Scaffold Characterization

I. Mechanical Characterization

1. Tensile Strength

- Load bearing upto which a scaffold could be elongated prior to breaking

- Tested for fibrous materials

Tensile strength = Pmax/ Ao

where Pmax = maximum load and Ao = original cross-sectional area

2. Compressive Strength

- It gives degree of compression of a material.

- The specimen is compressed and deformation at various loads is recorded.

- Tested for porous scaffold

Fig 1: Representative graph of stress-strain curve

Terminology

 Stress: An applied force or system of forces that tends to strain or deform a body

Stress (σ) = force/area = F/A

 Strain: change in dimension of a body under load (can be in terms of length, area or
volume)

Strain (ε) = original dimension/ change in dimension = ΔX/X

where X = length, area or volume

Load = weight/ force

applied

 Elastic limit: It is the limit where stress can be applied to an elastic body without
causing permanent deformation
 Break point: A point of discontinuity, change or cessation
 Yield stress/ Yield strength/ Yield point: It is defined as the stress at which a
material begins to deform plastically.
 Young's modulus- The slope of the elastic portion of stress-strain curve

Young’s modulus (Y) = stress/ strain = σ/ ε

 Ultimate strength- Maximum value of load bearing after which it may get
permanently deformed.

II. Physicochemical Characterization

1. Morphological Analysis

i) Scanning Electron Microscopy (SEM)

 Principle: Accelerated electrons in an SEM carry significant amounts of kinetic

energy, and this energy is dissipated as a variety of signals produced by electron-
sample interactions when the incident electrons are decelerated in the solid sample.
These signals include secondary electrons (that produce SEM images), backscattered
electrons (BSE), diffracted backscattered electrons (EBSD).
  Secondary electrons come from within a few nanometers of the sample surface, with a
lower energy compared to the backscattered electrons. They are very sensitive to
surface structure and provide topographic information. Secondary electrons, originate
from the atoms of the sample: they are a result of inelastic interactions between the
electron beam and the sample.
 Working: The electrons are emitted from a filament and collimated into a beam in the
electron source. The beam is then focused on the sample surface by a set of lenses in
the electron column.
 Applications:
- used to generate high-resolution images of shapes of objects (Precise measurement of
very small features and objects is also accomplished using SEM)
- to show spatial variations in chemical compositions.
- Pattern and diameter of pores and fibers can be determined

Fig 2: Working of SEM

ii) Transmission Electron Microscope (TEM)

• Principle: The TEM operates on the same basic principles as the light microscope but
uses electrons instead of light. What can be seen with a light microscope is limited by
the wavelength of light. TEMs use electron as “light source” and their much lower
wavelength makes it possible to get a resolution a thousand times better than with a
light microscope.
 • Working: electrons that are made to pass through the specimen and the image is
formed on the fluorescent screen, either by using the transmitted beam or by using the
diffracted beam.

• Applications:

- The transmission electron microscope is used to characterize the microstructure of

materials with very high spatial resolution.
- Used to determine morphology, crystal structure and defects
- Crystal phases and composition can be determined
- Information about magnetic microstructure can be obtained

Fig 3: Working of TEM

iii) Atomic Force Microscopy (AFM)

 Principle: The AFM consists of a cantilever with a sharp tip (probe) at its end that is
used to scan the specimen surface. When the tip is brought into proximity of a sample
surface, forces between the tip and the sample lead to a deflection of the cantilever.
 Deflection is measured using a laser spot reflected from the top surface of the
cantilever into an array of photodiodes
 The cantilever is typically silicon or silicon nitride with a tip radius of curvature in the
order of nanometers
 Forces that are measured in AFM include mechanical contact force, van der Waals
forces, capillary forces, chemical bonding, electrostatic forces, magnetic forces  etc.
  Advantages:
- provides a three-dimensional surface profile.
- samples viewed by AFM do not require any special treatments (such as metal/carbon
coatings) that would irreversibly change or damage the sample
- final image is free from charging artifacts.
- Most AFM modes can work perfectly well in ambient air or even a liquid
environment. This makes it possible to study biological macromolecules and even
living organisms

Probe

Sample surface

Fig 4: Working of AFM

2. Porosimetry

• “Porosimetry” includes the measurements of pore size, volume, distribution, density

and other porosity-related characteristics of a material.
• Porosity is especially important in understanding the formation, structure and
potential use of many substances.
• The porosity of a material affects its physical properties (adsorption, permeability,
strength, density) and, subsequently, its behaviour in its surrounding environment.

