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DOI: 10.3727/096368910X536608
E-ISSN 1555-3892
www.cognizantcommunication.com
Cell Transplantation, Vol. 20, pp. 767–774, 2011
Printed in the USA. All rights reserved.
Copyright  2011 Cognizant Comm. Corp.
Novel Cell Culture Model Using Pure Hydrostatic Pressure
and a Semipermeable Membrane Pouch
Shuichi Mizuno
Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
Cell constructs and culture methods are essential tools in tissue engineering. The cell construct should be
equivalent to the native cartilage it is intended to replace. Thus, three-dimensional cell constructs are usually
composed of a high density of cells and dense extracellular matrix. However, dense constructs suffer from
a lack of passive nutrient supply, gas exchange, and removal of degraded debris. We have developed a novel
hydrostatic pressure/perfusion culture system that improves the quality of neo-tissues, providing an automated and affordable system for clinical applications. We have also developed a semipermeable membrane
pouch that contains a fragile amorphous cell carrier. Although amorphous material is difficult to handle, it
is a useful medium in which to deliver cells to the desired site via injection. We evaluated phenotypes of
bovine articular chondrocytes embedded in a collagen type I gel enclosed within membrane pouches permeable to molecules of various sizes. Constant or cyclic hydrostatic pressure was externally applied to the
medium phase with a new culture system. Accumulation of cartilage specific matrix was promoted with a
500-kDa cutoff membrane pouch and cyclic hydrostatic pressure at 0.5 MPa, 0.5 Hz. This new method will
be useful in the delivery of engineered cells to a desired tissue in regenerative medicine.
Key words: Tissue engineering; Hydrostatic pressure; Semipermeable membrane pouch; Cell culture method;
Chondrocytes
INTRODUCTION
The first commercially available tissue engineering
product for cartilage repair was an autologous chondrocyte suspension (1,25). Advanced products have been
developed to improve methods of cell expansion and
delivery, and surgical approach (12). Cell culture is a
critical in vitro process for manufacturing cell/tissue
constructs. Technological advancements have made it
possible to implant cell constructs to replace damaged
cartilage and promote subsequent regeneration (13,16).
However, a three-dimensional (3D) construct creates its
own problems if it has high cell density and causes large
amounts of extracellular matrix (ECM) to accumulate;
this blocks the supply of necessary nutrients and gas exchange. To solve such problems, commercially available
or custom-made bioreactors have been used. Bioreactors
are designed to optimize culture conditions by controlling oxygen concentration (19,26), shear stress (8,17),
and hydrostatic pressure (HP). We and other researchers
have studied the ability of HP to promote chondrocyte
phenotypes (4,21,22) with a variety of pressure profiles:
continuous versus intermittent (10,29) and constant versus cyclic (2,3,5,7,11,30). The results of these studies
differed depending on the magnitude, duration, and profile of the HP.
Using our hydrostatic fluid pressure/perfusion culture
system, we previously reported that HP promoted accumulation of cartilage ECM by bovine articular chondrocytes (bACs) in a porous collagen sponge (21). In addition, the manufactured chondrocyte construct has been
studied for clinical applications (9). Besides the structural scaffold, injection of amorphous gel is another tool
that can be used to augment tissue in general. However,
amorphous gel is difficult to handle in culture and in
surgery, problems that must be solved in order to explore new applications of such gels (27). Therefore, we
developed a novel culture method using a semipermeable membrane pouch to handle amorphous gel and redesigned our culture system (23). We evaluated the accumulation of ECM by bACs seeded in the gel within
semipermeable membrane pouches of varied molecular
cutoff sizes and incubated with constant or cyclic HP.
Combining the HP and pouches will be explored to de-
Received February 26, 2010; final acceptance September 28, 2010. Online prepub date: November 5, 2010.
Address correspondence to Shuichi Mizuno, Ph.D., Orthopedic Research, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115,
USA. Tel: 617-732-6335; Fax: 617-732-6705; E-mail: [email protected]
767
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velop useful applications (e.g., augmentation of tissue
mass and minimally invasive cell/matrix delivery for tissue engineering).
