Lecture 2 Biological Response To Materials-Advance Biomaterials-2012

Lecture 2: Biocompatibility/interactions of cells with
surface
Lecturer:
Ivan Djordjevic, PhD (Materials

Department of Biomedical Eng.
Faculty of Engineering,
University of Malaya
Tissue Engineering Lab (Block U)

Tel: 037967 7616
Email: ivan.djordjevic@um.edu.my
Biocompatibility/interactions of cells with surface
Objective:
- outline basic principles behind human cellular/tissue response to artificial
surface implantation
- relate how tissue function and the body's response must be taken into
account in the design of a biomedical material
- outline principles behind cell/surface interactions
Outline:
 Biocompatibility: Definition
 Cell/biomaterial interface
 Biological responses to biomaterials
Biocompatibility and cell/surface interactions
Biocompatibility:
Biocompatibility is the ability of a material to perform with an appropriate host response

in a specific application. (Williams, 1987)
More generally: A material is considered “biocompatible” if it allows the body to function

without complications like allergic reactions or adverse side effects. Biocompatibility is
the “suitability” of a material for exposure to the body or bodily fluids.
The biocompatibility evaluation of biomaterials and biomedical devices is a very

complex and critical issue.
 According to FDA recommendations (Food and drugs administration) and the other
regulating bodies, tests must be performed as a function of:
– Nature of the implants and contact with the body: skin, mucous, bone, blood
– Site of implantation
– Duration
Biocompatibility: Type of Biomaterials
• Orthopedics: artificial hips, knees, wrists; spinal rods; bone grafts.
• Cardiovascular: heart valves, stents, pacemakers, catheters, grafts,

stents
• Dental: filings, prosthetics, orthodontics
• Soft tissue: wound healing, reconstructive and opthalmalgic
• Surgical/tools: staples, sutures, scalpels, catheters, etc.
• Micro/Nano-particles: Liposomes, dendrimers, etc.
• Biomedical devices: Biosensor, microarrays, etc

Biocompatibility: Duration
Time scale over which the host is exposed to the material or device must be
considered.
material contact time
syringe needle 1-2 s
tongue depressor 10 s
contact lens 12 hr - 30 days
bone screw / plate 3-12 months Need for

Biocompatible
total hip replacement 10-15 yrs
materials
intraocular lens 30 + yrs
Cell – an Introduction
 The cell is the structural and functional unit of all living

organisms.
 Some organisms, such as bacteria, are unicellular,
consisting of a single cell. Other organisms, such as
humans, are multicellular, (humans have an estimated
100,000 billion = 1014 cells).
The cell theory, first developed in the 19th century, Cells in culture,
states that stained for keratin
 all organisms are composed of one or more cells; (red) and DNA
 all cells come from preexisting cells;
(green)
 all vital functions of an organism occur within cells

 cells contain the hereditary information necessary for
regulating cell functions and for transmitting information
to the next generation of cells.
 The word cell comes from the Latin cella, a small room.
 Each cell is a self-contained and self-maintaining entity: it can
take in nutrients, convert these nutrients into energy, carry
out specialized functions, and reproduce as necessary.
All cells share several abilities:
 Reproduction by cell division.
 Metabolism, including taking in raw materials, building cell

components, creating energy molecules and releasing
byproducts. The functioning of a cell depends upon its ability
Cells in culture,
to extract and use chemical energy stored in organic
stained for keratin
molecules. This energy is derived from metabolic pathways.
(red) and DNA
(green)
 Synthesis of proteins, the functional workhorses of cells,
such as enzymes. A typical mammalian cell contains up to
10,000 different proteins.
 Response to external and internal stimuli such as changes in

temperature, pH or nutrient levels.
Type of cells
The cells of eukaryotes and prokaryotes.
 Eukaryotic cells (e.g. human): Nucleus (light blue), the

nucleolus (intermediate blue), mitochondria (orange), and
ribosomes (dark blue).
 Prokaryotic cell (Bacterium): Nucleoid containing the DNA

