1. Mestrado Integrado de engenharia Biomédica
1º Ano, Semestre 1 – Introdução à Engenharia Biomédica
Prof. Patrícia Figueiredo
Dr. ª Saba Oliveira da Silva; Prof. Dr. João Eurico da Fonseca
Structural and mechanical evaluation of the mineral and organic phases in osteoporotic bone using an animal model of osteoporosis
Osteoporosis
Beatriz Almeida, nº 73150; Dar'ya Khoroshylova, nº 73356; Elisa Pacheco, nº72993; Sara Pires, nº 72635

2. Introduction: Structure of the bone
Mestrado Integrado de engenharia Biomédica
Introduction: Structure of the bone
Bone structure
Cancellous
Compact
Organic
Phase
Extracellular
Matrix
Mineral
Phase

3. Mestrado Integrado de engenharia Biomédica
Organic
Mineral
Constituiton
Collagen type I fibrils
Hydroxyapatite crystals
Lay down
Paralel orientation
Between collagen fibrils
Biomechanical function in bone
Toughness
Ductility
Stifness
Strength
% weight of bone mass
30-40%
50-74%

4. How does bone maintain itself?
Bone Remodelling
Figure 1 – Bone Remodelling.

5. Mestrado Integrado de engenharia Biomédica
Building
Reabsortion
Healthy
Bone
Building
Reabsortion
Imbalance

6. Mestrado Integrado de engenharia Biomédica
Osteoporosis (OP)
Definition
OP is a disease marked by reduced bone strength leading to an increased risk of fractures, or broken bones. Usually tested for using the classical Bone Mineral Density method by DEXA
Incidence
Women after menopause
Men over the age 75
Common Locations
Fractures occur most often in bones of the: Hip, Wrist or Spine......but any bone can be affected!

7. Figure 2– Normal bone versus Osteoporotic bone.

8. Failure point Ultimate Stress Yield point Strain
Figure 3 – Stress-Strain Curve of the Bone

9. Mestrado Integrado de engenharia Biomédica
State of the Art
Atomic Force Microscopy (AFM)
A flexible cantilever acts as a “spring” to measure very small forces between the tip and the sample, generated when atoms or molecules come close together.
Figure 4 - Measurement of cantilever's deflection.

10. AFM : Advantages
Accurate quantitative analysis of the mechanical properties of the bone at the tissue level: collagen and mineral matrix.
Less destructive and high resolution in physiological medium and over a range of temperatures - Less sample preparation.
Reduced sample size (e.g 10 microns).

11. Three-point bending test
An external load is applied perpendicularly to a longitudinal axis of the material to determine its flexural strength and modulus.
Figure 5 – Three Point Bending Test

12. performance of the entire bone at a macro scale.
Advantages
Study the mechanical
performance of the entire bone at a macro scale.

13. Material & Methods Groups of Study Extraction of bone for testing
Animal model-Ovariectomy
Groups of Study
Control Group (SHAM)
Ovariectomized Group (OVX)
Sacrificies
After 2 months of disease
Extraction of bone for testing
Vertebra L4
Femures

14. Nanoindentation using AFM
Mestrado Integrado de engenharia Biomédica
Nanoindentation using AFM
- Machine JPK NanoWizard® AFM System
- Mice vertebra L4 (2 months after inducing OP)
Selection of a zone
Penetration of the sample
Analysis of the data
Histogram
(Frequency of each value of Young’s Modulus)

15. Nanoindentation using AFM: Formula
Figure 6 – Grid of Nanoindentation
Young’s Modulus

16. Three-point bending Test
-Instron 5566TM (Instron Corporation, Canton, USA), with a load cell of 10kN
- Mice femures (2 months after inducing OP)
The bone is placed on two supports, while only one contacts with the opposite surface, exactly at the mid point

17. Three-point bending Test: Formulas
Stress
Strain
Young’s Modulus

18. Development of the disease
Results & Discussion
Animal Model
Development of the disease
Control group
Ethical matter
Figure 7 – Extraction of mice femur

19. Nanoindentation using AFM
Young’s Modulus
One hundred indentation marks
OriginPro8
Histograms
Figure 8 – AFM Machine

20. Histogram 1 - Distribution of E values in SHAM2
in OVX2
Histogram 4- Distribution of E values
in OVX5

21. Three-Point bending Test Absorbed Energy until Fracture
Maximum Load (N)
Fracture
Point
Absorbed Energy until Fracture
(Area)
Young’s Modulus
Graph 1 – Force-Displacement Curve obtained with 3-Point-Bending Test performed in OVX1 left femur

22. Extension at Max Load (mm) 0,53 0,542828 ET (GPa) 1,9961 2,322258
Table 1 – Average results of 3-Point-Bending Test
Specimen
SHAM
(Average)
OVX
Max Load (N)
23,99
22,614
Max Stress (MPa)
188,279
186,708
Extension at Max Load (mm)
0,53
0,542828
ET (GPa)
1,9961
2,322258
Figure 9 – Three-Point
Bending Test
Figure 10 – Four-Point
Bending Test

23. Conclusion & Suggestions for Future Work
Mestrado Integrado de engenharia Biomédica
Conclusion & Suggestions for Future Work
3-Point-Bending Test
No noticeable changes in the mechanical properties
AFM
Differences in the mechanical properties
Bone’s ductility
Young’s Modulus VS

24. We can now say that Osteoporosis does lead to changes in the mechanical properties of the bone!

25. Mineralization Density Distribution (BMDD)
Mineralized component of bone
Fragility of bone
Bone
Mineralization Density Distribution (BMDD)
Quantitative Backscattered Electron Imaging (qBEI)

26. Enlarge the number of samples
Enlarge the period between sacrifice and osteoporosis induction