SMART BIOMATERIALS: Main Properties and Applications

resp

on

se

external variable

“all-or-nothing”

“0”

“1”

critical point

resp

on

se

external variable

high non-linear behaviour

Chemical stimuli= pH, ionic factors, chemical agents

Physical stimuli= Temperature, electric or magnetic fields, mechanical stress

Biochemical stimuli= antigens, enzymes, ligands

STIMULI-RESPONSIVE POLYMERS

+ stimulus

- stimulus

+ stimulus

- stimulus

+ stimulus

- stimulus

+ stimulus

- stimulus Based on: AS Hoffman, PS Stayton, Macromol. Symp. ‘04

solubility in aqueous solutions

adsorption control

collapse of grafted chains

swelling in hydrogels

STIMULI-RESPONSIVE POLYMERS

- Water swollen crosslinked polymers - Crosslinks may occur:

- by reaction of one or more monomers - hydrogen bonds - van der Waals interactions

HYDROGELS

Classification (based on preparation method) - homopolymer hydrogels (one type of hydrophilic mer) - copolymer hydrogels (two types of mers, at least one hydrophilic) - multipolymer hydrogels (more than three types of mers) -interpenetrating polymeric hydrogels (swelling a network of polmer1 in mer2, making intermeshing network of polymer1 and polymer2)

- Crosslinks may be induced during polymerization or by radiation following polymerization - Interchain connections can be:

- a carbon atom - chemical bridge - van der Waals - hydrogen bonds - molecular entanglements

- Crosslink structure:

A) ideal network with tetrafunctional

covalent crosslinks (rarely observed)

B) multifunctional junctions

C) molecular entanglements (could be

permanent or semi-permanent)

- Defects in crosslink structure:

D) unreacted functionality

E) chain loops

Mc: Molecular weight between crosslinks

HYDROGELS

HYDROGELS

Hydrogels prepared by crosslinking copolymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) (a) Homogeneous hydrogel (b) Example of popcorn formation (c) Heterogeneous (porous) hydrogel

Use of HEMA based hydrogels in rhinoplasty a) Patient before surgery, b) Patient after surgery

Soft contact lenses

J Biomed Mater Res 9, 675 (1975)

Natural polymers and synthetic monomers used in hydrogel fabrication

Adv Drug Del Rev 58, 1379 (2006)

HYDROGELS

HYDROGELS - APPLICATIONS

J Pharm Sci 96, 2197 (2007)

LCST Lower Critical Solution temperature

coil structure collapsed structure

TEMPERATURE RESPONSIVE HYDROGELS

LCST 32-33ºC in water

nCH

C O

NH

CH

CH3CH3

CH2

poly(N-isopropylacrylamide) - PNIPAAM

DESIGNS FOR LCST CONTROL

Acrylamide – more hydrophilic comonomer N-butylacrylamide – more hydrophobic comonomer

J Biomed Mater Res 52, 577 (2000)

TEMPERATURE RESPONSIVE HYDROGELS

Gelatin gels at ~35 ºC

Gelatin

Carrageenan

Carrageenan gels at between 25-80 ºC

Cellulose derivatives

MC gels between 40-50 ºC and HPMC between 75-90 ºC

TEMPERATURE RESPONSIVE HYDROGELS

TEMPERATURE RESPONSIVE HYDROGELS

Polyethylene oxide (PEO) – hydrophilic Polypropylene oxide (PPO) - hydrophobic

Amphiphilic block copolymers: PEO-PPO-PEO (Poloxamers)

Depending on the ratio and distribution along the chain of the hydrophilic and hydrophobic subunits different gelation temperatures (from 20 to 85 ºC) can be obtained

pH-RESPONSIVE HYDROGELS

H2

CHC

COOH

n

H2

CHC

COO-

n

-

+

OH

H

H2

CHC

C

n

H2

CHC

C

n

-

+

OH

H

CH2CH2N(CH2CH3)2

O

H

CH2CH2N(CH2CH3)2

O

+

LOW pH HIGH pH poly(acrylic acid) PAA

poly(N,N’-diethylaminoethyl methacrylate)

…consist of ionizable pendants that can accept and donate protons in response to the environmental change in pH.

