Hydrogels for Medical and Related Applications

11 Calcification and Bone Induction Studies in Heterogeneous

Downloaded by OHIO STATE UNIV LIBRARIES on October 14, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0031.ch011

Phosphorylated Hydrogels J. G. N. SWART Department of Oral Surgery, Free University, Amsterdam, The Netherlands A.A.DRIESSENandA.C.DEVISSER Department of Materials Science, Free University, Amsterdam, The Netherlands Development o f p o l y ( h y d r o x y e t h y l methacrylate) poly(HEMA) , h y d r o g e l s f o r a p p l i c a t i o n i n t h e m e d i c a l f i e l d has up t o date m a i n l y been d i r e c t e d towards t h e replacement o f s o f t - t i s s u e s t r u c t u r e s and o r g a n s , e.g. the s o f t c o n t a c t l e n s , Because o f i t s r e l a t i v e l y poor m e c h a n i c a l p r o p e r t i e s t h e h y d r o g e l as such can n o t be a p p l i e d as subs t i t u t e f o r h a r d t i s s u e . However, based on t h e f i n d i n g s t h a t c a l c i f i c a t i o n has o c c u r r e d i n heterogeneous poly(HEMA) h y d r o g e l s ( 1 - 6 ) , Calnan e t a l . (3) s u g g e s t e d t h a t t h e h y d r o g e l p o s s i b l y c o u l d promote t h e d e p o s i t i o n of c a l c i u m s a l t s i n i t s matrix, thus being a " c h a l l e n ger" f o r c a l c i f i c a t i o n . Some a u t h o r s ( 2 , 4 ) r e p o r t e d bone f o r m a t i o n f o l l o w ing the occurrence o f c a l c i f i c a t i o n . C a l c i f i c a t i o n and bone i n d u c t i o n appeared t o be a c c e l e r a t e d by t h e p r e s e n c e o f m e t h a c r y l i c a c i d (MAA) groups i n t h e g e l (£). S p r i n c l e t a l . (6), however, r e p o r t e d t h a t modif i c a t i o n o f a poly(HEMA) g e l by i n c o r p o r a t i o n o f MAA (up t o a mole r a t i o o f MAA/HEMA 1 : 5 ) o r 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 (DMAEMA) d i d n o t a f f e c t t h e c a l c i f i c a t i o n p r o c e s s , whereas C e r n i j e t a l . (2) found t h a t i n c o r p o r a t i o n o f 4 % MAA i n h i b i t e d c a l c i f i c a t i o n . From t h e above r e s u l t s i t i s e v i d e n t t h a t t h e e f f e c t o f MAA, i n c o r p o r a t e d i n t h e h y d r o g e l , on c a l c i f i c a t i o n o r bone f o r m a t i o n has n o t been unambiguously e s t a b l i s h ed y e t . P o s s i b l y , o t h e r f a c t o r s than c h e m i c a l m o d i f i c a t i o n such as pore s i z e o f t h e g e l , a n i m a l s p e c i e s and i m p l a n t a t i o n s i t e , p l a y a more dominant r o l e i n the o c c u r r e n c e o f t h e s e phenomena. A h y d r o g e l t h a t , i n a d d i t i o n t o c a l c i f i c a t i o n , c o u l d i n d u c e bone f o r m a t i o n i n i t s m a t r i x would have g r e a t p o t e n t i a l f o r r e s t o r a t i o n o f l a r g e d e f e c t s i n bone t i s s u e . Such a m a t e r i a l c o u l d f o r example be a p p l i e d t o f a c i l i t a t e t h e h e a l i n g p r o c e s s i n t h e p o s t e x t r a c t i o n a l v e o l u s o r bone i n 3

151

In Hydrogels for Medical and Related Applications; Andrade, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


