal antibody revealed the presence of a small quantity of desmin in the myoblasts on day 1 of incubation, but desmin content in the myotubes markedly increased upon long-term culture (11 days) (Fig. 3).
Fabrication of Disc-Type Hybrid Tissue The cold mixture of C2C 12 cells suspended in DMEM and type I collagen solution was poured into a 24-well plate and incubated at 37°C. A C2C12 cell-incorporated gel, which was transparent and fragile when prepared, shrank to form an opaque and round tissue with incubation time. Phase-contrast micrographs (Fig. 4) and scanning electron micrographs (Fig. 5) showed that myoblasts were homogeneously distributed and collagen fiber bundles were randomly oriented in the gel on day 1 of incubation (Figs. 4A and C and 5A). When the growth medium was substituted with the differentiation medium on day 4 of incubation, elongated myoblasts began to fuse, producing multinucleated myotubes. On day 14 of incubation, myotubes and collagen fiber bundles were densely accumulated. Both tended to be randomly oriented in the central region of the tissue and circumferentially oriented at the peripheral region (Figs. 4B and D and 5B). Figure 6 shows time-dependent changes in the diameter of the fabricated disc-type hybrid tissue (relative ratio to initial diameter; 16 mm). The C2C12 cellincorporated gel shrank rapidly for the first 4 days and slowly for the next 10 days of incubation. On day 14 of incubation, the diameter was approximately one-half of the initial diameter (relative ratio; 0.46 + 0.06).
Fabrication of Polyester Mesh-Reinforced Sheet-Type Hybrid Tissue A C2C 12 cell-incorporated gel reinforced with a polyester mesh was comprised of three layers: the polyester mesh was sandwiched between two cell-incorporated collagen gel layers. Similar to the disc-type hybrid tissue, the gel was transparent and fragile immediately following gelation. The gel became opaque with incubation time, but it shrank much less than the disc-type gel did (gel diameter decreased by approximately 10% in contrast with the 46% for the disc-type gel). Scanning electron micrographs showed that polygonal myoblasts were distributed throughout the network of the mesh and collagen fiber bundles were sparsely and randomly oriented on day 1 of incubation (Fig. 7A). When the growth medium was substituted with the differentiation medium on day 4 of incubation, elongated myoblasts began to fuse, producing multinucleated myotubes. On day 12 of incubation, elongated, spindle-shaped myotubes were densely distributed throughout the network of the mesh (Fig. 7B). Figure 7C and D shows phase-contrast micrographs of mesh-reinforced hybrid tissues on day 12 of incubation.
Fabrication of Tubular Type Hybrid Tissue The cold mixture of C2C12 cells suspended in DMEM and type I collagen solution was poured into a cylindrical mold with a mandrel (Fig. 8A). Subsequent incubation for 30 min produced a cylindrical, transparent gel (Fig. 8B). Upon culture, time-dependent shrinkage of the gel occurred and an opaque tissue was formed. On day 4 of incubation, the growth medium was substituted with the differentiation medium. On day 14 of incubation, a considerably shrunk tubular tissue was formed. The resultant tissue became tough. The length and wall thickness of the resultant tissue were approximately one-half of the original length and one-fifth of the original wall thickness, respectively (Fig. SC). Myoblasts were uniformly distributed throughout the gel and collagen fiber bundles were not yet well organized on day 1 of incubation (Fig.
Fig. 3. Micrographs of C2C12 cells on a tissue culture dish stained by antidestnin antibody. (A) On day 1 of incubation, desmin in myoblasts were hardly observed. (B) On day 11 of incubation, a large quantity of desmin in myotubes were observed. Arrow: desmins stained by antidesmin
antibody.
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Fig. 4. Micrographs of a disc-type hybrid tissue. (A, C) On day 1 of incubation, myoblasts were homogeneously distributed in a gel and collagen fiber bundles were randomly oriented. (B, D) On day 14 of incubation, elongated myotubes and collagen fiber bundles tended to become dense and to be randomly oriented in the central region of the tissue and circumferentially oriented at the peripheral region. Upper: phase-contrast micrographs. Lower: light micrographs stained with Masson-trichrome stain (horizontal section).
