Abstract
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.
Highlights
As a cell-based method for the treatment of full thickness cartilage defects, autologous chondrocyte implantation (ACI)—first described in 1994—promised the dawn of a new era [1]
Cartilage defects are known to represent an assignable cause of osteoarthritis (OA)—the most common musculoskeletal disease—which entails pain and immobilization due to progressive cartilage deterioration imposed to the affected joint [6,7]
The latter notion combined with the results presented here could provide further insights into the cascades of early healing response and/or tissue degradation following cartilage damage and potentially deterioration of joint homeostasis
Summary
As a cell-based method for the treatment of full thickness cartilage defects, autologous chondrocyte implantation (ACI)—first described in 1994—promised the dawn of a new era [1]. The first rush of enthusiasm was extenuated for complications such as graft hypertrophy and calcification soon to be redeemed by another hype coming up with the introduction of artificial cell carriers and novel culture methods [2,3,4]. Awareness occurred to potential limitations within the boundaries of in vitro culture such as rapid phenotypic changes of passaged chondrocytes [5], indicating in vitro settings to claim major influence on cell differentiation. Cartilage defects are known to represent an assignable cause of osteoarthritis (OA)—the most common musculoskeletal disease—which entails pain and immobilization due to progressive cartilage deterioration imposed to the affected joint [6,7]. Basic mechanisms of production and release as well as principles of multiple interactions among such biochemical mediators within initial and progressive OA remain rather unclear [9,10,11]
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