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Title: Effects of Calcium Concentration on Chondrocyte Maintenance in Chondrocyte-Alginate
Hydrogel Constructs
Authors: and Danyi Wu
Introduction
Cartilage is a connective tissue critical in joint function, as its strength and flexibility
allows for load transfer between bones and muscles and its role as a lubricant reduces the wear and
tear of bones and muscles. With traumatic injury and degenerative diseases such as osteoarthritis,
joint cartilage loses some of its essential mechanical functions. At present, the only cell-based
therapy for cartilage repair approved by the US Food and Drug Administration is autologous
chondrocyte implantation. However, donor-site morbidity and prolonged cell culture and surgical
times are commonly experienced downsides of this approach (Wan 2008). An alternative
treatment is the development of a functional substitute for cartilage using a chondrocyte-alginate
gel construct. Alginate hydrogels have previously been reported to facilitate the maintenance of
chondrocytic phenotype (Hauselmann 1994). Additionally, alginate mimics such soft tissue
characteristics of cartilage as elasticity and diffusivity. Final advantages of using alginate
hydrogels for cartilage tissue engineering include its biocompatibility, low toxicity, low cost, and
spontaneous gelation (Wan 2008). Alginate hydrogels are formed through the crosslinking of
alginate chains in the presence of cations such as calcium. In this experiment, we investigated the
effects of calcium concentration on chondrocyte maintenance in chondrocyte-alginate constructs.
We expected higher Ca2+ concentrations to lead to increased crosslinking, and therefore increased
gel density. This was expected, in turn, to decrease the porosity of the construct, making cell-cell
interactions more difficult and ultimately resulting in decreased chondrocyte maintenance. In this
study, the production of Collagen II was used as a marker of the chondrocytic phenotype.
Therefore, increased calcium concentration in alginate hydrogels was expected to result in
decreased Collagen II expression in chondrocyte-alginate constructs.
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Results
Chondrocytes were seeded in Protanal LF 10/60 alginate beads created under two
conditions: one sample of beads was created using 102 mM CaCl2, and the other sample using
510mM CaCl2. The Protanal alginate was selected because of its relatively high G/M ratio of
70/30, since only G-blocks can cross-link to form alginate gels. After 7 days, a live/dead
fluorescence assay was performed on one alginate bead from each sample to determine the
viabilities of our two cell cultures. Twelve days after cell seeding, RNA and protein samples were
collected from each cell sample to be measured for Collagen I&II transcript and protein
production, respectively. RNA samples were used to create cDNA through reverse transcription,
and the cDNA of interest (either Collagen I or Collagen II) was amplified through PCR. This
cDNA was then run through an agarose gel for transcript analysis. Protein samples were subject
to indirect ELISA assays for Collagen I and Collagen II protein analysis.
Live/Dead fluorescence assays revealed that cells were mostly alive based on microscope
observation, although fluorescence signals were dim at best and cells could barely be
distinguished from background in images taken. This is likely due to the fact that the fluorescence
microscope was used under sub-optimal conditions: while all room lights were turned off, the
environment around the microscope was not completely dark. When cells were viewed under the
microscope, some green fluorescence was observed in both samples, indicating that they were
alive. Additionally, in the 510mM CaCl2 sample, a few red spots were also observed, indicating
cell morbidity. However, images taken of microscope observations did not show clear distinctions
between cell signals and background, so quantitative analysis through ImageJ could not be carried
out.
Collagen I and Collagen II transcript analysis through semi-quantitative RT-PCR revealed
decreased Collagen II transcripts in cells cultured in alginate with higher CaCl2 concentrations.
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RNA isolated from cells was relatively pure, yielding A260:A280 values of 2.0 for both samples.
After mRNA was converted to cDNA through reverse transcription, GAPDH and either Collagen I
or Collagen II primers were used to amplify these gene transcripts. GAPDH was used as an
internal standard due to its relatively constant expression in all cells. Figure 1A shows bands of
relatively equal intensity for Collagen I expression in both samples, but a much brighter band for
Collagen II transcripts in the 102mM CaCl2 sample when compared to the band for Collagen II
transcripts in the 510mM CaCl2 sample. Quantitative analysis of band intensities using ImageJ
software confirms these observations, as shown in Figure 1B.
Collagen I expression was
approximately equal in both samples (3.093 for 102mM CaCl2 samples vs. 2.771 for 510mM
CaCl2 samples), and Collagen II expression was much higher in 102 mM CaCl2 samples (3.402 for
102mM CaCl2 samples vs. 0.924 for 510 mM CaCl2 samples).
Collagen I and Collagen II protein analysis through indirect ELISA assays revealed
decreased Collagen II protein production in cells cultured in alginate with higher CaCl2
concentrations. Standards used for ELISA assay ranged from 0.078-10g/mL, but only the 4
lowest concentrations (0.078, 0.156, 0.312, and 0.625g/mL) were used to create the calibration
curve since absorbance readings for remaining calcium concentrations were saturated, therefore
disrupting the linear fit correlating collagen concentration and absorbance. Calibration curves for
both Collagen I and Collage II standards had r2>0.99. Similar to collagen transcript results,
Collagen I protein expression was relatively equal in both samples (41.9ng/mL in 102mM CaCl2
samples vs. 39.6ng/mL in 510mM CaCl2 samples), and Collagen II protein expression was higher
in 102 mM CaCl2 samples (10.3 ng/mL for 102mM CaCl2 samples vs. 0.0 ng/mL for 510 mM
CaCl2 samples).
