Chondrocyte Culture Parameters for Matrix-Assisted Autologous Chondrocyte Implantation Affect Catabolism and Inflammation in a Rabbit Model

TY - JOUR

T1 - Chondrocyte Culture Parameters for Matrix-Assisted Autologous Chondrocyte Implantation Affect Catabolism and Inflammation in a Rabbit Model

AU - Sauerschnig, Martin

AU - Berninger, Markus T

AU - Kaltenhauser, Theresa

AU - Plecko, Michael

AU - Wexel, Gabriele

AU - Schönfelder, Martin

AU - Wienerroither, Valerie

AU - Imhoff, Andreas B

AU - Schöttle, Philip B

AU - Rosado Balmayor, Elizabeth

AU - Salzmann, Gian M

PY - 2019/3/27

Y1 - 2019/3/27

N2 - Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25 mm³) at three different densities (2 × 10⁵/matrix, 1 × 10⁶/matrix, and 3 × 10⁶/matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: ∅ 4 mm × 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-α, IL-1β, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1β, MMP-1, and MMP-3 compared to CSM-e. TNF-α expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-α. IL-1β expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1β expression decreased with prolonged membrane-holding time in CSM-e. For TNF-α, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-α expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular density was of no significant effect. We demonstrated cytokine and MMP expression levels to be directly influenced by in vitro culture settings in ACI. Linearity of expression-patterns between CSM-i and CSM-e may predict ACI regeneration outcome in vivo. Cytokine/protease interaction within the regenerate tissue could be guided via adjusting in vitro culture parameters, of which membrane-holding time resulted the most relevant one.

AB - Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25 mm³) at three different densities (2 × 10⁵/matrix, 1 × 10⁶/matrix, and 3 × 10⁶/matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: ∅ 4 mm × 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-α, IL-1β, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1β, MMP-1, and MMP-3 compared to CSM-e. TNF-α expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-α. IL-1β expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1β expression decreased with prolonged membrane-holding time in CSM-e. For TNF-α, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-α expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular density was of no significant effect. We demonstrated cytokine and MMP expression levels to be directly influenced by in vitro culture settings in ACI. Linearity of expression-patterns between CSM-i and CSM-e may predict ACI regeneration outcome in vivo. Cytokine/protease interaction within the regenerate tissue could be guided via adjusting in vitro culture parameters, of which membrane-holding time resulted the most relevant one.

KW - Animals

KW - Cells, Cultured

KW - Chondrocytes/cytology

KW - Cytokines/metabolism

KW - Disease Models, Animal

KW - Extracellular Matrix/metabolism

KW - Inflammation/metabolism

KW - Inflammation Mediators/metabolism

KW - Matrix Metalloproteinases/metabolism

KW - RNA, Messenger/genetics

KW - Rabbits

KW - Transplantation, Autologous

U2 - 10.3390/ijms20071545

DO - 10.3390/ijms20071545

M3 - SCORING: Journal article

C2 - 30934789

VL - 20

JO - INT J MOL SCI

JF - INT J MOL SCI

SN - 1661-6596

IS - 7

ER -