Porosity (%) = [(V1 – V3) / (V2 – V3)] x 100

where V1 = initial volume of liquid

V2 = Volume of liquid with immersed scaffold

V3 = residual volume of liquid

3. Hydrophilicity

i) Contact Angle Measurement

• The contact angle is the angle where a liquid/vapour interface meets a solid surface.
• Its conventionally measured through a liquid,
• It quantifies the wettability of a solid surface by a liquid. A given system of solid,
liquid, and vapour at a given temperature and pressure has a unique equilibrium
contact angle.
• The equilibrium contact angle reflects the relative strength of the liquid, solid, and
vapour molecular interaction.

• Significance:

- Determines amount of liquid a substance/scaffold can hold

- A scaffold implanted in the body, comes in contact with the body fluids
- Hydrophillicity of the scaffold is necessary for cell survival and cell attachment

ii) Swelling Behaviour and Water Uptake capacity

• The equilibrium swelling ratio (Es) can be measured by the conventional gravimetric
method.
• The dry weight (Wd) of scaffold is measured and then wet weight (Ws) by immersing
in simulated body fluid (SBF).
• The equilibrium swelling ratio of the scaffolds is defined as the ratio of weight
increase (Ws-Wd) with respect to the initial weight (Wd) of dry samples.
• Es is calculated using the following equation:

Es=

• Water uptake percentage (Wu) can be measured using the equation:

Wu= (Ws-Wd)/ Ws x 100

4. Viscosity

• Principle: The drag caused by relative motion of the fluid and a surface is a measure
of the viscosity.
• Viscosity of a fluid is measured by instrument known as Viscometer.
• For liquids with viscosities which vary with flow conditions, an instrument called a
rheometer is used. Viscometers only measure under one flow condition.
• In general, either the fluid remains stationary and an object moves through it, or the
object is stationary and the fluid moves past it.
• The flow conditions must have a sufficiently small value of Reynolds number for
there to be laminar flow.

5. Biodegradation

• When a scaffold is incorporated inside the body, it comes in contact with various
biological fluids and enzymes and thus degrades with time. And the space generated
by scaffold degradation facilitates new tissue formation.
• A scaffold should not only degrade, it should “Bio” degrade, i.e. its degradation
should be accompanied by its removal from the body, without generating any toxic
effect to the surrounding tissues.

Degradation rate of scaffold = Rate of tissue formation

• Degradation testing is done by giving enzymes like lysozyme and incubating in a

fluid medium (PBS or SBF) for particular time period and measuring change in
weight.

Degradation (%) = [(Wi – Wf)/ Wi] x 100

Where Wi = initial sample weight and Wf = final sample weight

Fig 5: Composition of SBF (Simulated Body Fluid)

III. Structural Characterization

1. X-ray Diffraction (XRD)

 Principle: Braggs Law of diffraction. A law stating that when a crystal is pictured as
a set of reflecting planes uniformly spaced at a distance d and a beam of X-rays of
wavelength λ strikes the crystal at an angle θ, reinforcement of the reflected waves
occurs when sin θ = n λ/2d, where n is an integer known as the order of reflection.

nλ = 2d sin θ

 XRD is a material characterization technique that can be used for analyzing the lattice
structure of a material.

 Applications
- Phase identification.
- Quantitative analysis.
- Crystal structure analysis.
- Microstructure of real materials.

2. Fourier Transform Infrared Spectroscopy (FTIR)

• Molecular bonds vibrate at various frequencies depending on the elements and the
type of bonds.
• Each bond vibrate at several specific frequencies.
• The data from the sample is collected in a wide spectral range and converted into
specific frequency which can be recorded as a function of transmittance.