A multifactorial approach for enhancing extracellular matrix deposited by human chondrocytes

Abstract

Event Abstract Back to Event A multifactorial approach for enhancing extracellular matrix deposited by human chondrocytes Valeria Graceffa1, Dimitrios Zeugolis1 and Abhay Pandit2 1 National University of Ireland, Galway NUIG, Regenerative, Modular & Developmental Engineering Laboratory (REMODEL), Ireland 2 National University of Ireland, Galway NUIG, Centre for Research in Medical Devices (CÚRAM), Ireland Introduction: Cartilage defects and osteoarthritis represent a big economic burden on individuals, health systems and social care systems. Despite autologous chondrocytes implantation and related techniques have shown good initial results, they have been demonstrated ineffective for a long term treatment of osteoarthritis[1]. Recently, it has been observed that culture of chondrocytes in alginate scaffolds under hypoxic conditions can increase collagen type II synthesis and decrease collagen type I deposition, resulting in the production of a hyaline-like cartilage[2]. Macromolecularly crowding (MMC) is a biophysical phenomenon that accelerates biological activities and it is able to increases tissue-specific extracellular matrix deposition by over 120 fold[3]. We hypothesize that a multifactorial approach combining MMC, hypoxia and a three dimensional culture system can increase extracellular matrix synthesised, resulting in the production of a hyaline-like cartilage, with histological properties similar of those of the native cartilage. Materials and Methods: Human articular chondrocytes were seeded in chondrocytes differentiation medium CDM (Lonza). 1, 7 days or 10 days after seeding, crowder medium was added (carrageenan 50 ug/mL, 100 ug/mL or 500 ug mL in CDM). After 3, 7 and 10 days, collagen type I and type II deposition were evaluated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), while matrix metalloproteinase activity was evaluated by zymography. Influence of MMC in cell viability and metabolic activity were determined using the Live/Dead assay and the Alamar Blue respectively. For assessing the combined effect of MMC, hypoxia and alginate scaffold system, human chondrocytes were seeded on alginate scaffold or in plastic tissue culture plates at 20% or 2% oxygen tension in CDM. Matrix deposition was evaluated by SDS-PAGE and immunfluorescence for Collagen type I, type II and aggrecan. Cells metabolic activity and cell viability were determined by the Live/Dead assay and the alamarBlue. Results: We first optimised CR concentration, duration and start time of MMC treatment in a monolayer system. We are now assessing the combined effect of MMC, hypoxia and alginate scaffolds. Conclusion: Extracellular matrix secreted by chondrocytes has been shown to be the main responsible of the biomechanical properties of a cartilage graft and indirectly of the clinical outcomes of the treatment[4]. Increasing matrix deposition, a multifactorial approach combining MMC, hypoxia and three dimensional culture of chondrocytes could improve overall quality of a engineered cartilage substitute, conferring a greater mechanical stability upon implantation and filling cartilage defects with matrix that is better integrated with the adjacent native cartilage. The authors would like to acknowledge SFI for financial support