(very light blue), cell membrane (black), cell wall
(intermediate blue), capsule (orange), ribosomes (dark
blue), and a flagellum (also black).
Cell membrane
The selectively permeable cell membrane (or plasma membrane) is a thin

and structured bilayer of phospholipid and protein molecules that
envelopes the cell. It separates a cell's interior from its surroundings and
controls what moves in and out.
Cell surface membranes often contain receptor proteins and cell adhesion
proteins. There are also other proteins with a variety of functions. These
membrane proteins are important for the regulation of cell behavior and the
organization of cells in tissues.
Cell membrane
Cells tend to stick to other cells or non-cellular components of their

environment.
Cell adhesion generally involves protein molecules at the surface of
cells, so the study of cell adhesion involves cell adhesion proteins and
the molecules that they bind to.
Relevant to the design of biocompatible biomaterials and many

biotech. applications.
Cell membrane
Transmembrane cell adhesion proteins extend across the cell surface membrane
and typically have domains that extend into both the extracellular space and the
intracellular space.
The extracellular domain of a cell adhesion protein can bind to other molecules
that might be either on the surface of an adjacent cell (cell-to-cell adhesion) or part
of the extracellular matrix (cell-to-ECM adhesion).
Adsorbed proteins at the surface of Biomaterials mediates cell adhesion (e.g.

platelets vs endothelial cells for blood contacting devices)
The molecule that a cell adhesion protein binds to is called its ligand. There are
families of cell adhesion proteins that can be characterized in terms of the
structure of the adhesion proteins and their ligands.
Family ligands
Selectins Carbohydrates
Integrins Extracellular matrix
Ig superfamily
proteins
Ig superfamily proteins Integrins
Ig superfamily
proteins
Cadherins Cadherins
Cell membrane
Example RGD peptide
Cell-material interactions are due to the

interactions of cell-adhesion proteins
(ligands) bound to a material surface
(adsorbed proteins) and cell surface
receptors (integrins).
An interesting property about these

receptor ligands is that they can be
mimicked by small synthetic linear or
cyclic oligopeptides.
One such oligopeptide is the three amino

acid sequence Arg-Gly-Asp acid (RGD)
which is present in many of the active
domains of various adhesion proteins
found within the extracellular matrix and
binds to many of the integrin receptors.
Non-fouling + ligands = specific

interaction on the surface of the
biomaterials
http://biomaterials.berkeley.edu/surfacemod.shtml
Cells and biomaterials – Experiment in laboratory
100 µm
I. Djordjevic et al. J of Biomater Sci Pol Ed, 2010

Protein structure and surface interactions
Cystine (Cys) Serine (Ser)

Aspartic acid (Asp)
Lysine (Lys)
Fibrin structure
Sequence of local events following

implantation:
• injury
• acute inflammation
• chronic inflammation
• granulation tissue
• foreign body reaction
• fibrous encapsulation
Host Response to
Implants:
Polymorphonuclear leukocytes
J.M. Anderson, Ann Rev Mater Res;

2001:31:81-110
J.M. Anderson, A. Rodriguez and D.T.
Chang, Semin Immunol 2008: 20:86-100
Host Response to
Implants:
J.M. Anderson, A. Rodriguez and

D.T. Chang, Semin Immunol
2008: 20:86-100
Scanning electron microscopy images of an Elasthane 80A Polyurethane surface from an in

vivo cage study showing the morphological progression of the foreign body reaction. The
sequence of events at the Polyurethane surface includes (A) monocyte adhesion (0 days), (B)
monocyte-to-macrophage development (3 days), (C) ongoing macrophage-macrophage fusion
(7 days), and (D) foreign body giant cells (14 days).
Inflammation:
• Inflammation is a non-specific response to tissue damage:
– arises in response to trauma, infection, intrusion of foreign materials, or

local cell death
– contain, neutralize, dilute, or wall-off the injurious agent or process
– heal and reconstitute the implant site
• This is done through the generation of new tissue via native parenchymal
cells or the formation of fibroblastic scar tissue.
Acute Inflammation
• Cellular terminology:
– granulocyte: any blood cell containing specific granules (e.g. neutrophils,

eosinophils, basophils)
– leukocyte: white blood cell capable of ameboid movement (formation of

pseudopods at the leading edge of a moving cell; e.g. lymphocytes,
monocytes, granulocytes)
– macrophage: large phagocytic mononuclear cell

