1. 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 dbollati@nobilbio.it Baveno, SIB Conference, 3-5 June 2013

2. Outline Introduction Goal of the study Materials and methods Results Comments and conclusions Baveno, SIB Conference, 3-5 June 2013

3. Introduction: bone substitutes  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 infectionsantibiotic delivery in the implant site Powder, granules or scaffolds used to fill up bone pits in different applications: - maxillofacial - dental - prosthetic surgery - spinal surgery Baveno, SIB Conference, 3-5 June 2013

4. Goal of the study Development of an innovative process of surface modification on a synthetic phosphatic bone substitute: Prevention of bacteria colonization Bone formation at the interface with tissue ‘’Multifunctional’’ synthetic bone substitute Baveno, SIB Conference, 3-5 June 2013

5. Materials and methods - β-TCP 75% - nano HAP 25% SymbolFormulaCa/P HAP Ca 10 (PO 4 ) 6 (OH) 2 1,67 Β-TCP Ca 3 (PO 4 ) 2 1,5 - Granules dimensions: 300 μm/ 2 mm 1 mm 10 µm Baveno, SIB Conference, 3-5 June 2013

6. 1. Collagen fibrillation with vancomycin and hyaluronic acid; Materials and methods Surface modification ‘’CVHA’’ 3. interaction of vancomycin with HA and crosslinking. ~ 200 nm 2. covalent linking of hyaluronic acid; collagen hyaluronic acid vancomycin βTCP/HAP granules Baveno, SIB Conference, 3-5 June 2013

7. Results: anti-bacterial effect Ctrl“CVHA” ‘’Agar germ’’ experiments Baveno, SIB Conference, 3-5 June 2013 Staph. Epidermidis growth is inhibited by vancomycin release from the granules

8. Results: anti-bacterial effect Vancomycin release Baveno, SIB Conference, 3-5 June 2013 Most of antibiotic is released within 3 days

9. 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

10. Gene expression of J774A.1 macrophages Results: biological response (macrophages) Baveno, SIB Conference, 3-5 June 2013 Expression of these genes at different timepoints Pro-inflammatory mediators, stimulate bone resorption, osteoclast formation, RANKL signaling and are involved in the response to bacteria and endotoxins - MCP-1 - IL-1β - IL-6 - IL-10 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

11. Gene expression of J774A.1 macrophages Results: biological response (macrophages) 4h24h Baveno, SIB Conference, 3-5 June 2013 Ctrl CVHA

12. Gene expression of SaOs-2 osteoblasts Results: biological response (osteoblasts) Baveno, SIB Conference, 3-5 June 2013 Expression of these genes at different timepoints Pro-osteogenic markers, generate free phosphates, involved in bone mineralization and calcium binding action - ALP - OCN - BSP RANK receptor,induces differentitation of pre-osteoclasts to osteoclasts - RANKL - 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

13. Gene expression of SaOs-2 osteoblasts Results: biological response (osteoblasts) 24h72h Baveno, SIB Conference, 3-5 June 2013 Ctrl CVHA

14. osteoblasts RANKL ALP, BSP IL-1, IL-6, MCP-1 IL-10 monocytes osteoclasts differentiation ‘’CVHA’’ surface modification could affect bone remodeling mechanisms Results: biological response Baveno, SIB Conference, 3-5 June 2013

15.  a synthetic biphasic bone substitute was developed  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  surface modification with collagen, vancomycin and hyaluronic acid (stable even after γ sterilization)  this multifunctional product could help to meet several clinical needs Comments and conclusions Baveno, SIB Conference, 3-5 June 2013 Thank you for attention