als PDF - AGA

27. AGA-Kongress 2010 - Wien
Res 4
The influence of cellular passage, quantity and in vitro 3-D matrix culturing time on gene
expression profiles before and after matrix-assisted chondrocyte transplantation (MACT) in a
leporine animal model
Salzmann G. 1 , Sauerschnig M. 2 , Berninger M. 2 , Schönfelder M. 3 , Schöttle P. 2 , Imhoff A. 2
1 Universitätsklinikum Freiburg, Department Orthopädie und Unfallchirurgie, Freiburg, Germany,
2 Klinikum rechts der Isar, Technische Universität München, München, Germany, 3 Technische
Universität München, Lehrstuhl für Sport und Gesundheitsförderung, München, Germany
Matrix-assisted chondrocyte transplantation (MACT) still lacks any standardization in its execution
when cell passage, cell yield and in vitro matrix-holding time are concerned. We hypothesize that
specific configurations stand out when optimal cartilage specific gene expression is intended.
Methods: Post puberty NZW Rabbit knee articular chondrocytes were seeded within 3-D matrices
(Chondrogide, Geistlich) at different passages (P 1, 3, 5); cellular yields (C: 2x10 5 /matrix=C1,
1x10 6 /matrix=C2, 3x10 6 /matrix=C3) and in vitro membrane-holding times (T: 24 hours=T1, 2
weeks=T2) to define 18 different groups. Each time, two cell-matrix-constructs (CMC, n=6/group) were
in identical duplicate whereof one was for in vitro (CMCi) analysis directly prior to re-implantation of
the other duplicate which was press-fit implanted (autologous) (CMCe) into trochlear full-thickness
chondral defects (n=2/animal) of the biopsy-contralateral knee. 12 weeks postimplantation the
regenerates were analysed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 mRNA expression. Data
were statistically compared using a mixed linear model, multiple regression analysis. Significance was
at P< 0.05 for all tests.
Results: Generally, CMCi values were higher than CMCe values for all differentiation targets
(Collagen-2, COMP, Aggrecan, Sox9), while the opposite was true for dedifferentiation targets
(Collagen-1,-10). There appeared a general linearity between CMCi and CMCe values to potentially
predict the CMCe outcome based on CMCi quality (CMCe values were dependent on CMCi values).
Typically, animals improved, target-specific, a disadvantageous CMCi profile into an advantegeous
CMCe expression, while generally advantageous CMCi values persisted within CMCes. CMCi values
were significantly different between groups for all targets analysed, while the difference was not
significant for Collagen-1,-10, Aggrecan among CMCes. The difference between CMCi and CMCe
was significant for all targets, except for COMP. Usually, interacting P and T resulted in significant
different results, while this was true with much lesser frequency for C.
A combination of low P as is P1, of medium C as is C2 and short T as is T1 appeared to result in an
optimal (strong differentiation, weak dedifferentiation) CMCi and CMCe outcome, while generally P3,
more pronounced P5 and foremost T2 (as well their combination) strongly impaired the outcome, with
much lesser impact of C.
Conclusion: The results demonstrate that both in vitro and in vivo performance are strongly affected
by cellular passage, density, membrane-holding time. Few passages, short holding result in an optimal
outcome, which could be useful information in the future to improve the clinical outcome when matrixassisted
transplanting cartilage cells.
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