Modification of Collagenous Surfaces by Grafting Polymeric Side

8 Modification of Collagenous Surfaces by Grafting Polymeric Side Chains to

Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

Collagen and Soft and Hard Tissues G. M. BRAUER and D. J. TERMINI Dental Research Section, National Bureau of Standards, Washington, D. C. 20234

Collagen, soft tissue, and bone can be modified at 37°C by allowing them to react with acrylic, methacrylic, or vinyl monomers usingcericions, persulfate-bisulfite or comonomers forming donor-acceptor complexes as initiators. The polymeric methacrylate side chain is chemically attached to collagen; similar bonding may occur on reaction with other monomers. With rat skin, the reaction takes place mainly at the surface whereas a higher yield of more homogeneous product is formed on grafting onto collagen. Grafting onto bone is best accomplished with persulfate-bisulfite initiator. Modification of the collagenous surface is indicated by changes in wettability, decreased water sorption, and improved resistance to mold growth; e.g., hydrophobic, oleophobic surfaces are obtained with fluorinated monomers. The modified surfaces could be useful as adhesion-promoting liners for restorative materials.

Grafting

of p o l y m e r i c s i d e chains offers

a n a t t r a c t i v e t e c h n i q u e for

a l t e r i n g the p r o p e r t i e s , e s p e c i a l l y the surface characteristics of the substrate.

M a n y studies h a v e d e a l t w i t h g r a f t i n g onto c e l l u l o s i c (1,

a n d p r o t e i n a c e o u s m a t e r i a l s s u c h as w o o l , silk, a n d c o l l a g e n ( 3 ) .

2)

Com-

p a r a t i v e l y little i n f o r m a t i o n has b e e n r e p o r t e d r e g a r d i n g the c h e m i c a l l y i n i t i a t e d g r a f t i n g of m o n o m e r s onto c o l l a g e n . G r a f t i n g onto collagenous surfaces, e s p e c i a l l y in vivo g r a f t i n g onto soft a n d h a r d tissues t o f o r m c o v a l e n t b o n d s b e t w e e n the collagenous m a t e r i a l a n d t h e p o l y m e r i c side c h a i n , is a n effective means to o b t a i n c h e m i c a l a d h e s i o n a n d to i m p r o v e 175 In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

176

APPLIED CHEMISTRY AT PROTEIN

t h e p h y s i c o - c h e m i c a l p r o p e r t i e s of the substrate.

INTERFACES

Such techniques, if

successful u n d e r c l i n i c a l c o n d i t i o n s , m i g h t find a p p l i c a t i o n s as soft tissue or b o n e cements i n s u r g i c a l p r o c e d u r e s or i n i m p r o v i n g a d h e s i o n of d e n t a l restoratives to d e n t i n a l surfaces.

O t h e r characteristics that c o u l d p r e -

s u m a b l y b e i m p r o v e d b y s u c h treatments are greater resistance of teeth to caries, i n c r e a s e d resistance of b o n e a n d s k i n to f u n g a l a n d b a c t e r i a l diseases, a n d i n c r e a s e d p r o t e c t i o n i n p h o t o c h e m i c a l reactions. F u r t h e r more, grafting procedures

h a v e i n d i c a t e d u p g r a d i n g c e r t a i n properties

of leather s u c h as w a t e r p e n e t r a t i o n a n d a b r a s i o n resistance

(3-13).

T h i s p a p e r s u m m a r i z e s c h e m i c a l g r a f t i n g t e c h n i q u e s e x p l o r e d i n this


l a b o r a t o r y that h a v e p o t e n t i a l b i o m e d i c a l a p p l i c a t i o n . T h e s e reactions, i n i t i a t e d b y eerie ions, p e r s u l f a t e - b i s u l f i t e r e d o x systems, or t h e presence of c o m o n o m e r s f o r m i n g donor—acceptor

complexes, w e r e c a r r i e d out i n

a n aqueous e n v i r o n m e n t u n d e r c o n d i t i o n s w h i c h , w i t h s u i t a b l e m o d i f i c a tions, m i g h t b e t o l e r a t e d in vivo.

G r a f t i n g onto tissue surfaces b y means

of i o n i z i n g r a d i a t i o n w i l l not b e discussed since t e c h n i q u e s for a v o i d i n g u n d e s i r a b l e side reactions h a v e not yet b e e n Grafting

onto

developed.

Collagen

D u r i n g the past f e w years, g r a f t i n g to c o l l a g e n u s i n g a v a r i e t y of initiators has b e e n r e p o r t e d .

R a o a n d co-workers g r a f t e d m e t h y l m e t h a -

c r y l a t e , a c r y l o n i t r i l e , a n d a c r y l a m i d e to c o l l a g e n u s i n g eerie a m m o n i u m nitrate

(CAN)

as i n i t i a t o r

R u s s i a n investigators t r e a t e d

(4-9).

col-

l a g e n p o w d e r w i t h ferrous a m m o n i u m sulfate a n d aqueous solutions or emulsions of v a r i o u s m o n o m e r s a n d h y d r o g e n peroxides u s u a l l y at 6 0 ° 80°C.

B y this p r o c e d u r e

a c r y l a m i d e (15),

g l y c i d y l m e t h a c r y l a t e (18, (21, 22, 23, 24)

they were

v i n y l acetate (16), 19, 20),

a b l e to graft a c r y l o n i t r i l e m e t h a c r y l i c a c i d (17),

and phosphorus-containing

side chains o n t o c o l l a g e n .

(14),

methyl and polymer

B e c a u s e of the e l e v a t e d t e m -

peratures u s e d i n these reactions, d e n a t u r a t i o n of the c o l l a g e n m a y occur. T h e same investigators u s e d m e t a v a n a d i c a c i d (25), p h o s p h a t e (26),

o z o n i z e d c o l l a g e n (27, 28),

manganese

and hydrogen peroxide

pyro(19,

29, 30) to graft m e t h y l m e t h a c r y l a t e onto c o l l a g e n . R e d u c i n g groups p r e s ent i n c o l l a g e n f o r m a redox system o n leather p o w d e r (29).

Acrylonitrile

c a n also b e g r a f t e d onto c o l l a g e n w i t h p o t a s s i u m persulfate as i n i t i a t o r i n t h e presence of a i r , u s i n g t e t r a k i s h y d r o x y m e t h y l p h o s p h o n i u m c h l o r i d e as o x y g e n scavenger (3). other v i n y l m o n o m e r s .

H o w e v e r , this m e t h o d p r o v e d u n s u i t a b l e w i t h G r a f t i n g e t h y l a n d b u t y l a c r y l a t e onto

chrome-

t a n n e d sheepskins a n d k a n g a r o o skins has b e e n m o r e successful u s i n g the p o t a s s i u m persulfate—sodium bisulfite r e d o x system as i n i t i a t o r i n a c a r b o n d i o x i d e atmosphere (10,11,12,

13).

In Applied Chemistry at Protein Interfaces; Baier, R.; Advances in Chemistry; American Chemical Society: Washington, DC, 1975.

8.

BRAUER AND TERMINI

Modification

of Collagenous

177

Surfaces

A graft c o p o l y m e r of c o l l a g e n ( as w e l l as other proteinaceous m a t e r i a l s ) w i t h m e t h y l m e t h a c r y l a t e has b e e n m a d e b y p o l y m e r i z a t i o n i n i t i ated b y tri-n-butylborane (31).

K u d a b a a n d c o - w o r k e r s also m o d i f i e d

c o l l a g e n b y t r e a t i n g i t w i t h e p i c h l o r o h y d r i n (32, 33) or e p o x y resins with B F · E t 0 3

2

as catalyst.