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HYDROGELS FOR MEDICAL AND RELATED APPLICATIONS

growth i n l a r g e c y s t s . On t h e o t h e r hand, c a l c i f i c a t i o n i s o f t e n an u n d e s i r a b l e phenomenon i n a h y d r o g e l s e r v i n g as s o f t t i s s u e s u b s t i t u t e s i n c e i t a f f e c t s t h e f u n c t i o n a l and e s t h e t i c p r o p e r t i e s o f t h e m a t e r i a l adversely. In t h i s case, i n h i b i t i o n o f c a l c i f i c a t i o n by c h e m i c a l m o d i f i c a t i o n would be b e n e f i c i a l . In view o f t h e above c o n s i d e r a t i o n s o u r o b j e c t i v e i s t o study c a l c i f i c a t i o n and bone f o r m a t i o n i n h e t e r o geneous h y d r o g e l s as f u n c t i o n o f t h e i r c h e m i c a l and p h y s i c a l p r o p e r t i e s and t o f i n d means t o c o n t r o l these p r o c e s s e s by s u i t a b l e m o d i f i c a t i o n o f t h e h y d r o g e l . The study d e s c r i b e d h e r e a f t e r d e a l s w i t h an experiment d e s i g n e d t o determine t h e e f f e c t o f i n c o r p o r a t i o n o f the phosphate group i n a heterogeneous poly(HEMA) g e l on i t s c a l c i f i c a t i o n - i n d u c i n g a b i l i t y . The h y d r o g e l s h o u l d be heterogeneous because i t was found t h a t a s u f f i c i e n t l y l a r g e pore s i z e (> 40ym) (A) i s a p r e r e q u i s i t e f o r ingrowth o f t h e s u r r o u n d i n g t i s s u e which i n t u r n can l e a d t o c a l c i f i c a t i o n and f o r m a t i o n o f bone. The phosphate group was s e l e c t e d t o be b u i l t i n t h e g e l because t h i s group i s one o f t h e b u i l d i n g b l o c k s o f c a l c i u m h y d r o x y a p a t i t e t h e main i n o r g a n i c c o n s t i t u e n t o f bone and, a l t h o u g h b e i n g p r e s e n t as HEMA-phosphate, s h o u l d have a h i g h a f f i n i t y towards calcium ions. M a t e r i a l and Methods HEMA-phosphate was p r e p a r e d by r e a c t i n g HEMA (Hydro Med S c i e n c e s , I n c . , U.S.A., p u r i t y min. 99.2%) w i t h phosphorus p e n t o x i d e (Merck,Darmstadt, Germany, p u r i t y min. 98%) i n a 1:1 mole r a t i o i n d i c h l o r o m e t h a ne a t 0 - 5°C. The r e a c t i o n proceeds almost q u a n t i t a t i v e l y . A f t e r removal o f s o l i d s by c e n t r i f u g a t i o n and e v a p o r a t i o n o f t h e s o l v e n t i n vacuo a t 20 C and 1 mm Hg, a m i x t u r e o f mono- and d i - e s t e r was o b t a i n e d i n a mole r a t i o o f 2 t o 1 assuming t h a t t h e amount o f t r i e s t e r formed i s n e g l i g i b l e . Ethyleneglycol d i m e t h a e r y l a t e (EGDMA) (Merck) was p u r i f i e d by d i s t i l l a t i o n a t 83 C and 1.5 mm Hg. HEMA was used as o b t a i n e d from Hydro Med S c i e n c e s . P r e p a r a t i o n o f t h e h y d r o g e l s was performed i n s e a l e d ampoules o f 9 mm i n n e r d i a m e t e r . The v a r i o u s monomers were d i s s o l v e d i n a Tyrode s o l u t i o n c o n t a i n i n g 1% (w/w) o f ammonium p e r s u l f a t e as i n i t i a t o r . Polymer i z a t i o n was c a r r i e d o u t a t 50 C d u r i n g 24 hours. o


11.

SWART E T A L .


