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In vitro evaluation of a multifunctional bactericidal synthetic bone substitute D. Bollati, M. Morra, C. Cassinelli, G. Cascardo Nobil Bio Ricerche srl, Via Valcastellana 26, 14037 Portacomaro (AT) Italy www.nobilbio.it [email protected] Baveno, SIB Conference, 3-5 June 2013 Outline • Introduction • Goal of the study • Materials and methods • Results • Comments and conclusions Baveno, SIB Conference, 3-5 June 2013 Introduction: bone substitutes Powder, granules or scaffolds used to fill up bone pits in different applications: - maxillofacial - dental - prosthetic surgery - spinal surgery These materials could be synthetic or of animal/human source ; a synthetic bone substitute should be biocompatible, osteoconductive and possibly osteoinductive; often used in presence of infections Baveno, SIB Conference, 3-5 June 2013 antibiotic delivery in the implant site Goal of the study Development of an innovative process of surface modification on a synthetic phosphatic bone substitute: Bone formation at the interface with tissue Prevention of bacteria colonization ‘’Multifunctional’’ synthetic bone substitute Baveno, SIB Conference, 3-5 June 2013 Materials and methods - β-TCP 75% - nano HAP 25% - Granules dimensions: 300 μm/ 2 mm Symbol Formula Ca/P HAP Ca10(PO4)6(OH)2 1,67 Β-TCP Ca3(PO4)2 1,5 1 mm 10 µm Baveno, SIB Conference, 3-5 June 2013 Materials and methods Surface modification ‘’CVHA’’ 1. Collagen fibrillation with vancomycin and hyaluronic acid; 2. covalent linking of hyaluronic acid; 3. interaction of vancomycin with HA and crosslinking. ~ 200 nm βTCP/HAP granules collagen Baveno, SIB Conference, 3-5 June 2013 vancomycin hyaluronic acid Results: anti-bacterial effect ‘’Agar germ’’ experiments Ctrl “CVHA” Staph. Epidermidis growth is inhibited by vancomycin release from the granules Baveno, SIB Conference, 3-5 June 2013 Results: anti-bacterial effect Vancomycin release 600 ug vancomycin 500 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 time (days) Most of antibiotic is released within 3 days Baveno, SIB Conference, 3-5 June 2013 20 22 Results: biological response “Osteoimmunology: crosstalk between the immune and bone system” T. Nakashima, H. Takayanagi ; J Clin Immunol (2009) 29:555–567 bone formation depends on the balancing between osteoblast and osteoclast activity osteoclast differentiation is enhanced by several cytokines produced by inflammatory cells and by specific signal molecules produced by monocytes and osteoblasts Gene expression analysis of macrophages and osteoblasts Baveno, SIB Conference, 3-5 June 2013 Results: biological response (macrophages) Gene expression of J774A.1 macrophages Expression of these genes at different timepoints - MCP-1 - IL-1β - IL-6 - IL-10 Pro-inflammatory mediators, stimulate bone resorption, osteoclast formation, RANKL signaling and are involved in the response to bacteria and endotoxins Anti-inflammatory mediator 1) M.Rauner et al “Osteoimmunology” Int. Arch. Allergy Immunol. 2007,143, 31-48 2) K. Steeve et al. “IL-6, RANKL, TNF alpha/IL1: interrelations in bone resorption pathophysiology” Cytokine and Growth Factor Reviews, 15 (2004), 49-60 Baveno, SIB Conference, 3-5 June 2013 Results: biological response (macrophages) Gene expression of J774A.1 macrophages 4h 24h Ctrl CVHA Baveno, SIB Conference, 3-5 June 2013 Results: biological response (osteoblasts) Gene expression of SaOs-2 osteoblasts Expression of these genes at different timepoints - ALP - OCN Pro-osteogenic markers, generate free phosphates, involved in bone mineralization and calcium binding action - BSP - RANKL RANK receptor,induces differentitation of pre-osteoclasts to osteoclasts - OPG Soluble RANK, its interaction with RANKL inhibits osteoclast formation 1) M.Rauner et al “Osteoimmunology” Int. Arch. Allergy Immunol. 2007,143, 31-48 2) K. Steeve et al. “IL-6, RANKL, TNF alpha/IL1: interrelations in bone resorption pathophysiology” Cytokine and Growth Factor Reviews, 15 (2004), 49-60 3) V. Pivodova et al. “Osteoblast and gingival fibroblast markers in dental implant studies” Biomed. Pap. Med. 2011, June, 155(2) 109-116 Baveno, SIB Conference, 3-5 June 2013 Results: biological response (osteoblasts) Gene expression of SaOs-2 osteoblasts 24h 72h Ctrl CVHA Baveno, SIB Conference, 3-5 June 2013 Results: biological response ‘’CVHA’’ surface modification could affect bone remodeling mechanisms monocytes osteoblasts ALP, BSP IL-1, IL-6, MCP-1 IL-10 RANKL differentiation osteoclasts Baveno, SIB Conference, 3-5 June 2013 Comments and conclusions a synthetic biphasic bone substitute was developed surface modification with collagen, vancomycin and hyaluronic acid (stable even after γ sterilization) antibiotic release is immediate and effective to inhibit bacterial growth in vitro the presence of collagen and hyaluronic acid seems to shift the balance of bone remodeling towards bone formation, instead of its resorption this multifunctional product could help to meet several clinical needs Thank you for attention Baveno, SIB Conference, 3-5 June 2013