POLYACIDICS SWELL AT HIGH pH

POLYABASICS SWELL AT LOW pH

pH-RESPONSIVE HYDROGELS

a) Poly(acrylic acid) PAAc, b) Poly(methacrylic acid) (PMAAc), c) Poly(2-ethyl acrylic acid) (PEAAc), d) Poly(2-propyl acrylic acid) (PPAAc)

pH-responsive polyacids

pH-responsive polybases

a) poly(N,N’-dimethyl aminoethyl methacrylate) (PDMAEMA), b) poly (N,N’-diethyl aminoethyl methacrylate) (PDEAEMA), c) poly(4 or 2-vinylpyridine) (PVP), d) poly(vinyl imidazole )

O

CH2OH

H

H

H

OH

NHCOCH3

HH

O

O

CH2OH

H

H

H

OH

NHCOCH3

HH

O

n

GlcNAc

O

CH2OH

H

H

H

OH

NH2

HH

O

O

CH2OH

H

H

H

OH

NH2

HH

O

n

CHITIN

Deacetylation

CHITOSAN

GlcNAc

+ CH3COONa

NaOH, temperature

pH-RESPONSIVE HYDROGELS

OH

H

HO

OH

H

H

C

O

H

H

O

H

HO

OH

H

C

H

OO

D-Mannuronic acid (M) residue

H

O

OH

O

OH

O

H

HC

H

HOH

H

O

HOH

OHH

O

H

H

C

O OH

OH

O

HOH

O

L-Guluronic acid (G) residue

n

1

1

1

1

4

4

4

4Alginic acid: source – e.g. brown algae (Phaeophyceae, mainly Laminaria)

DESIGNS FOR CRITICAL pH

Source: Adv Drug Del Rev 58, 1655 (2006)

DESIGNS FOR CRITICAL pH

Hydrogels of 1) PAAc and 2), 3), 4) copolymers with different fractions of acrylic acid and a n-alkyl acrylate (n=8)

Effect of the fraction of hydrophobic groups

Macromolecules 30, 8278 (1997)

T-RESPONSIVE HYDROGELS FOR TE APPLICATIONS

Laminin functionalized methylcellulose hydrogels for neural TE

LCST of the MC hydrogels

MC- methylcellulose OXMC- oxidized methylcellulose OXMC-LN: Laminin functionalized methycellulose

The LCST of the LN-functionalized MC gels do not exceed 37 ºC

LN-functionalized MC gels support

neuronal cell attachment

Attachment of cortical neurons to MC hydrogels

JBMRA 77, 718 (2006)

T-RESPONSIVE HYDROGELS FOR TE APPLICATIONS

Laminin functionalized methylcellulose hydrogels for neural TE

A significant increase in the

viability of cortical neurons

within the LN-functionalized MC

scaffolds when compared with

the unmodified MC scaffolds was

found Scale bar= 50 mm

Confocal Laser Microscopy Results

JBMRA 77, 718 (2006)

Chitosan--glycerophosphate salt formulations with bioactive glass (BG) nanoparticles

T-RESPONSIVE HYDROGELS FOR TE APPLICATIONS

a) 0% BG, b) 10% BG, c) 30% BG and d) 40% BG

Gelation point decreases with increasing BG content (from 41 ºC to 36.9 ºC) and gelation time less than 5 min

TE Applications: as injectable scaffolds and to deliver cells/bioactive agents

Daniela Couto, Zhongkui Hong, João Mano Acta Biomaterialia 2009

Enzyme-sensitive Hydrogels Enzymes= matrix metalloproteinases (MMPs)

AIM To guide tissue regeneration by mimicking the MMP-mediated

invasion of the natural provisional matrix Hubbell et al, PNAS 100 5413 (2003)

Polymer-Peptide Bioconjugates

ENZYME-RESPONSIVE HYDROGELS FOR TE APPLICATIONS

Hubbell et al, PNAS 100 5413 (2003)

A) Fibroblasts were demonstrated to proteolitically invade these networks, a process

that depends on MMP substrate activity, adhesion-ligand concentration and network

crosslinking density. Invading fibroblasts typically assumed spindle-like shapes - A) and

B) (A-image obtained by phase contrast microscopy and B-CLM image).

