In vitro evaluation of a multifunctional bactericidal synthetic bone substituteD. Bollati, M. Morra, C. Cassinelli, G. Cascardo
Nobil Bio Ricerche srl, Via Valcastellana 26, 14037 Portacomaro (AT) Italy
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
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
antibiotic 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
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
Materials and methods
- β-TCP 75% - nano HAP 25%
Symbol FormulaCa/P
HAP Ca10(PO4)6(OH)2 1,67
Β-TCP Ca3(PO4)2 1,5
- Granules dimensions: 300 μm/ 2 mm
1 mm
10 µm
Baveno, SIB Conference, 3-5 June 2013
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
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
Results: anti-bacterial effect
Vancomycin release
Baveno, SIB Conference, 3-5 June 2013
Most of antibiotic is released within 3 days
0
100
200
300
400
500
600
0 2 4 6 8 10 12 14 16 18 20 22
time (days)
ug
va
nc
om
yc
in
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
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
Gene expression of J774A.1 macrophages
Results: biological response (macrophages)
4h 24h
Baveno, SIB Conference, 3-5 June 2013
Ctrl
CVHA
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
- OPGSoluble 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
Gene expression of SaOs-2 osteoblasts
Results: biological response (osteoblasts)
24h 72h
Baveno, SIB Conference, 3-5 June 2013
Ctrl
CVHA
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
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
