Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Dr. Judit Pongrácz
Three dimensional tissue cultures and
tissue engineering – Lecture 9
SCAFFOLD
FABRICATION
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Basic criteria for
scaffolds I
• Biocompatibility – to avoid immune
reactions
• Surface chemistry – to support
cellular functions
• Interconnected pores – cell
infiltration and vascularization
support
• Controlled biodegradability – to aid
new tissue formation
TÁMOP-4.1.2-08/1/A-2009-0011
Basic criteria for
scaffolds II
• Mechanical properties – structure and
function maintenance after the implant
and during remodeling
• Drug delivery – suitable for
controlled delivery of drugs or
bioactive molecules
• ECM interaction – supporting the
formation of ECM after implantation
• ECM mimicking – ECM replacing role
after implantation
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Importance of scaffold
characteristics
• Scaffolds provide the 3D environment
for cells
• Scaffolds temporarily replace the ECM
after implantation
• Scaffolds are important in directing
cellular differentiation
• Scaffold structure determines cell
nutrition and mass transport into TE
tissues
Solvent casting and
particulate leaching (SCPL)
I
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• Pour the dissolved scaffold into a
mold filled with porogen
• Evaporation of solvent in order to
form scaffolds
• Dissolving pore-forming particles from
scaffolds
• Scaffold layers: dip the mold into the
dissolved scaffold material
• Simple, easy and inexpensive technique
• No special equipment is needed
• Organic solvents are often toxic,
Solvent casting and
particulate leaching (SCPL)
II
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Solvent
Polymer
Porous structure
is obtained
Mold
Porogen
Evaporation
of solvent
Porogen
is dissolved
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Phase separation methods
• Polymer is dissolved into the mixture
of 2 non-mixing solvents
• Saturated solutions at a higher
temperature
• Polymer-lean and polymer-rich phase
separates
• Lowering the temperature, the liquidliquid phase is separated and the
dissolved polymer is precipitating
• The solvent is removed (extraction,
evaporation, sublimation)
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Advanced techniques
Gas foaming
10,000
solid
supercritic
al fluid
1,000
Pressure
P (bar)
• Specialized equipment
needed
• Pressure chamber
filled with scaffold
material
• Scaffold is
„dissolved” in
supercritical CO2
• By lowering the
pressure, physical
condition turns to
gas
• Phase separation of
liquid
100
critical point
10
gas
triple point
1
200
250
300
350
Temperature
T (K)
400
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Electrospinning I
Syringe
Polymer or
composite
solution
High-voltage
V
power supply
Metallic needle
Electrified jet
Collector
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Electrospinning II
• Specialized equipment required
• Technique is very versatile
• No extreme conditions (heat,
coagulation, etc.) required
• Many types of polymers are applicable,
e.g. PLA, PLGA, silk fibroin,
chitosan, collagen, etc.
• Thickness, aspect ratio, porosity,
fiber orientation are easily regulated
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Advanced techniques
Fiber mesh
• Specialized equipment is needed
• Scaffold consists of (inter)woven
fibres
• 2D or 3D scaffold structure are both
available
• Pore size can be easily manipulated
• Versatile technique, scaffold
material is broadly applicable and
combinations can also be applied
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Fiber mesh
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Advanced techniques
Self assembly
• Self assembly is the spontaneous
organization of molecules into a
defined structure with a defined
function
• Amphiphilic peptides in solutions form
non-covalent bonds
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Design of peptide
ampholites
• Phosphoserine group to enhance
mineralization (bone)
• RGD groups to provide integrin
binding sites
• Cysteines to form intermolecular
bridges
• GGG linker between the head and tail
groups to increase flexibility
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Advanced techniques
Rapid prototyping
• Rapid prototyping is the automatic
construction of physical objects using
additive manufacturing technology.
• This technique allows fast scaffold
fabrication with consistent quality,
texture and structure.
• Expensive and specialized computercontrolled machinery needed.
Advanced techniques
Fused deposition modeling
(FDM)
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• Robotically guided
extrusion machine
• Extrudes plastic
filament or other
materials through a
nozzle
• Layers where the
object should be
solid and
• Cross-hatching (using
a different
substance) for areas
that will be removed
Advanced techniques
Selective laser sintering
(SLS)
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• Scaffold material in powder form,
slightly below melting temperature
• A computer-guided laser beam provides
heat for the powder particles to
sinter (weld without melting)
• More new powder layers will be
sintered as the piston moves downward
and
• The 3D structure of the object will be
formed layer-by-layer
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Selective laser sintering
(SLS)
1
Powder
delivery system
2
3
Scanner
Fabrication
powder bed
Roller
Build
cylinder
Fabrication
piston
Powder
Powder
delivery piston
delivery piston
4
5
6
7
Laser
Object being
fabricated
Manifestation of Novel Social Challenges of the
European Union
in the Teaching Material of
Medical Biotechnology Master’s Programmes
at the University of Pécs and at the University
of Debrecen
Identification number: TÁMOP-4.1.2-08/1/A-2009-0011
Dr. Judit Pongrácz
Three dimensional tissue cultures and
tissue engineering – Lecture 10
BIOCOMPATIBILITY
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Biocompatibility Definition
The ability of a material to perform with
appropriate host response in a specific
application.
