Introduction: · Web view2020. 1. 12. · RT-PCR), western blot and immunofluorescent staining...
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Early Stage Researcher (PhD Year 1) Post-Doctoral Researcher/Senior Researcher/PI X Entry for the Engineers Ireland Biomedical Research Medal Corresponding author has completed PhD and would like to review BinI abstract submissions X Please place an X in any appropriate categories INTRODUCTION: In osteoarthritis (OA), responses to proinflammatory cytokines and mechanical stressors drive increased extracellular matrix (ECM) catabolism and pathological cell phenotypes. Recent evidence links the hippo (Yap/Taz) pathway with canonical IL1 1 signalling, while mechanosensitive connexon membrane channels have roles in regulating cartilage development, homeostasis and degeneration. In other tissues, Yap directly interacts with connexins while the upregulation of cx43 (connexin-43; the predominant chondrocyte connexin) increases nuclear translocation of NFk and is increasingly implicated in the spread of proinflammatory signalling. Therefore, this study investigated the therapeutic potential of targeted cx43 knockdown to decrease OA- associated proinflammatory signalling during cartilage regeneration and restore a healthy ECM environment. Then, we functionalized previously developed chondrogenic collagen I- hyaluronic acid scaffolds 2 to develop a non-viral cx43-siRNA biomaterial delivery platform capable of targeting cx43-mediated proinflammatory responses related to OA. MATERIALS AND METHODS: Human articular chondrocytes (hACs) were cultured in 2D or in 3D scaffolds. Cx43 and Yap-mediated responses to IL1 were investigated using small molecule inhibitors/ promotors (cx43 channel inhibitor- gap27; Yap decrease- verteporfin, VP; Yap increase-lysophosphatidic acid, LPA). The effects of cx43 and Yap on cell viability, migration and proliferation in the presence of IL1 was determined. The role of cx43 and YAP in responses to IL1 was investigated by quantitative real- time pcr (qRT-PCR), western blot and immunofluorescent staining (IFS). For siRNA-activated scaffold development, cx43 siRNA was complexed with transfection GET-(glycosaminoglycan- binding domain) peptide 3 . Cx43 knockdown was determined through qRT- PCR and western blot. Get-siRNA complexes were then incorporated into type I collagen-hyaluronic acid scaffolds. The effect of scaffold- mediated cx43 knockdown in hAC responses to IL1 stimulation in scaffolds was assessed. RESULTS: Blocking cx43 channel activity and increasing Yap protein levels decreased cellular responses to IL1, promoting anabolic and decreasing catabolic gene/ protein expression. Cell viability, proliferation and migration in IL1 stimulated cells was significantly increased with cx43 channel inhibition and increased Yap, while decreasing Yap exacerbated proinflammatory cytokine decreases in proliferation and viability. Cx43 expression was found to be linked to yap expression and the proteins were found to co-localise and interact. In scaffolds, IL1 decreased healthy chondrogenic gene/ protein expression and glycosaminoglycan production over 28 days but this effect was reversed through increasing Yap or cx43 Bioengineering in Ireland 26 , January 17-18, 2020 Development of a siRNA-activated scaffold delivery system to ameliorate inflammatory responses during regeneration of osteoarthritic cartilage Hodgkinson, T 1 , Kelly, D 1 , Dixon, JE 2 , Shakesheff, KM 2 , O’Brien, FJ 1,3,4 . 1 Anatomy and Regenerative Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland. 2 School of Pharmacy, University of Nottingham, UK. 3 Trinity Centre for Bioengineering, Trinity College Dublin, Ireland. 4 AMBER Centre,
Transcript of Introduction: · Web view2020. 1. 12. · RT-PCR), western blot and immunofluorescent staining...