Composition follows :

Heterogeneous

Phosphorylated

Hydrogels

o f the p o l y m e r i z a t i o n mixtures

Hydrogel

HEMA

H 1 H 2

19.8 17.8

EGDMA HEMA( c r o s s l i n k e r ) phosphate 0.2 0.2

— 2.0

153

i s as

Tyrode s o l .

80% w/w 80% w/w

The g e l s were k e p t i n d i s t i l l e d water f o r s e v e r a l days and then b o i l e d t w i c e i n d i s t i l l e d water t o r e move low m o l e c u l a r weight s u b s t a n c e s . Then t h e g e l s were soaked i n s t e r i l e s a l t s o l u t i o n and c u t i n t o d i s c s about 2 mm t h i c k and 9 mm i n d i a m e t e r . These d i s c s were i n s e r t e d i n muscle p o c k e t s i n the back muscle o f r a t s . I n each r a t we i m p l a n t e d 3 d i s c s o f t h e same h y d r o g e l and one p o c k e t was f i l l e d w i t h g e l a t i n foam ( S p o n g o s t a n , F e r r o s a n , Will-Pharma N.V., H o l l a n d ) as a c o n t r o l . E x c i s i o n f o l l o w e d a f t e r p e r i o d s o f 3 days up t o 24 weeks. The s k i n was c u t away and t h e i m p l a n t s removed w i t h t h e s u r r o u n d i n g muscle t i s s u e . The removed t i s s u e b l o c k s were p l a c e d f o r 10 minutes between gauze s t r i p s s a t u r a t e d w i t h i s o t o n i c s a l i n e t o allow the c o n t r a c t i l e p r o p e r t i e s t o become q u i e s c e n t . T h e r e a f t e r t h e e x c i s e d t i s s u e was f i x e d i n 10% b u f f e r e d f o r m a l i n . A f t e r f i x a t i o n and d e h y d r a t i o n i n a b s o l u t e a l c o h o l t h e b i o p s i e s were embedded i n p a r a f f i n and c u t i n t o s e c t i o n s o f 6 ym. These s e c t i o n s were s t a i n e d w i t h t h e Haematoxylin and e o s i n s t a i n . C a l c i u m s a l t s were v i s u a l i z e d a c c o r d i n g t o Von Kossa. Pore s i z e o f t h e m a t e r i a l s was determined by scanning e l e c t r o n microscopy. R

R e s u l t s and D i s c u s s i o n M a c r o s c o p i c a l l y a l l i m p l a n t s were a c c e p t e d and w e l l t o l e r a t e d ; no s i g n s o f s e v e r e i n f l a m m a t i o n o r i m p l a n t r e j e c t i o n c o u l d be o b s e r v e d , ( f i g u r e 1 ) . Microscopic i n v e s t i g a t i o n revealed the following: 3 days a f t e r i m p l a n t a t i o n a s l i g h t edema and i n v a s i o n o f g r a n u l o c y t e s and mononuclear c e l l s i n t o a l l i m p l a n t s was o b s e r v e d . The p h o s p h o r y l a t e d hydrog e l s showed l e s s c e l l u l a r i n f i l t r a t i o n and s t a i n e d b a s o p h i l i c p r o b a b l y because o f t h e a c i d phosphate group. Some o f t h e c o n t r o l g e l a t i n foams (Spongostan ) showed i n v a s i o n o f f i b r o b l a s t s and mononuclear c e l l s as w e l l as hyperaemia around t h e i m p l a n t s as a s i g n o f acute inflammatory response. R