Acute Inflammation
Intravascular cells/white blood cells/leukocyte:
- Granulocyte:
neutrophils: the most common one, segmented
nuclei connected by thin strands of chromatin
eosinophils: bi-lobed nucleus
basophils: the least common one, large
cytoplasmic granules which obscure the nucleus
– Monocytes: the largest one, deeply indented or

U-shaped nucleus. In tissue: Macrophages
– Lymphocytes: or Killer T-cells: Cf immune

systems
Acute Inflammation
Clinical signs of inflammation: redness (rubor), swelling (tumor), pain (dolor),

heat (calor)
• Why rubor? Erythrocytes
• Why swelling? Permeability:

– pressure difference between capillary and external tissue bed
– endothelium is tight permits very slow flow of water and small
molecules into surrounding tissue
• NORMALLY: lymphatic vessels drain away this fluid
maintaining constant tissue volume
• INFLAMMATION: permeability increases and larger
molecules move into the tissue (increased fluid influx not
promptly balanced by the lymphatic system swelling)
Acute Inflammation
Why pain? edema and kinins

– local edema may activate local deep pain receptors (throbbing pain, peaks
with systolic pressure)
– kinins act directly on nerve ends to produce pain sensation (pain of bee sting:
the activation of a kininin bee venom)
Why heating? not clear! possible players:

– increased cellular metabolic activity
– possible generation of pyrogens which are known to cause systemic fever
– local disturbance of fluid flow
Acute Inflammation
• Lasts from minutes to days depending on the injury
• Initial stages:
– rapid dilation of local capillaries
– increase in the permeability of their endothelial cell linings
• Dilation?
– foreign protein or material  coagulation factor (factor XII) 
activation kinins  dilation and endothelial permeation
• Dilation leads to an increase in blood entry into the capillary beds

– loss of plasma through the capillary walls
– platelets and erythrocytes become sticky
– blood flow slower and sludgy
Acute Inflammation
First cells to appear at injury site are “neutrophils” ( white blood cells,
polymorphonuclear leukocytes)
– cells become sticky

– stick to capillary endothelium
– penetrate between the endothelial cells
– move into the surrounding damaged tissue
– neutrophil emigration (diapedisis) begins minutes to hours after insult and
may continue for as long as 24h
– neutrophil activates when engages foreign particle
How do leukocytes emigrate?

– adhesion molecules present on leukocyte and endothelial surfaces
– chemotaxis: unidirectional migration of cells along a chemical gradient
Acute Inflammation
• Monocyte emigration may continue for days to weeks.
• Chemotactic factors for monocytes are activated over longer periods of time.
• Following emigration from the vasculature, monocytes differentiate into

macrophages which are very long lived, up to months.
Acute Inflammation
Phagocytosis: Engulfing and degradation or digestion of fragments of tissue

or material
1. Attachment of the foreign particle to the long membrane evaginations,

called pseudopodia.
2. Ingestion of the particle forming a "phagosome," which moves toward
the lysosome.
3. Fusion of the lysosome and phagosome (phagolysosome), releasing
lysosomal enzymes into the phagosome.
4. Digestion of the ingested material.
5. Release of digestion products from the cell.
Acute Inflammation
Successful response to inflammatory challenge will reduce local tissue mass:

dead cells phagocytized and removed by neutrophils and macrophages
Engulfment and degradation of biomaterials may or may not occur
The disparity between the size of the biomaterial and the cells may lead to
“frustrated phagocytosis”
– leukocyte products are released extracellularly in an attempt to
degrade the biomaterial
Chronic Inflammation
Persistent inflammatory stimuli lead to chronic inflammation:

