Micro/Nanoporous Scaffolds

for Tissue Engineering

Applications
Department of Chemical Engineering and Materials Science
Amrita School of Engineering
Coimbatore – 641 105
March 2009
Second Review
By
Divya Haridas (CB105PE012)
Karthikeyan G (CB105PE023)
Krishna Priya C (CB105PE025)
Premika G (CB105PE028)

Guide
Dr. Murali Rangarajan. Ph.D

Co-Guide
Dr. Nikhil K Kothurkar. Ph.D
 Scaffolds - Overview
 Scaffolds are extra cellular matrix which acts as
platform onto which cells can attach, grow and
form new tissues

 Scaffolds play a critical role during cell adhesion,

proliferation and differentiation

 Modeling, design and fabrication of scaffolds are

always a difficult task in the regenerative tissue
engineering
 Cell adhesion - Overview

 Cell adhesion is the binding of cell to another cell

or to a surface or matrix
 Cell adhesion – interaction of specific cell
adhesion molecules with other molecules
 Fundamental of this process is the ability of the
cells to recognize ,communicate and work
together with other cells of similar type allowing
them to organize into a higher oriented
structures that ultimately form the body.
 Cells achieve these functions through the activity
of and interaction between 1000s of proteins
 Cell adhesion - Overview

 Receptor – ligand
interactions is the basis
of cell adhesion

 These interactions are

regulated by proteins,
catalyst, inhibitors
 Cell adhesion - Overview
 Extracellular matrix  Cell membrane
 Collagens  Nectins
 Fibronectins  Integrins
 Elastins  Cadherins
 Laminins  Selectins

Fibronectin promotes the attachment of cells to the

matrix via the members of the integrin family
 Cell adhesion - Overview
 The adhesion
involves two phases:
 Attachment phase
 Adhesion phase

 Model Considered:
 Peeling Model of cell
adhesion (long-term)

 Peeling Model:
 Attachment and
Evan A. Evans, 1985. Detailed
detachment of
mechanism of membrane- membrane
receptor – ligand adhesion and separation, Biophysical
bonds Journal, 48, 175-183
 Force governs extent
of adhesion
 Peeling Model
 Assumptions:
 Cell membrane –
rigid, elastic
 Scaffold – flat, rigid,
nonporous surface

 Two zones:
 Free zone –
membrane is not
subjected to any Dong Kong, Baohua Ji, Lanhong Dai,
forces 2008. Nonlinear mechanical model of
 Adhesive zone – cell adhesion, Journal of Theoretical
membrane is Biology 250, 75-84
subjected to
attractive forces
 Peeling Model – Solutions
 Linear Model:  Fn – max force at
which the bond will
break
 fn – sum of the
attractive and
repulsive forces
 lb – bond length (max
stretch required to
reach the peak force)
 ζ – distance of cell
membrane from
scaffold surface
 Peeling Model – Solutions
 Linear Model:
 ζ is the measure of the shape of the cell

 If the cell is entirely flat, ζ = 0  maximum

adhesion
 If ζ > l , there is no adhesion. The cell
b
membrane is peeled off
 In linear model, forces are linearly proportional
to ζ
 Peeling Model – Solutions
 Non-Linear Model:  L – bond extension
(empirical formula)  C – dimensionless
parameter
characterizing the
nonlinearity of
force–extension
relationship of bond
and the ability of bond
having large
deformation
 Model Parameters
 θα – Ratio of adhesion energy to bending modulus
 θ* - Microscopic angle
 θ0 – Macroscopic angle
 γ – adhesion energy
 δ – width of the boundary layer that are
stretched from the contact zone
 B – Bending modulus of the membrane (we
consider membrane to take high B, hence it is
stiff)
 s – arc length
 Results expected
 Weak adhesion:  Strong adhesion:

 For the given small θα,  For the given large θα,
we should get small θ* we should get large θ*
Result obtained
 Shape of the cell – result expected
 Weak adhesion:  Strong adhesion:

 At small θ*, the δ  At large θ*, the δ

should be large should be small
(i.e.) ζ vs. s plot (i.e.) ζ vs. s plot
should be shallow should be deep
Result obtained
 Inference
 Weak adhesion:  Strong adhesion:
 The result obtained  We cannot have –ve
by linear model is values for ζ
acceptable and  This implies that
works well the cell membrane
has penetrated the
rigid impenetrable
scaffold
 Realistic solution
 For small
displacement, force is
linear and linear
model works well
 But the real adhesion
happens only at large
forces, where the
profile is completely
non-linear
 Linear model fails here
Kim Hyonchol, Hideo Arakawa, Toshiya
Osada, Atsushi Ikai, 2002. Quantification
of fibronectin and cell surface interactions
by AFM, Colloids and surfaces:
Biointerfaces 25, 33-43
 Future Work on Modeling
 With this integrin-fibronectin force profile, we are
going to fit the profile to a nonlinear force-
displacement relationship

 This force-distance relationship will be used along

with the peeling model to predict how osteoblasts
will adhere to flat, rigid scaffold surfaces or
extracellular matrix.
 Experiments
 Preparation of Scaffolds for Adhesion of
Osteoblasts:

 Scaffold is a three-dimensional foam of poly

lactic acid prepared by solid-liquid phase
separation followed by sublimation of the
solvent under vacuum
 Preparation of Polymer
solution
 2.5 % (w/v) of Poly
(lactic acid) and Poly (lactic
dioxane solution is acid)
made
 Stir the mixture at Dioxane
50 °C for 2 hours

Polymer solution
 Design of Experimental setup
 Vial is made of borosil
glass
 Eight vials will be
placed in a custom
modified mixer jar
 This jar will be pre-
cooled to –10 °C
 The vials will be pre-
warmed to 50 °C
Mixer
jar
 Design of Experimental setup
 The prepared solution
is poured into the vials
and kept in freezer for
2 hours Polymer
 The setup is solution

immediately
transferred to ice bath
of -20 °C and freeze To vacuum
-20 °C
dried at 0.5 mm Hg for
4 days
 Characterization
 Density and porosity calculations:
 Accurately weighed sample (w) is immersed in a
graduated cylinder containing known volume (V1) of
ethanol
 The total volume of ethanol and the ethanol
impregnated foam (V2)
 Then the foam is removed from the container, and the
residual ethanol (V3)
 V2 – V1 = volume of skeleton of the foam
 V1 – V3 = void volume of the foam
 Characterization
 Total foam volume (V):
V = (V2 - V1) + (V1 – V3)

 Density of the foam (d):

d = W / (V2 – V3)

 Porosity of the foam (ε):

ε= (V1 – V3) / (V2 – V3)
 Status of the Experiments

 The vial in which scaffold will be synthesized is

under the process of fabrication

 All the raw materials have already been procured

 Time line - Experiments
 Experiments:
 Fabrication of the container – Next week
 Preliminary Experiments:
 Dry run – Next week (without PLA)
 Test if the vials withstand temperature shock, test if the vacuum is
sufficient to evaporate solvent in a porous medium
 Experiments with PLA and synthesis of scaffolds – 30th
March to 4th April 2009
 Density and Porosity measurements – 6th to 9th April
 SEM analysis – if possible, in the second week of April
 Time line – Modeling
 Modeling:

 Fitting the force data to a nonlinear function – by 30th

March 2009

 Development of nonlinear model with boundary

conditions – by 2nd April 2009

 Solution of the nonlinear model - 15th April 2009

Thank you