154

In the one week b i o p s i e s t h e r e was an i n c r e a s e i n c e l l u l a r ingrowth o f about 0.2 mm f o r the p o l y (HEMA) i m p l a n t s . The p h o s p h o r y l a t e d i m p l a n t s h a r d l y showed any ingrowth o r s i g n s o f i n f l a m m a t i o n . A f t e r two weeks we saw a f u r t h e r i n c r e a s e o f ingrowth up t o a maximum o f 1 mm i n t o the poly(HEMA) specimen. The i m p l a n t s were s l i g h t l y t h i c k e r (3 mm) and f o r the f i r s t time b a s o p h i l i c c l u s t e r s c o u l d be seen as the e a r l i e s t s i g n o f c a l c i f i c a t i o n ( f i g u r e 2 ) . The p h o s p h o r y l a t e d poly(HEMA) i m p l a n t s behaved q u i t e i n d i f f e r e n t l y towards the t i s s u e . There was p r a c t i c a l l y no f i b r o u s l i n i n g around the i m p l a n t , i t even seemed t o l a y d i r e c t l y upon the muscle f i b r e s . In the 4 week b i o p s i e s the "Spongostan " g e l a t i n foam i m p l a n t s showed no s i g n s o f acute i n f l a m m a t i o n : t i s s u e p r o l i f e r a t i o n with f i b r o b l a s t s , foreign-body, g i a n t c e l l s and c a p i l l a r i e s dominated the p i c t u r e . The g e l a t i n foam g r a d u a l l y d i s a p p e a r s as a r e s u l t o f p h a g o c y t o s i s by macrophages. In the poly(HEMA) h y d r o g e l s b a s o p h i l i c g r a n u l e s are s c a t t e r e d a l l through the i m p l a n t . The p h o s p h o r y l a t e d i m p l a n t s were w e l l t o l e r a t e d , and s t i l l showed p r a c t i c a l l y no ingrowth o r encapsulation. 8 weeks the "Spongostan " g e l a t i n foam has completely disappeared, with only small strands of f i b r o u s s c a r t i s s u e l e f t . The poly(HEMA) i m p l a n t s showed c o n s i d e r a b l y more f i b r o b l a s t s than 4 weeks bef o r e . The b a s o p h i l i c g r a n u l e s w i t h i n t h e s e i m p l a n t s formed l a r g e r c o l o n i e s . F r e q u e n t l y , a t h i n f i b r o u s c a p s u l e surrounded the p h o s p h o r y l a t e d i m p l a n t s , which s t i l l h a r d l y showed any ingrowth. A t 16 and 20 weeks whole g r a n u l a r f i e l d s were o b s e r v e d i n the poly(HEMA) i m p l a n t s . These f i e l d s p a r t i c u l a r l y appeared i n the edges o f the i m p l a n t . Where t h e s e g r a n u l a r f i e l d s were p r e s e n t , t i s s u e i n growth d i d not take p l a c e o r seemed t o be p r e v e n t e d . Some o f the poly(HEMA) i m p l a n t s showed l e s s b a s o p h i l i c g r a n u l e s , but c o n s i d e r a b l y more t i s s u e i n g r o w t h . C a l cium d e t e r m i n a t i o n by the Von Kossa s t a i n r e v e a l e d t h a t the s o c a l l e d b a s o p h i l i c g r a n u l e s were c a l c i f i e d ( f i g u r e 3). ^ 24 week b i o p s i e s , the l a s t p e r i o d e v a l u a t e d , showed some i n t e r e s t i n g f e a t u r e s . A rontgenogram (60 kv, 3 mA, 0.10 sec) o f b i o p s i e s from the 5 r a t s out o f t h i s group showed a r a d i o paque o u t l i n e i n the poly(HEMA) i m p l a n t s , p h o s p h o r y l a ted poly(HEMA) i m p l a n t s d i d not show t h i s phenomenon. R

A

T

f

t

e

r

e



SWART E T AL.

