A Comparison of High Resolution X-ray Micro-computed Tomography (micro-CT) with other techniques used in the characterization of scaffolds

1. Review
A Comparison of micro CT with other techniques
used in the characterization of scaffolds
Saey Tuan Ho, Dietmar W. Hutmacher
Biomaterials 27 (2006) 1362 - 1376

2. Contents
 Introduction
 Architectural and structural parameters
 Theoretical method and SEM analysis
 Mercury porosimetry
 Gas pycnometry
 Gas adsorption
 Flow porosimetry
 Micro CT
 A micro CT study
 Conclusion

3. Introduction
Crucial factors in scaffold design:
Structure
Architecture
Scaffold porosity and pore size:
Large surface area favors cell attachment and growth
Large pore volume is to accommodate and deliver
sufficient number of cells
High porosity is for easy diffusion of nutrients,
transport and for vascularization
Evaluation methodology:
Fast
Accurate
Non-destructive

4. Creation & Design
 Creation: Two methodology
 Design and fabrication
 Fabrication followed by design optimization
 Design: Two broad categories
 Precise geometrical layout
 Honeycombed scaffolds, woven textile meshes
 Deposition via non-precise ways
 Foams, Nano-fiber meshes

5. Various Scaffold Designs

6. Molecular Transport
 Vasculature growth & diffusion:
 Pore network optimization
 Main mode of transport
 Exchange of oxygen
 Nutrient
 Metabolic wastes
 Molecular signaling
 Key property: Porosity
 Cell seeding efficiency
 Diffusion
 Mechanical strength

7. Architectural and Structural Parameters
 Porosity
 Pore size
 Surface area to volume ratio
 Interconnectivity of pores
 Anisotropy
 Strut thickness
 Cross sectional area
 Permeability

8. Definition
Pore size : Average diameter of pores
Strut/Wall thickness : Average
diameter/thickness of scaffold struts
Anisotropy : A measure of the non -uniformity
in the alignment of scaffold struts
Cross-section area : A measure of the area in a
specified sectional plane of the scaffold
Permeability : A measure of the ease with
which fluid passes through the scaffold pores

9. Theoretical Method
 Most of the methods are capable of
estimating porosity
 There are two main approaches
 Unit cube analysis
 Mass technique
 Other approaches
 Archimedes Method
 Liquid displacement method

10. Unit Cube Analysis
Porosity = (1 – Vf / VA) x 100%
 Vf is scaffold material volume
 VA is apparent scaffold cube volume
 Vf=ПLd2
n1n2
 VA = Lwh
 d = Strut diameter
 L = Strut length
 w = Strut width
 h = Scaffold height
 n1 = Number of struts per layer
 n2 = Number of layers per scaffold

11. Merits & Demerits
 Commonly adopted for honeycombed scaffolds
 Calculation assume uniform struts and layers
 Cannot apply to scaffolds fabricated using extrusion
techniques
 Fused deposition modeling
 3D printing
 Stereolithography

12. Mass Technique
Porosity = (1 – Vg / VA) x 100%
 Vgis scaffold material volume
 VA is apparent scaffold cube volume
 Vg=mass / density of scaffold material
 VA = Lwh
 L = Strut length
 w = Strut width


13. Merits & Demerits
 Commonly adopted for scaffolds with controlled &
un-controlled geometries
 Dependent on accuracy of linear measurements (L, w
& h) of the cube
 Rough edges and inaccurate linear dimensions would
be a concern

14. Archimedes Method
Porosity = (M wet – M dry) / (M wet – M submerged)
• M dry = Dry mass of scaffold
• M wet = Mass of prewet scaffold
• M submerged = Mass of scaffold soaked in water
 Inappropriate for hydrophobic scaffolds

15. Liquid Displacement Method
Porosity = (V1 – V3) / (V2 – V3)
 V1 = Initial known volume of scaffold
 V2 = Volume sum of ethanol & submerged scaffold
 V3 = Volume of ethanol in bath after scaffold
removal

16. Scanning Electron Microscopy
 Complements theoretical calculation of
porosity
 Allows direct measurement of pore size and
wall thickness
 Qualitative - Provides visual estimation of
interconnectivity, cross-section area and
anisotropy
Restricted to surface analysis
Layer fusion & edge effects