S u c h treatments c o u l d b e

(34)

v a l u a b l e for

i m p r o v i n g c e r t a i n properties of leather, b u t the e x p e r i m e n t a l c o n d i t i o n s p r e c l u d e t h e i r use for c l i n i c a l a p p l i c a t i o n s . U V - i n d u c e d g r a f t i n g onto c o l l a g e n is the most advantageous m e t h o d for c o v e r i n g the substrate w i t h a p o l y m e r i c surface c o a t i n g . T h e l o w e n e r g y r a d i a t i o n does not d e g r a d e the c o l l a g e n to a n y a p p r e c i a b l e extent.


G e n e r a l l y , a - U V sensitive d y e or other p h o t o s e n s i t i z e r is a d d e d to the r e a c t i o n m i x t u r e to i m p r o v e the y i e l d of the c o p o l y m e r i z a t i o n .

This

p r o c e e d s via a free r a d i c a l a b s t r a c t i o n of a h y d r o g e n f o l l o w e d b y s u b sequent p o l y m e r c h a i n g r o w t h f r o m the free r a d i c a l site o n the substrate. T h e extent of g r a f t i n g depends o n the c o n c e n t r a t i o n of the sensitizer, its l i g h t a b s o r p t i o n characteristics, a n d the r a d i a n t energy c o r r e s p o n d i n g to the w a v e l e n g t h at w h i c h the sensitizer absorbs light. A c t i v a t i o n e n e r g y of g r a f t i n g d e p e n d s o n the t y p e of m o n o m e r used. Photosensitizers w h i c h h a v e b e e n u s e d to graft m e t h y l m e t h a c r y l a t e to c o l l a g e n i n c l u d e b e n zene (35, 36), b e n z i l (36, 37), b e n z o i n (38), eosin (37, 40),

a n d i o d o e o s i n (37, 41, 42).

benzophenone

(37, 38,

39),

B e n z i l a n d iodoeosin, w h i c h

a b s o r b strongly a r o u n d 260 n m a n d 546 n m , are m o r e effective

than

eosin a n d b e n z o p h e n o n e .

used

R i b o f l a v i n or

fluorescein

also c a n be

to graft a c r y l a m i d e , Ν,Ν-dimethylacrylamide, N - v i n y l p y r r o l i d o n e , a c r y l i c a c i d , a n d a c r y l o n i t r i l e onto c o l l a g e n (43, 44).

T h e q u a n t i t y of g r a f t e d

p o l y m e r i n t r o d u c e d i n t o the substrate d e p e n d s o n the c h e m i c a l n a t u r e and

c o n c e n t r a t i o n of m o n o m e r ,

photosensitizer, a n d

fibrous

substrate.

S o m e h o m o p o l y m e r i z a t i o n of m o n o m e r also takes place. I n the presence of o x y g e n , a n i n d u c t i o n p e r i o d occurs, b u t o x y g e n r e m o v a l retards the photopolymerizations. In

o u r studies w i t h c o l l a g e n , C A N w a s u s e d as a n i n i t i a t o r since

free r a d i c a l s are f o r m e d o n the side chains of the substrate, a n d thus a h i g h g r a f t i n g efficiency c o m p a r e d w i t h other r e d o x systems c a n be ex pected.

T h e f o r m a t i o n of h o m o p o l y m e r is k e p t to a m i n i m u m a n d there

is little d e g r a d a t i o n of t h e b a c k b o n e substrate. F u r t h e r m o r e , this system makes i t possible to c o n d u c t the r e a c t i o n u n d e r r e l a t i v e l y m i l d c o n d i tions at 37 ° C . T h e i n i t i a l objective of this i n v e s t i g a t i o n w a s to d e t e r m i n e w h i c h a c r y l i c or v i n y l m o n o m e r s c o n t a i n i n g a v a r i e t y of f u n c t i o n a l groups w o u l d react w i t h the c o l l a g e n substrate to y i e l d a graft c o p o l y m e r

(45).

A second objective w a s to d e t e r m i n e the o p t i m u m c o n d i t i o n s for g r a f t i n g onto soft a n d h a r d tissues. I n most of the experiments u s i n g c o l l a g e n , r a t s k i n , or b o n e , the f o l l o w i n g p r o c e d u r e was u s e d : a p p r o x i m a t e l y 1 g of


178

APPLIED CHEMISTRY

m a t e r i a l w a s s t i r r e d f o r 1 h r i n 50 m l of a 2%

AT PROTEIN

INTERFACES

aqueous d i o c t y l s o d i u m

sulfosuccinate s o l u t i o n . O n e m l 0 . 0 5 M C A N i n I N H N 0

was

3

added;

the m i x t u r e w a s d e a e r a t e d w i t h n i t r o g e n for 15 m i n a n d 2.25 m l of monomer was added. T h e reaction was continued i n an inert atmosphere at 37 ° C .

Homopolymer

was removed

b y thorough extraction w i t h

a

s u i t a b l e solvent. E f f i c i e n t r e m o v a l of o x y g e n is r e q u i r e d to o b t a i n o p t i m u m y i e l d s . E x p o s u r e to s w e l l i n g agents s u c h as 5 %

aqueous z i n c c h l o r i d e or potas-

s i u m t h i o c y a n a t e p r i o r to g r a f t i n g onto c o l l a g e n d i d not increase the y i e l d . H o w e v e r , t h e presence of a w e t t i n g agent p r o v e d b e n e f i c i a l .


Results of g r a f t i n g v a r i o u s m o n o m e r s onto p o w d e r e d s t e e r h i d e c o l l a g e n are g i v e n i n T a b l e I.

A s w a s e s t a b l i s h e d i n the p r e l i m i n a r y ex-

p e r i m e n t s , results d e p e n d o n the p h y s i c a l state ( p o w d e r or

film)

and

p r e t r e a t m e n t of the s p e c i m e n , l e n g t h of its storage i n w a t e r p r i o r to the r e a c t i o n , presence a n d c o n c e n t r a t i o n of w e t t i n g agent, a n d c o n c e n t r a t i o n a n d p u r i t y of the m o n o m e r .

A c o n s i d e r a b l e w e i g h t increase ( 2 1 % ) w a s

o b t a i n e d w h e n o n l y c o l l a g e n w a s a l l o w e d to r e a c t w i t h n i t r i c a c i d o r C A N s o l u t i o n i n the presence of w e t t i n g agent. Table I .

T h e a n i o n i c d i o c t y l so-

Grafting of Polymeric Side Chains to Collagenous Surfaces Reaction Times: 3 hours; Temperature: 3 7 ° C Avg. Weight Increase,

g/100 g substrate

0

Initiator :

Initiator :

K S Os-NaHSO

CAN Collagen

Monomer C o n t r o l (no m o n o m e r — C A N ) Control (methyl methacrylate, no C A N ) Acrylic acid Acrylates ethyl butyl b u t y l + 1 0 % acrylic acid isodecyl i s o d e c y l ( C 0 atmosphere) 2-ethylhexyl 2,2,2-trifluoroethyl hexafluoroisopropyl 1H,\H, 5 i / - o c t a f l u o r o p e n t y 1 pentadecafluorooctyl cyanoethyl Cellosolve 2 % aq. calcium 2 % a q . zinc Methacrylic acid 6

Ratskin

21

2

2 43

0.3 11

106 110 153

2

134 124 120 117 129 200 11 56

24 22 9 13 16 17 130 16 4

2

Bone -

2

z

Ratskin

Bone

9

—

—

5 -48

— —

-

-10 — — - 3

— 26 40 —

1 -52 4 6

6 3 9

-

-

5

—

9

201(212)' 8 48(45) 78 14 c

11

-38


8.

