POSTER ABSTRACTS - ISAKOS

period was 33.7 months, and the mean period of
time between surgery and second-look
arthroscopy was 15.0 months. In D, there were 3
men and 1 woman, 1 right and 3 left, 41.3 year old,
29.0 months and 15.5 months. The causes of P
were the protuberance at the surgery in our early
experience, the obliquely grafted plug at the
arthroscopic mosaicplasty and one protuberant
plug due to the large recipient site. The cause of
D was one depressed plug due to the multiple
grafted plugs into the recipient site.
[Results] In P, 2 patients had catching sensation
about 4 months after their surgery and sometimes
knee joints pain. In second-look arthroscopy, they
had fissuring of the plugs and fibrillation around
the recipient site. Another patient had depressed
area in the tibial joint surface, where was
contralateral of the recipient site. In D, there were
no symptoms due to the depressed plugs. In
second-look arthroscopy, the depressed areas
covered with fibrocartilage-like tissue, and the
smooth joint surface was obtained.
[Conclusion] In respect of the clinical results and
second-look arthroscopic evaluation, the
depression of localized and about 1 mm around
the cartilage was no problem. However, we
should avoid the protuberance of the plug at
mosaicplasty.
E-poster #730
Expression Profiles during Differentiation of
Chondrogenic Cells
Aki Osawa, Tokyo, JAPAN, Presenter
Masaki Kato, chiba, JAPAN
Masaki Takiguchi, Chiba, JAPAN
Naohiko Seki, Chiba, JAPAN
Hisashi Kurosawa, Tokyo, JAPAN
Department of Orthopaedics, Juntendo University,
Tokyo, JAPAN
INTRODUCTION
The differentiation of many mesenchyme-derived
cells, including cells that form the cartilage, is
regulated by gene transcription. However, many
factors involved in this regulation have not been
identified yet. Current advancements in molecular
biologic technology are facilitating the systematic
analysis of numerous genes. Using cDNA
microarray, systematic studies on expression
patterns of thousands of genes are now expected
to provide good understanding on the global
molecular basis of chondrogenesis. In this study,
we combined two powerful technologies. One is a
full-length enriched cDNA library derived from the
mouse chondrogenic ATDC5 cell line and the
mouse bone and cartilage tissues. The other is an
in-house cDNA microaray using these cDNAs, as a
high-throughput methodology to identify cDNA
clones that are differentially expressed in
differentiation in cultures of ATDC5 cells.. The
purposes of this study are to evaluate the validity
of this in-house cDNA microarray, and to
determine the expression profile during
differentiation of chondrogenic ATDC5 cells.
MATERIALS and METHODS
(1) Cell culture condition and RNA preparation:
The maintenance and differentiation of the ATDC5
cells were described previously. Total RNA was
extracted using TRIZOL reagent (Gibco-BRL,
Gaithersburg MD.) Total RNA was prepared from
the ATDC5 cells at 7, 14 and 28 days after
induction of chondrogenesis. The mouse cartilage
and bone tissues were collected from pregnant
mice on generation days of 14,15 and 17, and mice
aged 3 days .
(2) Construction of full-length enriched cDNA
library and in-house cDNA microarray: The fulllength
enriched cDNA library was constructed by
oligo-capped method as previously described
using 500-ug total RNA from various stages of
differentiated ATDC5 cells and the mouse
cartilage and bone tissues. The cDNA clones were
randomly picked up from the library, and their 5-
end nucleotide sequences determined using a Dye
Terminatior Cycle Sequencing Kit (Applied
Biosystems) and ABI 3700 DNA sequencer. The
cDNA microarray containing these cDNA clones
was constructed as previously described. We
constructed a full-length enriched cDNA library
using the oligo-capping method with RNA from
the mouse chrondrogenic ATDC5 cell line. We
determined 5-end sequence of 3,680 cDNA clones
from the library and the nucleotide sequences
were compared with the NCBI nucleotide
database using the BLAST programs. About 3,680
cDNA clone were assembled into 1,960 clusters
when overlapping clones were integrated.
Compared with the public databases, these clones
were judged to be full-length since 68% of the
cDNA clones had the first ATG codon. The average
length of the cDNA insert was approximately 2.0
kb pairs. Excluding overlapping clones, 1,960
independent clones were selected. In addition,
1,240 clones derived from mouse fetus cDNA
library were included in the production of the
microarray. Our in-house cDNA microarray was
used to analysis gene profiles of differentiation in