Heterogeneous

Phosphorylated

Hydrogels

Figure 1. Phosphorylated poly(HEMA) implants in the back of a rat 24 weeks after the implantation showing the excellent biocompatibility of this material

Figure 2. Photomicrograph of a poly(HEMA) implant section, 14 days after implantation. For the first time calcified granules (arrows) appear within the poly(HEMA) implants. (Haematoxylin and eosin stain, magnif. X39)




Figure 3a. Photomicrograph of a poly(HEMA) implant 20 weeks after insertion showing areas of dense calcification (1) and areas of massive tissue ingrowth (2) with only sparse calcification. The center (3) of the implant shows only a slight ingrowth of cells into the pores of the implant material. (Haematoxylin and eosin stain, magnification X20)

Figure 3b. Same section stained according to Von Kossa for calcium determination. With this method calcified tissues stain black and uncalcified tissues red, which shows plain grey on this photomicrograph.



11.

SWART E T A L .

Heterogeneous

Phosphorylated

Hydrogels

157

The Von Kossa s t a i n proved the p r e s e n c e o f c a l c i u m w i t h i n the poly(HEMA) i m p l a n t s which showed r a d i o paque l i n e s i n the roentgenogram and the absence o f c a l c i u m s a l t d e p o s i t s i n the non-radiopaque specimen. The p h o s p h o r y l a t e d i m p l a n t s showed o n l y a t h i n f i b r o u s l i n i n g and i n g r o w t h , i f any, p r e d o m i n a n t l y i n the o u t e r margin ( f i g u r e 4 ) . In one r a t we o b s e r v e d a remarkable macroscopic d i f f e r e n c e between two poly(HEMA) i m p l a n t s t h a t were c h e m i c a l l y i d e n t i c a l ; one i m p l a n t had a w h i t e a s p e c t and a normal s i z e ; the o t h e r i m p l a n t showed an i n c r e a s e i n s i z e and the same c o l o u r as the s u r r o u n d i n g t i s s u e . The m i c r o s c o p i c p i c t u r e r e v e a l e d t h a t the white i m p l a n t was more c a l c i f i e d compared t o the o t h e r i m p l a n t and showed l e s s t i s s u e i n g r o w t h . T h i s d i f f e r ence might be the r e s u l t o f a d i f f e r e n t b l o o d s u p p l y (figure 5). In o r d e r t o r e l a t e the r e s u l t s w i t h the p h y s i c a l s t r u c t u r e o f the g e l s , the pore s i z e o f the v a r i o u s i m p l a n t s was determined by s c a n n i n g e l e c t r o n m i c r o s copy. The poly(HEMA) i m p l a n t s had pores from 30 - 70 ym, w h i l e the p h o s p h o r y l a t e d h y d r o g e l s p o s s e s s e d pores from 70 t o o v e r 100 ym ( f i g u r e 6 ) . The i n h i b i t i o n o f t i s s u e i n f i l t r a t i o n i n the phosphate c o n t a i n i n g g e l s i s s u r p r i s i n g because one would e x p e c t t h a t s u b s t a n t i a l i n g r o w t h would o c c u r i n an i m p l a n t h a v i n g such a l a r g e pore s i z e . Wether t h i s phenomenon i s due t o the h i g h e r a c i d i t y i n t h e s e g e l s , as compared t o the poly(HEMA) g e l s , o r the n a t u r e o f the phosphate group i t s e l f i s not known and needs f u r t h e r study. Conclusions Summarizing the r e s u l t s we can c o n c l u d e t h a t : 1. I n c o r p o r a t i o n o f the phosphate group i n h e t e r o g e neous poly(HEMA) h y d r o g e l s when i m p l a n t e d i n t r a m u s c u l a r l y i n r a t s , does not promote c a l c i f i c a t i o n o r bone f o r m a t i o n , on the c o n t r a r y i t seems t o p r e v e n t c a l c i f i c a t i o n and t i s s u e i n g r o w t h . T h i s may make the p h o s p h o r y l a t e d poly(HEMA) h y d r o g e l s a better material for soft tissue substitution than r e g u l a r poly(HEMA), a l t h o u g h i t i s t o e a r l y y e t t o make d e f i n i t e c o n c l u s i o n s and recommendations. 2. None o f the i m p l a n t s showed bone o r c a r t i l a g e f o r mation. The c a l c i f i c a t i o n o c c u r i n g w i t h i n the p o l y (HEMA) i m p l a n t s i s most l i k e l y due t o c a l c i f i c a t i o n o f degenerate t i s s u e ( d y s t r o p h i c c a l c i f i c a t i o n ) as the r e s u l t o f i n s u f f i c i e n t b l o o d s u p p l y .




