Micro/nanoporous Scaffolds For Tissue Engineering Applications
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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
Collagens Fibronectins Elastins Laminins
Cell membrane
Nectins Integrins Cadherins 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:
Model Considered:
Attachment phase Adhesion phase
Peeling Model of cell adhesion (long-term)
Peeling Model:
Attachment and detachment of receptor – ligand bonds Force governs extent of adhesion
Evan A. Evans, 1985. Detailed mechanism of membrane- membrane adhesion and separation, Biophysical Journal, 48, 175-183
Peeling Model
Assumptions:
Cell membrane – rigid, elastic Scaffold – flat, rigid, nonporous surface
Two zones:
Free zone – membrane is not subjected to any forces Adhesive zone – membrane is subjected to attractive forces
Dong Kong, Baohua Ji, Lanhong Dai, 2008. Nonlinear mechanical model of cell adhesion, Journal of Theoretical Biology 250, 75-84
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: (empirical formula)
L – bond extension 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, we should get small θ*
For the given large θα, we should get large θ*
Result obtained
Shape of the cell – result expected
Weak adhesion:
At small θ*, the δ should be large (i.e.) ζ vs. s plot should be shallow
Strong adhesion:
At large θ*, the δ should be small (i.e.) ζ vs. s plot should be deep
Result obtained
Inference
Weak adhesion: The result obtained by linear model is acceptable and works well
Strong adhesion: We cannot have –ve values for ζ This implies that 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 forcedisplacement 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 dioxane solution is made Stir the mixture at 50 °C for 2 hours
Poly (lactic acid) Dioxane
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 precooled to –10 °C The vials will be prewarmed to 50 °C
Mixer jar
Design of Experimental setup
The prepared solution is poured into the vials and kept in freezer for 2 hours The setup is immediately transferred to ice bath of -20 °C and freeze dried at 0.5 mm Hg for 4 days
Polymer solution
To vacuum
-20 °C
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
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
Collagens Fibronectins Elastins Laminins
Cell membrane
Nectins Integrins Cadherins 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:
Model Considered:
Attachment phase Adhesion phase
Peeling Model of cell adhesion (long-term)
Peeling Model:
Attachment and detachment of receptor – ligand bonds Force governs extent of adhesion
Evan A. Evans, 1985. Detailed mechanism of membrane- membrane adhesion and separation, Biophysical Journal, 48, 175-183
Peeling Model
Assumptions:
Cell membrane – rigid, elastic Scaffold – flat, rigid, nonporous surface
Two zones:
Free zone – membrane is not subjected to any forces Adhesive zone – membrane is subjected to attractive forces
Dong Kong, Baohua Ji, Lanhong Dai, 2008. Nonlinear mechanical model of cell adhesion, Journal of Theoretical Biology 250, 75-84
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: (empirical formula)
L – bond extension 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, we should get small θ*
For the given large θα, we should get large θ*
Result obtained
Shape of the cell – result expected
Weak adhesion:
At small θ*, the δ should be large (i.e.) ζ vs. s plot should be shallow
Strong adhesion:
At large θ*, the δ should be small (i.e.) ζ vs. s plot should be deep
Result obtained
Inference
Weak adhesion: The result obtained by linear model is acceptable and works well
Strong adhesion: We cannot have –ve values for ζ This implies that 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 forcedisplacement 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 dioxane solution is made Stir the mixture at 50 °C for 2 hours
Poly (lactic acid) Dioxane
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 precooled to –10 °C The vials will be prewarmed to 50 °C
Mixer jar
Design of Experimental setup
The prepared solution is poured into the vials and kept in freezer for 2 hours The setup is immediately transferred to ice bath of -20 °C and freeze dried at 0.5 mm Hg for 4 days
Polymer solution
To vacuum
-20 °C
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
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