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Cell Interactions with Biomaterials

Topics:

•Cell Structure and Components

•Properties of Cell Components

•Interaction of Cells with Extracellular Material (ECM)

•Cell Adhesion and Migration

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A successful biomaterial implant must support all the required functions of the attached (or neighboring) cells, including

• Viability All cell types• Communication All cell types• Protein synthesis All cell types• Proliferation Some cell types• Migration Some cell types• Activation/differentiation Some cell types• Programmed cell death Some cell types

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2 types of cells:

•Differentiated: perform specific tissue functions

•Undifferentiated: progenitors for many different cell types

Cell Structure

Cell tasks are compartmentalized in various organelles. Organelles in all mammal cells include the plasma cell membrane, mitochondria, Golgi apparatus, cytoplasm, lysosome, cytoskeleton, nucleus, and smooth and rough endoplasmic reticulum

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The cell membrane separates the cytoplasm, or cell interior, from

the aqueous external environment. It is a bilayered structure made up of phospholipids, or fatty acids

with a polar (hydrophilic) head and nonpolar tail.

Transmembrane proteins span the cell membrane to channel ions into and out of cell and maintain proper

cell chemistry

Other proteins target specific extracellular molecules

The mitochondria produce the energy for cell functions via a

process called oxidative phosphorization. Surrounded by

a phospholipid membrane, the mitochondria contains enzymes that help break down molecules

In oxidative phosphorization ATP (adenosine triphosphate) is

converted to ADP (adenosine diphosphate, an exothermic

reaction that releases energy to drive the cellular process.

Phospholipid

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The cytoskeleton is made up of three filaments:

•Actin microfibrils,~6-8 nm diameter

•Intermediate filaments ~10 nm diameter •Microtubules ~25 nm diameter

Made up of proteins, the cytoskeleton

•Gives the cell shape

•Can provide cell locomotion •Aids in separation/duplication of DNA

The Golgi apparatus modifies, sorts and packages

proteins for their final destination

Lysosomes are specialized vesicles

with digestive enzymes

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The nucleus is the control center for the cell. It

contains:

DNA – (deoxyribonucleic acid) that is

condensed into chromatin, a complex combination of DNA and

protein that makes up chromosomes

The nuclear envelope, a bilayer of phospholipid membranes that

surrounds the nucleus

The outer membrane of the nuclear envelope is contiguous with the endoplasmic reticulum and is

connected with the inner membrane at specific locations called nuclear

pores. The pores are composed of proteins that form gates to allow only specific molecules in and out of the

nucleus

Ribosomes, minute round particles composed of RNA and protein that are

found in the cytoplasm of living cells and catalyze reactions in which mRNA is used

to synthesize proteins. Ribosomes are located in the nucleolus

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DNADNA (deoxyribonucleic acid) is a polymer of nucleic acid subunits. Each nucleic acid has a phosphate group, a sugar, and a base.

DNA contains genes, and forms the template for all proteins synthesized by the cell

When a gene is expressed, the cell is actively producing the protein encoded by the gene

Genes contain codons which determine the structure of the protein

The bases are directed toward the interior, where they form hydrogen bonds with other bases.

Bases can be either double ring structures (e.g., purines Adenine (A) or guanine (G)), or single ring (pyrimidines, e.g., thymine (T) or cytosine (C))

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RNARNA participates in DNA synthesis and protein production.

Three types of RNA:

Messenger or mRNA

Transfer, or tRNA

Ribosomal, or rRNA

RNA is similar to DNA, but the sugar in the backbone contains an additional O2, and thymine (T) in DNA has been replaced with uracil (U).

RNA is single stranded , and does not form the helical

structure of DNA

mRNA: messenger RNA; DNA is unzipped, and mRNA strands are synthesized that are complementary to DNA

tRNA: serves as an adaptor to combine mRNA strands in the rough ER

rRNA: the central component of the ribosome, the function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation

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The outer membrane of the nucleus is connected to the

endoplasmic reticulum (ER). The ER is the site of protein

synthesis.

The ER is made of long, flattened sheets of phospholipids, and may

be either rough or smooth.

The rough ER has ribosomes attached to the surface that act as

catalysts for protein synthesis.

The smooth ER is more tubular and does not contain ribosomes.

It packages the proteins produced in the nucleolus and rough ER in phospholipids for delivery to the

Golgi apparatus.

Vesicles transport proteins from the ER to the Golgi, or from the Golgi to the

target destination (exocytosis). Specialized vesicles (lyosomes) may also take in and digest particles from

the ECM

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Schematic of the endoplasmic reticulum (ER), which is responsible for protein synthesis. The rough ER, which contains a large number of

ribosomes, is attached to the nuclear envelope. The rough ER transforms into the smooth ER away from the nucleus. Pieces of the ER will then split off and transfer to the Golgi apparatus, where the

proteins created in the ER are further modified and transported ot their final destinations.