9A and C). On day 4 of incubation, proliferated myoblasts were induced to fuse upon substitution of the medium. On day 14 of incubation, elongated, spindle-shaped myotubes and collagen fiber bundles became dense and were circumferentially oriented (Fig. 9B and D).
spindle-shaped myotubes and very densely accumulated collagen fiber bundles were oriented parallel to the direction of the stretch (Fig. 10).
Orientation and Morphological Response to Stress Loading A mesh-reinforced sheet-type hybrid tissue, which had been incubated for 7 days in FBS-DMEM, was subjected to periodical stretching and recoiling (5% above and below the resting tissue length at 60 RPM frequency) for 7 days in HS-DMEM. In dynamically stressed tissues, compared with nonstressed tissues, both elongated
Skeletal muscles are unique tissues that are composed of bundles of highly oriented and highly dense muscle fibers, each of which is a multinucleated cell derived from myoblasts. The muscle fibers in native skeletal muscle are closely packed together in an extracellular matrix composed mainly of collagen to form an organized tissue with high cell density and cellular orientation. These uniaxially structured cellular assemblages are
DISCUSSION
Fig. 5. Scanning electron micrographs of a disc-type hybrid tissue. (A) On day 1 of incubation, polygonal myoblasts were distributed throughout the network of collagen fibers. (B) On day 14 of incubation, both elongated cylindrical myotubes and collagen fiber bundles tended to be oriented.
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Incubation Period (days)
Fig. 6. Time-dependent change in the diameter of disc-type hybrid tissue. (Relative to initial diameter when prepared.) The disc-type hybrid tissue shrank markedly for first 4 days, followed by reduced shrinking rate. On day 14 of incubation, the diameter of the resultant tissue was approximately one-half of the diameter of the initial tissue.
essential of skeletal muscle tissues. A large vector force is generated due to contraction of highly dense and highly oriented muscle fibers when skeletal muscle is subjected to an appropriate stimulus. Muscle fibers can-
not regenerate (29) but during repair of injured muscles, myoblasts (satellite cells) scattered in skeletal muscles (20), which remain in a quiescent and undifferentiated state, can enter the mitotic cycle which induces to proliferate and to fuse to each other to form multinucleated and elongated myotubes, which self-assemble to form a more organized structure, namely a muscle fiber (3). Although there is a similarity in terms of contractibility between skeletal muscle and cardiac muscle, in contrast to skeletal muscle cells, cardiac muscle cells (cardiomyocytes) are incapable of regeneration due to the absence of satellite cells in cardiac muscle (21). Therefore, it has been expected that myoblasts transfer to diseased cardiac muscles may contribute to the restoration of cardiac function. Muscular tissue engineering has been focusing on repair of damaged myocardial tissues upon intramural or transventricular injection of skeletal myoblasts (6,16,18,28). When satellite cells were implanted into injured myocardium, they survived and differentiated to form cardiac-like muscle cells, thus repairing damaged heart muscle in an appropriate environment (‘‘milieu-dependent’ ’ differentiation) (6). The implantation of genetically engineered myoblasts has been utilized as a potential therapy for genetic muscle diseases such as Duchenne muscular dystrophy (2,7,17,27). These findings and stimuli-responsive functional capabilities of skeletal muscle cells could have important clinical implications such as myoblast transfer
Fig. 7. Morphological characteristics of polyester mesh-reinforced sheet-type tissue. (A, B) Scanning electron micrographs. (A) On day 1 of incubation, myoblasts and collagen fibers were sparsely distributed in the mesh. (B) On day 12 of incubation, elongated myotubes and collagen fiber bundles became dense. (C, D) Phase-contrast micrographs on day 12 of incubation. (C) Elongated myotubes were distributed throughout the network of polyester mesh (visualized as black bold lines). (D) At higher density of myotubes was present close to the surface of the tissue than deeper into the tissue. P: polyester mesh. Mb: myoblast. Mt: myotube.
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Fig. 8. Fabrication of a tubular type hybrid tissue. (A) A mold was assembled from an outer and an inner glass tube (Inner diameter, 1.5 mm; outer diameter, 7 mm; length, 7 cm). (B) A transparent and opaque gel formed by 30-min incubation at 37°C after a mixed solution of collagen and C2C12 cells was poured into the interstitial space of the mold. (C) A tubular type hybrid tissue on day 14 of incubation and removal of glass rod.