Figure 4a. Photomicrograph of a phosphorylated poly(HEMA) imphnt section 24 weeks after insertion showing the thin fibrous lining of two or three cell layers and rather superficial tissue ingrowth.

Figure 4b. Photomicrograph of a section of the same material which has been excised after 14 days shows almost the same picture. (Haematoxylin and eosin stain, magnification X 85)



SWART ET AL.

Heterogeneous Phosphorylated Hydrogels

Figure Sa. Poly(HEMA) implants out of the same rat 24 weeks after implantation. In one implant (1) there is a diffuse ingrowth of tissue practically without calcification. The other implant (2) shows colonies of calcification and only tissue ingrowth in the outer margin of the implant. (Haematoxylin and eosin stain, magnification X 21)

Figure 5b. Comparable section, stained according to Von Kossa, proving the presence of calcium salts which stain black (arrows)




Figure 6a. Scanning electron micrograph of poly(HEMA) having pore sizes from 80-70 μ

Figure 6b. Scanning electron micrograph of phosphorylated poly(HEMA) having pores from 70 to over 100 μ


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SWART E T A L .

Heterogeneous Phosphorylated Hydrogels

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3. No lymphocytes were seen in the implants which indicates that poly(HEMA) and phosphorylated poly (HEMA) are immunologically inert materials. 4. A large variation in calcification within the regular poly(HEMA) implants has been observed. Variation in blood supply with implant site possi bly plays an important role in this phenomenon. Downloaded by OHIO STATE UNIV LIBRARIES on October 14, 2014 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0031.ch011

Abstract The effect of chemical modification of a hetero geneous poly(hydroxyethyl methacrylate) hydrogel on calcification in its matrix has been studied by intra muscular implantation in Wistar rats. Ten percent phosphorylated hydroxyethyl methacrylate, based on total amount of monomer, was incorporated in hydrogels containing 80% (w/w) water. These gels which exhibit poor mechanical properties can not be used as direct replacement for hard tissue, but may have potential as calcification "challenger" for living tissue. Scanning electron micrographs show that the gels have pores with an average diameter 70 - 100 μm. Histological examination of the implants after 3 days up to 24 weeks revealed that the poly(HEMA) and the phosphorylated poly(HEMA) gels were very well tolerated by the organism. Tissue ingrowth and calcification occurred in the poly(HEMA) gels, but bone formation was not found. The phosphorylated gels were encapsulated by a thin fibrous lining whereas ingrowth only sporadically and to a slight extent was observed; calcification did not take place. Literature Cited 1. Kliment, Κ., Stol, Μ., Fahoun, K., Stockar, Β., J . Biomed. Mater. Res. (1968), 2, 237. 2. Winter, G.D., and Simpson, B . J . , Nature (1969), 223, 88. 3. Calnan, J . S . , Pflug, J.J., Chhabra, A.S., Raghypatli, N., Brit. J . Plastic Surgery,(1971) ,24, 113. 4. Smahel, J.,Proserova, J.,Behounkova, Ε . , Acta Chirurgiae Plasticae (1971), 13, 193. 5. Sprincl, L.,Vacik, J.,Kopecek, J., J. Biomed. Mater. Res. (1973), 7, 123. 6. Sprincl, L.,Kopecek, J.,Lim, D., Calc.Tiss.Res. (1973), 13, 63. 7. Cernij, Ε . , Chromeĉek, R., Opleta, Α., Papousek, F. Otoupalová, J., Scripta Medica (1970).


Hydrogels for Medical and Related Applications

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