MATERIALS AND METHODS
Performance of Semipermeable Membrane Pouches
Pouches were constructed of semipermeable membrane tubing made of polyvinylidene difluoride (PVDF;
8-mm diameter, Spectra/Por Biotech, Spectrum Laboratories, Rancho Dominguez, CA). We tested two tubings with different molecular weight cutoffs: 250 and
500 kDa. The tubing was cut into 15-mm pieces and
folded 1–2 mm from one end (leaving the other end
open), and the folded end was sealed with two stainless
steel clips to construct a tube. The tubes were rinsed
with culture-grade water (Sigma-Aldrich, St. Louis,
MO), submerged in the water, and autoclaved at 121°C
for 15 min.
For performance evaluation, the tubes were filled
with sterile culture-grade water. The open end of the
tube was folded at 90° from the opposite closed end and
sealed with two stainless steel clips to construct a pouch
like a tetra pack (Fig. 1A).
The pouches were placed in a pressure-proof culture
chamber unit (Fig. 1B) connected to a medium bag
(Transfer Pack Container, Baxter, Deerfield, IL) with
gas-permeable silicon tubing. The bag was filled with
100 ml of 10 mg/ml sterile bovine serum albumin (BSA,
Sigma-Aldrich). The culture chamber unit and a medium
bag were installed in the culture system (Fig. 1C, TEP02, Takagi Industrial, Shizuoka, Japan). In a series of
experiments, the pouches were incubated at 1) static
conditions (no pressure and no perfusion) in the BSA;
2) perfusion alone at 100 µl/min with continuous replenishment of the BSA; 3) constant HP at 0.5 MPa with
100 µl/min continuous replenishment of the BSA; or 4)
cyclic HP at 0.5 MPa, 0.5 Hz, with 100 µl/min continuous replenishment of the BSA using the culture system
(Fig. 1D). All incubation was conducted under the same
conditions as regular cell culture, at 37°C and 5% CO2.
Four pouches each were harvested after 3, 6, 12, and 24
h of incubation. The outside of each harvested pouch
was quickly flushed with water, and excess water on the
outside of the pouch was absorbed with Kimwipes. One
end of the pouch was cut, and the sample was immediately aspirated. The protein content of each sample was
measured with a protein assay kit (BCA protein assay,
Bio-Rad, Hercules, CA).
Chondrocyte Isolation
A bovine forelimb (from a calf 2–3 weeks old) was
purchased from a local abattoir. Pieces of articular cartilage (5 × 5 × 2–5 mm) were harvested from the weight-
Figure 1. A pouch device and hydrostatic pressure (HP) culture system. Pouches were constructed of semipermeable
membrane tubing. (A) Both ends of the tubing were folded
and sealed with stainless steel clips to construct a pouch like
a tetra pack. (B) The pouches were placed in a pressure-proof
culture chamber unit. (C) The culture chamber unit and a medium bag were installed in the culture system. The culture unit
has three components: a pump unit (1), a pressure-proof culture chamber unit (2), and a backpressure regulator unit (3).
Medium is replenished with gas-permeable silicon tubing (4)
and kept in a medium bag (5). The culture system is automatically controlled with a built-in computer control system. The
backpressure regulator unit is fitted with a needle valve and
attached to a spring-operated actuator of the control system.
The set pressure is regulated by changing the distance between
the valve and the actuator. The culture chamber unit has a
flexible silicon film (500 µm thick) that separates the medium
in the culture chamber from the adjacent water-filled compression chamber. The compression chamber has a piston connected to a spring-attached actuator. The piston compresses
the water in the compression chamber and indirectly the medium through the silicon film. (D) The HP profile at 0.5 MPa,
0.5 Hz, 100 µl/min.
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NOVEL CELL CULTURE METHOD
bearing area of the humeral chondyle (18). Slices of the
middle zone (MZ) layer were isolated by removing a
layer of surface zone (⬃100 µm thick from the top surface) and a layer of deep zone (⬃300 µm thick from
subchondral bone) using a scalpel (#15, BD, Franklin
Lakes, NJ) under a stereomicroscope (Nikon Instruments, Melville, NY). The MZ slices were digested with
0.15% collagenase (CLS-1, Worthington, Lakewood,
NJ) dissolved in Ham’s F-12 medium with 100 U/ml
penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C with gentle shaking for 12 h. The
bACs of the MZ were collected through a cell strainer
(70-µm mesh, BD), followed by rinsing with D-PBS.
