Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-β1 and G-CSF

Formation of chondrocyte clusters is not only a morphological sign of osteoarthritis but it is also observed in cell culture. Active locomotion of chondrocytes is controlled by integrins in vitro. Integrins bind to Laminin-A4 (LAMA4), a protein that is highly expressed in vivo in clusters of hypertrophic chondrocytes. We tested if LAMA4 is relevant for cluster formation. Human chondrocytes were cultured in a 2D matrigel model and treated with different concentrations of a monoclonal inhibitory anti-LAMA4-antibody. Migration and cluster formation was analysed using live cell imaging technique. Full genome gene expression analysis was performed to assess the effect of LAMA4 inhibition. The data set were screened for genes relevant to cell motility. F-actin staining was performed to document cytoskeletal changes. Anti-LAMA4 treatment significantly reduced the rate of cluster formation in human chondrocytes. Cells changed their surface morphology and exhibited fewer protrusions. Expression of genes associated with cellular motility and migration was affected by anti-LAMA4 treatment. LAMA4-integrin signalling affects chondrocyte morphology and gene expression in vitro, thereby contributing to cluster formation in human osteoarthritic chondrocytes.

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“…Motility of chondrocytes is essential during growth, development, wound healing, and in the manufacturing of scaffolded cartilage implants …”

mentioning

confidence: 99%

Effect of Laminin‐A4 inhibition on cluster formation of human osteoarthritic chondrocytes

Moazedi-Fuerst

Gruber

Stradner

et al. 2015

Journal Orthopaedic Research

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“…Although higher cell yields have been previously observed in auricular cartilage compared with NC, the ability to produce tissue in an age independent manner makes them an ideal cell source (Rotter et al, 2001(Rotter et al, , 2002Tay et al, 2004). Minimal donor site morbidity with a nasal biopsy may facilitate the use of autologous cells and could circumvent concerns surrounding disease transmission, immune rejection and ethical considerations associated with allogeneic tissue (Marmotti et al, 2013). Improved proliferative kinetics of NC cells, 25% greater than AC, is also advantageous with reduced time and costs involved in cell amplification.…”

Section: Discussionmentioning

confidence: 99%

In vitro extracellular matrix accumulation of nasal and articular chondrocytes for intervertebral disc repair

Vedicherla

Buckley

2017

Tissue and Cell

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Chondrocyte based regenerative therapies for intervertebral disc repair such as Autologous Disc Cell Transplantation (ADCT, CODON) and allogeneic juvenile chondrocyte implantation (NuQu®, ISTO Technologies) have demonstrated good outcomes in clinical trials. However concerns remain with the supply demand reconciliation and issues surrounding immunoreactivity which exist for allogeneic-type technologies. The use of stem cells is challenging due to high growth factor requirements, regulatory barriers and differentiation towards a stable phenotype. Therefore, there is a need to identify alternative non-disc cell sources for the development and clinical translation of next generation therapies for IVD regeneration. In this study, we compared Nasal Chondrocytes (NC) as a non-disc alternative chondrocyte source with Articular Chondrocytes (AC) in terms of cell yield, morphology, proliferation kinetics and ability to produce key extracellular matrix components under 5% and 20% oxygen conditions, with and without exogenous TGF-β supplementation.Results indicated that NC maintained proliferative capacity with high amounts of sGAG and lower collagen accumulation in the absence of TGF-β supplementation under 5% oxygen conditions. Importantly, osteogenesis and calcification was inhibited for NC when cultured in IVD-like microenvironmental conditions. The present study provides a rationale for the exploration of nasal chondrocytes as a promising, potent and clinically feasible autologous cell source for putative IVD repair strategies.

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“…Cartilage regeneration potential of a one-stage procedure is likely dependent on multiple parameters, such as size and loading density of minced fragments, method of harvest, culture time/method and material properties of scaffolds [19]. This may be further modified by the use of growth factors such as transforming growth factor (TGF)-β1 and granulocyte colony-stimulating factor (G-CSF) [22]. Our short-term (four month) animal study demonstrated no difference in histology scores, tissue filling (35 %) or repair tissue stiffness between cell-based regeneration and MC technique.…”

Section: Discussionmentioning

confidence: 99%

Particulate cartilage under bioreactor-induced compression and shear

Wang¹,

Grad²,

Stoddart³

et al. 2013

International Orthopaedics (SICOT)

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Purpose Our aim was to explore the effect of varying in vitro culture conditions on general chondrogenesis of minced cartilage (MC) fragments. Methods Minced, fibrin-associated, bovine articular cartilage fragments were cultured in vitro within polyurethane scaffold rings. Constructs were maintained either free swelling for two or four weeks (control), underwent direct mechanical kneejoint-specific bioreactor-induced dynamic compression and shear, or they were maintained free swelling for two weeks followed by two weeks of bioreactor stimulation. Samples were collected for glycosaminoglycan (GAG)/DNA quantification; collagen type I, collagen type II, aggrecan, cartilage oligomeric matrix protein (COMP), proteoglycan-4 (PRG-4) messenger RNA (mRNA) analysis; histology and immunohistochemistry. Results Cellular outgrowth and neomatrix formation was successfully accomplished among all groups. GAG/DNA and collagen type I mRNA were not different between groups; chondrogenic genes collagen type II, aggrecan and COMP revealed a significant downregulation among free-swelling constructs over time (week two through week four). Mechanical loading was able to maintain chondrogenic expression with significantly stronger expression at long-term time points (four weeks) in comparison with four-week control. Histology and immunohistochemistry revealed that bioreactor culture induced stronger cellular outgrowth than free-swelling constructs. However, weaker collagen type II and aggrecan expression with an increased collagen type I expression was noted among this outgrowth neotissue. Conclusions The method of MC culture is feasible under in vitro free-swelling and dynamic loading conditions, simulating in vivo posttransplantation. Mechanical stimulation significantly provokes cellular outgrowth and long-term chondrogenic maturation at the mRNA level, whereas histology depicts immature neotissue where typical cartilage matrix is expected.

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Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-β1 and G-CSF

Cited by 41 publications

References 64 publications

Effect of Laminin‐A4 inhibition on cluster formation of human osteoarthritic chondrocytes

Effect of Laminin‐A4 inhibition on cluster formation of human osteoarthritic chondrocytes

In vitro extracellular matrix accumulation of nasal and articular chondrocytes for intervertebral disc repair

Particulate cartilage under bioreactor-induced compression and shear

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