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: StructureArchitecture

Scaffold porosity and pore size:Large surface area favors cell attachment and growthLarge pore volume is to accommodate and deliver sufficient number of cellsHigh porosity is for easy diffusion of nutrients, transport and for vascularization

Evaluation methodology:FastAccurateNon-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 – Vf / VA) x 100%

� Vf is scaffold material volume� VA is apparent scaffold cube volume

� Vf =ПLd2n1n2

� 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

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 – Vg / VA) x 100%

� Vg is scaffold material volume

� VA is apparent scaffold cube volume

� Vg = mass / density of scaffold material

� VA = 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 = (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

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 - PrincipleWashburn 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

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

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

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

� Does not account closed pores

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

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

SEMTheorectic-al Method

Parameters

Q = Qualitative S = Dependent on software

A Micro CT Study

00/600/1200

00/900/1800

Angle

1009.0775.4570.60 + 0.67

Copolymer of PEG, PCL & PLA

1008.6574.9972.05 + 0.41

Copolymer of PEG, PCL & PLA

Interconnectivity (%)

Surface area/volume –

Mimics (mm2/mm3)

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