OBJECTIVE: To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. METHODS: Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. RESULTS: Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. CONCLUSION: Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.
OBJECTIVE: To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. METHODS: Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovinecartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with humantissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. RESULTS: Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. CONCLUSION: Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.
Authors: Anja Weimer; Henning Madry; Jagadeesh K Venkatesan; Gertrud Schmitt; Janina Frisch; Anna Wezel; Jochen Jung; Dieter Kohn; Ernest F Terwilliger; Stephen B Trippel; Magali Cucchiarini Journal: Mol Med Date: 2012-05-09 Impact factor: 6.354
Authors: Teresa C Coughlan; Aileen Crawford; Mary B Goldring; Paul V Hatton; Michael D Barker Journal: J Tissue Eng Regen Med Date: 2010-12 Impact factor: 3.963
Authors: Heenam Kwon; Lin Sun; Dana M Cairns; Roshni S Rainbow; Rucsanda C Preda; David L Kaplan; Li Zeng Journal: Acta Biomater Date: 2013-01-16 Impact factor: 8.947