Discussion
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Because both Collagen II transcript and protein production decreased in cells cultured in
alginate with higher concentrations of CaCl2 and Collagen II is a marker of the chondrocytic
phenotype, our results suggest that increased Calcium concentration in chondrocyte-alginate
constructs leads to decreased chondrocyte maintenance. This agrees with our expectations for the
experiment, since increased calcium concentration was expected to result in increased crosslinking, and therefore decreased porosity and decreased ability of cells to receive signals to
maintain the chondrocyte phenotype. However, our experiment used only two different calcium
concentrations and only two samples per calcium concentration. To determine if this indirect
relationship between calcium concentration and chondrocyte maintenance actually exists and is
not simply due to chance, it would be desirable to use a wider range of calcium concentrations and
to create more samples for each calcium concentration so the statistical significance of the
observed relationship can be determined. In addition, because our collagen sample protein
concentrations for the ELISA were very low, with absorbance readings just between those found
for background and the first standard concentration, one might consider allowing cells to grow for
longer periods of time so they can produce more protein. In general, it is desirable to have
absorbance readings in the middle of standard calibration curves, whereas our absorbance reading
did not even fall within the calibration curve range.
The specific aim of our experiment was to assess the effects of calcium concentration on
chondrocyte maintenance. However, the broader implication of this experiment would be to
determine the best calcium concentration to be used for chondrocyte-alginate hydrogel constructs
for cartilage repair. Since the chondrocyte-alginate construct will deliver directly into the body,
the effects of calcium concentration on the biocompatibility of the construct should be investigated
in future experiments. Once a biocompatible range of calcium concentrations has been
determined, one could then look at other properties of chondrocyte-alginate constructs affected by
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calcium concentration. In the case of cartilage repair where we aim to maintain the chondrocyte
phenotype, it is more desirable to use alginate with lower concentrations of calcium. However, the
alginate hydrogel must still be strong enough to fulfill the load transfer function of cartilage tissue.
Gel strength is directly related with calcium concentration since increased crosslinking generally
leads to increased density, so it might be more desirable to use higher concentrations of calcium.
Thus, there is a tradeoff between chondrocyte maintenance and chondrocyte-alginate strength in
variations of calcium concentrations in alginate hydrogels. Future experiments could investigate
this relationship to determine the optimal calcium concentration for a chondrocyte-alginate
construct that will both maintain chondrocyte phenotype and be strong enough (both in terms of
tensile strength and compressive strength) to function as cartilage tissue does. In addition, since
the ultimate goal of tissue engineering is generate new tissue in vivo, it would be desirable for the
alginate scaffold to eventually degrade once enough cartilage tissue is generated. Thus the effects
of calcium concentration on the degradability of alginate scaffolds should also be investigated.
References
Wan, Leo, Jie Jiang, Diana Arnold, X. Guo, Helen Lu, and Van Mow. Calcium Concentration Effects on the
Mechanical and Biochemical Properties of Chondrocyte-Alginate Constructs. Cellular and Molecular
Bioengineering 2008; 1:93-102. (http://www.springerlink.com/content/g6r10wh512843027/)
Hauselmann, H.J., J. Fernandes, S.S. Mok, T. M. Schmid, J.A. Block, M.B. Aydelotte, K.E. Kuettner,
and E.J. Thonar. Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate
beads. Journal of Cell Science 1994; 107:17-27. (http://jcs.biologists.org/cgi/content/abstract/107/1/17)
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Module 3 Figures
Ladder
102 mM
CaCl2
CNI
510 mM
CaCl2
CNI
102 mM
CaCl2
CNII
510 mM
CaCl2
CNII
A
102 m M
CaCl2
CNI
B
Effects of [Calcium] on Collagen I/II
Transcript Production
4.000
Normalized Expression
3.500
3.000
2.500
102 mM CaCl2
510mM CaCl2
2.000
1.500
1.000
0.500
0.000
Collagen I
Collagen II
Figure 1. Effects Of Calcium Concentration On Collagen I/II Transcript Production, As
Measured By RT-PCR. RT-PCR was carried out to create cDNA from cell lysate mRNA and to
amplify the cDNA of interest, either Collagen I (CNI) or Collagen II (CNII). GAPDH
(represented by lower band in all lanes) was used as an internal standard used for normalization of
collagen expression. (A) cDNA created from samples of cells cultured in alginate with 102mM
CaCl2 and 510mM CaCl2 was run through an agarose gel, along with a standard ladder, to
measure relative expression of Collagen I and Collagen II in each sample. (B) ImageJ analysis of
band intensities on agarose gel reveal normalized expression levels of: 102mM CaCl2—CNI
3.093, CNII 3.402, and 510mM CaCl2—CNI:2.771, CNII 0.924.
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Effects of [Calcium] on [Collagen I/II] Protein Production
45
40
[Collagen] (ng/mL)
35
30
25
102mM CaCl2
510mM CaCl2
20
15
10
5
0
Collagen I
Collagen II
Figure 2. Effects Of Calcium Concentration On Collagen I/II Protein Production, As
Measured Through ELISA. Alginate beads from each sample, one sample containing alginate
made with 102mM CaCl2 and another with 510mM CaCl2, were dissolved and assessed for
Collagen I/Collagen II protein concentration through an indirect ELISA assay. Collagen protein
concentrations were measured as follows: 102mM CaCl2—CNI 41.9ng/mL, CNII 10.3ng/mL, and
510mM CaCl2—CNI 39.6ng/mL, CNII 0.0ng/mL.