an
The biocompatibility of a scaffold or matrix
for tissue-engineering products refers to the
ability to perform as a substrate that will
support the appropriate cellular activity,
including the facilitation of molecular and
mechanical signaling systems, in order to
optimize
tissue
regeneration,
without
eliciting any undesirable effects in those
cells, or inducing any undesirable local or
systemic responses in the eventual host.
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Biocompatibility - Recent
views
Old concept: use of inert biomaterials
that do not interact with the host
tissues
New aims in biomaterial design:
• Biomaterials actively interacting with
host tissues
• Biomaterials provoking positive
physiological responses
• Biomaterials supporting cell growth
and differentiation
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Biocompatibility of
biomaterials
• Natural derived materials are inherently
biocompatible (e.g. collagen, fibrin,
hyaluronic acid)
• Xenogenic biomaterials have to be modified
to achieve biocompatibility (e.g. bovine
collagen has to be slightly digested before
human application to remove the immunogenic
sequences)
• Nowadays recombinant human collagen is
available
• Other xenogenic materials (e.g. plantderived polysaccharides have to be tested
for biocompatibility
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Biocompatibility Terminology
Biodegradable: in vivo macromolecular
degradation; no elimination of
degradation products from the body
Bioabsorbable: macromolecular
components enter in the body without
metabolic change
Bioresorbable: macromolecular
components are degraded and
metabolized, reduction in molecular
mass and excretion of the final product
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Biocompatibility testing
• Blood/material or tissue/material interface must be
minimal.
• Resistance to biodegeneration must be high.
• The biomaterial must interact as a natural material would
in the presence of blood and tissue.
• Implantable materials should not:
– Cause thrombus-formations
– Destroy or sensitize the cellular elements of blood
– Alter plasma proteins (including enzymes) so as to
trigger undesirable reactions
– Cause adverse immune responses
– Cause cancer
– Cause teratological effects
– Produce toxic and allergic responses
– Deplete electrolytes
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Complications from
incompatibility
• Immune reaction towards the implanted
material
• Chronic inflammation
• Scar tissue formation
• Increased blood clotting (vascular
graft incompatibility)
• Graft insufficiency
• Rejection
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Normal wound healing
Wound healing may be divided into
phases characterized by both cellular
population and cellular function:
1. Blood clotting
2. Inflammation
3. Cellular invasion and remodeling
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Foreign Body Reaction I
The presence of the implant changes the healing response, and
this is called the Foreign Body Reaction (FBR) consisting of:
• Protein adsorption
• Macrophages
• Multinucleated foreign body giant cells
• Fibroblasts
• Angiogenesis
Continuing presence of an implant may result in the
attainment of a final steady-state condition called
resolution.
There are 3 possible outcomes for the implant:
• Resorption
• Integration
• Encapsulation (fibrosis)
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Foreign Body Reaction II
Adsorbed plasma proteins mediate
Frustrated phagocytosis results in
granulocyte and macrophage response
macrophage activation and giant cell formati
Bloodvessel
Endothelium
Monocyte
Cell-migration
Foreign body
giant cell
Macrophages
Layer containing
fibroblasts and
collagen
Layer containing
macrophages
Biomaterial
Biomaterial
TÁMOP-4.1.2-08/1/A-2009-0011
Biomaterials
Temporary implants:
• Temporary support of tissue
regeneration and repair
• Bone grafts, bioabsorbable surgical
sutures
Permanent implants:
• Long term physical integrity and
mechanical performance
• Long term replacement of organ
function (heart valves, joints, etc.)
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Bioinert materials
Poly-tetrafluor-ethylen (PTFE, Teflon®)
• Inert in the body
• Extremely low friction coefficient
(0.05-0.10 vs. polished steel)
• Biologically inert, no interaction
with living tissue
• Surface coating of joint prostheses
and artificial heart valves
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Silicone derivates
• Silicones are polymers that contain Si
besides of common C, H, N, O elements
of biocompatible polymers.
