1
CD44 antibody does not prevent, but upregulates dedifferentiation of adult
chondrocytes into fibroblast
Albert Chi, Mark Shteyn
BACKGROUND
Joint conditions such as arthritis pose a significant challenge for biological
engineering due to the number of people affected by such diseases and the unique
problems of repairing or forming new cartilage. Chondrogenesis, the production of the
primary cells called chondrocytes that are found in cartilage, occurs infrequently in vivo
due to the avascular nature of chondrocytic growth sites and the low turnover rate of old
chondrocytes. The lack of blood vessels and minimal access to growth factors make in vivo
regeneration or growth-promotion difficult. Growing new chondrocytes in vitro for in vivo
implantation may provide a therapeutic solution to joint disease.1
There are currently several problems associated with in vitro production of
chondrocytes including the difficulty of obtaining a high enough yield of singular
chondrocytes to produce cartilage due to the sensitivity of growing chondrocytes in
culture. One major problem is that chondrocytes tend to dedifferentiate into fibroblast in
vitro.2 Current methods for growing chondrocytes include using hydrogels such as those
made of alginate to create a 3D scaffold to prevent dedifferentiation and hence provide
higher yields. Studies have shown that although this certainly helps, dedifferentiation still
occurs.3 One reason this could occur is that, although using a 3D scaffold is meant to
minimize cellular interaction, the chondrocytes within the scaffold become closely packed
together, which can cause dedifferentiation.4
2
One way to prevent dedifferentiation is by adding proteins or other biochemical
factors.5 CD44 is a cell-surface protein that is involved with cell-cell interaction and
adhesion and has been implicated in the dedifferentiation/redifferentiation pathway of
chondrocytes into fibroblast.6-7 In the dedifferentiated state, CD44 levels have been shown
to be elevated, whereas in the chondrocytic state, a decrease in CD44 concentration occurs.
Recently, CD44 antibody has also been shown to affect differentiation during
chondrogenesis from mesenchymal stem cells.8
Stem cells, however, are difficult to
maintain and produce lower yields than adult chondrocytes. We therefore investigated the
addition of CD44 antibody to adult chondrocytes grown in a 3D scaffold to see if this
method of direct CD44 inhibition will produce better yield during in vitro chondrogenesis.
DESIGN
We used a 1% alginate gel formed in beads and grown in standard medium. We
added bovine CD44 antibody at least twice a week for 2 weeks. Cell viability assay was
done using the LIVE/DEAD test kit. RT-PCR was performed before transcript analysis on a
1.2% agarose gel, checking for type 1 collagen and type 2 collagen in a sample containing
CD44 against a control without CD44. We analyzed cell viability and gel data using the
image program ImageJ. ELISA protein assay was performed to check for CN1 and CN2
relative to CD44.
RESULTS
Cell Viability
3
(B)
(A)
(C)
1
2
3
4
5
Avg
Area
XStart
YStart
22.015
1968
481
17.541
1626
534
92.512
2075
810
21.986
741
1144
116.347
1379
1537
54.0802
Figure 1 – Cell viability assay. Cell growth was not
affected by the addition of CD44. Cells were stained with
STYO10 (green) and ethidium homodimer-2 (red) using
the LIVE/DEAD kit and inspected under a fluorescence
microscope. (A) An unfiltered photo of cells excited for
green fluorescence. (B) Image of viable cell outlines as
analyzed by ImageJ with an adjustment for contrast and
threshold to decrease noise. (C) Table of cells detected
and their areas.
Cell growth was decreased by the addition of CD44 antibody, although the bead
itself was particularly delicate and brittle to pipetting. Cells were assayed with LIVE/DEAD
test kit, analyzed through a fluorescence microscope at FITC excitation for lives cells and
EthD-1 excitation for dead cells (Figure 1a). Images from microscopy were analyzed
through ImageJ to give us cell counts and cell sizes (Figure 1b, c).
RT-PCR and Gel Electrophoresis
Ratios
CN1/GAPDH
CN2/GAPDH
CD44
Control
2.435897 1.99322
0.660108 0.855944
Figure 2 – Transcript
assay.
RNA
RT-PCR
fragments were run through
gel electrophoresis in a
1.2% agarose gel at 125V for
45 minutes and analyzed
with ImageJ. Samples run
are CNI control, CNI+CD44,
CNII control, and CNII+CD44
(labeled above).
ImageJ
analysis
showed
that
whereas the CN1/GAPDH
ratio
for
CD44
was
heightened versus control,
CN2/GAPDH was decreased.
Transcript assay showed that
dedifferentiation occurred even with
the addition of CD44 antibody, in fact
increasing the rate of fibroblast
formation relative to control. GAPDH
was our control signal (Figure 2,
image).
Comparing
Collagen
1
4
(CN1)/GAPDH ratios and Collagen 2 (CN2)/GAPDH ratios between samples with CD44anbtibody added and controls showed that CD44 antibody increased type 1 collagen
expression (fibroblast) and decreased type 2 collagen expression (chondrocytes) (Figure 2,
table).