– chemical and physical properties of biomaterial
– motion in the implant site
Confined to the implant site
Characterized by:
– the presence of macrophages, monocytes, and lymphocytes
– proliferation of blood vessels and connective tissue
– no exudates
Chronic Inflammation
Macrophages produce great number of biologically active products

– proteases
– chemotactic factors
– coagulation factors
– growth promoting factors
– cytokines
Growth factors (e.g. PDGF, FGF, TGF-b, IL-1, TNF) are essential for:
– the growth of fibroblasts and blood vessels and the regeneration of epithelial
cells
– stimulate the production of a wide variety of cells
– initiate cell migration and differentiation
– tissue remodeling and wound healing
Granulation Tissue
Granulation tissue is the hallmark of healing inflammation:
– derives its name from the pink, soft granular appearance on the surface of
healing wounds
– may be seen as early as 3-5 days following implantation of a biomaterial
New small blood vessels are formed by budding or sprouting of preexisting

vessels in a process known as “neovascularization” or “angiogenesis”
Angiogenesis involves proliferation, maturation, and organization of endothelial

cells into capillary tubes
Granulation Tissue
Fibroblasts:
– develop the granulation tissue by synthesizing collagen (especially type
III) and proteoglycans
– some fibroblasts may have the features of smooth muscle cells
(myofibroblasts) which are considered to be responsible for wound contraction
Wound healing response is dependent on the extent of injury:

– Wound healing by primary union (or first intention):
• clean surgical incisions in which the wound edges have been
approximated by surgical sutures
• occurs without significant bacterial contamination and minimal loss of
tissue
– Wound healing by secondary union (or second intention):
• a large tissue defect that must be filled
• there is an extensive loss of cells and tissue
• the original architecture is not reconstituted
• much larger amounts of granulation tissue are formed that result in larger
areas of fibrosis or scar formation
Foreign Body Reaction
Foreign body reaction (FBR) consists of:
– multinucleated foreign body giant cells

– macrophages
– fibroblasts
– capillaries
Multinucleated foreign body giant cells form upon coalescence of macrophages

Foreign Body Reaction
FBR depends on the geometry and the form of the implant:
– flat and smooth surfaces such as those found on breast prostheses; FBR is
composed of a layer of macrophages one to two cells in thickness
– relatively rough surfaces such as those found on the outer surfaces of

vascular prosthesis; FBR composed of multiple layers of macrophages and
foreign body giant cells at the surface
– rough surfaces such as fabric-type materials;

composed of macrophages and foreign body giant cells with varying degrees of
granulation tissue
Fibrosis and Fibrous Encapsulation
• End stage of healing response
• Usually four or more weeks after implantation
• A relatively acellular fibrous capsule:

– spindle shaped fibroblasts
– small number of macrophages
• Presence of neutrophils suggests persisting inflammatory challenge
• Presence of foreign body giant cell suggests production of small particles by

corrosion, depolymerization, dissolution or wear
Fibrosis and Fibrous Encapsulation
• Presence of lymphocytes suggests specific immune response
• Thickness of the capsule depends on the chemical activity (rate of release) of

the material:
– metals which corrode freely
– polymers with leachable constituents
• Capsule thickness increase with increasing relative motion between the

implant and the tissue
• Shape of the implant: capsule will be thicker over sharp edges

Resolution
• Overall aim of healing is to reach the status quo ante (the reestablishment of
homeostasis)
• Presence of implant prevents attainment of original condition but a steady

state can be attained which is termed as resolution
• After the attainment of the resolution no more biological changes occur
• Acute and chronic responses generally end with a resolution
• Immune response may or may not reach a resolution:

– ends with the formation of a granuloma (a benign tumorous
condition due to continued elaboration without remodeling)
• Neoplastic response does not resolve and results in the
failure of the implant
Resolution
• Possible outcomes for the implant:
– resorption: if the implant is resorbable then the implant site eventually

resolves to a collapsed scar or, in the case of bone, may completely disappear
– integration: very limited occurrence in practice; close approximation of normal
host tissue to the implant without an intervening capsule (e.g. implantation of
pure titanium in bone)
– incapsulation: the most usual response
• Possible outcomes for the injured tissue:
– replacement of injured tissue with by parenchymal cells of the same type