BRAUER AND TERMINI

Modification T a b l e I.

of Collagenous

Continued

Avg. Weight Increase," Initiator


Methacrylates methyl m e t h y l (no w e t t i n g agent) ethyl isobutyl lauryl 2-chloroethyl 2,2,2-trifluoroethyl hexafluoroisopropyl ΙΗ,ΙΗ, 5H-oc t a f l u o r o p e n t y 1 hydroxyethyl glycidyl i-butylaminoethyl dimethy lami noethy 1 dimethylaminoethyl (acidi fied to p H 2.5) Ethylene dimethacrylatemethyl methacrylate Ethylene dimethacrylate 1,3-Butylene d i m e t h a c r y l a t e Acrylonitrile a-Chloroacrylonitrile V i n y l acetate Styrene Vinyltoluene Divinylbenzene iV-Vinyl-2-pyrrolidone 4-Vinylpyridine D i a l l y l phosphite T r i a l l y l phosphate Butenediol α 6 c

Col lagen

Ratskin

187 51 182 91 148 96 118 134 30 226 239 2 12

14

— — —

Initiator

4

—

24 23

27 0 29 25 18

17

—

-

—

-

— — — — — — — — — — — —

45

—

12 1 10

— — — —

85(38) 138

7

—

— — — — — — — — — — — —

—

-

3

-14

—

1 -10

— —

Bone

—

-4

— — —

— — — — —

3

Ratskin

-5

—

9

16 16 13

1

-10

—

13 15

—

3 1 1

—

11

—

— — — —

2

28

-11 61

4

—

Bone

14 11 12 4 20

34

104 58 74 77 28 33 25

2

16

—

:

K S 0s-NaIIS0

-

—

g/Ί00 g substrate

:

CAN Monomer

179

Surfaces

142(16)

—

49

-

—

5 37

— -

63 9

—

-14

— —

A f t e r extraction of h o m o p o l y m e r w i t h a p p r o p r i a t e solvent. C e r i c a m m o n i u m nitrate. R e a c t i o n t i m e : 15 m i n .

d i u m sulfosuccinate

w e t t i n g agent w h i c h is present

i n a fairly

c o n c e n t r a t i o n ( 2 % ) is strongly s o r b e d b y t h e p o s i t i v e l y c h a r g e d

high

collagen

m o l e c u l e s i n t h e a c i d i c r e a c t i o n m i x t u r e . It is p r o b a b l y i n s o l u b i l i z e d i n the presence of t h e n i t r i c a c i d electrolyte a n d thus is n o t r e m o v e d

by

w a s h i n g the substrate w i t h w a t e r . Increase

i n weight

(after

e x t r a c t i o n of

soluble homopolymer)

in

excess of the increase i n w e i g h t o b t a i n e d i n the absence of m o n o m e r w a s


180

APPLIED CHEMISTRY AT PROTEIN

INTERFACES

u s e d as the c r i t e r i o n for successful g r a f t i n g . Y i e l d ( b a s e d o n

monomer

a d d e d a n d a s s u m i n g t h a t the w e i g h t increase after e x t r a c t i o n results solely f r o m graft p o l y m e r f o r m a t i o n ) v a r i e d w i d e l y , b u t it w a s highest for acrylates a n d m e t h a c r y l a t e s . Y i e l d s d i d not c h a n g e greatly w i t h the h i g h e r homologues. e r a l l y i n the 4 0 - 9 0 %

Y i e l d s for these t w o h o m o l o g o u s series w e r e r a n g e ( 9 0 - 2 3 9 % increase i n w e i g h t ) .

gen-

Monomers

c o n t a i n i n g h y d r o x y l or g l y c i d y l groups s u c h as h y d r o x y e t h y l or g l y c i d y l m e t h a c r y l a t e w e r e c o n v e r t e d n e a r l y q u a n t i t a t i v e l y to the p o l y m e r i c f o r m . W i t h a c r y l i c a n d m e t h a c r y l i c a c i d , the y i e l d s w e r e c o n s i d e r a b l y l o w e r t h a n those o b t a i n e d w i t h t h e i r esters. W i t h m o n o m e r s c o n t a i n i n g b a s i c g r o u p s , no a p p a r e n t g r a f t i n g took p l a c e . H o w e v e r , some g r a f t i n g o c c u r r e d Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

w h e n d i ( m e t h y l a m i n o e t h y l m e t h a c r y l a t e ) h y d r o c h l o r i d e was a l l o w e d to react i n an acid environment.

F l u o r i n a t e d acrylates a n d m e t h a c r y l a t e s

also g r a f t e d onto the c o l l a g e n substrate, g e n e r a l l y w i t h g o o d y i e l d s . Graft

copolymerization

with

monomers

containing

2-chloroethyl,

h y d r o x y e t h y l , or g l y c i d y l groups leaves r e s i d u a l p o t e n t i a l r e a c t i o n centers for f u r t h e r c h e m i c a l m o d i f i c a t i o n of the p r o d u c t . G r a f t c o p o l y m e r s w i t h e t h y l e n e or butylène d i m e t h a c r y l a t e are p r o b a b l y c r o s s l i n k e d a n d s h o u l d s h o w r e d u c e d s o l u b i l i t y a n d i n c r e a s e d c h e m i c a l resistance. M o s t v i n y l m o n o m e r s other t h a n those c o n t a i n i n g a c r y l i c or m e t h a c r y l i c groups w e r e not as r e a d i l y g r a f t e d onto c o l l a g e n i n a n aqueous e n v i ronment.

L i t t l e or no w e i g h t increase c o m p a r e d w i t h the C A N b l a n k

took p l a c e w i t h 4 - v i n y l p y r i d i n e , v i n y l t o l u e n e , t r i a l l y l p h o s p h a t e , or b u t e n e d i o l . G r a f t i n g c o n d i t i o n s for these m o n o m e r s are m o r e s u i t a b l e w h e n the r e a c t i o n is c o n d u c t e d i n a p p r o p r i a t e non-aqueous solvents. T h e presence of p o l y m e r o n the c o l l a g e n w a s c o n f i r m e d f r o m spectra o b t a i n e d after r e m o v a l of s o l u b l e h o m o p o l y m e r . noncollagenous

IR

A p p e a r a n c e of

b a n d s w a s most p r o n o u n c e d for c o l l a g e n t r e a t e d w i t h

the l o w e r h o m o l o g u e s of the a c r y l i c or m e t h a c r y l i c esters.

I n general,

collagenous p r o d u c t s that i n c r e a s e d m o r e t h a n 2 5 % i n w e i g h t h a d m o r e d i s t i n c t i v e spectra possessing s h a r p e r peaks a n d a d d i t i o n a l a b s o r p t i o n b a n d s w h e n c o m p a r e d w i t h the o r i g i n a l c o l l a g e n p o w d e r . F o r e x a m p l e , a l l esters ( a c r y l a t e s , m e t h a c r y l a t e s , acetate) gave the C = 0 a r o u n d 1740 c m

- 1

stretching b a n d

a n d m a n y h a d another strong ester — C — Ο s t r e t c h i n g

b a n d b e t w e e n 1140 a n d 1160 c m " . 1

I n one series of experiments, c o l l a g e n films (0.0013 c m a n d 0.005 c m t h i c k ) m a d e f r o m c o l l a g e n fibrils of a b o u t 9 9 % p u r i t y w e r e s u b s t i t u t e d for the c o l l a g e n p o w d e r s i n the s t a n d a r d g r a f t i n g e x p e r i m e n t . A l t h o u g h i t was not p o s s i b l e to measure w e i g h t increase i n the t h i n n e r film s p e c i mens, t h e I R spectra of the treated a n d solvent-extracted films i n d i c a t e d that p o l y m e r was p r o b a b l y g r a f t e d onto the substrate. Under

the

experimental conditions,

surface

t h a n 30 m i n after a d d i t i o n of the m o n o m e r .

g r a f t i n g occurs

less

T h u s , after a 3 0 - m i n reac-


8.