ELISA Protein Assay
ELISA assay showed that CN1 was thoroughly expressed, whereas CN2
measurements were insignificant (yielding negative values) (Figure 3).
The control
concentration gradient data was
regressed linearly to calculate the concentrations of CN1 and CN2. CN1 concentration was
Protein Concentration vs. Absorbance
350
Collagen I
Standards
Collagen II
Standards
Collagen I
Experimental
Collagen I
Control
Collagen II
Experimental
Collagen II
Control
Linear (Collagen
I Standards)
Linear (Collagen
II Standards)
Protein Concentration (ng/mL)
300
250
200
150
100
50
0
0
-50
0.1
0.2
0.3
Absorbance
0.4
0.5
0.6
y = 717.03x + 4.9289
R² = 0.9995
y = 541.76x - 10.14
R² = 0.9399
Figure 3 – ELISA results. ELISA ran at 26.4˚C to detect type 1 and type 2 collagen absorbance in samples
containing CD44 and compared to a standard gradient curve (the line of best fit and its equation is shown).
Through this analysis we were able to see that CN1 was more highly expressed in samples containing CD44
whereas both CD44+ and CD44- results showed a lack of significant results for CN2 concentration.
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much higher in samples with CD44 added than control with a difference of 177.4 ng/mL,
from 40.5 ng/mL in CD44- control to 224.0 ng/mL in CD44+ (about a five-fold increase).
DISCUSSION/FUTURE WORK
CD44 antibody is ineffective in decreasing the rate of chondrocyte dedifferentiation
into fibroblast, in fact seemingly increasing the rate of dedifferentiation. The gel analysis
from RT-PCR shows that CN1 was more highly expressed while CN2 was downregulated
relative to control. The increased concentration of CN1 in samples with CD44 antibody also
indicates that dedifferentiation was promoted, rather than inhibited by the addition. The
low detection of CN2 concentration in our samples shows that there was much more
fibroblast than chondrocytes. The cell viability tests show that CD44 antibody addition did
not affect cellular growth, but did affect cell differentiation as very few cells were observed.
One possible explanation for the lack of regulation of dedifferentiation by the
addition of CD44 antibody is that the CD44 antibody did not affect CD44 functionality.
CD44 is a receptor that is perhaps better inhibited by the use of a ligand inhibitor. The
attachment of antibody to the receptor could block the active sites, but it could also not.
CD44 antibody could have also increased the concentration of CD44 in the culture by
exposing it more, which would increase dedifferentiation. To measure this effect, we could
have used the CD44 antibody to run a direct ELISA to detect CD44 concentrations. We
should have also tested for CD44 antibody concentrations to be able to make relations
between CD44 antibody concentrations to CD44 concentrations and finally to CN1 and CN2
concentrations. Further experimentation on the possible regulation of dedifferentiation via
CD44 could be done by the trial of certain inhibitors, rather than antibodies, which would
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better permeate through the alginate scaffold by nature of their smaller size. Another
interesting experiment could be to silence CD44 expression using siRNA instead of a
chemical substrate.
REFERENCES
1. Gomez-Camarillo MA, Almonte-Becerril M, Vasquez Tort M, Tapia-Ramirez J, Kouri Flores
JB. Chondorcyte proliferation in a new culture system. Cell Prolif. Apr 2009; 42(2):207-18.
2. Gomez-Camarillo MA, Almonte-Becerril M, Vasquez Tort M, Tapia-Ramirez J, Kouri Flores
JB. Chondorcyte proliferation in a new culture system. Cell Prolif. Apr 2009; 42(2):207-18.
3. Lee CSD, Gleghorn JP, Choi NW, Cabodi M, Stroock AD, Bonassar LJ. Integration of layered
chondrocyte-seeded alginate hydrogel scaffolds. Biomaterials 2007; 28:2987-2993.
4. Genes NG, Rowley JA, Mooney DJ, Bonassar LJ. Effect of substrate mechanics on
chondrocyte adhesion to modified alginate surfaces. Biochemistry and Biophysics 2004;
422:161-167.
5. Hoben GM, Willard VP, Athanasiou KA. Fibrochondrogenesis of hESCs: growth factor
combinations and cocultures. Stem Cells Dev Mar 2009; 18(2):283-92.
6. Yoshida M, Yasuda T, Hiramitu T, Ito H, Nakamura T. Induction of apoptosis by anti-CD44
antibody in human chondrosarcoma cell line SW1353. Biomed Res. Feb 2008; 29(1):47-52.
7. Yanada S, Ochi M, Adachi N, Nobuto H, Agung M, Kawamata S. Effects of CD44 antibody –
or RGDS peptide – immobilized magnetic beads on cell proliferation and chondrogenesis of
mesenchymal stem cells. J Biomed Mater Res A 2006 Jun 15; 77(4):773-84.
8. Albrecht C, Schlegel W, Eckl P, Jagersberger T, Sadeghi K, Berger A, Vécsei V, Marlovits S.
Alterations in CD44 isoforms and HAS expression in human articular chondrocytes during
the de- and re-differentiation processes. Intnat’l Journal of Mol Med Feb 2009; 23(2):253259.