A Comparison of micro CT with other techniques used in the characterization of scaffolds

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

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

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

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

Various Scaffold Designs

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

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

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

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

Unit Cube Analysis
Porosity = (1 – V f / V A ) x 100%
V f is scaffold material volume
V A is apparent scaffold cube volume
V f = П Ld 2 n 1 n 2
V A = Lwh
d = Strut diameter
L = Strut length
w = Strut width
h = Scaffold height
n 1 = Number of struts per layer
n 2 = Number of layers per scaffold

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

Mass Technique
Porosity = (1 – V g / V A ) x 100%
V g is scaffold material volume
V A is apparent scaffold cube volume
V g = mass / density of scaffold material
V A = Lwh
L = Strut length
w = Strut width
h = Scaffold height

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

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

Liquid Displacement Method
Porosity = (V 1 – V 3 ) / (V 2 – V 3 )
V 1 = Initial known volume of scaffold
V 2 = Volume sum of ethanol & submerged scaffold
V 3 = Volume of ethanol in bath after scaffold
removal

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

Layer Fusion & Edge Effects in SEM

Mercury Porosimetry

Mercury Porosimetry - Principle
Washburn Equation : DP = - 4 γ 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

Gas Pycnometry
V x = (P E V c + P E V r – P C V C - P r V r ) / (P E - P C )
V x is scaffold volume
V c is chamber volume
P c is initial chamber pressure
V r is reference chamber volume
P r is reference chamber pressure
P E is pressure at equilibrium

Merits & Demerits
Measures scaffold material volume
Accuracy depends on absence of moisture and volatile substances
Sample pre-treatment in vacuum oven
Porosity to be calculated using unit cube approach
Error in linear measurement may concern

Gas Adsorption
Gas Adsorption Process

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

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

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

Micro CT - Principle

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

2D Slicing & Reconstruction

3D Visualization

Various Specimens

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

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

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

A Micro CT Study 0 0 /60 0 /120 0 0 0 /90 0 /180 0 Angle 100 9.07 75.45 70.60 + 0.67 Copolymer of PEG, PCL & PLA 100 8.65 74.99 72.05 + 0.41 Copolymer of PEG, PCL & PLA Interconnectivity (%) Surface area/volume – Mimics (mm 2 /mm 3 ) Porosity – Mimics (%) Porosity – Pycnometer (%) Material

3D Analysis of Copolymers

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

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A Comparison of micro CT with other techniques used in the characterization of scaffolds

by Sree Chitra Tirunal Institute for Medical Sciences and Technology

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