Constructing Cell/Collagen Gel in a Semipermeable
Membrane Pouch
One tenth volume of 10× Dulbecco’s modified Eagle
medium (DMEM, Invitrogen) and 1/10 volume of 0.1 N
NaOH were added to 8/10 volume of 0.3% pepsin-digested
acid-soluble collagen solution from bovine skin (PureColTM, Cohesion, Palo Alto, CA). This collagen solution
was neutralized (7 < pH < 8) with additional 0.1 N
NaOH, confirming the pH using pH paper (EM Science,
Gibbstown, NJ). Cells were suspended in the neutralized
collagen solution (750,000/50 µl), and 50 µl of the cell
suspension was injected into the semipermeable membrane tube. The tube was incubated at 37°C for 1 h to
let the collagen form an amorphous cell/gel. Finally, the
tube was filled with serum-free DMEM/F12 to eliminate
air bubbles, and the open end of the tube was folded at
90° from the opposite end, constructing a tetra pack, and
sealed with two stainless steel clips (Fig. 1A).
Optimization of Culture Conditions with HP
We assessed culture conditions with two profiles of
HP treatment: 1) constant HP and 2) cyclic HP (Fig.
1D). For a static-controlled culture, the amorphous cell/
gel was ejected from each pouch and incubated in
DMEM/Ham’s F-12 medium (F-12, Invitrogen) with
10% fetal bovine serum (FBS, Invitrogen), 100 U/ml
penicillin, and 100 µg/ml streptomycin (Invitrogen) at
37°C and 5% CO2 for 7 days. Pouches were incubated
with continuous medium replenishment at 100 µl/min
(designated “perfusion”), with constant HP at 0.5 MPa
with continuous medium replenishment at 100 µl/min,
and with cyclic HP at 0.5 MPa, 0.5 Hz with continuous
medium replenishment at 100 µl/min. The magnitude
and cycle of HP were programmed and automatically
controlled with our new culture system. Ten milliliters
of medium was used for each pouch. After combinations
of multiple culture conditions were evaluated, static control, constant HP, and cyclic HP culture conditions were
evaluated simultaneously without normalization.
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Biochemical Evaluation
After 7 days of culture, the pouches were harvested
for biochemical and histological evaluation. Both ends
of each pouch were cut, and the cell/gel was ejected and
digested in 450 µl of 125 µg/ml papain (Sigma-Aldrich)
at 60°C for 8 h (14). Four cell/gels from each group
were measured for DNA and sulfated glycosaminoglycan (S-GAG) content. For DNA content, the samples
were incubated with 10 ng/ml bisbenzimide H 33258
(Hoechst 33258, Molecular Probes) and diluted in TNE
buffer (Tris-EDTA sodium chloride, pH 7.4). Fluorescence intensity of the Hoechst 33258 was measured at
ex 365 nm and the em blue region with a fluorometer
(TBS-380, Turner, Sunnyvale, CA). Salmon sperm DNA
(Sigma-Aldrich) was used as a standard. S-GAG was
measured with 1,9-dimethyl-methylene blue (DMB,
Sigma-Aldrich) (6). DMB (200 µl) was added to 2 µl of
sample, and the optical density was immediately measured at 540 and 570 nm (Bio-Rad 550). Chondroitin
sulfate C from a shark (Sigma-Aldrich) was used as a
standard.
Histological Evaluation
Two samples of cell/gel from each test condition
were fixed with 2% paraformaldehyde in 0.1 M cacodylic acid, pH 7.4 (Polysciences, Warrington, PA), and
embedded in paraffin. Rehydrated sections (5 µm) were
stained with safranin-O (Saf-O) to identify negatively
charged ECM. Accumulation of collagen type II (Col 2)
and keratan sulfate (KS) was evaluated immunohistochemically. The immunohistochemistry sections were
stained with an anti-Col 2 antibody (1:20, Chemicon,
Temecula, CA) and then with a biotinylated second antibody kit following the manufacturer’s instructions (Vectastain ABC Elite kit, Vector Laboratory, Burlingame,
CA). For KS staining, the sections were digested with
0.1 U/ml chondroitinase ABC (Seikagaku America) for
1 h at 37°C before staining. The sections were stained
with an anti-KS antibody (Seikagaku America, Falmouth, MA) and then a biotinylated second antibody kit
following the manufacturer’s instructions (Vectastain
ABC Elite kit). For color development, the sections
were incubated with DAB (DAB substrate kit, Vector
Laboratory). Counterstaining was performed with Harris’s hematoxylin (Sigma-Aldrich).
Semiquantitative Assessment of Amount
of Saf-O-Positive Matrix and Cell Number
in Histological Section
We randomly selected five square areas (200 × 200
µm) at the exterior, periphery, and interior of each section stained with Saf-O under 33× optical magnification.
