Chondrocyte Viability in 1-D Diffusion Systems for Gradient Limited Studies in 3-D Cultures
1
Lin, D.D.W.; 2Babalola, O.M.; 1Dorvee, J.R.; 1Bodereau, A.; 1Eckes, K.M.; 3Boskey, A. L.;2Bonassar, L.; +1Estroff, L.A.
+1Department of Materials Science & Engineering, Cornell University, Ithaca, NY, 2Department of Biomedical Engineering, Cornell University, Ithaca, NY,
3
Mineralized Tissue Research, Hospital for Special Surgery, New York, NY.
+lae37@cornell.edu
INTRODUCTION:
Hydroxyapatite (HAP) mineral can be grown in gels by diffusing
calcium and phosphate ions through a gel [1-2]. Experimental models
have been established to study HAP formation and growth using these
techniques. Until now, these models have been acellular systems [3].
Viable cell cultures in such systems would allow both the study of cell
activity in the presence of ionic gradients and the effect of cells on the
mineralization process. Such studies can lead to potential models of
bone growth in the growth plate and at the bone-cartilage interface. This
study demonstrates a procedure for introducing bovine articular
chondrocytes into a modified double diffusion system. The cell activity
and viability in such an environment has been examined over a period of
12 days. An average viability above 90% was maintained throughout
the experiment.
This system represents an approach towards
mineralization studies under cellular regulation.
METHODS:
Articular Chondrocytes. Cartilage tissue was harvested from
condyles of 1-3 day old bovine knee joints. The articular chondrocytes
were isolated from the tissue via collagenase digestion. Chondrocytes
were suspended at 50e6 cells/mL of 2X concentration of calcium-free
and phosphate-free Dulbeco’s Modified Eagle Medium (DMEM) with
10% fetal bovine serum (FBS), and 1% antibiotics/antimycotics
(AB/AM). Cell viability and density were determined by staining cells
with trypan blue and calculated from a hemocytometer. Only cell
solutions with viability exceeding 89% were used for these studies.
Tube Construct. Tubes were constructed from 7.5 cm Biopharm®
tubing (gas-permeable silicone) with 3 cm glass tube insets on either
side (Figure 1). Tubes were coated with 0.5% polyethylene-imine (PEI)
on the interior for 24 hrs. A
Figure 1A
0.4 cm length of the interior
of the tubing was left
uncoated for the chondrocyte
seeded
section.
Tube
constructs were sterilized in
70% ethanol before use.
Figure 1B
Tube-Cell-Gel Assembly.
Articular chondrocytes were
incorporated into the tube
construct in a layer-by-layer
assembly process.
CellFigure 2
culture grade agarose (BP165-25, Fischer Scientific)
was made into 2 wt% gels
with DMEM. The tube was
plugged on the end so that
only the Biopharm portion of
the tube construct came in
contact with the agarose. The
first layer of gel was injected into the tube to the PEI-free inner surface
of the tube and allowed to set. The next layer is a 0.4 cm layer of 25e6
chondrocytes/mL seeded gel in the PEI-free section of the tube which is
allowed to set. The final chondrocyte-free layer of agarose was added to
the tube for a total gel construct of 6 cm in length after it had set. The
chondrocyte-seeded layer was placed at the center of the tube, 2.8 cm
from the end (Figure 1A) or 4 cm from the end (Figure 1B).
Single Tube Reservoirs. Gel-filled tubes were inserted between
two modified 250 mL media bottles under sterile conditions (Figure 2).
The media bottles were filled to 200 mL and the system placed into an
incubator at 37 °C with 5% CO2.
Tube Construct Assessment. The tube construct was assessed for
bypass leaks and mineralization through the agarose gel. Bypass leaks
were determined with colorometric assay by connecting the tube
construct to two reservoirs, one filled with dye, the other without dye.
To assess mineralization in the tube construct, a reservoir of 100 mM
CaCl2 and another of 100 mM NaH2PO4 were connected to the tube and
mineralization bands in the gel were examined at 5 days. All solutions
were buffered at pH 7.4 with Tris buffer.
Chondrocyte Analysis. At days 0, 3, 6, 9, and 12, triplicates of the
tubes were removed from the single-tube diffusion system. The cellseeded portion of the gel is removed and sectioned.
Viability. Chondrocyte viability in sectioned gels was determined
using Invitrogen Alive/Dead® stain.
RESULTS:
The single tube diffusion system has shown itself to be a viable
system for the study of bovine articular chondrocyte activity through 12
days. An average viability
above 90% was successfully
maintained
through
the
course of 12 days in the
single tube reservoir system
(Figure 3). Comparing the
viability of cells seeded at the
center of the tube construct
and near the end of the tube
construct (see Fig. 1), there
was no major change, indicating that sufficient nutrients can reach the
chondrocytes in the system from the media despite their location in the
tube construct. Chondrocyte cell morphology is retained from Day 0
(Figure 4A) through Day 12 (Figure 4B). Cell division is observable in
later days (Figure 4B).
The colormetric assay demonstrated that the tube construct with
PEI coating suffers no bypass leaks. The dye
Figure 4A
was only able to diffuse through the agarose
and not between the agarose gel and tube
walls. Interdiffusing calcium and phosphate
through the tube also confirms that no bypass
leaks occurred and that mineral bands could
75 µm
be formed across the gel in the tube
construct.
Figure4B
DISCUSSION:
The single tube diffusion system has
been shown to be a good environment for
maintaining articular chondrocyte viability
and health.
The tube construct design
75 µm
requires diffusion to be the vehicle of
transporting material from the reservoirs to the chondrocytes. The
agarose has sufficient binding to the tube walls so that nutrients, ions,
and other growth factors can reach the chondrocytes only via diffusion
through the gel. For larger scale experiments, the tube construct can be
inserted into a multiple tube system connected to two reservoirs, as done
by Boskey for mineral formation [1]. Spatial gradients of Ca, or other
ions or proteins, can be introduced to the single tube diffusion system to
assess cell activity and viability. Diffusion of multiple interacting ions
such as phosphate and calcium would allow the study of mineralization
in gel systems as affected by cellular processes.
REFERENCES:
1. Boskey, A. L., Hydroxyapatite Formation In A Dynamic Collagen
Gel System - Effects Of Type-I Collagen, Lipids, And Proteoglycans.
J. Phys. Chem. 1989, 93 (4), 1628-1633.
2. Hunter, G.K., Goldberg, H.A., Nucleation of hydroxyapatite by bone
sialoprotein. Proc. Natl. Acad. Sci., 1993, 90, 8562-8565
3. Hunter, G., Poitras, M., Under hill, T., Grynpas, M., Goldberg, H.,
Nucleation and inhibition of hydroxyapatite formation by mineralized
tissue proteins, Biochem. J., 1996, 317, 59-64.
ACKNOWLEDGEMENTS:
This work was supported by the Cornell Center for Materials Research
(CCMR, NSF MRSEC Program No. DMR 0520404), Cornell
Engineering Learning Initiatives (ELI), Nanobiotechnology Center
Cornell (NBTC, NSF STC Program No. ECS-9876771).
Poster No. 812 • 56th Annual Meeting of the Orthopaedic Research Society