Lecture B4 - Biosurface Engineering Practical aspects …pg.gda.pl/info/mech/katedra/imis/wp-content/blogs.dir/49/files/... · Lecture B4 - Biosurface Engineering Practical aspects

Lecture B4 - Biosurface EngineeringPractical aspects of bioactive coatings

Faculty of Mechanical Engineering, Institute of Materials Science, Max Bergmann Center of biomaterials

Teaching Gdansk October 2012

Cornelia Wolf-Brandstetter

[email protected]

Teaching Gdansk 2012 - C. Wolf-Brandstetter Slide 2 of 65

Why Surface Engineering?

BiocompatibilityThe ability of a material to perform with an appropriate host response in a specificapplication. (Chester Consensus Conference 1986)

Structural biocompatibility

Adaptation of the implant structure to the mechanical behaviour of the surrounding tissue (host tissue). This includes the design as well as the ‘inner’ structure (i.e. placement of fibres in anisotropical materials). Goal is mimicry of structure.

Surface biocompatibility

Adaptation of the • morphological,• physical,• chemical, and • biological surface propertiesof the implant to the specific needs of the surrounding tissue aiming at a clinical desired interaction.


Biomaterials and cells

All healing is due to cells: from blood cells (coagulation, inflammation) to stem cells (source of undifferentiated cells that form new tissue specificcells).

The ideal biomaterials has to give the right cues for cellbehavior and differentiation!

OH-Groups+ -

Ions Proteins

Cells

Tissue

0,1 1 10 100 1000 104 105 106

1 10d ds s s s

time

Implant / Biomaterial


(Bio)-Surface Engineering

Morphology ChemistryPhysico-chemical Biochemical

Examples Examples ExamplesMicrostructures to improve•Adhesion•Proliferation•Differentiation

of cells

•Mechanical interaction with surrounding tissue – inter-looking

Adaptation of•Surface energy•Isoelectric point•ionic/electronic conductivity

Improved properties rela-ted to•wear / abrasion•corrosion behaviour

Inorganic layers (i.e. HAP)• thermical• electrochemical assisted• Sol Gel

Organic layers / Immobili-zation of organic molecu-les

• Adsorption• Covalent coupling

Layers as ‘drug delivery systems’ for

• Antibiotics• Stimulating factors (GF)


(Bio)-Surface Engineering for Bone

What does this mean in thespecial case of bone?

Aim: Engineer the surface to resemble the microenvironment of cells! Method: By using the main components of their surroundings!

Mineral (HAp) Protein* H2O

0 20 40 60 80 100 %

Structural protein collagen type I

Other collagens, proteoglycans, glycosaminoglycans, growth factors,…


Adhesion and the biomaterial surface

Aim: Engineer the surface to resemble the microenvironment of cells! Method: By using the main components of their surroundings!

Possible approaches: Inorganic coatings (bone)

hydroxy apatiteOrganic coatings

Peptides (i.e. RGD)ECM proteins (i.e. collagen)Synthetic matrices mimicingthe ECM (ie. )

Aim to incorporate ECM signals into biomaterialsby using motifs of thenatural ECM

ECM motifson thebiomaterial

Influence: • cell adhesion/ migration• matrix remodelling• cytokine signalling•….

biomaterial


Bone forming cells

OsteoblastsBone froming cellsProduce calcifying ECMOriginate frommesenchymal stem cellsMarker enzyme: alkalinephosphatase

OsteocytesMature from osteoblastsEmbedded in the calcifiedmatrix

Markers of differentiationMesenchymal stem cells: STRO-1+, AP-Pre-osteoblast: STRP-1+, AP+Osteoblast: STRO-1-, AP+

• Alkaline phosphatase• Osteonectin, osteopontin, bone

sialoprotein• Osteocalcin• Calbindin

osteocyte

Low differentiation

High differentiation


Bone degrading cells

OsteoclastsBone degrading cellsLysate and phagocyte boneOriginate from heamatopoeticcells (macrophage line)Marker enzyme: tartrateresistant acid phosphatase(TRAP) Characteristics

• 4-20 nuclei• Ruffled border and sealing zone• Calcitonin receptor positive• Vitronectin receptor positive• Cathepsin K positive• Matrixmetalloproteinase 9 (MMP-9)

Activity test• Resorption pit formation• Collagen degradation products(hydroxyproline)


Inorganic coatings –for boneimplants


Inorganic coatings

Hydroxyapatite

main component of bone is the mineral phase

Biological adaptation to surrounding tissue by using inorganic layers:Deposition of Calcium Phosphate Phases (CPP)

