#FIRM2013 Jonathan Dawson University of Southampton, UK Self-organising clay gels for regenerative microenvironments Jon Dawson and Richard Oreffo Clays for regeneration Structure & properties of clay Laponite: a synthetic clay used in cosmetics for 25 years Clay gels self-organise Protein localisation 1.6 1.2 Control 1.2 Alginate r2 =0.95 0.8 0.8 0.4 0.4 Laponite 20 40 60 80 1.6 1.2 Control 1.2 Alginate 0.8 0.8 0.4 0.4 r2 =0.96 0 20 40 800 Adsorbed Protein 600 Ambient Protein 400 200 60 Laponite 80 Control Alginate r2 =0.95 Laponite 0 1.6 Protein Conc (mg/ml) Protein in Media (mg/ml) 0 Protein Uptake Lysozyme 1.6 20 40 60 80 Control Alginate r2 =0.97 0 Time (hrs) 20 800 Protein Conc (mg/ml) Albumin Protein in Media (mg/ml) Protein Release 600 400 200 40 Adsorbed Protein Ambient Protein 60 Laponite 80 Clay-films for surface modification Auto-flourescence FITC-BSA Clay-films for BMP2 delivery 1ul drops of 1% clay *** 10-1 10-2 * TCP + BMP2 10-3 Clay-gel dot – ve Clay-gel dot + BMP2 10-4 TCP -ve TCP +BMP2 Clay -ve Clay +BMP2 1.2 1 0.8 0.6 0.4 0.2 0 Mean branch-points per cell HUVEC tubule assay Mean branch-points per cell Clay-bound VEGF Function 2.5 ** *** 2 1.5 1 0.5 +ve 0 Laponite only -ve Laponite + fibronectin Unbound Bound Residual VEGF (ng/ml) Clay-bound VEGF Function In vivo Collagen Scaffold Scaffold + Clay Gel Following incubation in media +/- VEGF 200 100 *** 0 VEGF Control Collagen alone Collagen + laponite ** Vessel volume (%) 3 2 1 0 Collagen alone Collagen alone Collagen Collagen + laponite + factors Collagen + laponite + factors Collagen + laponite Vessel number (per mm) Clay-bound VEGF Function In vivo * 0.3 0.2 0.1 0 Collagen alone Collagen + factors Collagen Collagen + laponite + factors Collagen + laponite + factors Collagen + laponite factors + Conclusions • Clay gels can provide a one-step procedure for the encapsulation and delivery of agents without the need for complex chemical / physical approaches and methods. • Retention of proteins and microcapsule recovery provides a versatile mode of generating spatially-controlled biological microenvironments. • We have provided proof of concept of the ability clay-gels to stimulate angiogenesis via localisation of VEGF and osteogenesis by enhancing the activity of BMP2 • Clay films may allow functionalisation of implant surfaces for localised/enhanced activity of biomolecules Acknowledgements Bone and Joint Research Group, University of Southampton Surgeons at Southampton General Hospital BMP2 binding? Effect of BMP2 vs. Serum Concentration Effect of cell adhesion/proliferation? Preserved BMP2 activity…
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