Two sections were also randomly selected from both
samples. We measured optical density of the area at
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fixed video mode (100 ns) with a longpass filter (590
nm, Omega), a CCD camera (ORCA-AG, Hamamatsu,
Bridgewater, NJ), and the OpenLabTM image acquisition
system (Improvision, Waltham, MA). The filter emphasized the red color of Saf-O and minimized the interference of other colors (shorter wavelength). The optical
density of each area was automatically converted to
pixel number. The pixel number/area of the periphery
and interior of a section was subtracted from the mean
number of the exterior (background) of each section.
The number of nuclei in the same area was counted and
converted to the number per mm2. Because these measurements were taken from only two samples, statistical
analysis was not performed. However, the data were
used to support our understanding of the heterogenic
spatial distribution of Saf-O-positive matrix and cells.
Data Analysis
The performance of the semipermeable membrane
pouch was assessed by a mean value and SD representing four samples. S-GAG and DNA data were analyzed
using one-way analysis of variance (ANOVA) followed
by Dunnett’s test for comparing all cutoff size and HP
profiles versus control (static) with p < 0.05 considered
statistically significant (GraphPad InStat ver. 3.00, San
Diego, CA). The optical density of Saf-O-positive matrix and the cell number in the randomly selected five
areas in each sample were measured and counted, respectively.
RESULTS
Performance of the Semipermeable Membrane Pouch
Infiltration of BSA (MW 70 kDa) was used to measure the performance of semipermeable membrane
pouches in two MW cutoff sizes and culture under static
conditions (no pressure), with cyclic HP/perfusion, with
constant HP/perfusion, and perfusion alone. Under static
conditions, less than 5% of BSA infiltrated the pouches
(regardless of cutoff size) in 24 h (Fig. 2). With perfusion, 18% of BSA infiltrated the 500-kDa pouch in 24
h, but less than 10% of BSA infiltrated the 250-kDa
pouch. With constant HP for 24 h, 40% and 20% of
BSA infiltrated the 500- and 250-kDa pouches, respectively. With cyclic HP for 24 h, 99.5% and 15% of BSA
infiltrated the 500- and 250-kDa pouches, respectively.
Biochemical Evaluation
The amount of S-GAG produced by bACs under
static conditions without a pouch (used as a control) was
85.4 ± 5.4 µg/gel (Fig. 3A). With cyclic HP, bACs in
the 500-kDa pouch accumulated significantly more SGAG (101.6 ± 7.7 µg/gel, p < 0.01) than the static control. S-GAG in the 250-kDa pouch was slightly less than
in the control. With constant HP, the amounts of S-GAG
in pouches of the 250 and 500 kDa cutoff sizes were
similar to the static control. With perfusion, S-GAG in
pouches of the 250 and 500 kDa cutoff sizes was significantly less than the control (p < 0.01).
The optical density of the Saf-O-positive matrix was
converted to pixel number and expressed as an index
(Fig. 3B). In all cutoff sizes, regardless of HP profiles
and static control, the density at the interior section was
greater than at the periphery. With perfusion, the periphery and interior of the 250-kDa pouch had similar density. However, there were differences between the interior and periphery of the 500-kDa pouch.
DNA content of bAC/gel after 7 days of culture under static conditions was 4.0 ± 0.5 µg/gel (Fig. 4A).
Figure 2. Performance of semipermeable membrane pouches with cutoff of (A) 250 kDa and (B)
500 kDa. The pouches were incubated in 0.1% BSA at static, perfusion at 100 µl/min, constant
HP at 0.5 MPa, or cyclic HP at 0.5 MPa, 0.5 Hz, each with replenishment at 100 µl/min. BSA
concentration within each pouch was converted to a percentage in a culture chamber. Data represent the mean and SD of four samples.
