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

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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

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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

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Solvent casting and particulate leaching (SCPL) II

Evaporationof solvent

Porogenis dissolved

Solvent

Polymer PorogenMold

Porous structureis obtained

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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 liquid-liquid

phase is separated and the dissolved polymer is precipitating

• The solvent is removed (extraction, evaporation, sublimation)

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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

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Electrospinning I

V

Syringe

Collector

Metallic needle

Polymer or composite solution

Electrified jetHigh-voltagepower supply

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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 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

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Fiber mesh

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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

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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 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.

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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

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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

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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. plant-derived

polysaccharides have to be tested for biocompatibility

• Synthetic materials have to be tested for biocompatibility

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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

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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– 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

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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.)

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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: non-implantable, 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-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

+

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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)

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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)

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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, non-inflammatory• 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.

• 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.

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Types of degradation in biomaterials

TimeDegradation

Bulk erosionSurface erosion

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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 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

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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