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Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s P rogrammes at the University of Pécs and at the University of Debrecen Identification number : TÁMOP-4.1.2-08/1/A-2009-0011. - PowerPoint PPT Presentation
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Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
SCAFFOLD FABRICATION
Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 9
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
Solvent casting and particulate leaching (SCPL) I• 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, difficult to
eliminate contaminations
TÁMOP-4.1.2-08/1/A-2009-0011
Solvent casting and particulate leaching (SCPL) II
Evaporationof solvent
Porogenis dissolved
Solvent
Polymer PorogenMold
Porous structureis obtained
TÁMOP-4.1.2-08/1/A-2009-0011
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 liquid-liquid
phase is separated and the dissolved polymer is precipitating
• The solvent is removed (extraction, evaporation, sublimation)
TÁMOP-4.1.2-08/1/A-2009-0011
Advanced techniques Gas foaming• 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 dissolved scaffold occurs
10,000
1,000
100
10
1200 250 300 350 400
TemperatureT (K)
Pres
sure
P (b
ar)
solid
liquid
gas
critical point
supercritical fluid
triple point
TÁMOP-4.1.2-08/1/A-2009-0011
Electrospinning I
V
Syringe
Collector
Metallic needle
Polymer or composite solution
Electrified jetHigh-voltagepower supply
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
Advanced techniquesFiber 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
TÁMOP-4.1.2-08/1/A-2009-0011
Fiber mesh
TÁMOP-4.1.2-08/1/A-2009-0011
Advanced techniquesSelf 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
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
Advanced techniquesRapid 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 computer-controlled machinery needed.
TÁMOP-4.1.2-08/1/A-2009-0011Advanced techniquesFused deposition modeling (FDM)• 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 later.
TÁMOP-4.1.2-08/1/A-2009-0011
Advanced techniques Selective laser sintering (SLS)• 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
TÁMOP-4.1.2-08/1/A-2009-0011
75
3
Selective laser sintering (SLS)
4
Laser
Fabricationpowder bed
Object beingfabricated
Scanner1
Powderdelivery piston
Roller
Fabricationpiston Powder
delivery piston
Powderdelivery system
2
6
Buildcylinder
BIOCOMPATIBILITY
Dr. Judit PongráczThree dimensional tissue cultures and tissue engineering – Lecture 10
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Biocompatibility - DefinitionThe ability of a material to perform with an appropriate host response in a specific application.
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.
TÁMOP-4.1.2-08/1/A-2009-0011
Biocompatibility - Recent viewsOld concept: use of inert biomaterials that do not interact with the host tissuesNew aims in biomaterial design: • Biomaterials actively interacting with host
tissues• Biomaterials provoking positive physiological
responses• Biomaterials supporting cell growth and
differentiation
TÁMOP-4.1.2-08/1/A-2009-0011
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. plant-derived
polysaccharides have to be tested for biocompatibility
• Synthetic materials have to be tested for biocompatibility
TÁMOP-4.1.2-08/1/A-2009-0011
Biocompatibility - TerminologyBiodegradable: in vivo macromolecular degradation; no elimination of degradation products from the bodyBioabsorbable: macromolecular components enter in the body without metabolic changeBioresorbable: macromolecular components are degraded and metabolized, reduction in molecular mass and excretion of the final product
TÁMOP-4.1.2-08/1/A-2009-0011
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– Be affected by sterilization
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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 healingWound healing may be divided into phases characterized by both cellular population and cellular function:1. Blood clotting2. Inflammation3. Cellular invasion and remodeling
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Foreign Body Reaction IThe 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 IIAdsorbed plasma proteins mediate
granulocyte and macrophage responseFrustrated phagocytosis results in
macrophage activation and giant cell formation
Biomaterial
Monocyte
Macrophages
Bloodvessel
Endothelium
Cell-migration
Layer containingfibroblasts andcollagenLayer containingmacrophages
Biomaterial
Foreign bodygiant cell
TÁMOP-4.1.2-08/1/A-2009-0011
BiomaterialsTemporary implants:• Temporary support of tissue regeneration and
repair• Bone grafts, bioabsorbable surgical suturesPermanent implants: • Long term physical integrity and mechanical
performance • Long term replacement of organ function
(heart valves, joints, etc.)
TÁMOP-4.1.2-08/1/A-2009-0011
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: non-implantable, short- and long-term implantable
• Silicone is used for catheters, tubing, breast implants, condoms
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
Degradation of poly-a-hydroxy-acids
Most frequently used poly-a-hydroxy-acids:• Poly-lactic acid (PLA)• Poly-glycolic acid (PGA)• Poly-capronolactone (PCL)Degradation products enter into the citric acid
cycle.
Polyester Hydroxi-terminal Carboxy-terminal
H2O(CH2)nCO(CH2)n CO O
HO(CH2)n COO
(CH2)COHO
+
TÁMOP-4.1.2-08/1/A-2009-0011
Biodegradation of poly-a-hydroxy-acids
PGA
PLA
H2OGlycolic acidGlycineSerine
Lactic acidPyruvic acid
CO2
Acetyl-CoA
Citrate
Citric acidcycle
Oxidative phosphorylation
CO2
b-Hydroxybutyricacid
Acetoacetate
H2O
H2O
PDS
PHBEsterase
Urine
H2O
ATP
PGA = poly(glycolic acid)PLA = poly(lactic acid)PDS = poly-(d-dioxane)PHB = poly(hydoroxy butyrate)
TÁMOP-4.1.2-08/1/A-2009-0011
Application of poli-a-hydroxy-acidsClass Polymer Current application
Polyester
Polylactides
Poly(L-lactide), [PLLA]
Poly(D, L-lactide), [PDLLA]
• Resorbable sutures • Bone fixtures • Tissue engineering scaffolds for
bone, liver, nerve • Drug delivery (various)
Polyester Poly(lactide-co-glycolide), [PLGA]
• Controlled release devices (protein and small molecule drugs)
• Tissue engineering scaffolds • Drug delivery (various) • Gene delivery
Polyester Poly( -ε caprolactone), [PCL]• Slow controlled release devices –
drug delivery (e.g. > 1 year)
TÁMOP-4.1.2-08/1/A-2009-0011
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)
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
Biodegradation of polylactides• Generally involves random hydrolysis of ester
bonds• Type and duration of degradation depends on
composition• Products are non-toxic, non-inflammatory• In case of larger orthopedic implants acidic
degradation may produce toxic metabolites• Small particles may break off the implant
inducing inflammation
TÁMOP-4.1.2-08/1/A-2009-0011
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
TÁMOP-4.1.2-08/1/A-2009-0011
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.
• Surface erosion of the polymer occurs when the rate at which the water penetrating the device is slower than the rate of conversion of the polymer into water soluble materials.
TÁMOP-4.1.2-08/1/A-2009-0011
Types of degradation in biomaterials
TimeDegradation
Bulk erosionSurface erosion
TÁMOP-4.1.2-08/1/A-2009-0011
Degradation I • Biodegradable hydrogels: cleavage of
chemical cross-links between water soluble polymer chains
• Surface erosion is typical• Mass loss upon degradation is linear
TÁMOP-4.1.2-08/1/A-2009-0011
Degradation IICleavage of the polymer backbone leading to water soluble monomers
−(CH − C − O − CH − C − O −)x−(CH2 − C − O − CH2 − C − O)y−−HO − CH − C − OH + OH − CH2 − C − OH
CO2 + H2O
H2O
Krebbs cycleO
CH3
O
CH3
O O
CH3
O O
TÁMOP-4.1.2-08/1/A-2009-0011
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