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Interactions between a cell and its environment can result in cell

spreading, migration, communication, differentiation

and activation. This is called “outside-in” signaling.

Conversely, a cell may secrete molecules or rearrange contacts to

alter the ECM. This is called “Inside-out” signaling

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Types of cell contacts

Tight junctions: cells adhere fast to each other – no

molecular transport

Gap junctions: small hydrophilic channels between

cells and membranes

Desmosomes: mechanical attachment between two cells.

Cells can attach to ECM via hemidesmosomes or focal

adhesion. These form strong adhesion to the ECM

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Cell membrane receptors & ligandsCellular interactions are facilitated by cell membrane receptors, each of which is specific for a small range of target molecules, or ligands.

Common receptor molecules are:

Cadherins: responsible for demosomes; a cadherin molecule on one cell binds to a cadherin on another cell. This is homophilic binding

Selectins: selectins are like cadherins, but bind to other types of receptors, or heterophilic binding

Mucins: participate in heterophilic binding to selectins.

Integrins: transmembrane proteins involved in both cell-cell and cell-matrix contacts.

Location of cadherins in epithelial cells.. They link to each other to bind cells, and their cytoplasmic regions attach to intermediate filaments linking the ECM to the intracellular environment

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Cell membrane receptors & ligands

Various types of cell membrane receptors: mucins, integrins, selectins, and Ig-cell adhesion

molecules (Ig-CAMs).

Integrins have two distinct and subunits, and are called heterodimers.

Variations in the composition of the and chains results in selective adhesion to different ligands

Other cell adhesion molecules (CAMs) comprise a large group of membrane proteins that mediate cell-cell interactions via both homophilic and heterophilic binding.

An example of these other CAMs is the immunoglobulin (Ig) family in the picture at right.

The receptors described consist mainly of protein with a small attached carbohydrate (sugar). Similar molecules with small protein and large sugar content are called proteoglycans.

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Extracellular Environment

Understanding the cell-ECM interaction and response is key to designing new biomaterials

The ECM may be thought of as a fiber reinforced composite with fibers made of collagen or elastin, and a

matrix made of glycoproteins and proteoglycans

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Collagen

Collagen is the most abundant protein in mammals. It is responsible for tissue

tensile strength.

Collagen is made up of -polypeptides, or amino acids in a gly-x-y pattern (above). Gly is a small molecule

resulting in tight packing

X and Y molecules are often proline and hydroxyproline

Cell excretes procollagen molecules that self-assemble into fibers (right)

Upon secretion of procollagen from the cell into the ECM, small peptide sequences are cleaved to from the molecule to allow for more efficient packing of collagen molecules into fibrils (10-300 nm). Individual fibers

are then assembled into larger fibers (0.5 – 3 m diameter)

Properties of collagen may be manipulated by controlling crosslinking of molecules

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Elastins

Elastin is responsible for the resiliency and elasticity of the

ECM.

Elastin is made up of 85% hydrophobic amino acids

When relaxed, elastin molecules coil up. When a tensile load is applied they unfold into long chains. The chains are crosslinked to adjacent

elastin molecules

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Additional ECM molecules include proteoglycans and glycoproteins. These proteins are mainly carbohydrate, with some

protein side chains.

These molecules attract and interact strongly with water.

Carbohydrates in proteoglycans form long chains of polysaccharides called

glycosaminoglycans (GAGs)

Proteoglycans have several GAGs attached to a protein core.

An aqueous environment is favorable to transport and store bioactive molecules.

Example of the bottle-brush structure of a large proteoglycan including areas of keratan sulfate and chrondroitin sulfate that are

attached to a protein core

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Two molecules that represent glycoproteins are fibronectin and

laminin.

Each consists of peptide subunits held together by disulfide bonds and contain many sites for binding to various ECM

molecules.

Laminin (right) consists of three disulfide linked peptide chains in a loosely woven

structure with multiple binding sites

Glycoproteins are important in blood clotting (coagulation) since they posses binding domains for

heparin and fibrin

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Cell-Environment Interactions that Affect Cellular Functions

Interactions between cells and cell/ECM can alter cells function and affect gene expression in the nucleus

Alterations in gene expression affect four major functions:

1. Cell viability

2. Proliferation

3. Differentiation

4. Protein synthesis and communication

Changes in ECM can cause cell death via necrosis or

apoptosis due to changes in the local chemistry (e.g. pH),

factors that attach to cell membrane leading to cell

death.