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NOVEL CELL CULTURE METHOD
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condition showed empty spaces and dense Saf-O-positive ECM (Fig. 5A). With a pouch under the static condition, Saf-O-positive ECM had less color intensity than
that without a pouch (data not shown). In addition, nuclei of the bACs in the pouch were pyknotic. Therefore,
the bAC/gel under static conditions was always incubated without a pouch in further studies, and that was
defined as the static control. With perfusion, bAC/gel
accumulated Saf-O-positive ECM at much lower levels
in the 250-kDa pouch (Fig. 5B) than in the 500-kDa
pouch (Fig. 5C). In addition, the volume of the gels with
perfusion was smaller than under static (without pouch)
or HP conditions. In order to compare a complete set of
all cutoff sizes, perfusion culture was eliminated from
further studies because necessary metabolic activity was
not shown with the 250-kDa cutoff pouch. With both
constant and cyclic HP, bACs accumulated dense SafO-positive ECM in gels within pouches of both 250-
Figure 3. (A) S-GAG accumulation by bACs cultured in gel
within pouches for 7 days. Bars represent the mean ± SD of
four samples. *Significant difference relative to static control,
p < 0.01. (B) Optical density of Saf-O-positive matrix by
bACs cultured in gel within pouches for 7 days. The optical
density in randomly selected 200 × 200 µm square at periphery (blank bar) and interior (closed bar) of each section was
measured with a CCD camera. The density was converted to
pixel number, the background subtracted, and expressed as an
index.
With cyclic HP, constant HP, and perfusion alone, DNA
was 4.1–4.7, 3.8–4.2, and 3.7–4.7 µg/gel, respectively.
DNA contents were similar across cutoff sizes and HP
profiles.
The cell number within randomly selected areas at the
periphery and interior of each section were counted and
normalized per mm2 (Fig. 4B). The cell density at the periphery and interior was similar under all HP profiles.
However, the cell number in the 250-kDa pouch with perfusion alone was greater than in the 500-kDa pouch.
Histological Evaluation
Characteristics of accumulated ECM differed notably
between constant and cyclic profiles of HP. After 7 days
of culture, bAC/gel without a pouch under the static
Figure 4. (A) DNA content in gel within pouches at day 7.
The pouches are exposed to cyclic HP 0.5 MPa, 0.5 Hz; 0.1
ml/min, constant HP 0.5 MPa, 0.5 Hz, 0.1 ml/min, and perfusion alone 0.1 ml/min. Bars represent the mean ± SD of four
samples. (B) Cell density in gel within pouches at day 7. The
cell number in randomly selected 200 × 200 µm square at periphery (open bars) and interior (filled bars) of each section
was counted under a microscope. The density was converted
to cell number per area (mm2).
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sizes and the profiles of fluid HP to which they are subjected. The best performance was found in the 500-kDa
cutoff pouch with cyclic HP at 0.5 MPa, 0.5 Hz, allowing infiltration of 99.5% of BSA in 24 h. Although performance was evaluated for only 24 h and was limited
to a typical serum molecule, we speculate that essential
nutrients infiltrated the pouch under the aforementioned
culture conditions.
Most bioreactors were designed to promote nutrient
supply to a 3D cell construct (28,31). However, it is
Figure 5. Photomicrographs of bACs in collagen gel after 7
days of culture. (A) A static culture control without a pouch,
(B, C) perfusion at 0.1 ml/min, (D, E) constant HP at 0.5 MPa,
0.1 ml/min, and (F, G) cyclic HP at 0.5 MPa, 0.5 Hz, 0.1
ml/min. Intensity of red indicates S-GAG accumulation (a bar
indicates 100 µm, Safranin-O, 5-µm-thick section).
and 500-kDa cutoff sizes. Less accumulation was seen
with constant (Fig. 5D, E) than with cyclic HP (Fig. 5F,
G). In particular, notably large amounts of Saf-O were
found within the 500-kDa pouch with treatment of cyclic HP at 0.5 MPa, 0.5Hz (Fig. 5G). The 500-kDa
pouch showed both intense and fibrous accumulation of
Saf-O-positive ECM (Fig. 5G).
bACs accumulated Col 2 in the gel with treatment of
constant or cyclic HP (Fig. 6A–D). Both constant and
cyclic HP showed dense Col 2 accumulation within the
500-kDa pouch, particularly pericellularly.
The bACs accumulated KS, one of the components of
aggrecan, with treatment of constant or cyclic HP (Fig.
6E–H). Constant HP elicited dense and fibrous accumulation of KS in the 500-kDa pouch. Cyclic HP produced
dense and fibrous accumulation of KS in the 500-kDa
pouch.
DISCUSSION
Semipermeable membrane pouches have varied permeability to BSA depending on their molecular cutoff
Figure 6. Photomicrographs of immunohistochemistry of
(A–D) collagen type II (Col 2) and (E–H) keratan sulfate (KS)
in gel after 7 days of culture. Intense brown indicates accumulation of each matrix component. Nuclei were counterstained
with hematoxylin (a bar indicates 100 µm, 5-µm-thick section).