Use selected ECM components to create a matrix that resembles the microenvironment of the respective cells


Proposed mechanism:- Supersaturation of implant surrounding with calcium and phosphate ions

deposition of biological apatite (high specific surface area)- Adsorption of endogenous proteins

Methods: Plasma SprayingSputter depositionSol-Gel coatingsElectrophoretic depositionBiomimetic approaches

Inorganic coatings

Mainly used calcium phosphate phases (CPP)Hydroxyapatite (HAp)BrushiteOctacalciumphosphate (OCP)Tricalcium phosphate (TCP)


Specific Adsorption

from http://www.cup.uni-muenchen.de/ac/kluefers/homepage/L/biominerals/osteocalcin.gif and Q. Q. Hoang et al Nature 2003, 425, 977–980

glutamic acid residues (Gla)of osteocalcin

…..of calcium phosphate binding proteins (e.g. osteocalcin)

and calcium ions of hydroxyapatite

specific interactionbetween


Impact of coating properties

HAp crystallites can differ in morphology, size and solubility from those of bone

X 10

Plasma sprayed HAp on titanium


Load bearing but insufficient bone interlock possible delamination of layers

Delaminations can be enhanced by thermal decomposition:Ca10(PO4)6(OH)2 ⇔ Ca10(PO4)6(OH)2-2xOx > 850 °CCa10(PO4)6(OH)2 ⇒ 2 β-Ca3(PO4)2 + Ca4(PO4)2O + H20 > 1050 °Cβ-Ca3(PO4)2 ⇒ α-Ca3(PO4)2 > 1350 °C

HA plasma spray coating on Titanium grit blasted surface

Coating has to be load bearing

Titanium grit blasted surface

Topographical interlock between bone and implant supports load transfer

slow and inhomogenous degradation which can lead to particle release

Impact of coating properties


Impact of mineral size

From: M. Espanol, Biointerphases (2012) 7:37

Crystal size of applied coatings is able to override the intrinsic affinity of proteins for a substrate

Preferred binding of big proteinwith high affinity

Impaired binding of big proteinsby steric hindrance


Thin coatings - ECAD method

ECAD HA coating on titanium grit blasted surface

• 5 µm thick• 60 nm crystallites of

structure related to bone

Details of ECAD method see lecture A1


• Deposition of thin, not monolithic layers with a composition, crystal size and chemical history close to that of bone mineral possible

• Thickness and chemical composition of coatings can be controlled to the sub-µm level• Generation of coatings with high specific surface area possible - specific surface of about

50 g/cm2, thus for 100 µg/cm2 coatings surface enlargement by factor of about 40• Use of appropriate conditions results in CPP with a composition and structure close to

mineral phase of bone• Low energy process• Low cost process (with respect to production costs as well as waste management)• No adverse effect of heat on substrate material• Compared to incubation in SBF more defined and higher relative supersaturation at the

interface, resulting in shorter processing times• Due to possible physiological processing parameters (pH, temperature, aqueous solution)

the deposition of CPP can be combined with the immobilisation/incorporation of organic components like proteins (peptides)

• Excellent homogeneity of coatings on structured and porous surfaces as well as on irregularly formed structures as it is no line of sight process.

Advantages of the ECAD method


Organic coatings

PeptidesODN

NH

O

O

OH

O

OH

NHNH

NH2

NH

NH

NH

NH

O

O

O

NH

O

O

N

OHO


Peptides

The most reduced form uses only the receptor adhesion motifs without therest of the protein.

Advantages:• Stable• Comparatively cheap• Reproducible• Characterizable

Disadvantages:• Immobilization complex due to small size• Only one motif loss of more complex

interactions

Controlling cell response to biomaterials by building in ECM cues on a ‘blank slate’background


RGD peptide as ligand for osteoblasts

GR

KNH

Df

BioactiveRGD

component

R= ArgininG= GlycinD= AspartatK= Lysinf= D-Phenylalanin

A

A

A

ASurface anchor

A= Phosphonat

(AHX)3

Spacer

K

K

KAHX= Aminohexoic acid

Osteoblast

Integrin


Effect of ligand density

0,01 µM 0,1 µM 1 µM

10 µM 100 µM 1000 µM

Osteoblasts on RGD coatings Increasing RGD density enhances adhesion

Cells respond to control of ligand density at the surface:


BUT: too much RGD reduces cell motility!