• Medical grade silicones: nonimplantable, short- and long-term
implantable
• Silicone is used for catheters,
tubing, breast implants, condoms
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Biocompatible metals
• Titanium alloys for joint replacement
and dental implants
• Excellent mechanical properties
• Non-toxic and non-rejected
• Uniquely capable of osseointegration
• Hydroxyapatite coating before
implantation enhances osseointegration
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Hydroxyiapatite ceramics
• Hydroxyapatite (HA) is naturally
occurring in the bones and teeth
• HA crystals are often combined with
other polymers to form scaffolds
• Microcrystalline HA is sold as a
nutrition supplement to prevent bone
loss
• It is superior to CaCO3 in preventing
osteoporosis
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Poly-a-hydroxy-acids:
bioabsorbable polymers
• Most frequently used biomaterials
• Main uses are resorbable sutures, drug
delivery scaffolds and orthopedic
fixtures
• Polyester chains
• Degradation by simple hydrolysis
• The resulting a-hydroxy-acids are
eliminated via metabolic pathways
(e.g. citric acid cycle) or excreted
unchanged with the urine
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Degradation of poly-ahydroxy-acids
(CH2)nCO(CH2)n C
O
Polyester
O
H2O
HO(CH2)n CO
+
(CH2)COH
O
O
Hydroxi-terminal Carboxy-terminal
Most frequently used poly-a-hydroxyacids:
• Poly-lactic acid (PLA)
• Poly-glycolic acid (PGA)
• Poly-capronolactone (PCL)
Degradation products enter into the
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Biodegradation of poly-ahydroxy-acids
PHB
Esterase b-Hydroxybutyric
acid
PDS
Serine
Glycine
Glycolic
acid
H2O
Lactic
acid
H2O
Pyruvic acid
PGA
PLA
CO2
H2O
Urine
Acetoacetate
Acetyl-CoA
PGA
PLA
PDS
PHB
Citrate
Citric acid
cycle
CO2
H2O
Oxidative phosphorylation
ATP
=
=
=
=
poly(glycolic acid)
poly(lactic acid)
poly-(d-dioxane)
poly(hydoroxy butyra
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Application of poli-ahydroxy-acids
Class
Polymer
Current application
Polylactides
•
Polyester Poly(L-lactide), [PLLA]
Poly(D, L-lactide), [PDLLA]
Resorbable sutures
• Bone fixtures
• Tissue engineering scaffolds for
bone, liver, nerve
• Drug delivery (various)
Controlled release devices (protein
and small molecule drugs)
• Tissue engineering scaffolds
• Drug delivery (various)
• Gene delivery
•
Polyester Poly(lactide-co-glycolide), [PLGA]
Polyester Poly(ε-caprolactone), [PCL]
•
Slow controlled release devices –
drug delivery (e.g. > 1 year)
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Poly-(Glycolic Acid), (PGA)
• PGA is a rigid, highly crystalline
material
• Only soluble in highly apolar organic
solvents
• Main use as resorbable sutures
(Dexon®)
• SCPL method for scaffold fabrication
• Bulk degradation
• Natural degradation product (glycolic
acid)
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Poly-(Lactic Acid), PLA and
PGA co-polymers
• D, L isoform and racemic mixture
• Most often the L isoform is used
together with PGA → PLGA copolymer
• PLGA is one of the few polymers
approved for human use
• Copolymer mixtures of PGA and PLLA
have various features thus allowing
versatile application range in tissue
engineering
• Degradation rate and type depends on
the composition of the co-polymers
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Biodegradation of
polylactides
• Generally involves random hydrolysis
of ester bonds
• Type and duration of degradation
depends on composition
• Products are non-toxic, noninflammatory
• In case of larger orthopedic implants
acidic degradation may produce toxic
metabolites
• Small particles may break off the
implant inducing inflammation
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Poly-(caprono-lactone),
(PCL)
• Semicrystalline polymer
• Very slow degradation rate (pure PCL
degrades in 3 years, copolymers with
other caprones can be degraded more
readily)
• Used for drug delivery for longer
periods
• PCL is considered non-toxic and
biocompatible material
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Polymer erosion
• Water penetrates the bulk of the device,
attacking the chemical bonds in the
amorphous phase and converting long
polymer chains into shorter water-soluble
fragments.
• This causes a reduction in molecular
weight without the loss of physical
properties as the polymer is still held
together by the crystalline regions. Water
penetrates the device leading to
metabolization of the fragments and bulk
erosion.
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Types of degradation in
biomaterials
Surface erosion
Bulk erosion
Degradation
Time
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Degradation I
• Biodegradable hydrogels: cleavage of
chemical cross-links between water
soluble polymer chains
• Surface erosion is typical
• Mass loss upon degradation is linear
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Degradation II
Cleavage of the polymer backbone
leading to water soluble monomers
CH3
H 2O
−(CH − C − O − CH − C − O −)x−(CH2 − C − O − CH2 − −HO
C − −O)CH
y− − C − OH + OH − CH2 − C
CH3
CH3
O
O
O
O
O
O
Krebbs cycle
CO2 + H2O
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Degradation III
• Polymer hydrophobicity: stability
increases with increased
hydrophobicity
• Bulky substitutes (e.g. methyl group
in PLA) increase degradation time
(PGA<PLA)
• Glass transition: Rubbery polymers
above Tg have more chain mobility thus
easier access for water
• Crystallinity decreases, amorphous
structure increases degradation time