Interactions of copolymeric poly(glyceryl methacrylate)-collagen hydrogels with neural tissue: effects of structure and polar groups

Interactions of copolymeric poly(glycerylmethacrylate)-collagen hydrogelswith neural tissue: effects of structure and polar Doups S. Woerlyand R. Marchand Centre de recharche en neurobiologie, Hopital de I Enfant-J&us,

Centre de recherche en sciences et en genie des macromol&ule.s, Presented at Biointeractions 90, Oxford, UK 2 l-23 August 1990

1401

18e rue, QuBbec, Canada G1J 124

Universith Laval, Q&bee,

Canada GlK 7P4

In a previous study we developed copolymeric glyceryl methacrylate-collagen hydrogels for implantation in surgical lesions of the rat brain. Such materials provide porous matrices that can serve as support systems for oriented growth of scar tissue and axonal growth. In the present work, we have investigated the effect of structural modifications (studied by mercury porosimetry) of polymeric matrices and the effect of polar groups on the response of the brain tissue. The findings show that the fractional porosity and the pore size distribution of matrices are critical for tissue ingrowth and that negative charges, i.e. carboxylic acid groups, incorporated in the polymer have a strong influence on reactive astrocytosis. Keywords: Methacvlate-collagen

(co)polymer hydrogels. charged hydrogels, intracerebral implantation, porosimetw, regeneration

Extracellular matrices (ECM) characteristically involve threedimensional networks composed of polymers and macromolecules and provide the necessary substrates for cell attachment, cell locomotion and mediating cell interactions’3. With the availability of ECM-like substructures, the gliosis and the collageneous scarring which develop after any injury of the neural tissue are expected to have some degree of organization that could provide efficient migratory pathways and promote growth of nerve fibres. In a previous study, we developed copolymeric poly(glyceryl methacrylate)-collagen hydrogels4. The combination of synthetic polymer with natural polymer yields properties of both materials mechanical stability together with biological acceptability by the host tissue. Such materials consist of cross-linked hydrophilic polymers. They form a three-dimensional network which is well tolerated by the neural tissue and serve as support systems for cell attachment and tissue ingrowth in the central nervous system (CNS)4. However, the extent to which the host tissue interacts with polymeric matrices is directly affected by physical and chemical properties of the polymer, such as porosity, hydrophilicity and presence of collagen substrate. Correspondence to Dr S. Woerly, presently at Department of Biological Sciences, University of Keele, Keele, Staffordshire ST5 5BG. UK. 0 199 1 Butterworth-Heinemann

In this work, the effect of structure modifications of polymeric matrices, and the effect of the expression of anionic and cationic chemical groups on the host rat brain tissue response were investigated. For this purpose, the fractional porosity and the pore size of poly(glyceryl methacrylate) (pGMA) gels were varied by adding glycidyl methacrylate (GdMA), as GdMA decreases the cross-linking density of the polymer4. Ionizable groups have been introduced in pGMA, carboxylic acid groups with methacrylic acid and tertiary amino groups with (dimethylamino) ethyl methacrylate.

MATERIALS Preparation

AND METHODS of hydrogels

The following chemical were used: glycidyl methacrylate (GdMA, 2,3_epoxypropyl methacrylate), 2-(dimethylamino) ethyl methacrylate (DMAEMA), methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), ammonium persulphate and sodium metabisulphite, purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). Type I collagen was obtained from Collagen Corporation (Palo Alto, CA, USA).

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Neural tissue jntera~tjon with syntheric hydroge/s: S. Woer~ et al

Table 1

Nomenclature

F,-Ivv\I

and composition of hydragels

Hs

“vvG(CH,-

GMA

F CO-DCHz

-

EGDMA

DMAEMA

MAA

GdMA

CH- OH

ionic structure

I

GMA

CHz-OH

pGMA20 ~MA20-Gd2 pGMA20-Gd6 pGMA30 pGMA30+ pGMA30-

1.23 ‘2: 20 30 30 30

2 6 1.23 1.23 1.23

10 15

Neutral Neutral Neutral Neutral Cationic Anionic

The concentrations of EGDMA, MAA, DMAEMA and GdMA are expressed in % weight ratio to GMA and GMA in v/v (final volume 1 ml). GMA, glyceryl methacrylate; EGDMA, ethylene giycol dimet~ac~late: DMAEMA, 2-(dimethylamino) ethyl methacn/iate; MAA, methacryiic acid; GdMA, glycidyl methacrylate.