Effect of ligand density


Other peptide adhesion motifs

aMb2fibrinogen, C3bQKRLDGS

αvβ1, α4β7fibronectin, VCAMEILDV

β3 integrinsfibrinogenKQAGDV

Elastase receptorelastaseVAPG

α2β1collagenDGEA

N-cadherinN-cadherinHAV

?lamininRNIAEJIKDI

α1β1, α3β1lamininYIGSR

α3β1, α4β1, αvβ1, αIIbβ3, αvβ5, αvβ3, ..

fibronectin, laminin, collagen, vitronectin, vWF, bone sialo protein osteopontin, thrombospondin

RGD

LB110lamininIKVAV

ReceptorOriginPeptide


Collagen

Artificial ECM


Multi-component coatings Artificial ECM

Main functional componentStableEase of use


Basic idea – what is the biological target? What mechanism of response do I anticipate?

Example: coating of titanium implants for use as bone substitute

Making a bioactive coating


Replacement of type III by type I; Generation of mature bone

Phases of Bone Healing

Hiltunen et al., Clinical Orthopaedics and Related Research, 297(1993) 23.

In vitro: Proliferation In vitro: Matrix deposition In vitro: Mineralisation

Synthesis of collagen III + fibronectin; Migration of osteoprogenitor cells

Production of type I to type III; Start of mineralization

Ca deposition

Alkalinephosphatase

Collagen

Type IIIType I

Inflammation Regeneration Remodeling


Basic idea – what is the biological target? What mechanism of response do I anticipate

Can I produce the desired coating?What are the best conditons?What are the characteristics of mycoating?



fibril morphologyfibril amountdeposition on titanium (quantity, conformation/ activity)


0 200 400 600 8000.0

0.2

0.4

0.6

0.8

OD

313

time [min]

100% I

20% III

50% III

100% III

0 % 2.5 % 5 % 10 % 20 % 50 %100 %0

20

40

60

80

100

depo

sitio

n in

per

cent

percentage type III

fibrillogenesis collagen deposition in fibrils

Fibrillogenesis of heterotypic fibrils

Collagen I/III fibrils can be formed, but …


2,5 µm 2,5 µm

Morphology of collagen I and III fibrils

Type I Type I/III (1:1) Type III

S. Bierbaum et al., J. Biomed. Mater. Res. 67A(2003), 421-30.

2,5 µm

Decrease in fibril diameter with increasing content of type III collagen

Decrease in incorporation into fibrils with increasing content of type III collagen (90 → 70 %)

…there is a significant effect ont fibril morphology




How does my coating influence cells?(Which cell type?)




adhesionproduction of marker substances(enzymes, calcium phosphate)expression of marker genes


Influence on cell shape

Collagen I Collagen I and III Collagen III

100 µm 100 µm 100 µm

Osteoblasts 24h after adhesion

obvious impact of collagen type on cell morphology


collagen synthesisproliferation

0 2 4 6 8 10 12 14 160,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5 I I /II I I II PS

abso

rban

ce 5

70 n

m

days in culture2 3 4 5 6 7 8

50

100

150

200

250

300 I I/ II I II I PS

prol

ine

inco

rpor

atio

n [c

mp/

1000

cel

ls]

days in culture

Influence on early healing events

Markers for early events are stronger on type III


activity of ALP extracellular calcium

21 22 23 24 25 26 27 28 29 30

1

2

3

4

5

6

7

8

Ca

- µm

ol/ c

m²

I I /II I I II PS

days in culture3 4 5 6 7 8 9 10 11 12

0,05

0,10

0,15

0,20

0,25 I I/I II III PS

ALP

act

ivity

[U/m

g pr

otei

n]

days in culture

Markers for late events are stronger on type I

In vitro cell behavior on different matrices differs and reflects eventsassociated with such a matrix in vivo

Influence on late healing events




How does my coating influence cells?(Which cell type?)

How does my coating behave in vivo?




adhesionproduction of marker substances(enzymes, calcium phosphate)expression of marker genes

inflammatory reactiontissue regeneration


Collagen

Artificial ECM



Syntheticmatrices


Synthetic matrices

Synthetic matrices consist of polymers that do not occurnaturally in the ECM

- gelatine- chitosan

Synthetic polymers- polylactide- polyethylene glycol

Advantages:• Cost• Reproducability• Pathogens• Antigenicity

Disadvantages:• (Often) no natural degradation

mechanisms• No cell recognition sites


Tailoring material degradation

… by using degradablematerials


… or by including motifs thatcan be enzymaticallydegraded

Tailoring material degradation


Cross-linking the material

For stability, many biomaterials, both synthetic and natural, arecross-linked

Advantages: • higher stability• lower degradation rates

Disadvantages:• lower degradation rates

(depends on application!)• Usually chemical treatment

necessary– changes proteins– can be cytotoxic– …

Use enzyme-sensitivecrosslinks

Use enzymatic cross-linking


Enzym cross-linking and enzyme-sensitive cross-links


Collagen

Artificial ECM


Type I

FibronectinDecorinBiglycan

Chondroitin sulfateHeparin

Heparan sulfate

(Type II)