BRAUER AND TERMINI

Modification

tion time, weight increased

of Collagenous

181

Surfaces

(after r e m o v a l of h o m o p o l y m e r s )

100%

w h e n m e t h y l m e t h a c r y l a t e a n d i s o d e c y l a c r y l a t e w e r e g r a f t e d onto c o l lagen powder.

S i m i l a r l y , the increase i n w e i g h t o n g r a f t i n g g l y c i d y l

m e t h a c r y l a t e onto 0.005-cm c o l l a g e n films d e m o n s t r a t e d c o n c l u s i v e l y that m e a s u r a b l e amounts of surface graft w e r e f o r m e d w i t h i n 30 m i n s . Spontaneous

grafting under

high

swelling conditions

has

been

a c h i e v e d w i t h t h e w o o l - e t h y l a c r y l a t e system w i t h o u t u s i n g a n y of t h e n o r m a l means of i n i t i a t i n g g r a f t i n g reactions (46).

T h i s r e a c t i o n is p r e -

s u m e d to b e i n i t i a t e d b y t h e free r a d i c a l f o r m a t i o n as a r e s u l t of t h e strong anisotropic swelling brought about initially b y water.

W i t h this

mechanc—chemical technique, polystyrene a n d P M M A could be grafted Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

to c o l l a g e n (47)

i n states t h a t w e r e not e x t r a c t e d b y b o i l i n g t o l u e n e ,

c h l o r o f o r m , or acetone. W h e n the r e a c t i o n w a s c o n d u c t e d u n d e r n i t r o g e n for r e l a t i v e l y short p e r i o d s , t h a t i s , u p to 24 h r s , g r a f t i n g d i d n o t take place.

A m o r e d r a s t i c s w e l l i n g r e a c t i o n appears to b e r e q u i r e d w h i c h

w o u l d l i m i t t h e usefulness of this t e c h n i q u e f o r m a n y a p p l i c a t i o n s . Grafting

onto Soft Tissues

S i n c e m a n y m o n o m e r s of v a r y i n g p o l a r i t y c o u l d b e g r a f t e d onto c o l l a g e n , i t is d e s i r a b l e to e x p e r i m e n t w i t h g r a f t i n g onto soft tissue. I f s u c cessful, i t m i g h t b e possible to v a r y w i d e l y a n d thus c o n t r o l surface p r o p e r t i e s of s u c h a substrate. D e f a t t e d r a t skins w e r e l y o p h i l i z e d a n d t h e n g r a f t e d i n t h e presence of C A N (48).

C o n t r o l runs i n d i c a t e d that

a d d i t i o n of w e t t i n g agent a n d C A N i n i t i a t o r o r m o n o m e r results i n n o a p p r e c i a b l e increase i n w e i g h t o f t h e treated r a t s k i n ( T a b l e I ) . A w e i g h t increase after e x t r a c t i o n of s o l u b l e h o m o p o l y m e r w a s o b t a i n e d o n r e a c t i o n of the r a t skins w i t h m o s t m o n o m e r s

investigated

( T a b l e I ) . Y i e l d of side c h a i n p o l y m e r g r a f t e d onto t h e substrate w a s m u c h l o w e r f o r r a t s k i n t h a n f o r c o l l a g e n w i t h most w e i g h t increases being 15-25%

( a v e r a g e of t w o o r m o r e r u n s ) . T h e h i g h l y

fluorinated

p e n t a d e c a f l u o r o c t y l a c r y l a t e y i e l d e d b y f a r t h e largest a m o u n t of i n s o l u b l e p o l y m e r ( 1 3 0 % w e i g h t i n c r e a s e ) . O n t h e other h a n d , m o n o m e r s c o n t a i n ing

h y d r o x y l or ethoxy groups d i d n o t f o r m a n y a p p r e c i a b l e a m o u n t of

graft p o l y m e r w i t h r a t s k i n despite t h e fact that these m o n o m e r s

gave

n e a r l y q u a n t i t a t i v e y i e l d s of p o l y m e r w i t h steerhide c o l l a g e n . W i t h m o n o mers c o n t a i n i n g b a s i c groups, o n l y a s m a l l w e i g h t increase w a s o b s e r v e d even w h e n these m o n o m e r s w e r e a l l o w e d to react after c o m p l e t e n e u t r a l i z a t i o n of t h e b a s i c groups.

A p p a r e n t grafting took place not only w i t h

a c r y l a t e o r m e t h a c r y l a t e m o n o m e r s b u t also w i t h other v i n y l

monomers

s u c h as v i n y l acetate a n d styrene. R e a c t i o n o f d i a l l y l p h o s p h i t e o r t r i a l l y l p h o s p h a t e w i t h s k i n p r o d u c e d m o d i f i e d surfaces c o n t a i n i n g p h o s p h i t e or p h o s p h a t e groups.

A l l f l u o r i n a t e d acrylates a n d m e t h a c r y l a t e s c o u l d


182

APPLIED CHEMISTRY

AT PROTEIN

INTERFACES

b e g r a f t e d onto the rat s k i n substrate. T r e a t m e n t of r a t s k i n w i t h e t h y l e n e d i m e t h a c r y l a t e also r e s u l t e d i n a n increase i n w e i g h t .

No

appreciable

c h a n g e i n y i e l d o c c u r r e d w h e n c a r b o n d i o x i d e w a s s u b s t i t u t e d for n i t r o g e n to deaerate the r e a c t i o n m i x t u r e . R e d u c i n g the r e a c t i o n t i m e b e l o w 3 hrs decreased the a m o u n t polymer formed on rat skin.

of

H o w e v e r , w i t h a r e a c t i o n t i m e as l o w as

20 m i n , significant w e i g h t increases w e r e r e c o r d e d w i t h s u c h d i v e r s e m o n o m e r s as m e t h y l m e t h a c r y l a t e , i s o d e c y l a c r y l a t e , a n d g l y c i d y l m e t h a crylate. H e n c e , r e l a t i v e l y short r e a c t i o n times m a y be sufficient to m o d i f y significantly c h e m i c a l a n d b i o l o g i c a l characteristics of soft tissue surfaces.


A s m a y b e e x p e c t e d for p o l y m e r i z a t i o n reactions of this t y p e , the use of rat skins as substrates decreased the r e p r o d u c i b i l i t y of the p o l y m e r yield.

T h e coefficients

of v a r i a t i o n f o r the w e i g h t increases

averaged

2 8 % a n d w e r e a b o v e 5 0 % for a f e w m o n o m e r s , g e n e r a l l y w h e n t h e y i e l d of p o l y m e r f o r m e d w a s l o w .

T h e l o w e r graft p o l y m e r y i e l d o b t a i n e d

w i t h r a t s k i n c o m p a r e d w i t h steerhide c o l l a g e n m a y b e a t t r i b u t e d to ( a ) the different c h e m i c a l c o m p o s i t i o n ( s u c h as k e r a t i n ) of the e p i d e r m i s , a n d ( b ) the c o h e r e n t n a t u r e of the r a t s k i n p r o v i d i n g a m u c h s m a l l e r surface area t h a n the c o l l a g e n p o w d e r .