– replacement by connective tissue that constitutes the fibrous capsule
Resolution
• These processes are controlled by:

– the proliferative capacity of the cells in the tissue or organ receiving the
implant
– the extent of injury
– persistence of the tissue framework of the implant site
• Classification of cells based on regenerative capacity:

– labile: continue to proliferate throughout life (skin, bone)
– stable: can retain the proliferation capacity but do not normally replicate
(parenchymal cells of liver, kidney and pancreas)
– permanent: cannot reproduce themselves after birth (nerve cells,
skeletal muscle cells, and cardiac muscle cells)
Infections/Sterilization:
One of the greatest challenges for devices is ensuring sterility of implants

• Minimizes bacterial contamination
• Reduces likelihood for infection
Sterility Definition: the state in which the probability of any one bacterial
endospore surviving is 10-6 or lower
• Manufacturing issues:
– What is the best sterilization method?
– What is shelf life for the device?
– Effect on the devices? Many in-vivo degradation schemes have been linked to
loss of mechanical properties due to post-sterilization aging.
Infections
• Infected implant is misnomer, it is the tissue that is infected!
• Infection is the invasion and multiplication of microorganisms in tissue
• 60’s and 70’s infection incidence rates ~ 5%, currently < 1%
• Duration of surgical operation is highly correlated with rate of infection

Infections
• Standard operating room has 50-500 cfu/m3 (cfu: colony forming unit,
minimum # of cells to grow a cluster of cells)
• Precautions reduce airborne bacterial levels to 1-5 cfu/m3:
– air-tight gowns and hoods

– body exhaust systems
– laminar flow away from patient
Infections: Antibiotic-Impregnated Implants
• Infection is considered as the failure of the implant since the need to change
the implant arises
• Bacterial growth in the presence of implants is tenacious and difficult to deal

with
• Antibiotic treated implants used against late or delayed infection
• Diffusion or dissolution controlled delivery system that is an integral part of an

implant:
– PMMA cements in total joint replacements mixed with antibiotics

Infections Antibiotic-Impregnated Implants
• Drawbacks:
– difficult to select the appropriate antibiotic since late infection is produced by

a range of bacteria
– patient may develop antibiotic sensitivity that would require the removal of the
implant
– antibiotic may not be able to penetrate efficiently to the infected tissue
– chronic use of antibiotics leads to more resistant virulent strains

Infections
Geometric factors are important for the interactions between implants and
infecting microorganisms
– geometry should render the cells of blood-borne inflammatory response

accessible to the implant
– avoid dead spaces: a volume filled with cell-free fluid rather than tissue
represents a special hazard; fluid can act as in vivo culture medium for bacteria
– bacteria may form their own defense about themselves or around the implant
by forming a slimy coating termed s glycocalyx
– porous bodies may initiate and support infection; bacteria may evade host-
defense mechanisms within the pores
Infections
• Types of infection:
– superficial immediate infection:
• growth of organisms on or near the skin in association with the
implant (e.g. suture infections, burn dressings)
• skin dwelling bacteria (e.g. staphylococcus aereus or staphylococcus
epidermidis) or airborne bacteria responsible
– deep immediate infection:

• low-frequency infections seen immediately after surgery;
– skin dwelling bacteria carried inside during implantation
– airborne (less frequent)
– bacteria already present in the operation site but inactive (even less
frequent)
• physical disruption of a cyst or sequestered area during operation
may facilitate bacterial growth; extremely infrequent; patients with
previous history of infection at the operation site
Infections
• Types of infection (cont):
– late infection: months to years after surgery in sites of no prior history of

infection
• believed to be caused by the transport and seeding of bloodborne
bacteria from an established infection at a remote site (e.g. tooth root
abcess, urinary tract infection)
– delayed infection: happens within three months; slow development of

intraoperative bacterial contamination
• delayed and late infections are rarely seen in total joint replacement or heart
valve implantation; difficult to treat once it is established in or around the
implant
Assessing Biocompatibility
• In vitro tests
• In vivo/Usage tests
• Clinical Trials
In Vitro Tests:
• Diverse
• Cell number, growth rate, metabolic rate, cell function, protein
expression
• Simple, repeatable, inexpensive, rapid
Techniques for In Vitro Analysis:

