INTRODUCTION



left1438275Samuel, S1, McDonnell, E1,2, Buckley, CT1,2,3,41 Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland,2 School of Engineering, Trinity College Dublin, Ireland, 3 AMBER Centre, Royal College of Surgeons in Ireland & Trinity College Dublin, Ireland, 4 Department of Anatomy and Regenerative Medicine, Royal College of Surgeons in Irelandcbuckle@tcd.ie00Samuel, S1, McDonnell, E1,2, Buckley, CT1,2,3,41 Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland,2 School of Engineering, Trinity College Dublin, Ireland, 3 AMBER Centre, Royal College of Surgeons in Ireland & Trinity College Dublin, Ireland, 4 Department of Anatomy and Regenerative Medicine, Royal College of Surgeons in Irelandcbuckle@tcd.ie01047750Effect of Growth Factor Priming to Promote a Discogenic Phenotype of Tissue Spheroids for Intervertebral Disc Regeneration00Effect of Growth Factor Priming to Promote a Discogenic Phenotype of Tissue Spheroids for Intervertebral Disc RegenerationINTRODUCTIONMost cases of chronic low back pain is associated with intervertebral disc (IVD) degeneration, which predominantly occurs due to the degeneration of nucleus pulposus (NP). Hence, treatment of IVD degeneration focusses on repopulating the NP and thus augmenting the repair process. Currently, mesenchymal stromal cells (MSCs) and articular chondrocytes (ACs) are the predominant cell sources being investigated. However, a recent study showed that nasal chondrocytes (NC) had superior potential in the treatment of disc degeneration [1]. Culturing of these cells in monolayer using growth factor like transforming growth factor β (TGF-β) has been shown to play a significant role in IVD growth and homeostasis. Previous studies have used growth factors GDF and TGF-β1 to differentiate stem cells to NP-like cells, with the aim to regenerate the intervertebral disc [2]. However, no consensus exists as to the optimum growth factor needed to drive differentiation towards a discogenic phenotype. This study investigated the effect of supplementing NCs with TGF-β3 to promote a discogenic phenotype of tissue spheroids (formed in polydimethylsiloxane (PDMS) concave microwell plates) for IVD regeneration. Self-assembled tissue spheroids may offer an attractive approach to form microtissues, which can be primed in vitro using a cocktail of growth factors and are compatible with injectable delivery.Materials and methodsNasal chondrocytes were isolated from porcine nasal septa. Concave PDMS microwell molds having an array of 10 x 10 wells were used to form spheroids. The effect of growth factor was evaluated on three different cell densities - 1000, 2500 and 5000 cells per spheroid. The spheroids were cultured in either TGF-β3 supplemented media (CDM+) or TGF-β3 free media (CDM) and harvested after 7 days. Spheroids were digested with papain and DNA was quantified using Quant-iT picogreen dsDNA assay kit. Proteoglycan (sulphated glycosaminoglycan, sGAG) content was quantified using the dimethylmethylene blue dye-binding assay, with a chondroitin sulphate standard. Total collagen was determined by measuring the hydroxyproline content.resultsSEM image of the concave microwell plate showed microwells had an average diameter of 504 μm (A). Thesize of the cell aggregates formed in the microwell plates was found to decrease from 143 μm to 127 μm by day 7 compared to day 1 (B). Figure 1C showed increased cell death in spheroids cultured in CDM+ media with higher cell death occurring towards the center of spheroids. Concentration of sGAG/cell was high in spheroids with a density of 1000 cells/spheroid. Figure 1(A) SEM image shows the shape and dimensions of concave microwells. (B) Graph shows changes in the spheroid diameter after day 1 and day 7 of culture in CDM and CDM+ media. * indicates significance compared to 1000 cells for the same experimental group (p<0.05). (C) Cell viability assay showing live and dead cells after 7 days of culture in CDM and CDM+ media. Scale bar: 20 um. (D) Concentration of sGAG normalized to DNA. DiscussionThis study compared the effect of low cell density spheroids with the aim of their application as an injectable cell source for disc repair. The use of concave PDMS molds resulted in the formation of microspheroids with low size variability. Previous studies have shown that PDMS resists cells adhesion that facilitates spheroid formation. This also offers the advantage of less spheroid size variability. Increased cell death was observed in higher cell density spheroids which may be due in part to limited diffusion of oxygen and nutrients required for cell survival and proliferation [3]. Cell death was also comparatively high in spheroids cultured in CDM+ media. This was in accordance with a study that reported the detrimental effect of TGF-β supplementation in low oxygen (5% O2) conditions for agarose encapsulated porcine articular chondrocytes [4]. References[1] Gay MH (et al.), Eur Cell Mater 2019;37:214-32.[2] Clarke LE (et al.), Arthritis Res Ther 2014;16:R67.[3] Yen SE (et al.), PLoS One 2013;8:e73345.[4] Buckley CT (et al.), Osteoarthritis Cartilage 2010;18:1345-54. ................
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