Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model

Lübke, Carsten; Ringe, Jochen; Krenn, Veit; Fernahl, Gabriele; Pelz, S; Kreusch-Brinker, Rüdiger; Sittinger, Michael; Paulitschke, M

doi:10.1016/j.joca.2004.01.009

Cited by 35 publications

(39 citation statements)

References 40 publications

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“…6,12,19 For this purpose, primary chondrocytes have been cultivated in highdensity cartilage pellets and then co-cultivated with human RASFs. These activated RA-SFs are believed to play a major role in the destruction of joint cartilage during the pathogenesis of RA.…”

Section: Discussionmentioning

confidence: 99%

“…12,32 Nevertheless, automated media exchange led to the same or higher mRNA expression level of all cartilagespecific markers, showing that automated cultivation preserves the chondrocyte differentiation level to a greater extent than manual cultivation does.…”

Section: Automated Pellet Cultivationmentioning

confidence: 99%

“…[9][10][11] The detailed growth characterization of high-density cartilage pellets in microplates and their use in the interactive pannus model was previously reported by the authors. 12 Pellet-cultured chondrocytes form cartilage-specific ECM by expression of proteoglycans and collagen type II. 12,13 This 3-D ECM reflects the specific cellular environment of the chondrocytes, allowing functional interactions between the cells and their ECM.…”

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confidence: 99%

“…12 Pellet-cultured chondrocytes form cartilage-specific ECM by expression of proteoglycans and collagen type II. 12,13 This 3-D ECM reflects the specific cellular environment of the chondrocytes, allowing functional interactions between the cells and their ECM. For co-cultivation, 2 SV40T antigen-transformed human SF cell lines are available: 1) The hepatic sinusoidal endothelium (HSE) cell line was derived from an RA patient and shows an aggressive invasive behavior in vitro, whereas 2) the K4IM cell line was established from a healthy donor and represents the noninvasive phenotype.…”

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confidence: 99%

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Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

et al. 2007

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The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBi™-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time. (Journal of Biomolecular Screening 2007:956-965)

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Section: Discussionmentioning

confidence: 99%

Section: Automated Pellet Cultivationmentioning

confidence: 99%

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Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

et al. 2007

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“…One approach is to seed cells in a high-density pellet culture. 11 Chondrocyte pellet cultures were originally applied as a growth plate model for cartilage differentiation, hypertrophy, and degradation. 12 After addition of interleukin 1b (IL1b), catabolic effects on ECM content and gene expression similar to the response of native cartilage were observed.…”

Section: Introductionmentioning

confidence: 99%

Tissue‐engineered cartilage of porcine and human origin as in vitro test system in arthritis research

Göhring

Lübke

Andreas

et al. 2010

Biotechnology Progress

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The increasing prevalence of cartilage destruction during arthritis has entailed an intensified amount for in vitro cartilage models to analyze pathophysiological processes and to screen for antirheumatic drugs. Tissue engineering offers the opportunity to establish highly organized 3D cell cultures facilitating the formation of in vitro models that reflect the human situation. We report the comparison of porcine chondrocyte pellet and alginate bead cultures as model systems for human cartilage and the further development into a human system that was applied in an arthritis model. In porcine pellet and alginate cultures, formation of cartilage matrix similar to human matrix was verified by histology and PCR. As alginate beads could be cultivated batch-wise in one well of a multiwell plate, we further developed this setting into a human system. In contrast, each pellet had to be cultivated individually in one well of a multiwell plate, which is time consuming. Following stimulation of human chondrocyte alginate cultures with conditioned media from human synovial fibroblasts derived from arthritis patients, microarray analysis verified the induction of genes related to cartilage destruction (like MMP10, -12) and inflammation (like IL6, -8 and chemokines). Several genes are coding for proteins that are members of inflammatory and catabolic pathways. Belonging to the most affected pathways, we identified the focal adhesion, cytokine-cytokine receptor interaction, ECM-receptor signalling, Jak-STAT signalling, and toll-like receptor signalling pathways, all relevant in arthritis. Therefore, we demonstrate that engineered cartilage of porcine and human origin represents a powerful in vitro model for cartilage in vivo.

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Parathyroid hormone/parathyroid hormone‐related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation

Harrington

Roddy

West

et al. 2007

The Anatomical Record

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Parathyroid hormone (PTH; 10 À7 to 10 À15 M) decreased terminal chondrogenesis in the avian sterna. During the first half of an 8-day culture, 100 nM PTH (1-34) significantly increased sternal length and downregulated the deposition of type X collagen and its mRNA expression. However, it remains unclear how PTH increased cartilaginous growth. In this study, we examined growth by both cell proliferation and analysis of cyclin d1 and collagen mRNA. Types II, IX, and X collagens and cyclin d1 mRNA were quantified through real-time RT-PCR, while Ki-67 was used as an immunohistochemical proliferation marker. Extracellular matrix content was measured through mRNA quantification of types II, IX, and X collagen and observing deposition of the same collagens. PTH significantly increased the proliferation marker Ki-67 in the sternal cephalic region. There was less type II and X collagen in PTHtreated sterna with concomitant decreases in mRNA production, suggesting that proliferation was the major contributor to cartilage growth in the presence of PTH/PTH-related peptide receptor activation. In conclusion, these experiments demonstrated that PTH increased cartilage growth by upregulating cell proliferation or other extracellular matrix components. Anat Rec, 290:155-167, 2007Rec, 290:155-167, . 2007 Wiley-Liss, Inc.

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Growth characterization of neo porcine cartilage pellets and their use in an interactive culture model

Abstract: Pellet formation of freshly isolated chondrocytes followed a reproducible developmental kinetics and showed typical immature hyaline cartilage properties. Such uniform cartilage pellets are very useful as a substrate for interactive cell culture models that simulate diseases like RA.

Cited by 35 publications

References 40 publications

Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

Tissue‐engineered cartilage of porcine and human origin as in vitro test system in arthritis research

Parathyroid hormone/parathyroid hormone‐related peptide modulates growth of avian sternal cartilage via chondrocytic proliferation

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