T h u s , t h e n u m b e r of sites

for g r a f t i n g is g r e a t l y r e d u c e d .

F u r t h e r m o r e , w e t t i n g agent, i n i t i a t o r ,

accessible

a n d m o n o m e r diffuse o n l y s l o w l y i n t o the i n t e r i o r of the substrate.

With

t h e t h i c k e r rat s k i n specimens, e q u i l i b r i u m c o n d i t i o n s m a y not b e r e a c h e d readily.

W i t h rat s k i n , the g r a f t i n g takes p l a c e m a i n l y at the surface,

whereas a m o r e h o m o g e n o u s p r o d u c t is f o r m e d b y g r a f t i n g t h r o u g h o u t the collagen

powder.

P e r s u l f a t e - b i s u l f i t e - i n i t i a t e d graft p o l y m e r i z a t i o n w a s a c c o m p l i s h e d ( T a b l e I ) u s i n g the p r o c e d u r e of F e a i r h e l l e r et al. (11).

Lyophilized

rat skins w e r e soaked i n 100 m l of a 0 . 4 % aqueous s o l u t i o n of a n o c t y l p h e n y l e t h e r of groups.

polyethylene

glycol containing 9-10

ethylene

A f t e r 1 h r , 0.4 g p o t a s s i u m p e r s u l f a t e a n d 0.135

bisulfite w e r e a d d e d , a n d C 0

2

g

oxide sodium

w a s passed t h r o u g h the s o l u t i o n before

5 m l of m o n o m e r w e r e a d d e d . a c c e p t o r m o n o m e r (A) -+- L e w i s A c i d ( Z n C l ) 2

(methacrylate, maleic anhydride)

wZnCl

2

+

^

^

adduct ( A — Z n C l ) 2

+ donor monomer (styrene)

[donor-acceptor] < x

u s u a l l y 1:1 alternating copolymer

[donor

acceptor · Z n C l ] 2

complex


8.

BRAUER AND TERMINI

Modification

of Collagenous

183

Surfaces

Y i e l d s w i t h m e t h y l m e t h a c r y l a t e w e r e s o m e w h a t l a r g e r t h a n those o b t a i n e d i n the c o m p a r a b l e C A N - i n i t i a t e d p o l y m e r i z a t i o n . E v e n greater increases i n w e i g h t w e r e o b t a i n e d w i t h b u t y l a c r y l a t e , e s p e c i a l l y

on

a d d i t i o n of 1 0 % a c r y l i c a c i d b o t h for 3-hr a n d 2 0 - m i n r e a c t i o n times. Electron-acceptor monomers

s u c h as acrylates, m e t h a c r y l a t e s , a n d

a c r y l o n i t r i l e b e c o m e stronger acceptors o n c o m p l e x i n g w i t h L e w i s acids s u c h as m e t a l h a l i d e s . I n t e r a c t i o n of this c o m p l e x w i t h a s t r o n g e l e c t r o n d o n o r m o n o m e r , e.g., styrene, leads to the f o r m a t i o n of a charge-transfer ( donor—acceptor )

complex.

This complex

radical-initiated polymerization, propagating

undergoes

spontaneous

as a m o n o m e r i c

or

u n i t to

y i e l d e q u i m o l a r a l t e r n a t i n g c o p o l y m e r s , i r r e s p e c t i v e of the i n i t i a l m o n o Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

m e r c o m p o s i t i o n (49, 50, 51, 52).

A r a d i c a l i n i t i a t o r s u c h as C A N causes

a c o m p e t i n g r a d i c a l - i n i t i a t e d h o m o p o l y m e r i z a t i o n of the charge-transfer c o m p l e x c o n c u r r e n t l y w i t h the spontaneous r e a c t i o n . D o n o r - a c c e p t o r p o l y m e r i z a t i o n takes p l a c e o n r e a c t i n g r a t s k i n w i t h e q u i m o l a r amounts of m e t h a c r y l a t e ( o r i s o d e c y l a c r y l a t e ) , z i n c c h l o r i d e , and

styrene e v e n for as short a p e r i o d as 20 m i n s . W i t h a l a r g e excess of

z i n c c h l o r i d e , p o l y m e r f o r m a t i o n o n c o l l a g e n takes p l a c e w i t h t h e m a l e i c a n h y d r i d e - s t y r e n e c o m o n o m e r system.

I n the reactions w h e r e

donor-

acceptor p o l y m e r i z a t i o n o c c u r r e d , y i e l d s w e r e of the same o r d e r as those o b t a i n e d solely w i t h C A N i n i t i a t o r . T h u s the g r a f t i n g r e a c t i o n onto soft tissues is h i g h l y versatile. It takes p l a c e w i t h m a n y m o n o m e r s a n d different i n i t i a t o r systems s u c h as C A N , persulfate-bisulfite, and donor-acceptor

monomers.

A l t h o u g h the p o l y -

m e r i z a t i o n is i n h i b i t e d b y o x y g e n a n d thus a i r , i t is c o n d u c t e d r e a d i l y i n a n i t r o g e n or c a r b o n d i o x i d e atmosphere. Grafting

onto Hard

Tissue

Bone. M o n o m e r s w e r e also g r a f t e d onto b o n e . P o w d e r e d b o n e m a r r o w w a s p u l v e r i z e d i n a W i l e y m i l l i n the presence of d r y i c e a n d w a s subjected to the s t a n d a r d g r a f t i n g p r o c e d u r e u s i n g C A N as i n i t i a t o r S i g n i f i c a n t w e i g h t increases of the substrate, before e x t r a c t i o n of p o l y m e r , r a n g i n g u p to 8 0 %

(53). homo-

( a v e r a g e for t w o or m o r e r u n s ) w e r e

t a i n e d w i t h a f e w selected m o n o m e r s .

H o w e v e r , a considerable

ob-

amount

of the a d d e d p o l y m e r was r e m o v e d as h o m o p o l y m e r o n solvent extraction.

C o n s i s t e n t g r a f t i n g was e v i d e n c e d o n l y w i t h m e t h y l m e t h a c r y l a t e ,

g l y c i d y l m e t h a c r y l a t e , a n d 2,2,3-trifluoroethyl a c r y l a t e ( T a b l e

I).

M u c h m o r e successful w a s the r e a c t i o n of v a r i o u s m o n o m e r s

with

bone, u s i n g the p e r s u l f a t e - b i s u l f i t e r e d o x system as i n i t i a t o r i n a c a r b o n d i o x i d e atmosphere. monomers

O v e r 10 a c r y l a t e a n d m e t h a c r y l a t e a n d a f e w v i n y l

p o l y m e r i z e d o n the substrate, some i n n e a r l y q u a n t i t a t i v e

y i e l d . R e a c t i o n times of 15 m i n w e r e sufficient for g r a f t i n g . T h e a m o u n t


184

AT PROTEIN

INTERFACES


APPLIED CHEMISTRY

Figure 1. IR absorption spectra ( KBr pellets): Curve 1, bone; Curve 2, bone to which glycidyl methacrylate had been grafted after extraction of the product with acetone of s o l u b l e h o m o p o l y m e r w a s q u i t e l a r g e for some p r o d u c t s .

However,

w e i g h t increases of o v e r 1 3 8 % w e r e o b t a i n e d (after e x t r a c t i o n ) i n the reaction w i t h cyanoethyl acrylate, g l y c i d y l methacrylate, a n d ethylene dimethacrylate.