The length and wall thickness of the resultant tissue were approximately one-half of the original length and one-fifth of the original wall thickness, respectively. or transplantation. On the other hand, studies on hybrid muscular tissues have only recently began. When C2C 12 myoblasts were injected into a fibrous, supple collagen disc, seeded myoblasts exhibited reasonable cellular distribution and myotube formation in the differentiation medium, which was studied to develop autologous seeded/cultured patches for abdominal wall reconstruction (31). Very recently, an attempt to prepare a myoblast-incorporated polyurethane sponge was also reported (25). Our working principle for the preparation of a functional, vital hybrid muscular tissue is that when myoblasts in collagen gels that are cultured at high cell density in the growth medium are induced to fuse to each other in the differentiation medium, multinucleated muscle fiber-incorporated collagen gels are formed, and subsequent continuous external mechanical stress loading may generate reinforced cellular alignment. This may produce a hybrid muscular tissue biomimicking natural ones. When such a well-organized hybrid tissue is subjected to pulsed electrical stimulation, it may contract or
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relax in response to the electrical stimulation. A disctype or a sheet-type hybrid tissue may be useful as a transplantation vehicle to repair damaged and diseased skeletal and cardiac muscle, while a tubular type hybrid tissue may be useful as a vascular prosthesis. The currently available technique of using a collagenase treatment method for harvesting satellite cells from living muscles did not give us a 100% pure preparation of satellite cells, i.e., one not contaminated with cells of other types such as fibroblasts. Also in our experience, more than two or three passages of a culture of harvested satellite cells derived from newborn rats eventually resulted in alteration from a myoblast-rich tissue to a fibroblast-rich tissue. Therefore, a more reliable harvesting method is needed for a 100% pure preparation of satellite cells. In this study, we used C2C 12, which is an established cell line of satellite cells from skeletal muscle of C3H mouse. As reported previously (33), these cells rapidly proliferate to form a monolayer tissue on a tissue culture dish in the growth medium at containing a high concentration of FBS, and subsequently differentiate to form multinucleated myotubes in the differentiation medium at containing a low concentration of HS (Fig. 2). An increased intracellular content of desmin was observed upon differentiation, which was verified by immunohistochemical staining of muscle-desmin-specific monoclonal antibody. A basic feature of the hybrid skeletal muscle tissues which we designed was C2C12 cell-incorporated collagen gel. The preparation procedure was very similar to those used for living tissue substitutes for skin (l), vessels (8,9,15,19,22-24,32), corneas (26), and ligaments (11). We prepared three different types of hybrid muscular tissues as prototype hybrid muscular tissues (Fig. 1): a disc-type, a tubular type and a polyester meshreinforced sheet-type. The former two were free of a synthetic scaffold, and the latter was structurally reinforced with a polyester mesh. C2C12 cell-incorporated collagen gels were prepared by thermal gelation of a cell-mixed collagenous solution in respective vessels upon incubation at 37°C. Irrespective of tissue type, C2C12 cells differentiated with time to form myotubes in the collagen gels in the differentiation medium. Transparent and fragile gels when prepared shrank time dependently to form opaque and dense tissues. C2C12 cells and collagen fiber bundles in the hybrid muscular tissues tended to become denser and to be highly oriented with time. Such a self-organized tissue formation process is driven by contraction of cells interacting with collagen fibers. The rapid reduction of the size of the hybrid tissues incorporated with mesenchymal cells has been generally observed. This phenomena is common to mesenchymal cells such as fibroblast and smooth muscle cell (1,13,14,30). However, little study on immobilization of
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Fig. 9. Micrographs of a tubular type hybrid tissue. (A, C) On day 1 of incubation, myoblasts were uniformly distributed throughout the tissue and collagen fiber bundles were not yet well organized. Top shows luminal surface. (B, D) On day 14 of incubation, elongated, spindle-shaped myotubes and collagen fiber bundles tended to become dense and to be circumferentially oriented. Upper: light micrographs stained with Masson-trichrome stain (cross-section). Lower: scanning electron micrographs. Arrow: circumferential direction. Mb_ myoblast. Mt: myotube. myoblasts