ENZYME-RESPONSIVE HYDROGELS FOR TE APPLICATIONS

Preparation of alginate+PNIPAAM semi-IPN Beads Preparation of alginate+PNIPAAM semi-IPN Beads with a Polyelectrolyte Complex Coating

Constant stirring

CaCl2

PNIPAAM +Alginate

Constant stirring

PNIPAAM +Alginate

CaCl2

Chitosan

Alginate+CaCl2

PNIPAAM-Alginate Core

Chitosan-Alginate complex coating

Uncoated beads

Coated beads

SEM

pH- and thermo-responsive semi-IPN Beads for Drug Release

water uptake of the uncoated beads : effect of temperature and pH

0

10

20

30

40

50

60

Sw

elli

ng r

atio (

%)

A B C D E

at pH2.1, 37oC

at pH7.4, 37oC

at pH7.4, 25oC

Increasing PNIPAAm content

SWELLING RESULTS - UNCOATED BEADS

Jun Shi, Natália Alves, João Mano, Macromol. Biosc. 2006

0 100 200 300 400

0

20

40

60

80

100

Sample C

Dru

g r

ele

ase

(%

)

time (min)

pH=2.1

pH=7.4

0 100 200 300 400

0

20

40

60

80

100

Sample B

Dru

g r

ele

ase

(%

)

time (min)

25 ºC

37 ºC

pH=7.4

37 ºC

EFFECT OF pH EFFECT OF TEMPERATURE

Jun Shi, Natália Alves, João Mano, Macromol. Biosc. 2006

DRUG RELEASE RESULTS - UNCOATED BEADS

T pHpH

PNIPAAMalginate

Ca2+

drug

Drug= indomethacin

0

10

20

30

40

50

60

Sw

elli

ng

Ra

tio

(%

)

A B C

25oC, pH7.4

37oC, pH7.4

25oC, pH2.1

37oC, pH2.1

SWELLING/DRUG RELEASE RESULTS - COATED BEADS

0 100 200 300 400 500

0

20

40

60

80

100

Time (min)

Dru

g r

ele

ase(%

)

Sample A

Sample B

Sample C

pH 7.4, 37oC

Water uptake– effect of temperature and pH

Drug= Indomethacin

Drug release - effect of coating

Jun Shi, Natália Alves, João Mano, JBMRB 2008

A- uncoated bead B and C - coated beads

DRUG RELEASE RESULTS – COATED BEADS

Release of Indomethacin previously incorporated into the beads

EFFECT OF pH

0 100 200 300 400 500

0

20

40

60

80

100

37 oC

Time (min)

Dru

g r

ele

ase (

%)

pH 7.4, Sample B

pH 2.1, Sample A

pH 2.1, Sample B

pH 2.1, Sample C

0 100 200 300 400 500

0

20

40

60

80

100

Time (min)

Dru

g r

ele

ase

(%)

37 oC, Sample B

25 oC, Sample A

25 oC, Sample B

25 oC, Sample C

pH 7.4

EFFECT OF TEMPERATURE

Jun Shi, Natalia Alves, João Mano, JBMRB 2008

SMART BIOACTIVE SURFACES: T-RESPONSIVE

PLLA+BG

SBF NIPAAm solvent

casting

plasma activation (10 min, 30W)

Immersion in NIPAAm sol. (8h, 40 ºC) Immersion in

SBF (2 weeks)

bioactivity tests

Hypothesis: the change in roughness and surface energy across the LCST of surface modified substrates with PNIPAAM could influence the ability for apatite formation in SBF.

EXPERIMENTAL PROTOCOL

PNIPAAm grafting

PLLA+BG substrate grafted with PNIPAAM

50 mm 50 mm

10 mm 10 mm

50 mm 50 mm

50 mm 50 mm

25 ºC 37 ºC

PNIPAAM grafted PLLA film with 20% of BG

T-RESPONSIVE SURFACES: BIOACTIVITY RESULTS

SEM images of control samples after immersion in SBF during 2 weeks

unmodified film with 10% of BG

PLLA/10%BG film after plasma modification

PNIPAAm grafted PLLA film.

[too much BG]

[sample with 10% BG not bioactive]

[plasma modification does not promote bioactivity]

[PNIPAAm modification without BG does not promote bioactivity]

25 ºC

10 mm

37 ºC

50 mm 50 mm

50 mm

PLLA+10%BG+PNIPAAm modification n

o s

urf

. mo

d.

PN

IPA

Am

mo

dif.

CA=51.92.4º CA= 58.82.4º

T-RESPONSIVE SURFACES: BIOACTIVITY RESULTS

Jun Shi, Natalia Alves, João Mano, Adv Funct Mater 2007

2000 2500 3000 3500 4000

10

20

30

40

50

60

70

80

90

Tra

nsm

itta

nce

wavenumber (cm-1)

PLLA

chitosan

grafted PLLA

82.4 ± 1.3 81.7 ± 0.5

82.6 ± 2.7 81.5 ± 2.5

67.6 ± 2.3 88.9 ± 4.1

non modif. PLLA

non modif. PLLA+30%BG

CHT modif. PLLA+30%BG

pH 7.4 pH 5.4

PLLA+BG

chitosan

solvent casting

Plasma activation (60 sec, 32W, Ar) Air for 30 min 1% cht sol.

