Scaffolds For Tissue Engineering Applications - Final Review (8 May 2009)

Scaffolds for Bone-Tissue Engineering: Preparation, Characterization, Modeling Department of Chemical Engineering and Materials Science Amrita School of Engineering Coimbatore – 641 105 May 2009

Final Review Divya Haridas Karthikeyan G Krishna Priya C Premika G

By (CB105PE012) (CB105PE023) (CB105PE025) (CB105PE028)

Guide Dr. Murali Rangarajan Co-Guide Dr. Nikhil K Kothurkar

Polymeric Scaffolds

Three dimensional scaffolds play important roles as extracellular matrices onto which cells can attach, grow, and form new tissues and degrade either during or after healing

Concept of Tissue Engineering n

Life science and Engineering dealing with the development of biological substitutes that restore, maintain and improve tissue functions or a whole organ

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Experiment: Synthesis and Characterization of PLLA and PU Scaffolds  Synthesis: Solid/Liquid Phase Separation/Freeze Drying  Characterization: Density and Porosity Measurements, Surface and Pore Morphology using Optical Microscope 

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Modeling of Cell Adhesion on Scaffolds

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One-dimensional Peeling Model

Linear Model: Linear force- bond displacement profile Nonlinear Model: Ligand-receptor force interactions

Characterization of Scaffolds

Characterization of Scaffolds Porous PLLA and PU scaffolds at 100X magnification characterized by optical microscopy

P LLA

P U

Characterization of Scaffolds Porous PLLA and PU scaffolds at 200X magnification characterized by optical microscopy

P LLA

P U

Inferences n

Both PLLA and PU scaffolds are highly porous

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PLLA scaffolds are lighter compared to PU scaffolds

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High density of PU scaffolds are suitable for implantable urinary tracts and bladders

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PLLA scaffolds can serve as a substrate for osteoblasts adhesion, provided these scaffolds are modified with ECM proteins such as collagen which improves the strength and toughness of the scaffolds.

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Modifying the scaffold by adding HAp will improve growth of bone tissue

Modeling of cell adhesion on scaffolds n n n n n n n n n

Importance of modeling cell adhesion General introduction to Cell adhesion Forces involved in cell adhesion One-dimensional Peeling Model and its assumptions Linear Peeling model Results Inferences Nonlinear peeling model Results

Bone Formation on Scaffolds

None of these steps will proceed if the cells do not adhere well to the scaffold

Cell adhesion n

Cell adhesion is the contact and firm interaction between cells, between cells and extra cellular matrix (ECM) and between cells and synthetic materials such as scaffolds in tissue engineering

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During cell adhesion there is specific binding between the cell surface receptors and the counter adhesion molecules

Forces involved in cell adhesion n

Forces that contribute to biological interactions are n

Non-specific forces/interactions n n n

n

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Electrostatic forces van der Waals forces Steric interactions

Specific forces/interactions

Adhesion involves two phases n n

Attachment phase Adhesion phase

Leckband D., 2000. Measuring the forces that control protein interactions, Annual Review Biophysics Biomolecular Structure 29, 1-26

One-Dimensional Peeling model n

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Peeling is opposite of adhesion. At equilibrium, the structure of the cell surface is the same since the process is reversible Assumptions: n

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Cell membrane – rigid and elastic Scaffold – flat, rigid, nonporous surface

Dong Kong, Baohua Ji, Lanhong Dai, 2008. Nonlinear mechanical model of cell adhesion, Journal of Theoretical Biology 250, 75-84

Linear Peeling model n

Linear, where the cell detachment occurs when the bond force at the leading edge of the cell exceeds the maximum bond force

Force is assumed to be a linear function of the displacement of bond length (ζ) from

Evan A. Evans, 1985. Detailed mechanism of membrane- memb adhesion and separation, Biophysical Journal, 48, 175-183

Governing Equations

Free Zone

Adhesive Zone Free

Zone

s = arc length bond k= local curvature

f = adhesive bond force of a single n = bond density

Solution for free zone

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Local contact angle - θ External force - Tex

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Macroscopic angle - θ0

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Solution for adhesive zone

Ɵα - ratio of adhesion and bending energies t - ratio of tension and bending s- arc length σn - adhesive stress

Results expected for strong and weak adhesion Strong adhesion

Weak adhesion

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More number of ligandreceptor bonds

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Less number of ligandreceptor bonds

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Long adhesive zone, short free zone

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Short adhesive zone, long free zone

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Microscopic contact angle is large

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Microscopic contact angle is small

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More adhesion energy is required

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Less adhesion energy is required

Results Obtained –Strong/Weak adhesion

Results expected for shape of the cell Strong adhesion n

The effective width δ of the boundary layer for which bonds are stretched at the edge of the contact zone is small Relationship between δ and θα ratio of adhesive energy(γ) to bending modulus (B)

Weak adhesion n

The effective width δ of the boundary layer for which bonds are stretched at the edge of the contact zone is large

Results obtained for shape of the cell

Inferences Weak adhesion n

The result obtained by linear model is acceptable and works well

Strong adhesion n

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We cannot have – ve values for ζ This implies that the cell membrane has penetrated the rigid impenetrable scaffold

Nonlinear Peeling model n

Nonlinear model for osteoblasts adhesion is carried out by considering the integrin-fibronectin force profile

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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

Force displacement profile of integrin and fibronectin

Li et al., 2003. Force Measurements of the α5β1 Integrin– Fibronectin Interaction, Biophysical Journal 84(2), 1252-1262

Curve fitting for force displacement profile The above graph is fitted to Weibull distribution as,

Where x > 0 a = 0.8107 ± 0.192 ; b = 2.147 ± 0.303 The above equation can rewritten in terms of force f, and displacement ζ as

Where a = 0.8107 ± 0.192, b = 2.147 ± 0.303

Solution for adhesive zone – Nonlinear peeling model n

The work done to either break or form cross bridge when the membranes are bought together from a large separation distance to planar, equilibrium contact is given by,

n

Total adhesion energy Γ = n W

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Adhesion (normal) stress

Solution for adhesive zone – Nonlinear peeling model n

Governing equations

These equations need to be solved to obtain the contour of the membrane for osteoblast adhesion

Conclusions Three dimensional, interconnected, highly porous PLLA and PU based scaffolds – fabricated Porosity, density and pore morphology of scaffolds – characterized. Membrane contours of cell, based on adhesive force assumptions - studied (Modeling). Solutions for linear peeling model - obtained. Basic equations for non linear model - derived.

Future work n

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The shape of the membrane for an osteoblasts adhesion can be determined by solving the nonlinear peeling model Nonlinear model can be further extended to curved scaffolds The properties of the PLLA and PU scaffold can be further enhanced by modifying the them with suitable extracellular matrix proteins like collagen In-vitro cell culture can be done on the scaffolds to study biocompatibility

Acknowledgement n n n n n n n n

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Prof. R. R. Subba Rao Dr. Murali Rangarajan Dr. Nikhil K Kothurkar Mr. K. Jayanarayanan Ms. Meera B. Sasikumar Mr. M. Kannan Dr. G. Prema (Dept. of Mathematics) Mr. Sanjivi Arul (Dept. of Mechanical Engineering) Mr. R. Padmanaban (Dept. of Mechanical Engineering)