Since the chains obtained on polymerization w i t h the

latter t w o m o n o m e r s are l i k e l y to b e h i g h l y c r o s s l i n k e d , h o m o p o l y m e r e x t r a c t i o n is less efficient, a n d i t becomes i n c r e a s i n g l y difficult to ascer-


8.

BRAUER AND TERMINI

Modification

of Collagenous

Surfaces

185

t a i n i f c o v a l e n t b o n d i n g to h a r d tissue o c c u r r e d . N e v e r t h e l e s s , the surface properties of the r e s u l t i n g p r o d u c t s w e r e m o d i f i e d . A n i n t e r e s t i n g r e a c t i o n is that of c a l c i u m or z i n c a c r y l a t e w i t h bone. T h e r e s u l t i n g p r o d u c t s after acetone a n d w a t e r e x t r a c t i o n h a d a 4 0 % and

7 6 % increase i n w e i g h t , r e s p e c t i v e l y .

A p o t e n t i a l graft of this c a l

c i u m - c o n t a i n i n g m o n o m e r onto h i g h l y m i n e r a l i z e d b o n e o r d e n t i n m a y i n c o r p o r a t e c a l c i u m i n defective tissues a n d t h e r e b y i m p r o v e b o n e h e a l i n g . M o n o m e r s w h i c h c o u l d n o t b e g r a f t e d o n t o b o n e w e r e those c o n t a i n i n g basic or a c i d i c side groups. T h e large loss i n w e i g h t o n r e a c t i o n of b o n e w i t h a c r y l i c o n m e t h a c r y l i c a c i d is i n d i c a t i v e of d i s s o l u t i o n of the b o n e u n d e r the e x p e r i m e n t a l c o n d i t i o n s . Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

IR

spectra of the p r o d u c t s i n d i c a t e d t h e presence

m a t e r i a l o n the b o n e ( F i g u r e 1 ) .

of

polymerized

T h u s , for the g l y c i d y l m e t h a c r y l a t e

c o p o l y m e r i n i t i a t e d w i t h C A N , I R spectra s h o w e d the o x i r a n e r i n g of t h e g l y c i d y l g r o u p (899 c m "

1

a n d 843 c m " ) 1

as w e l l as the C = 0

stretching

b a n d at 1730 c m " , a C — Ο s t r e t c h i n g b a n d at 1270 c m " , a n d a n a b s o r p 1

1

t i o n b a n d at 747 c m " w h i c h is c h a r a c t e r i s t i c of the m e t h a c r y l a t e g r o u p . 1

Dentin.

P r e l i m i n a r y studies h a v e b e e n c o n d u c t e d to d e t e r m i n e the

f e a s i b i l i t y of g r a f t i n g o n t o d e n t i n (53). fication

W i t h C A N as i n i t i a t o r , some m o d i

of d e n t i n o c c u r r e d o n treatment w i t h m e t h y l m e t h a c r y l a t e .

other m o n o m e r s , no increase i n w e i g h t was f o u n d .

With

S i n c e d e n t i n is the

most h i g h l y m i n e r a l i z e d collagenous substrate that has b e e n s t u d i e d , its l a c k of r e a c t i v i t y t o w a r d s g r a f t i n g is expected. T h u s , the r e l a t i v e ease of g r a f t i n g m o n o m e r s

onto w a t e r - i n s o l u b l e

collagenous substrates u s i n g C A N as i n i t i a t o r decreases i n the f o l l o w i n g order: p o w d e r e d c o l l a g e n > c o l l a g e n film >

e p i d e r m a l rat s k i n ,

bone > powdered dentin A p p a r e n t g r a f t i n g to d e n t i n a n d other proteinaceous

m a t e r i a l s has

been r e p o r t e d t h r o u g h p o l y m e r i z a t i o n of m e t h y l m e t h a c r y l a t e w i t h t r i - n b u t y l b o r a n e (31, 54, 55, 56, 57). has b e e n

T h e m e c h a n i s m o n the top of p. 186

suggested.

Complexes

of t r i b u t y l b o r a n e w i t h a m m o n i a or p r i m a r y a n d sec

o n d a r y a m i n o groups are m o r e stable i n a i r a n d m a y also b e used. are e a s i l y a c t i v a t e d b y isocyanates a n d a c i d c h l o r i d e s .

They

B o n d formation

of the b o r a n e - c u r e d r e s i n to d e n t i n is e n h a n c e d b y m o i s t u r e , a n d b o n d strength is r e t a i n e d f a i r l y w e l l after w a t e r i m m e r s i o n . C o m m e r c i a l d e n t a l restoratives c o n t a i n i n g b o r a n e i n i t i a t o r s h a v e b e c o m e a v a i l a b l e . p a r e d w i t h other restorative

filling

a d h e s i o n to d e n t i n (58, 59, 60, 61). collagen whereas Therefore,

materials, they showed

Com

improved

B o n d i n g is o n l y to t h e d e n t i n a l

a n y r e t e n t i o n to e n a m e l is of a m e c h a n i c a l n a t u r e .

c a v i t y p r e p a r a t i o n s u s i n g c o n v e n t i o n a l u n d e r c u t s to r e t a i n


186

APPLIED CHEMISTRY

R

3

B - ^ R

2

B O O R

2 R 3

' > B

R'. + — N H — C H — C — N H — C H — C

II

I

II

0

R'

0

Ι CH

3

AT PROTEIN

2R .+R BOBR ,

2

,

+other

2

products.

>—NH—C—C—NH—CH—Ο

II

I

CH 0 3

methvl

J (MMA) —NH—C

I

II

R"

0

methacrylate (MMA)

n

C—NH—CH—Ο

II

I

II

0

R"

0

Ι Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

INTERFACES

CH

3

the d e n t a l restorations m u s t be used. F u r t h e r m o r e , c e r t a i n pretreatments a n d c l e a n s i n g processes of the c a v i t y , as w e l l as a p p l y i n g too viscous a l i q u i d , diminish adhesion. Changes in Properties of Modified Surfaces S u r f a c e b e h a v i o r of s o l i d m a t e r i a l s a n d b i o l o g i c a l tissues d e p e n d s almost solely o n the n a t u r e a n d t h e p a c k i n g d e n s i t y of t h e or exposed atoms a n d f u n c t i o n a l groups.

outermost

E v e n a unimolecular layer

g r a f t e d to a c t i v e sites m a y s i g n i f i c a n t l y change s u c h properties as w a t e r s o r p t i o n , w e t t a b i l i t y , a n d c r i t i c a l surface tension, thus affecting the ease of a d h e s i o n to the surface.

T h e r e l a t i v e l y short r e a c t i o n times f o u n d to

y i e l d s u b s t a n t i a l w e i g h t increases i n the v a r i o u s substrates s h o u l d

be

sufficient to m o d i f y g r e a t l y t h e p h y s i c a l , c h e m i c a l , a n d b i o l o g i c a l c h a r acteristics of the t r e a t e d surfaces. Table II.

Water Sorption of Graft Copolymers Water Sorption at 50% Relative g/100g substrate

Monomer Substrate C o n t r o l (no m o n o m e r — C A N ) Control (methyl methacrvlate, no C A N ) Acrylic acid Acrylates ethyl butyl isodecyl 2-ethylhexyl 2,2,2-trifluoroethyl hexafluoroisopropyl

Collagen

a

Ratskin

a

19.2 11.6

16.8 7.1

17.4 9.4

8.0 8.9

4.6 4.4 4.7 3.8 3.8 4.3

12.1

Humidity, Bone

—

9.4 9.4 7.4

—


8.1

— —

7.9

7.4 6.9 7.5 6.3

— —

8.