2x

grafted chitosan

FTIR

SMART BIOACTIVE SURFACES: pH-RESPONSIVE

50 mm 50 mm

10 mm 10 mm

Bioactivity results – PLLA+30%BG : no surface modification

pH 7.4 pH 5.4

7 d

ays

in S

BF

21

day

s in

SB

F

Bioactivity results – PLLA+30%BG+chitosan modification

50 mm 10 mm

pH 5.4 pH 7.4

pH 5.4

50 mm 10 mm

7 d

ays

in S

BF

21

day

s in

SB

F

Catarina Dias, João Mano, Natália Alves J Mater Chem 2008

50 mm

polycarbonate mask

PLLA/10%BG composite

PATTERNING THE APATITE FORMATION

PATTERNING THE APATITE FORMATION

plasma activation

activated surface

PATTERNING THE APATITE FORMATION

polymerization

SBF

PATTERNING THE APATITE FORMATION

500μm500μm

50μm50μm50μm

PATTERNED MODIFIED SURFACES: BIOACTIVITY RESULTS

Jun Shi, Natália Alves, João Mano, Adv Funct Mater 2007

T=37 ºC, immersion time in SBF= 2 weeks

SMART BIOACTIVE SURFACES

Surface modification with pH- and T-responsive polymers can be used to

obtain a smart surface where the ability for apatite formation can be switched

depending on temperature or on pH. We thus propose the idea of “smart” or

”stimuli-responsive” mineralization. The concept could be extended to other

external stimuli and other kind of mineral deposited.

By patterning the modification of the surface, it is possible to combine

stimuli and spatial control of the biomimetic apatite formation.

Films of Chitosan-graft-NIPAAm (GRAFT) and alginate (ALG)

(a) chitosan, (b) PNIPAAm (c) GRAFT.

Dual-responsive Coatings

FTIR

T<LCST T>LCST

GRAFT

pH 5.5, 0.15 M NaCl

QCM-D- GRAFT/ALG)5GRAFT films

In-situ detachment of SaOs-2 cell sheets after 10 min (A) and 15 min (B) of incubation at 4 ºC from (GRAFT/ALG)5GRAFT films Microscope images of a

detached cell sheet re-cultured in a new tissue culture plate on time 0 (C) and after 45 min (D)

Dual-responsive Coatings – Cell behaviour

The developed dual-responsive films could be potentially used for cell sheet engineering:

[Okano] Gabriela Martins, João Mano, Natália Alves, Langmuir 2011

Mussels are promiscuous fouling organisms and have been shown to attach to virtually all types of inorganic and organic surfaces (including PTFE – Fig. A) Clues to mussels’ adhesive versatility may lie in the amino acid composition of proteins found near the plaque-substrate interface (Fig. B to D), which are rich in 3,4-dihydroxy-L-phenylalanine (DOPA) and lysine amino acids. DOPA contains the ortho-dihydroxyphenyl –catechol- functional group In addition to participating in reactions leading to bulk solidification of the adhesive, DOPA forms strong covalent and noncovalent interactions with substrates

H. Lee (…) P.B. Messersmith, Science ‘07

Bioinspired adhesive coatings

Hyaluronic Acid (HA) HA-DN

EDC Dopamine

PBS pH 5.5

2 hr

Hypothesis

Chitosan and hyaluronic acid (HA) are very popular biomaterials, and have been often combined using the LbL technique to form nanostructured coatings HA can be modified using the carbodiimide chemistry in order to incorporate dopamine domains that present catechol groups – HA-DN conjugate We hypothesize that the resulting conjugate can form LbL films with chitosan and produce surfaces with distinct properties with respect with the conventional CHT/HA ones, including improved adhesive properties and enhanced biological performance

Mechanical tests of CHT/HA-DN films: adhesion

12,7mm

LbL film

127+L mm

177,8+L mm

25,4mm 25,4mm

25,4mm

2 mm

Area in

test

grips

standard procedure ASTM D1002 – tensile shear strength

The adhesion strength of the CHT/HA-DN film is significantly higher when compared with the control film

Biological tests : direct contact tests using L929 cells

Biological tests : direct contact tests using L929 cells

HA-DN/CHT films presented enhanced cell response

These films could be potentially used as adhesive coatings of distinct implants in order to improve both cell response and adhesion strength in a simple and versatile way.

Isabel Neto,…, Natália Alves, João Mano, Small 2014