Early Stage Researcher (PhD Year 1) Post-Doctoral Researcher/Senior Researcher/PI X
Entry for the Engineers Ireland Biomedical Research Medal
Corresponding author has completed PhD and would like to review BinI abstract submissions X
Please place an X in any appropriate categories
INTRODUCTION: In osteoarthritis (OA), responses to proinflammatory cytokines and mechanical stressors drive increased extracellular matrix (ECM) catabolism and pathological cell phenotypes. Recent evidence links the hippo (Yap/Taz) pathway with canonical IL11
signalling, while mechanosensitive connexon membrane channels have roles in regulating cartilage development, homeostasis and degeneration. In other tissues, Yap directly interacts with connexins while the upregulation of cx43 (connexin-43; the predominant chondrocyte connexin) increases nuclear translocation of NFk and is increasingly implicated in the spread of proinflammatory signalling. Therefore, this study investigated the therapeutic potential of targeted cx43 knockdown to decrease OA-associated proinflammatory signalling during cartilage regeneration and restore a healthy ECM environment. Then, we functionalized previously developed chondrogenic collagen I-hyaluronic acid scaffolds2 to develop a non-viral cx43-siRNA biomaterial delivery platform capable of targeting cx43-mediated proinflammatory responses related to OA.
MATERIALS AND METHODS: Human articular chondrocytes (hACs) were cultured in 2D or in 3D scaffolds. Cx43 and Yap-mediated responses to IL1 were investigated using small molecule inhibitors/ promotors (cx43 channel inhibitor- gap27; Yap decrease- verteporfin, VP; Yap increase-lysophosphatidic acid, LPA). The effects of cx43 and Yap on cell viability, migration and proliferation in the presence of IL1 was determined. The role of cx43 and YAP in responses to IL1 was investigated by quantitative real-time pcr (qRT-PCR), western blot and immunofluorescent staining (IFS). For siRNA-activated scaffold development, cx43 siRNA was complexed with transfection GET-(glycosaminoglycan-binding domain) peptide3. Cx43 knockdown was determined through qRT-PCR and western blot. Get-siRNA complexes were then incorporated into type I collagen-hyaluronic acid scaffolds. The effect of scaffold-mediated cx43 knockdown in hAC responses to IL1 stimulation in scaffolds was assessed.
RESULTS: Blocking cx43 channel activity and increasing Yap protein levels decreased cellular responses to IL1, promoting anabolic and decreasing catabolic gene/ protein expression. Cell viability, proliferation and migration in IL1 stimulated cells was significantly increased with cx43 channel inhibition and
increased Yap, while decreasing Yap exacerbated proinflammatory cytokine decreases in proliferation and viability. Cx43 expression was found to be linked to yap expression and the proteins were found to co-localise and interact. In scaffolds, IL1 decreased healthy chondrogenic gene/ protein expression and glycosaminoglycan production over 28 days but this effect was reversed through increasing Yap or cx43 channel inhibition. Get-siRNA complexes successfully delivered cx43 siRNA and knocked-down cx43 expression in 2D and in an advanced 3D scaffold delivery system.
Figure 1 (A) GET-Cx43siRNA activated scaffold knockdown of Cx43 in collagen I-hyaluronic acid scaffolds (B) IFS showing that Cx43 knockdown increases nuclear Yap localization. *p<0.05;**p<0.01;***p<0.001. Scale bar = 200m.
DISCUSSION: This study determines the role cx43 in OA-associated proinflammatory responses, confirms cx43 and cx43-mediated signalling as promising therapeutic targets. The development of the siRNA-activated scaffold delivery system for cx43 knockdown potentially offers a new paradigm in biomaterials in cartilage repair.
REFERENCES
1. Deng, Y et al. (2018) Nat. Commun. 9(1): 4.2. Levingstone, TJ et al. (2014) Acta Biomater 10(5):1996. 3. Dixon, JE et al. (2016) PNAS 113(3): E291.
Bioengineering in Ireland26, January 17-18, 2020
Development of a siRNA-activated scaffold delivery system to ameliorate inflammatory responses during regeneration of osteoarthritic cartilage
Hodgkinson, T 1 , Kelly, D1, Dixon, JE2, Shakesheff, KM2, O’Brien, FJ1,3,4.1 Anatomy and Regenerative Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland.
2 School of Pharmacy, University of Nottingham, UK. 3 Trinity Centre for Bioengineering, Trinity College Dublin, Ireland. 4AMBER Centre, Dublin, Ireland.
Early Stage Researcher (PhD Year 1) Post-Doctoral Researcher/Senior Researcher/PI X
Entry for the Engineers Ireland Biomedical Research Medal
Corresponding author has completed PhD and would like to review BinI abstract submissions X
Please place an X in any appropriate categoriesACKNOWLEDGEMENTS: Science Foundation Ireland (SFI) European Regional Development Fund 13/RC/2073.
Bioengineering in Ireland26, January 17-18, 2020
