Transcript synthetic bone substitute Baveno, SIB Conference, 3

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