• Cell/Tissue Culture
• Cell adhesion, growth, metabolic assays
• Flow Cytometry (FCM)
• Immunocytochemistry
• Molecular Techniques, RT-PCR
• Gene array technology
• Live cell imaging
In Vivo tests:
• Relevant mammalian model
• Comprehensive biological response
• Ethical concerns
• Expensive & time-consuming
Clinical trial:
• Most relevant test
• All other tests measured against this
• Expensive, logistically complicated
• Difficult to interpret results

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Lecture 2: Biocompatibility/interactions of cells with

Ivan Djordjevic, PhD (Materials

Tissue Engineering Lab (Block U)

Biocompatibility is the ability of a material to perform with an appropriate host response

More generally: A material is considered “biocompatible” if it allows the body to function

The biocompatibility evaluation of biomaterials and biomedical devices is a very

Biocompatibility: Type of Biomaterials

• Orthopedics: artificial hips, knees, wrists; spinal rods; bone grafts.

• Cardiovascular: heart valves, stents, pacemakers, catheters, grafts,

• Dental: filings, prosthetics, orthodontics

• Soft tissue: wound healing, reconstructive and opthalmalgic

• Surgical/tools: staples, sutures, scalpels, catheters, etc.

• Micro/Nano-particles: Liposomes, dendrimers, etc.

• Biomedical devices: Biosensor, microarrays, etc

material contact time

syringe needle 1-2 s

contact lens 12 hr - 30 days

bone screw / plate 3-12 months Need for

 The cell is the structural and functional unit of all living

 all vital functions of an organism occur within cells

All cells share several abilities:

 Reproduction by cell division.

 Metabolism, including taking in raw materials, building cell

 Response to external and internal stimuli such as changes in

The cells of eukaryotes and prokaryotes.

 Eukaryotic cells (e.g. human): Nucleus (light blue), the

 Prokaryotic cell (Bacterium): Nucleoid containing the DNA

The selectively permeable cell membrane (or plasma membrane) is a thin

Cells tend to stick to other cells or non-cellular components of their

Relevant to the design of biocompatible biomaterials and many

Adsorbed proteins at the surface of Biomaterials mediates cell adhesion (e.g.

Cell-material interactions are due to the

An interesting property about these

One such oligopeptide is the three amino

Non-fouling + ligands = specific

Cells and biomaterials – Experiment in laboratory

I. Djordjevic et al. J of Biomater Sci Pol Ed, 2010

Cystine (Cys) Serine (Ser)

Sequence of local events following

J.M. Anderson, Ann Rev Mater Res;

J.M. Anderson, A. Rodriguez and

Scanning electron microscopy images of an Elasthane 80A Polyurethane surface from an in

• Inflammation is a non-specific response to tissue damage:

– arises in response to trauma, infection, intrusion of foreign materials, or

– granulocyte: any blood cell containing specific granules (e.g. neutrophils,

– leukocyte: white blood cell capable of ameboid movement (formation of

– macrophage: large phagocytic mononuclear cell

Intravascular cells/white blood cells/leukocyte:

– Monocytes: the largest one, deeply indented or

– Lymphocytes: or Killer T-cells: Cf immune

Clinical signs of inflammation: redness (rubor), swelling (tumor), pain (dolor),

• Why rubor? Erythrocytes

• Why swelling? Permeability:

Why pain? edema and kinins

Why heating? not clear! possible players:

• Lasts from minutes to days depending on the injury

• Dilation leads to an increase in blood entry into the capillary beds

– cells become sticky

How do leukocytes emigrate?

• Monocyte emigration may continue for days to weeks.

• Following emigration from the vasculature, monocytes differentiate into

Phagocytosis: Engulfing and degradation or digestion of fragments of tissue