~~ Figure 1 Chemical structure of the various monomers used in this study. GM.4 giyceryl methacryiate; GdMA glycidyl methacrylate; MA.4 methacrylic acid; 2-(dimethylamino]eihyl methacrylate. The charges of MAA and O~AE~A rafer to the ionjzation state of the carboxyl and amino groups at physiological pfi. CH3

7H3

CH3

1

I

a

I

CHZ----_CH2d=O I 0 Ltia0~0H

I

CH~~H

.

CHs-C-

C-

I 7=0

t n

d=O I

0

‘:cH2

LH&HOH

I

I

CH~

I 0 CH3

CHz-

A=0

d; -0H:,-;

-

CH3 f

CHa -

I

‘c =0

AH3

ZI &lHpCHOH

I

OH,OH

7 n c =o I 0 I CHaCHOH

I

CH*~H

Figure 2 Chemical structure of basic cross-linkedpoly(glyceryl methacrylate) (pGMA) polymer network.

GMA was obtained by hydrolysis of glycidyl methacrylate in aqueous sulphuric acid according to the method described by Refojo’. In addition, the GMA was further purified by distillation under dynamic vacuum (1.33 X 1 0e3 Pa) to remove epoxy-type residues and traces of polymer. Hydrogels based on pGMA were prepared by radical polymerization with the cross-linking agent EGDMA in distilled water. Polymerization was initiated in small glass tubes with the redox initiator ammonium persulphate (6% v/v) and sodium me~bisulphite (12% v/v) added in a 0.37 wt% ratio to GMA, at 60°C during 12 h. Six series of hydrogels were then prepared from the two basic hydrogels: pGMA2Oand pGMA30 (Figures ? and Z), the nomenclature

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199 I, Vol 12 March

and the composition of which are given in Table I. At 10 and 15% (wt/wt), DMAEMA and MAA have been shown to influence the reactivity of non-neural tissues without toxicit$. Glycidyl methacrylate was used as cross-linking agent and it has been shown that GdMA affects the structure of pGMA at these concentrations5. The hydrogels were washed in distilled water for a mjnimum of 4 wk and kept in water solution. They were sterilized by boiling in distilled water.

Characterization

of hydrogels

The hydrogels were characterized using scanning electron microscopy (SEM) for examination of topography and morphology of surfaces, and by mercury porosimetry for quantitative analysis of porosity. For SEM, the gels were lyophilized and coated with gold and analysed with a Jeol 1300 model at 15 kV. The porosity of pGMA and p(GMA-GdMA) was measured on freeze-dried samples by mercury intrusion using an American Instrument Company porosimeter model 414 MPa. The pore diameter, d, was calculated as: d = 4#cos@/p (p, applied pressure: 8, contact angle (130”); and (0, tension of mercury (4.73 mN/cm)). The intrusion volume was recorded for pressures ranging from 0 to 27.6 MPa. The intrusion is the ratio of injected mercury volume to the volume of the gel and the normalization is the slope of the intrusion graph, calculated from the logarithm of the porosity.

Preparation of pGMA-collagen

hydrogels

A collagen solution was prepared by neutralizing a sterile arid-soluble type I collagen with a concentrated phosphate solution (1.3 M NaCl in 0.2 M monophosphate) and with 0.1 M NaOH in the volume ratio 8: 1: 1 giving a collagen concentration of 2.4 mg/ml and a pH of 7.2. The collagen solution was kept on ice to prevent spontaneous gelation. Dehydrated pGMA were re-equilibrated by swelling in the collagen solution for 2 d at 4°C. The gels were then maintained at 37°C in a humidified incubator to allow polymerization of collagen.

implantation

surgery

Twenty-four young female Sprague-Dawley rats (250300 g) were used. The animals were anaesthesized with an intramuscular (i.m.) injection of ketamine (Rogarsetic, 60 mg/kg) followed by an i.m. injection of sodium pentobarbital (Somnotol, 21 mg/kg) and placed in a stereotactic head frame. Under a surgical microscope and through a midline incision, a small craniotomy was performed

Neural tissue interaction with synthetic hydrogels: S. Woerly et al.

