Bone mineralisation during osteoporosis

Bone mineralisation during
osteoporosis
RCSI Tissue Engineering Research Group
Lead PI: Prof. Fergal J. O‘Brien, PhD
Dept. of Anatomy, Royal College of Surgeons in Ireland
Trinity Centre for Bioengineering, Trinity College Dublin

Osteoporosis
• Osteoporosis is a skeletal disorder where bone strength is
reduced resulting in increased risk of fracture. Every 3 minutes
someone has a fracture due to osteoporosis.
• Cost to European Economy: €17 billion annually
• Aging population worldwide. 30% of people will have a hip
fracture by 90yrs (IOF). In Ireland, 3-fold increase in hip
fractures since 1990
• Affects most commonly post- menopausal women (14-18%
risk v 3-6% in males)
• Detected clinically by measuring bone quantity (BMD) using
DEXA scanning. Treated clinically with drugs (eg:
bisphosphonates) which prevent osteoclast activity & maintain
bone density

Osteoporosis and Bone Fracture
Bone Quantity + Bone Quality = Bone Strength
Bone Mineral
Density (BMD)
DEXA Scan
Architecture
Microstructure
Mineralisation
Bone Turnover
Microdamage
Cells
Half of all patients with fractures have a BMD value above the
diagnostic threshold of osteoporosis (Garnero and Delmas, 2004)
Evidence indicates that bone mass alone is insufficient to explain the
skeletal fragility of osteoporosis (Bouxsein, 2003)

Bone Hierarchy
To gain a comprehensive understanding of bone, different structural levels need to be analysed

Bone for Life Project
Ovine model used to simulate postmenopausal osteoporosis in women.
Examined the effects of estrogen depletion and bisphosphonate therapy on BMD,
bone quality and bone strength in osteoporosis.
Funded by: HEA PRTLI III & HRB 2004 & 2007

Examined
12
Months
31 Months Zoledronic Acid
Bone Mineral Density - - -
Architecture ↓ ↓ ↑
Microstructure ↓ ↓ No data
Mineralisation ↓ ↓ ↑
Microdamage - ↑ ↑
Bone Turnover ↑ ↑ ↓
Osteocyte Apoptosis ↑ ↑ ↓
Strength
Material ↓ ↓ ↑
Unit ↑ ↑ No data
Specimen ↓ ↓ ↑
Whole Bone ↓ ↓ No Data
Brennan et al, J Orthop Res
(In Press).
Brennan et al, J Biomech (In
Press).
Healy et al, J Orthop Res
2010.
Brennan et al, J Biomech,
2009.
Kennedy et al, J Anat 2009.
Keeley et al, Vet Comp
Orthop Traumatol 2008,.
Kennedy et al, Spine. 2008.
Kennedy et al, J Orthop Res
2008.
Kennedy et al, Bone. 2008.
Kennedy et al, Stud Health
Technol Inform. 2008.
DEXA does not adequately predict fracture risk in an ovine osteoporosis model
Collaborators: RCSI, UCD, TCD, NUIG, Olin College, University of Adelaide

Bone Repair
• 2.5 million bone grafts procedures annually worldwide
- Non-union fractures
- Implants, plates & screws
- Spinal fusion
- Arthritis
orthopaedic
- Regeneration following injury, infection & disease eg: osteosarcoma, osteomyelitis
- Reconstructive surgery eg: maxillofacial and dental applications
• Second most transplanted tissue after blood
• Current clinical treatment:
Autografts: lack of tissue & donor site morbidity
Allografts: lack of donors & risk of infection
Bone graft substitutes and other approaches: no ‗gold standard‘

Tissue Engineering Focus
• Biomaterial scaffolds
- Collagen-glycosaminoglycan (tailored)
- Collagen-ceramic composites
• Cells
– Cell types
• Mesenchymal stem cells
• Amniotic fluid-derived stem cells
• Osteoblasts
• Chondrocytes
• Smooth muscles cells, endothelial cells
• Cell signalling
- Bioreactors
- role of biophysical stimuli in regulating stem cell
differentiation and tissue formation
- Genes & Growth factors
Tissue Engineering
Triad
Angiogenesis
+VEGF
+Ephrin- B2
+SMC & ECs
+ Hypoxia
+ Flow perfusion
Funded by: SFI, HRB, IRCSET, Enterprise Ireland & European Research Council (FP7)

Collagen-based Scaffolds
Most abundant protein in
mammals
Found in:
Blood vessels
Intestines
Cartilage
Ligaments
Tendons
Bone
Skin
Triple helix
Collagen slurry
Chamber
shelf
(-40 o C)
Freeze-drying
Chamber
Lyophilisation
Stainless steel
tray
O‘Brien et al. Biomaterials 2004; 25: 1077-1086, Biomaterials 2005; 26: 433-441.
Technology & Healthcare 2007; 13: 1-15.