BRAUER AND TERMINI

Modification Table II.

187

of Collagenous Surfaces Continued

Water Sorption at 50% Relative g/100g substrate


Monomer

Collagen

0,

A c r y l a t e s (continued) 1H, 1H , 5 # - o c t a f l u o r o p e n t y l pentadecafluorooctyl cyanoethyl Cellosolve 2 % aq. calcium M e t h a c r y l i c acid Methacrylates methyl m e t h y l (no w e t t i n g agent) ethyl isobutyl lauryl 2-chloroethyl 2,2,2-trifluoroethyl hexafluoroisopropyl \H,\H, 5 # - o c t a f l u o r o p e n t y 1 hydroxyethyl glycidyl 2-butylaminoethyl dimethylaminoethyl dimetb^laminoethyl (acidified to p H 2.5) Ethylene dimethacrylate 1,3-Butylene d i m e t h a c r y l a t e Acrylonitrile a-Chloroacrylonitrile V i n y l acetate Styrene Vinyltoluene Divinylbenzene A^-Vinyl-2-pyrrolidone 4-Vinylpyridine D i a l l y l phosphite T r i a l l y l phosphate Butenediol a

6

4.1

—

4.2 5.5 14.2 10.8 5.8 8.7 4.4 6.1 4.2 5.4 4.3 6.0 7.8 7.4 4.4 18.4 15.9 8.1 6.9 9.0 9.3 5.3 7.7 7.8 9.4

—

7.2 16.4 8.3 10.1 9.3

Humidity, Bone

Ratskin

0

0

— —

8.6 3.1 7.4 12.5

2.8 7.0 9.1 9.2

—

14.0 7.9

6.0

— — —

— — —

—

— — — —

8.7

6.7

7.5 8.0 7.8 10.4

4.3 3.0

— — —

—

7.9

8.6 9.7

—

—

—

—

— —

4.5

7.2

5.5

8.5 7.4

5.7

—

—

7.2 8.5

3.8

— — — — —

—

7.4 7.2 9.8

Initiator: Ceric a m m o n i u m nitrate. I n i t i a t o r : P o t a s s i u m p e r s u l f a t e - s o d i u m bisulfite.

T h e appearance

of the p r o d u c t s

of the r e a c t i o n of c o l l a g e n

and

various m o n o m e r s often v a r i e d c o n s i d e r a b l y f r o m the o r i g i n a l c o l l a g e n p o w d e r (45,

48).

W i t h some m o n o m e r s , the graft c o p o l y m e r

of p o w d e r s w h i l e w i t h others mats or films w e r e f o r m e d .

The

consisted modifica-

t i o n of surface properties c o u l d often be d e t e c t e d b y v i s u a l i n s p e c t i o n .


188

APPLIED

CHEMISTRY AT PROTEIN

INTERFACES

T h u s w i t h i s o d e c y l , 2 - e t h y l h e x y l a c r y l a t e , or l a u r y l m e t h a c r y l a t e , r u b b e r y mats w e r e f o r m e d .

S i m i l a r l y , rat skins t r e a t e d w i t h i s o d e c y l or p e n t a -

d e c a f l u o r o o c t y l a c r y l a t e possessed a v e r y t a c k y , r u b b e r y surface d i f f e r i n g s i g n i f i c a n t l y f r o m the o r i g i n a l substrate. W a t e r s o r p t i o n of c o l l a g e n p o w d e r , w h i c h at 5 0 % r e l a t i v e h u m i d i t y w a s 19.2 g H O / 1 0 0 g c o l l a g e n , was a l w a y s l o w e r e d o n t r e a t m e n t of the 2

collagen ( T a b l e I I ) .

T h i s e v e n a p p l i e d for p r o d u c t s c o n t a i n i n g h y d r o -

p h i l i c groups i n the side c h a i n . I n s p e c t i o n of T a b l e s I a n d I I i n d i c a t e s t h a t w a t e r s o r p t i o n d e p e n d s not o n l y o n the presence of h y d r o p h i l i c groups i n the side c h a i n s b u t also to a v e r y m a r k e d extent o n the a m o u n t of c o p o l y m e r i n c o r p o r a t e d i n the p r o d u c t . Downloaded by UNIV LAVAL on September 30, 2015 | http://pubs.acs.org Publication Date: June 1, 1975 | doi: 10.1021/ba-1975-0145.ch008

4%

L o w e s t w a t e r u p t a k e of a b o u t

w a s o b t a i n e d for p r o d u c t s c o n t a i n i n g

fluorinated

acrylates or the

h i g h e r m o l e c u l a r w e i g h t esters of a c r y l i c or m e t h a c r y l i c a c i d . W a t e r s o r p t i o n at 5 0 % r e l a t i v e h u m i d i t y c h a n g e d f r o m 17 g H 0 / 2

100 g substrate for u n t r e a t e d r a t s k i n to 7 - 1 0 g H O / 1 0 0 g substrate 2

for the t r e a t e d s p e c i m e n .

T h e o n l y e x c e p t i o n w a s the

pentadecafluoro-

o c t y l a c r y l a t e - m o d i f i e d rat s k i n w h i c h h a d b y f a r the largest increase a n d a g r e a t l y r e d u c e d w a t e r u p t a k e ( 3 % ).

weight

It is not s u r p r i s i n g

that w a t e r s o r p t i o n , w h i c h is p r e d o m i n a n t l y a f u n c t i o n of the

water

a b s o r p t i o n t h r o u g h o u t the s p e c i m e n , is not a l t e r e d as m u c h f o r the rat skins as for c o l l a g e n p o w d e r .

T h e h i g h y i e l d s for g r a f t i n g onto c o l l a g e n

i n d i c a t e that the r e a c t i o n takes p l a c e t h r o u g h o u t the s p e c i m e n .

Even

c o n s i d e r i n g the r e d u c e d a m o u n t of c o l l a g e n present p e r u n i t w e i g h t of s k i n substrate, the m u c h l o w e r w e i g h t increase o n g r a f t i n g onto s k i n is a p p a r e n t l y c a u s e d b y r e s t r i c t i o n of the graft process to the exterior surface. T h e w e t t i n g b e h a v i o r of the collagenous g r e a t l y o n r e a c t i n g w i t h most m o n o m e r s . slowly

(Figure 2).

However,

surface is also

changed

C o l l a g e n is w e t t e d b y w a t e r

the r e a c t i o n p r o d u c t

of

collagen

a c r y l i c a n d m e t h a c r y l i c a c i d w a s h y d r o p h i l i c a n d gave a zero a n g l e w i t h i n 15 secs. t a i n i n g grafts of

A m a j o r i t y of the m a t e r i a l s , s u c h as those c o n -

fluorinated

acrylates or m e t h a c r y l a t e s or the

a c r y l a t e or m e t h a c r y l a t e h o m o l o g u e s , b e c a m e c o m p l e t e l y (Figure 2).

with

contact

E x a c t contact angles w e r e difficult to d e t e r m i n e

of t h e p o r o u s a n d u n e v e n surface of the specimens.

higher

hydrophobic because

T h e collagen—lauryl

m e t h a c r y l a t e a n d c o l l a g e n - 2 - e t h y l h e x y l a c r y l a t e substrates h a d a p p r o x i m a t e contact angles of 85° w h e r e a s those of the

fluorinated

a n d m e t h a c r y l a t e s h a d contact a n g l e values over 9 0 ° .

acrylates

O n introduction

of sufficient fluorine content, t h e graft p o l y m e r s also b e c a m e o i l r e p e l l e n t . A f t e r r e m o v a l of drops of w a t e r a n d o i l t h a t c o n t a i n e d dyes, the mats s h o w e d no s i g n of stains ( F i g u r e 2 ) , thus e s t a b l i s h i n g not o n l y t h e w a t e r a n d o i l r e p e l l e n c y of the m a t e r i a l s b u t also t h e i r stain resistance. W a t e r - r e p e l l e n t rat s k i n surfaces w e r e also o b t a i n e d w i t h

fluorinated

acrylates or m e t h a c r y l a t e s w h e r e a s p o l a r groups i n the side c h a i n i m -


BRAUER AND TERMINI

Modification

of Collagenous

Surfaces

189


8.