Type III


• dimeric protein of 240.000 kD

• occurs in plasma and on the cell surface

• important role in woundhealing

• binds cells primarily via RGD-sequence

• binds native and denaturedcollagen

collagenbinding site

cellbinding

site

heparinbinding site

Glycoproteins: Fibronectin


Decorin (130 kd, 40 kd core)• Small leucine rich

proteoglycan• CS in bone, DS in soft tissue• Binds TGF-ß and collagen,

influences fibril formation, found in bone, cartilage, skin

Proteoglycans: Decorin


Heparan sulfate, heparin, chondroitinsulfate

• basement membrane, cell surface, part of PGs

• Sulfated, binds growth factors (i.e. VEGF)

• Heparin: higher sulfation degree

Hyaluronic acid• No core protein, unsulfated

Glycosaminoglycans (GAGs)

Chondroitin-4-sulfate

Hyaluronic acid

Glucuronic acid

Glucuronic acid N-acetyl-glucosamine

N-acetyl-galactosamine


GAGs during fibrillogenesis

0 200 400 600 800 1000

0,0

0,1

0,2

0,3

1: 4 1: 8 1: 16 1: 32 1: 64 1: 128 coll

OD 30

3

time

0 200 400 600 800 1000

-0,2

-0,1

0,0

0,1

0,2

0,3

coll 1: 4 1: 8 1: 16 1: 32 1: 64

OD

303

time

Chondroitinsulfate

Heparansulfate

Heparin

0 200 400 600 800 1000

0,0

0,1

0,2

0,3

0,4

0,5

0,6 coll 1: 4 1: 8 1: 16 1: 32 1: 64 1: 12 8

OD

303

time

GAGs can be introduced to collagen fibrilsduring in vitro fibrillogenesis

Influence on kinetics of fibril formation


Organization of the cytoskeleton

Collagen Collagen/Chondroitin sulfateCollagen/Decorin

Red: ActinGreen: Vinculin

Osteoblast adhesion after 2 h incubation


Differentiation markers

Osteoblasts: Expression of osteopontin

dex-coll

dex-CS

SF-coll

SF-CS

exp-coll

exp-CS

day 5

day 280,0

0,2

0,4

0,6

0,8

1,0

[a.u

.]

relative ALP activitydonor 1

hMSC: Expression of ALP


dex-coll dex-CS SF-coll SF-CS exp-coll exp-CS0

250

500

750

4000

5000

6000

7000

OC

rela

tive

to G

APD

H

donor 1 day 5 day 15 day 28

M. Wollenweber et al. Tissue Engineering 12(2006)2, 345-59.

ECM type stronglyinfluences

differentiationbehaviour

hMSC: Transcription (mRNA synthesis) of osteocalcin(quantitative RT-PCR)

Differentiation markers


Tailoring GAGs

CH2

C

O

O -

S

O

O

O -

Chondroitin 6-sulfateHyaluronate

Carboxymethyl groupSulfate group

First sulfatation

Modification of properties(GF binding and release)….

Natural sulfatationsite for CS-4


Cell response to tailored GAGs

Cell culture wihout any osteogenic supplements - upper rowAddition of dexamethasone (corticoal growth hormone) - lower row

Increased amount of sulfate groupsleads to increased activity of ALP (even without osteogenic induction)


Collagens

aECM and soluble mediators


Type IType II


Type III

non-collagenouscomponents

fibronectindecorinchondroitin sulfateheparan sulfateheparin

growth factors

TGF-βBMP-4


Soluble mediatorsgrowth factorsmorphogenetic factorscytokineschemokineshormones

They are usually recognized by specific receptors in the cell membraneand are very potent regulators of cell fuctions.

For which reason they are of interest in biomaterials applications

But …

Diverse functionsInduce/stop cell migrationInduce/stop cell growthInduce/ stop celldifferentiationUp/downregulate tissue-specific functionsCharacteristicsAct at pmol concentrationsSynergize with otherreceptor signals, i.e. integrins

What soluble mediators are there?


What soluble mediators are there?