17. Layer Fusion & Edge Effects in SEM

18. Mercury Porosimetry

19. Mercury Porosimetry - Principle
Washburn Equation : DP = - 4γ cos θ
 D = Pore diameter
 γ = Surface tension of mercury
 P = Applied pressure
 θ = Contact angle between pore wall and mercury
 Provides bulk volume, total open pore volume and
porosity
 Measurable pore size range: 0.0018 to 400 µm
 Does not account closed pores
 Excess pressure may compress the sample
 Calculation assume cylindrical pores
 Destructive analysis

20. Gas Pycnometry
Vx = (PE Vc + PEVr – PCVC - PrVr) / (PE- PC)
 Vx is scaffold volume
 Vc is chamber volume
 Pc is initial chamber pressure
 Vr is reference chamber volume
 Pr is reference chamber pressure
 PE is pressure at equilibrium

21. Merits & Demerits
 Measures scaffold material volume
 Accuracy depends on absence of moisture
and volatile substances
Sample pre-treatment in vacuum oven
Does not account closed pores
Porosity to be calculated using unit cube
approach
Error in linear measurement may concern

22. Gas Adsorption
Gas Adsorption Process

23. Gas Adsorption - Principle
• Based on adsorption of gas molecules due to Van der
Waals & electrical forces
• Surface area is calculated using BET theory
• Pore size is derived using BJH method
 Measurable pore size range: 0.35 to 400 nm
 Relevant to nano - featured & nano - modified
scaffolds
 From isotherms and hysteresis loop several key
parameters are elucidated
 Does not account closed pores and scaffold volume
 Not suitable for scaffolds with low specific surface
area

24. Flow Porosimetry
 Non - destructive method
 Compression in pore size is measurable
 Measurable pore size range: 0.013 to 500 µm
Should be coupled with other techniques to
determine porosity
Does not account closed pores

25. Micro CT
History & Developments:
 Feldkamp et al pioneered the system in
early 70’s
 Used extensively to study trabecular
architecture
 Explored for assessment of scaffolds,
regenerated tissue and vasculature
networks

26. Micro CT - Principle

27. Micro CT Scanning
Specimen is divided into series of 2D slices
Emergent x-rays are captured by detector
2D pixel map is created
Attenuation coefficient correlated to material
density
3D modeling program visualize the object
Accuracy depends on software & hardware

28. 2D Slicing & Reconstruction

29. 3D Visualization

30. Various Specimens

31. Merits
 Non - destructive method
 No sample pre-preparation
 Precise quantitative & qualitative
information on 3D morphology
 Finite element modeling as an alternative
to mechanical testing

32. Beam hardening
Artifacts created by metals
Thresholding is not always accurate
More time consumption for higher
resolution
Storage and processing of large data sets
Demerits

33. Summary of Individual Techniques
Parameters Theorectic
-al
Method
SEM Mercury
Porosimetry
Gas
Pycnometry
Gas
Adsoprtion
Flow
Porosimetry
Micro
CT
Porosity + Q + + + - +
Surface area - - + - - - +
Interconnect
-ivity
- Q - - - - +
Pore size - + + - + + +
Wall
thickness
- + - - - - +
Anisotropy - Q - - - - +
Cross-
section area
- Q - - - - +
Permeability - - + - - - S
Q = Qualitative S = Dependent on software

34. A Micro CT Study
Material Angle Porosity –
Pycnometer
(%)
Porosity –
Mimics
(%)
Surface
area/volume –
Mimics
(mm2
/mm3
)
Interconnectivity
(%)
Copolymer
of PEG,
PCL & PLA
00
/900
/1800
72.05 + 0.41 74.99 8.65 100
Copolymer
of PEG,
PCL & PLA
00
/600
/1200
70.60 + 0.67 75.45 9.07 100

35. 3D Analysis of Copolymers

36. Conclusion
Evaluation of scaffold architecture is necessary
Potential and concerns of technique is crucial
Micro CT is a rather new technique but possess it’s
own merits & demerits
New technique and future advancements are
anticipated to address the demerits