Article
The effect 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 leprine animal model
Search Medline for
Authors
-
G.M. Salzmann
- Universitätsklinik Freiburg, Abt. für Orthopädie und Traumatologie, Freiburg, Germany
-
M. Sauerschnig
- Klinikum Rechts der Isar der TU München, Abteilung für Orthopädie und Sportorthopädie, München, Germany
-
M. Berninger
- Klinikum Rechts der Isar der TU München, Abteilung für Orthopädie und Sportorthopädie, München, Germany
-
M. Schönfelder
- Technische Universität München, BFTS, München, Germany
-
P. Schöttle
- TU München, Klinikum Rechts der Isar, Abteilung f. Sportorthopädie, München, Germany
-
A.B. Imhoff
- TU München, Klinikum rechts der Isar, Abteilung und Poliklinik für Sportorthopädie, München, Germany
| Published: | October 21, 2010 |
|---|
Outline
Text
Objective: 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: Harvested, digested, post puberty New Zealand White Rabbit knee articular chondrocytes were seeded within 3-D matrices (Chondrogide, Geistlich, Switzerland) at different passages (P 1, 3, 5); cellular yields (C: 200.000/matrix=C1, 1.000.000/matrix=C2, 3.000.000/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 (strictly autologous) (CMCe) into trochlear full-thickness (strictly chondral) defects (n=2/animal) of the biopsy-contralateral knee, reproducing a MACT situation. 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<.05 for all tests.
Results and conclusions: 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.
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 matrix-assisted transplanting cartilage cells.