into
dependent
dimensional
A polyester designed
collagen
or other change
mesh-reinforced
to improve
matrix
and
its time-
have not been reported. sheet-type
the mechanical
hybrid
strength
tissue,
of the hy-
brid tissue, shrank markedly less than the mesh-free disctype tissue. Polyester mesh significantly improved mechanical strength but produced a minimal degree of gel shrinkage. Some difference of the state of C2C12 in the mesh-reinforced and mesh-free tissue was observed. Myotubes in mesh reinforced-tissue were more stretched than in the mesh-free disc-type tissue. In the tubular type hybrid tissue, circumferential orientation of cells and collagen fibers proceeded with time particularly at the luminal surface, similar to observations in our previous study of SMC-incorporated hybrid vascular medial tissue (8,9,13,14). If contraction and relaxation of the tubular type hybrid tissue occur upon pulsed electrical stimulation, this may cause narrowing and enlargement of the tube, resulting in the generation of pulsatile flow. To accelerate the orientation of skeletal muscle cells, the mesh-reinforced hybrid tissue was subjected to periodic stress loading. The C2C12 cells in the dynamically stressed tissue became elongated and spindle-shaped, collagen fiber bundles became densely accumulated with time, and both were aligned to the direction of the stretch in contrast to those in the nonstressed tissue (Fig. 10). This is in good agreement with our previous finding (13,14) in which accelerated accumulation and orienta-
tion of SMCs in collagenous tissues occurred with time under dynamic stress loading. The periodic stretching of the hybrid tissues extended the collagen fiber bundles in the direction of the stretch, which increased the orientation of the collagen fibers, resulting in more prominent elongation and orientation of skeletal muscle cells in the direction by the contact guidance mechanism, as was previously proposed (14). Although myoblasts differentiated to form myotubes in the three-dimensional collagen gels, cellular density and orientation of cells and collagen fibers in the hybrid muscular tissues were still much lower than those of native skeletal muscle. In addition, individual muscle fibers in a native tissue are surrounded by rich capillary beds, via which oxygen and nutritive elements are supplied to maintain metabolism. Therefore, a much higher density of oriented muscle fibers as well as incorporation of capillary networks (or microvascularization) into a hybrid tissue is essential for a transplantation vehicle for repair of damaged and diseased muscular tissues. To this end, the following factors should be taken into account when preparing of hybrid muscular tissue: 1) high cell density and high degree of orientation of myotubes and 2) angiogenesis. Use of an optimal design incorporating biomechanical and tissue engineering principles may yield a transplantation vehicle for repair of damaged and diseased skeletal and cardiac muscle and a muscularpowered vascular prosthesis for tubular type tissues.
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OKANO AND T. MATSUDA
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t
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IOOpm
stress
Fig. 10. Morphological characteristics of periodically stretched mesh-reinforced sheet-type hybrid tissues. (A-C): Masson-trichrome staining of cross-section parallel to the direction of the stretch. (A) On day 1 of incubation (prior to stress loading), myoblasts were homogeneously distributed throughout the tissue. (B) On day 14 of incubation in the nonstressed tissue, spindle-shaped myotubes were distributed in the tissues. (C) On day 14 of incubation (7 days after stress loading) in the dynamically stressed tissues, elongated and spindle-shaped myotubes were closely accumulated and oriented in the direction of the stretch. (D-F): Scanning electron micrographs of hybrid tissue surfaces. (D) On day 1 of incubation, collagen fibers were coarse and randomly oriented. (E) On day 14 of incubation, in the nonstressed tissues, collagen fibers remained coarse and randomly oriented. (F) On day 14 of incubation, in the dynamically stressed tissues, densely collagen fibers were oriented parallel to the direction of the stretch.
Such
a study
reported
is now underway,
and the results
will be
in the near future. 7.
Acknowledgments - The authors thank Dr. Shigeko Takaichi of the Department of Etiology and Pathophysiology of the National Cardiovascular Center, who kindly instructed us in electron microscopic techniques. The first author (T.O.) is grateful for the continuous encouragement of Professor Takahiro Oka of the Second Department of Surgery, Kyoto Prefectural University of Medicine, from which T.O. was on leave.
8.
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