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

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

SCAFFOLD

FABRICATION

Dr. Judit Pongrácz

Three dimensional tissue cultures and tissue engineering – Lecture 9

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

• 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

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

• 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

leaching (SCPL) II

Evaporation of solvent

Porogen is dissolved Solvent

Polymer Mold Porogen

Porous structure is obtained

• 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)

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

1

200 250 300 350 400

Temperature T (K)

Pressure P (bar)

solid

liquid

gas

critical point supercritical

fluid

triple point

V

Syringe

Collector

Metallic needle Polymer or

composite solution

Electrified jet High-voltage

power supply

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

• 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

Fiber mesh

• Self assembly is the spontaneous organization of molecules into a defined structure with a defined function

• Amphiphilic peptides in solutions form non-covalent bonds

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

• 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.

Fused 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.

• 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

7 5

3

Selective laser sintering (SLS)

4

Laser

Fabrication powder bed

Object being fabricated Scanner

1

Powder delivery piston

Roller

Fabrication

piston Powder delivery piston Powder

delivery system

2

6

Build cylinder

BIOCOMPATIBILITY

Dr. Judit Pongrácz

Three dimensional tissue cultures and tissue engineering – Lecture 10

Biocompatibility - Definition

The 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.

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

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

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

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

• Immune reaction towards the implanted material

• Chronic inflammation

• Scar tissue formation

• Increased blood clotting (vascular graft incompatibility)

• Graft insufficiency

• Rejection

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

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)

Foreign Body Reaction II

Adsorbed plasma proteins mediate granulocyte and macrophage response

Frustrated phagocytosis results in

macrophage activation and giant cell formation

Biomaterial Monocyte

Macrophages Bloodvessel

Endothelium

Cell-migration

Layer containing fibroblasts and collagen

Layer containing macrophages

Biomaterial

Foreign body giant cell

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.)

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

• 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

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

• 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

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

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 C

O O

HO(CH₂)_n CO O

(CH₂)COH O

+

acids

PGA

PLA H₂O

Glycolic acid Glycine

Serine

Lactic acid Pyruvic acid

CO₂ Acetyl-CoA

Citrate

Citric acid cycle

Oxidative phosphorylation CO₂ b-Hydroxybutyric

acid

Acetoacetate

H₂O H₂O PDS

PHB Esterase

Urine

H₂O

ATP

PGA = poly(glycolic acid) PLA = poly(lactic acid) PDS = poly-(d-dioxane)

PHB = poly(hydoroxy butyrate)

Class 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)

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)

• 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

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

• 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

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.

Time

Degradation

Bulk erosion Surface erosion

Degradation I

• Biodegradable hydrogels: cleavage of chemical cross-links between water soluble polymer chains

• Surface erosion is typical

• Mass loss upon degradation is linear

Cleavage 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 cycle

O CH₃

O CH₃

O O

CH₃

O O

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