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NOVEL CELL CULTURE METHOD
vital that the neo-tissue build the ECM within the cell
construct, and the high rate of medium flow creates the
risk of washing away the ECM. A semipermeable membrane pouch was exposed to fluid-flow shear stress
through replenishing the medium. However, the stress
was thought to be negligible, because the replenishment
rate was 0.1 ml/min, and the culture chamber has an
obstacle platen to prevent jet flow through the injection
of medium. Thus, using a semipermeable membrane
pouch is a useful method to reduce such risk.
Histological evaluation indicated that nutrient supply
to the pouches was probably restricted by the semipermeable membrane in both static and perfusion culture.
On the other hand, constant and cyclic HP apparently
allowed for the maintenance of cell viability and ECM
production by bACs in a pouch. The ECM in constant
and cyclic HP showed slightly different morphology, indicating that interstitial fluid motion due to each HP profile affected both the assembly and the amount of ECM
(31). Medium volume shrinks by 0.022% with HP at 0.5
MPa. Even though this change in volume is quite small,
the cyclic HP seems to increase permeability of soluble
molecules through the semipermeable membrane. HP
application involves medium perfusion at the same replenish rate as perfusion alone. Thus, HP should be recognized as a major stimulator to promote ECM accumulation by bACs. Although determining the mechanism
of the effects of HP was not our aim in this study, extended studies using pouches and HP indicate that HP
stimulates both anabolic and catabolic mRNA expression (20). The mechanism of HP stimulation is under
investigation. Besides direct effects of the HP, we speculate that eventually nutrients and gases could be supplied, allowing large-molecule products to be retained
within the pouch and small-molecule debris to be exuded (Fig. 7). This speculation is partially supported by
Klein and Sah’s model by diffusion or convection using
a semipermeable membrane (15). The amount of S-GAG
in the 500-kDa pouch with cyclic HP was 18% (p < 0.01)
greater than in the static control without a pouch. However, the small difference in the amounts was hard to
correlate with histological changes (Fig. 5F, G).
The cell density of the cell/gel construct with perfusion was higher than with other culture conditions (Fig.
4B). The properties and shape of the gel were not considered in this study, because it was amorphous and
formed within a flexible plastic tube. It was difficult to
determine whether the gel was degraded or shrank. One
possibility was that the gel shrank and made the cell
density appear higher. Thus, we have to carefully evaluate cell density and determine the optimal culture conditions for secure clinical applications.
Recently, we developed a structural 3D construct to
replace damaged and/or defective cartilage, because the
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Figure 7. Schematic drawing of a semipermeable membrane
pouch device.
goal of engineering tissue was to build a cell construct
similar to native cartilage in vitro. By extending the concept from replacement to repair of tissues, the clinical
applications can be broadened. One proposal was to inject cells/gel into fibrocartilage to repair insufficient regeneration of hyaline cartilage or to augment defective
cartilaginous tissue (e.g., ears and nose). The injected
chondrocytes/gel are expected to release cytokines and
recruit appropriate cells from adjacent tissue. Other
ideas may include injecting cell/gel into the vertebral
space for intervertebral disc repair. By combining new
methods, even uncontrollable and fragile amorphous cell
carriers can be used to develop further applications. In
this study, we focused on a profile of HP and the permeability of semipermeable membrane pouches for tissue
engineering applications. Recently, we demonstrated
that S-GAG accumulation by chondrocytes or chondrogenic adipose-derived stem cells increased with cyclic
HP at 0.5 MPa, 0.5 Hz followed by static culture for 3
weeks (15,24). Beyond our current studies, applications
using a semipermeable membrane pouch and HP will be
explored for other applications. In addition, deformation
of the amorphous construct within a semipermeable
membrane can be made with a strain module. Cartilage
physiology in response to weight bearing and joint loading will be reproduced with those apparatus.
In conclusion, we developed an HP/P culture system
that applies HP by compressing the medium fluid (23).
We also developed a semipermeable membrane pouch
to incubate chondrocytes in amorphous gel. We found
that constant or cyclic HP maintained viability and increased dense ECM by bACs in gel. Combining the HP
and pouches will be explored to develop useful applications for tissue engineering.
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ACKNOWLEDGMENTS: This research was partially supported by CIMIT (Boston, MA) and Takagi Industrial, Co.,
LTD. (Shizuoka, Japan). Special thanks to Dr. Toshimi Murata
for technical assistance.
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