A NEW CONCEPT OF CHONDROCYTES CULTURE USING A SEMIPERMEABLE MEMBRANE POUCH AND
PHYSICAL STIMULI
+*Mizuno, S; Vrahas M.S.
+Brigham and Women’s Hospital, Boston, MA
email: [email protected]
INTRODUCTION
Tissue engineering has faced many challenges in repairing or replacing
damaged tissue to restore biomechanical and metabolic functions. For
orthopedic applications, rigidity and stiffness are critical, as implants
must withstand weight-bearing, joint-loading, and stretching.
Characteristics of the cell construct vary according to materials and
structure. But, if the cartilage defect was filled with surface layer or with
migrated cells, chondrocytes with their own matrix can be injected
underneath of the layer. We designed an injectable cell/matrix to
maximize chondrogenic activity in vitro using a semipermeable
membrane and physical stimuli and to promote regeneration at the host
tissue. Molecular weights of the newly synthesized ECMs e.g., mainly
chondrocyte-specific proteoglycan, aggrecan, collagen type II are >500
kD. Thus, the ECMs can be maintained within a semipermeable
membrane pouch (cut-off size 500 kD). However, essential nutrients
and gases (<500 kD) can be exchanged through the membrane. By
manipulating hydrostatic pressure (HP) and its algorithms combining
with static (resting or free swelling) condition, cell viability,
proliferation, and phenotypic expression may be altered. At optimal
aforementioned physical stimuli, chondrocytes may start the
regenerative process in vitro. Chondrogenic activity of bovine articular
chondrocytes (bACs) isolated from a middle zone was evaluated with
cell viability and biosynthesis histologically and biochemically.
MATERIALS AND METHODS
A. Culture device preparation.
A semi-permeable membrane pouch was made from PVDF tubing (1
mm ID, 1.2 mm OD, 30 mm length) with defined cut-off size (500 kD).
One end of the tubing (5 mm) was folded and sealed with a stainless
steel clip. The tubings were autoclaved in water.
B. Chondrocyte isolation and culture.
A bovine shoulder (2 -3 week old calf) was purchased from a local
abattoir. Cartilage cubes (5x5x2~5 mm) with subchondral bone were
harvested from the weight-bearing region or area of the femoral condyle
of the forelimb. Middle zone cartilage slice was isolated by removing
the surface and deep zone under a stereomicroscope. Chondrocytes
were enzymatically isolated from the slices at 37˚C for 12 hr. 3 x 105
cells in a 15-µl aliquot of DMEM/Ham’s F-12 were suspended
neutralized collagen type I solution, injected into the semipermeable
membrane tubing, incubated at 37˚C overnight to allow gel formation.
Another end of the tubing was sealed to make a pouch. The pouches
were incubated under varied culture conditions: static at 19% O2, 3%
O2, constant HP (0.5 MPa), cyclic HP (0.5 MPa, 0.5 Hz), and combined
HP/static (4/20 hr a day; 12/12 hr a day). Cells were incubated in
DMEM/Ham F-12 or F-12 with 10% FBS, 100 units/ml penicillin, and
100 µg/ml streptomycin for 7 days.
C. Histological and Biochemical evaluation.
After 7 days, the cells/gels were ejected with flushing PBS. Live/dead
was determined with fluorescent dyes with a confocal microscopy. 3
pouches were harvested for histological evaluation and 4~5 pouches
were for biochemical assays. 2 samples were fixed with 2%
paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4°C and were
embedded in either glycolmethacrylate for metachromasia staining with
0.2% toluidine blue-O, pH 4, or in paraffin for collagen type II
immunostaining. Each of 3~5 replicates was digested in 200 µl of 125
µg/ml papain for 12 h at 60°C and were used for measurement of
sulfated glycosaminoglycan (S-GAG) by DMB color metric assay and
DNA by fluorescent assay using Hoechst 33258.
RESULTS
Histological evaluation. bACs were distributed homogeneously
throughout the gel. After 7 days of exposure to 19% O2, bACs
accumulated by fine fibrous metachromatic matrix. At 3% O2, denser
fibrous metachromatic matrix was seen uniformly in a gel. With constant
HP and cyclic HP, there were greater S-GAG accumulation than without
HP, but less collagen type II than without HP. This suggested that HP
enhanced S-GAG production but declined collagen type II production.
Static 3% O2
Cyclic HP, 3% O2
Constant HP, 3% O2
Fig 1 S-GAG accumulation. Toluidin blue stain. The cells were
incubated with DMEM/F12+10%FBS+antibiotics. Medium was
continuously replenished at 100 µl/min except the static condition.
Significant differences in both cell shape and structure were noted
among algoriythms: Intermittent 4/20 hr/day, 12/12 hr/day, and
continuous 24 hr/day static, S-GAG accumulation at 24 hr/day was
homogenious compare to intermittent. Empty pockets were seen in the
gels treated with intermittent HP.
Fig 2
Biochemical evaluation of ECM. Since a cells/gel was so fragile
resulting in fragmentation of the samples, S-GAG production was
expressed per DNA. Data from overlapping multiple groups were
normalized with 3% O2 and no pouch condition of each experiment.
4/20 hr and 12/12 hr algorithms significantly enhanced S-GAG/DNA
accumulation (Fig 2, p < 0.01). But 24 hr enhanced 1.1-fold compared
to static but significantly enhanced compare to 19% O2 static (P<0.05).
F12 medium reduced S-GAG and type II collagen accumulation.
DISCUSSION
A new concept to incubate chondrocytes was demonstrated using a
collagen gel, a semi-permeable membrane pouch, and a bioreactor. A
semipermeable membrane pouch maximized accumulation of S-GAG
and type II collagen. Without direct influences by medium flow, effects
of pure HP were evaluated. At 3% O2, DNA decreased compare to the
initial although more S-GAG accumulated. Medium flow promoting
mass transfer was critical to maintain viability for longer period of static
culture. Constant or cyclic HP with medium flow using a bioreactor was
useful stimuli for S-GAG production but not for collagen type II. A
static culture period may be more favorable for accumulation of S-GAG.
However, there is less type II collagen at a middle zone. HP may be
useful to maintain histogenesis of middle zone. S-GAG/DNA does not
indicate quality of S-GAG accumulation. HP at 24 hr/day allowed
uniform distribution of S-GAG (Fig 1). Resting period after HP or post
continuous HP stimuli may have potential to promote S-GAG
production. Based on optimized culture methods, our hypothesis is that
injectable cell/matrix treated with physical stimuli will facilitate
regeneration of cartilage in situ. Clinical applications may involve
injection of the cell/gel underneath of incomplete repair tissue e.g.,
fibrocartilage. This new injectable cell/gel pretreated with physical
stimuli may rescue failed cases of resurfacing articular cartilage
attempted by other means e.g., autologous chondrocyte implantation
(transplantation) and microfracture.
53rd Annual Meeting of the Orthopaedic Research Society
Poster No: 1492