Apoptosis: cell shrinkage followed by fragmentation into vesicles containing small groups of organelles. No inflammatory response

Necrosis: cell death from membrane permeability and enzyme leakage, leading to disintegration

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Cell classifications

Labile: replicate continuously

Terminal: terminally differentiated

Stable: don’t change once differentiated but can be induced to proliferate

The cell cycle is divied into two phases: Mitosis (the M phase) and interphase (G1,S,G2)

Mitosis: cell division

Interphase: cellular DNA and organelles replicated in preparation for mitosis

Go: quiescent phase of stable cells

G1: general cell growth

S: DNA replicated

G2: proteins and structures enabling cell division ar assembled

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Mitosis is divided into several characteristic periods:

ProphaseMetaphaseAnaphaseTelophase

Prophase: dissipation of the nucleolus and formation of the mitotic spindlesMetaphase: chromosomes are aligned between two mitotic spindles

Anaphase: chromosomes are pulled apart by the spindle microtubules and arrange themselves at the spindle poles

Telophase: nuclear envelope begins to reform and the cell starts to undergo cytokinesis

Cytokinesis: division of cell cytoplasm

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Cell Differentiation

Progenitor or stem cells can form more than one type of cell

The cells produced may be committed or differentiated and can be labile,

stable or permanent

Or

Create additional pluripotent or totipotent cells (produce other or all

cell types)

Red blood cells generated from hematopoietic stem cell can either replicate itself of

differentiate into various cell types. These cells

are pluripotent

Embryonic stem cells are an example of a totipotent cell

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Mesenchymal stem cells (MSCs) can differentiate into bone, cartilage, muscle, tendon,

ligament, and adipose tissue.

The commitment and progression of an MSC to and through a specific lineage involves the action of bioactive molecules such as growth factors and cytokines.

In the process of differentiation and maturation the cell increases its production of tissue-specific molecules.

Terminally differentiated cells may alter their levels of synthesis of matrix molecules to play an increased role in tissue maintenance and homeostasis

Differentiation stages can be initiated and controlled by soluble and insoluble elements in their environment and is directly applicable to

tissue engineering

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Protein SynthesisReceptor-ligand binding can change the

function of committed cells which is associated with alterations in the amount of protein

synthesized

Block diagram of the steps for the creation and modification of

proteins.

Transcription: Chromatin becomes less compact; RNA “unzips” DNA and synthesizes mRNA strands that are complimentary to DNA then moves into the endoplasmic reticulum (ER)

Translation: the process of converting codons from the mRNA to a polypeptide. This takes place in the ER through complex interaction with tRNA.

Post Translation: fully formed proteins are combined into various molecules

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Summary of the steps of collagen synthesis.

Transcription, translation, synthesis to create the -chain and the joining of three of

thee chains (post translational

modification) to create the collagen triple helix occur within the cell.

The procollagen molecule is then

secreted and assembled into fibrils

and finally fibers.

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Models for Adhesion, Spreading and MigrationThe DLVO theory: (Derjaguin, Landau, Verway and Overbeek)

Based on thermodynamics

Particles potential energy the sum of attractive and repulsive forces: U = UA + UR

Particles approaching a surface reduce their potential energy, and are loosely attached at the secondary minimum – long range electrostatic/van der Waals forces

Particles overcoming primary minimum become firmly attached through short range electrostatic forces

Model shortcomings: does not include steric repulsion, surface topography/roughness or ligand-receptor interactions

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Spreading & MigrationCell spreading: After attachment, cells extend finger-

like pseudopodia along surface. The integrin receptors in the cell membrane interact with ligands on the material

surface to firmly anchor the cell in place.

Cell spreading includes cytoskeleton rearrangement and production/adsorption of adhesive proteins on surface

Cell migration: extension of the cell membrane in long pseudopodia is directed by polymerization of actin

microfibrils near the leading edge of the cell. (b) the membrane then attaches to the substrate via integrin

receptors. (c,d) After the pseudopodia are firmly adhered, there is generation of a contractile force along

with a release of rear receptors, leading to forward motion. (e) the integrin receptors are recycled to the

leading edge so they may be used again as the process continues.

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Tracking cell movements

Trajectories of bovine pulmonary artery endothelial cells migrating in a uniform environment. Symbols represent the location of the centroid of each cell at 30 minute intervals. Arrows indicate starting points.

Plots of cell trajectory, such as the one at right, provide information on cell

movement in the form of translocation speed (s) and persistence time (t).

Translocation speed is the speed of cell movement over any straight-line

portion of the graph, between changes in direction

Persistence time is the length of time that the cell moves along the substrate without a drastic change in direction.

Measurements of this type are used in mathematical models developed to

model cell migration

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The End

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