… they are:

expensiveunstable (both in vivo and in vitro)recombinant growth factors often far less effectivethan their physiological counterpartssoluble

Soluble mediators are used in combination with carriers toprevent quick diffusionprotect them from degradation


Strategies for Biomaterial Presentation of Growth Factors

1. Chemical conjugation of GFs to scaffold materialsa) non-covalent incorporation

direct adsorption via direct charge-charge or hydrophobic interactions

indirect interaction via specific biological sites that are coated on the surface(heparin, fibronectin, gelatin, small oligopeptides)

b) covalent incorporation

conjugation for the surface via functional groups

2. Physical encapsulation of GFs with pre-programmed release, and diffusioninto surrounding tissue

Adapted from Lee, K et al., 2010


Chemical conjugation of growth factors to biomaterials

Direct adsorption Indirect interactionfor example artificial ECM

(collagen/GAG)

GF GF GFGF GF GF

Inactivation by denaturationdue to binding

GF presented in waycomparable to the natural situation


Interactions between matrix, soluble factor and cell

cell

Direct:engagement of cell adhesion receptors by ECM

Indirect: Matrix interactionswith growth factors

• Storage• Accumulation in specific areas (gradients)• Conformational changes and dimerisation

(presentation for receptors)• Protection from proteolytic degradation

Direct:engagement of cell receptors bysoluble factors


BMP-4

Desorption from different matrices

-20 0 20 40 60 80 100 120 140 160

-0,20,00,20,40,60,81,01,21,41,61,82,02,2

collagen coll + chondroitin sulfate coll + decorin

ng d

esor

bed

h

0 20 40 60 80 100 1200,00,20,40,60,81,01,21,41,61,82,02,22,42,6

collagen coll + chondroitin sulfate coll + decorin

ng d

esor

bed

hTGF-βDecorin

2 µm

Chondroitin sulfate

2 µm


Using 3d matrix structure

osteoblast

collagen/ decorin/ (TGF-ß)

collagen/ chondroitinsulfat/ (BMP-4)

titanium

A

collagen/ collagen-chondroitin sulfate (collagenred, CS green).

Modulation of growth factor activity through influencing the temporal release pattern


Osteoblastic cells on layered aECM

Message: ‚construction principle‘ of aECM stronglyinfluences cell reactions

0

10

20

30

40

50

coll/CS/BMPcoll/dec/TGF

coll/CScoll/dec/TGF

coll/CS/BMPcoll/dec

coll/CScoll/dec

prol

in in

tegr

atio

n [c

pm/1

000

cells

]

0

50

100

150

200

250

300

350

coll/CS/BMPcoll/dec/TGF

coll/CScoll/dec/TGF

coll/CS/BMPcoll/dec

coll/CScoll/dec

ALP

act

ivity

[mU

/mg

prot

ein]

Collagen synthesis ALP activityday 4


Control of GF-binding

Highest amount of bound GF for high sulfatated GAGsMethyl group decreases affinity for GF

… by suitable modification of GAGs (sulfatation degree and pattern)

From: Hintze et al.


… and control of GF-release

High GF release rates from highly sulfated GAGsAfter 4d still highest remaining GF on highly sulfated GAGs(lowest percentage of delivered GF)


Cellular response

Highest relative increase in collagen synthesis for highly sulfated GAGs

Collagen synthesis by osteoblasts due to immobilized TGF-b1related to collagen synthesis on uncoated matrices (100%),

Light columns 1dDark columns: 8d(from Hempel et al. Acta Biomaterialia2012


Increase in complexity of the artificial matricesallows for better adaptation to natural systems,

but …..

…..increases also complexity of the in vitro characterization of thea-ECM coatings:

• Interference of several quantification methods by othercompounds (e.g. collagen quantification is strongly affected bypresence of GAGs)

• Desorption of growth factors can be accompanied by simultaneousdelivery of GAGs from a-ECM, which mayimpair the detectability of the GFas well as influence the activity of the mixture free GF/GAG-GF

Challenges in a-ECM characterization


Summary

Within certain limits growth factors release can be influenced byinclusion of GF binding components, which also influences cellbehavior

Using modified GAGs, growth factor binding can be increased(and their presentation altered)

A matrix can be/should be „tailored“ for different growth factors

Layering differently composed matrices can allow for time differences in growth factor release, which influences cellresponses


Finished !!! …

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Lecture B4 - Biosurface Engineering Practical aspects …pg.gda.pl/info/mech/katedra/imis/wp-content/blogs.dir/49/files/... · Lecture B4 - Biosurface Engineering Practical aspects