Figure 2. Water and oil repellency of graft polymers: left, acrylic acidcollagen; center, collagen; Hght, hexafluoroisopropyl methacrylate-collagen mat. Top and center, spreading of water and oil drops placed respectively on left and right of each mat; bottom, mats after blotting off drops with filter paper. parted h y d r o p h i l i c characteristics. O i l repellency was incorporated i n the

s u b d e r m a l surface o n p o l y m e r i z a t i o n w i t h h i g h l y

fluorinated

mono-

mers, b u t efforts to o b t a i n o l e o p h o b i c e p i d e r m a l surfaces w e r e u n s u c cessful.

T h i s different b e h a v i o r of the s k i n t o w a r d n o n - p o l a r l i q u i d s


190

APPLIED CHEMISTRY

AT PROTEIN

INTERFACES

results f r o m the k e r a t i n o u s n a t u r e of the e p i d e r m a l surface a n d its r o u g h texture e v e n after r e m o v a l of h a i r .

G e n e r a l l y , m o d i f i c a t i o n of w e t t i n g

characteristics w a s m o r e easily a c c o m p l i s h e d w i t h c o l l a g e n p o w d e r t h a n w i t h rat skin. U n d e r m o l d - g r o w i n g c o n d i t i o n s , g r o w t h w a s o b s e r v e d o n the o r i g i n a l rat s k i n substrate, b u t not o n skins t r e a t e d w i t h a v a r i e t y of

monomers.

A l t h o u g h no d e t a i l e d m y c o l o g i c a l studies h a v e b e e n u n d e r t a k e n , these results agree w i t h the resistance to m i c r o b i a l a n d f u n g a l attack r e p o r t e d f o r graft p r o d u c t s of other n a t u r a l l y o c c u r r i n g m a t e r i a l s . Some

growth

took p l a c e at the edges w h e r e the rat skins h a d b e e n c u t after treatment,


i n d i c a t i n g that g r a f t i n g o c c u r r e d p r e d o m i n a n t l y at the surfaces. M o d i f i c a t i o n of b o n e b r i n g s about some decrease i n its w a t e r s o r p t i o n behavior.

T h e decrease i n s o r p t i o n at 5 0 %

pronounced

r e l a t i v e h u m i d i t y w a s less

t h a n that f o u n d for c o l l a g e n or rat s k i n .

However,

when

h i g h y i e l d s of p o l y m e r w e r e f o r m e d o n the substrate s u c h as i n the react i o n of b o n e w i t h c y a n o e t h y l a c r y l a t e , h y d r o x y e t h y l a n d g l y c i d y l m e t h a c r y l a t e , ethylene, d i m e t h a c r y l a t e , or d i v i n y l b e n z e n e , w a t e r s o r p t i o n at 5 0 % r e l a t i v e h u m i d i t y w a s r e d u c e d to 3.0-4.5 g H 0 / g bone. 2

G r a f t i n g w a s c o n s i d e r e d successful i f the w e i g h t increase after ext r a c t i o n of s o l u b l e h o m o p o l y m e r i n the absence of m o n o m e r .

w a s greater t h a n the w e i g h t

It is r e c o g n i z e d that some

increase

homopolymer,

e s p e c i a l l y h o m o p o l y m e r of the b i f u n c t i o n a l m e t h a c r y l a t e s that crosslink d u r i n g the r e a c t i o n , is not r e m o v e d f r o m the substrate b y this treatment. A

separate, d i s t i n c t i n t e r p e n e t r a t i n g p o l y m e r n e t w o r k

entwined

with

m a c r o m o l e c u l e s of the c o l l a g e n substrate m a y be f o r m e d i n the r e a c t i o n . R e m o v a l of s u c h a phase b y solvent e x t r a c t i o n m a y not be possible. E v e n i f t h e substrate a n d p o l y m e r chains are not c o v a l e n t l y b o n d e d , the surf a c e properties of t h e r e s u l t i n g p r o d u c t s h o u l d differ f r o m those of the o r i g i n a l substrate.

S u c h m o d i f i c a t i o n m a y be u s e d a d v a n t a g e o u s l y

to

d e v e l o p p r o d u c t s w i t h i m p r o v e d properties. Characterization

of Graft

Copolymers

A n u m b e r of studies to d e t e r m i n e the m e c h a n i s m s of g r a f t i n g onto c o l l a g e n i n i t i a t e d b y C A N , i n c l u d i n g c h a r a c t e r i z a t i o n of the r e a c t i o n p r o d u c t s , h a v e b e e n r e p o r t e d ( 5 , 8, 9, 53, 62).

C e r i c a m m o n i u m nitrate

forms a n effective r e d o x system w i t h alcohols, a l d e h y d e s , amines, a n d thiols. A l c o h o l s f o r m a c e r i c ion—alcohol c o m p l e x , a n d the d i s s o c i a t i o n of this c o m p l e x is the r a t e - d e t e r m i n i n g step

Ce

I V

(63):

+ RCH OH C e 2

I I I

+H+ +

RCHOH


8.

BRAUER AND TERMINI

Modification

191

of Collagenous Surfaces

C o l l a g e n contains a l c o h o l i c groups i n the h y d r o x y p r o l i n e , serine, t h r e o n i n e , a n d h y d r o x y l y s i n e moieties.

F r e e r a d i c a l s are p r o b a b l y

formed

at s u c h sites w h i c h , i n the presence of a v i n y l m o n o m e r , serve to i n i t i a t e g r a f t e d side chains. S i n c e the free r a d i c a l s are f o r m e d o n t h e side chains of the substrate, a h i g h g r a f t i n g efficiency a n d a m i n i m u m a m o u n t of homopolymer

formation

compared

with

other r e d o x

systems

can

be

expected. T o d e t e r m i n e t h e p r o b a b l e l o c a t i o n w h e r e g r a f t i n g is i n i t i a t e d b y CAN

o n the c o l l a g e n m o l e c u l e , the rate of p o l y m e r i z a t i o n of m e t h y l

m e t h a c r y l a t e i n the presence of a m i n o acids f o u n d i n c o l l a g e n w a s s t u d i e d


(62).

P o l y m e r f o r m a t i o n ( h o m o - a n d graft p o l y m e r i z a t i o n ) took p l a c e

i n the presence of a l l a m i n o acids s t u d i e d w i t h the e x c e p t i o n of tyrosine and

m e t h i o n i n e . T h e r e l a t i v e y i e l d s after the 3-hr r e a c t i o n p e r i o d w e r e

of the f o l l o w i n g o r d e r : tyrosine, m e t h i o n i n e < glycine, alanine, hydroxylysine

Modification of Collagenous Surfaces by Grafting Polymeric Side

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