Collagen-based Scaffolds
Excellent
Biocompatibility
Homogeneous Pore
Size
50µm - 500µm
Biodegradable
High Porosity
~ 99.5%
Non- Toxic
degradation
products
O‘Brien Lab
Optimised composition and pore structure for specific applications
Improved mechanical properties
SFI PIYRA 2004

Composite Collagen-Ceramic Scaffolds
Collagen
scaffold
SFI PIYRA 2004
SFI RFP 2006
Enterprise Ireland
Hydroxyapatite scaffold
- High stiffness
- High compressive strength
But
- Low porosity & permeability
- Low tensile strength
- Issues with degradation

Effect of nano-HA addition & crosslinking on
mechanical properties of collagen scaffolds
Cunniffe G. et al. Journal of Biomedical Materials Research: Part A. 2010; Sep 28. [Epub ahead of print]

Addition of nano-HA on cellular behaviour
and osteogenesis
{
{
Increase in cell
number
Increase in
osteogenesis
Cunniffe et al. (2010) Journal of Materials Science: Materials in Medicine 21(8): 2293- 2298.

Gene-activated scaffolds: nano-HA particles as
non-viral delivery vectors
Relative Luminescence .
12
10
8
Collagen
s100
s500
6
4
2
0
1 2 3 7 10
Time (Days)
Image of GFP-transfected MSCs (green) on
composite coll-nHA scaffold
Sustained gene expression on composite
scaffolds
Curtin C, Cunniffe G et al. (2010)

Assessment of in vivo healing
1
8mm calvarial defect
Alhag M. et al. Oral and Maxillofacial Surgery (2010) Jul 20. [Epub ahead of print]
Lyons F., Kieran S. et al. (2010) Biomaterials 31: 9232-9243.

Control (8 weeks)
1
Collagen-CP (8 weeks) - Cell Free
Lyons F., Kieran S. et al. (2010) Biomaterials 31: 9232-9243.
Alhag M. et al. Oral and Maxillofacial Surgery (2010)
Jul 20. [Epub ahead of print]

Commercial Orthopaedic Applications (1)
HydroxyColl
• Bone Tissue Regeneration
• Collagen-HA Scaffold
— Load-bearing composite scaffold
— Patent filed (WO2008096334)
— Undergoing regulatory approval
process (FDA 510k /CE mark)
Bioengineering in Ireland 16 (2010) ―Best Overall Paper‖
HydroxyColl
Rabbit radial defect
Week 6
RAMI Bronze Medal
CHA Scaffold
Autogenous
Bone: Week 6
Surgical
Defect
week 0
• Translational Research
Real ―bedside‖ benefits
Clinical trials sooner than later
Enterprise Ireland Commercialisation Fund
POC PC/2007/331 & CFTD TD/2007/112

Commercial Orthopaedic Applications (2)
RCSI ―Big Ideas‖ winner 2009
• Osteochondral Tissue Repair
Roche Researcher of the Year Nomination 2009
• Multi-layer Scaffold
(ChondroColl)
— ―Seamless Integration‖
• Optimised regions for
— cartilage, calcified cartilage,
bone
• Novel IP (WO2010084481)
— Pre-clinical trial (2010)
— Femoral condylar defect
Enterprise Ireland Commercialisation Fund
POC PC/2005/226 & CFTD TD/2009/104

Science Foundation Ireland
Enterprise Ireland
Health Research Board
Higher Education Authority PRTLI
RCSI Research Committee
Irish Research Council for Science, Engineering
and Technology
Integra Life Sciences
Siemens
Novartis
European Research Council (2010-2014)

European Research Council Investigator Award
Fergal O‘Brien (2010- 2014)
• Role of biophysical stimuli in regulating MSC
differentiation
- computational predictions of optimal flow
conditions
- applied to experimental models of:
- osteogenesis
- angiogenesis
• Scaffold as delivery systems for bone formation
- cells
- tissue
- genes (non-viral technologies)
- growth factors (sustained release)

Tissue Engineering Research Group
Principal Investigators:
Fergal O‘Brien, Clive Lee, John Gleeson, Garry Duffy
Postdoctoral Associates
Orlaith Brennan, Grainne Cunniffe, Caroline Curtin, David Hoey,
Siti Ismail, Tanya Levingstone, Ciara Murphy, Ryan McCoy, Sonia Partap
Graduate Research Students
Mohamed Alhag Claire Brougham Sarah Louise Gill
Conn Hastings Ian Hutchison Michael Keogh
Stephen Kieran Frank Lyons Peter Mauer
Lauren Mulcahy Amos Matsiko Lyiah Subramaniam
Emmet Thompson Erica Tierney Amro Widaa
Niamh Walsh
Research Technician
John O‘Brien