(3 x 3 mm). The dura was incised and folded to expose the left parietalcor-tex. Across-shaped incision about 2-2.5 mm deep was performed in an avascular region of the cortex. After haemostasis with thrombin-soaked gelfoam, hydrogel samples (1 mm3) were introduced with jeweler’s forceps through the cross-shaped incision. A slight pressure was applied over the parietal cortex for 2 min to allow the gel to settle. The bone flap was immediately replaced and the scalp clipped.

Histochemistry At intervals from 1 wk to 3 month, animals were transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. After overnight fixation, the brains were transferred in 30% buffered sucrose and coronally cut at 30pm using a freezing microtome. Some animals were perfused with Bouin’s fixative and blocks of brain containing the hydrogel were embedded in a resin embedding medium (Historesin, LKB). The plastic blocks were cut at 3 and 10 pm on a glass knife using a microtome. The brain sections mounted on slides were stained using the phosphotungstic acid haematoxylineosin method (PTAH) for glial cells and fibres, Bodian’s method for nerve fibres and neuronal cell bodies, and the picrosirius polarization method for collagen7. In addition, plastic sections were stained with Coomassie blue.

RESULTS Morphology

and structure

of hydrogels

The hydrogels were transparent and exhibited different capacities to swell in water according to their composition. The gels copolymerized with GdMA showed particular features as they had a spongy appearance and displayed a considerable degree of swelling. Analysis of the gels by SEM showed orientated structures with pores of variable size, either opened to the interior of the gel or closed (Figure 3a). Analysis of fracture of the gel structure revealed channel systems that showed a spatial orientation (figure 36). The fractionai porosity (porous percentage of volume), the pore size and the cumulative pore size distribution (Gaussian distribution of the logarithm of diameter) were studied by mercury porosimetry (figure 4). The values of pore parameters of hydrogels pGMA and p(GMA-GdMA) are given in Table 2. The hydrogels were considered hyperporous from a value of pore size when the fractional porosity was at least 50% of the gel volume from this value: a hydrogel hyperporous from 2,~rn means that 50% of its volume consists of pores larger than 2 pm. The effect of increasing the concentration of GMA or adding GdMA on the structure of hydrogels is shown with the intrusion curves and the poresizedistribution (Figure 4). The GMA and the GdMA increase both the pore size and the pore size distribution towards macroporous domains while they have an opposite influence on the fractional porosity of the hydrogels, GdMA increasing the fractional porosity (Table 2). The hydrogel pGMA20-Gd6 is the one that has the highest fractional porosity and the gel pGMA30 the one that has the larger pores. In all cases, the sigmoid shape of the intrusion curves indicates that the internal structure of hydrogels is fairly homogeneous corresponding to a Gaussian distribution of the logarithm of the pore size.

Figure 3 Scanning electron micrographs showing some examples of the structure of poly(glyceryl methacrylate) (pGMAJ-based hydrogels. a, the surface of the gel shows a highly porous structure with pores of variable size. most of which are open to the inner structure of the gel. b, the Internal structure of hydrogel consists of oriented microchannels. The arrows show collagen that has been introduced into the polymer nehvork. This hydrogel has been used for introducing embryonic nerve cells in the matrix p) (unpublished data).

Findings

at autopsy

All animals survived the surgery. There were no physical or behavioural signs suggestive of neurotoxicity or bacterial infection. Macroscopic examination of the operative zone showed that the implants were encased in the host tissue without dead spaces, constituting a continuity with the neural parenchyma. There were no signs of rejection, oedema or necrosis of the surrounding parenchyma. Neither did we notice any phenomena of hypervascularization around the gels.

Histology General. The implants were entirely contained within the cortex. Examination of the neural tissue around the polymer implants by light microscopy and after staining with the techniques used in this study showed that the polymeric matrices were well tolerated and that the surrounding parenchyma had a fairly normal morphology. Signs of chromatolysis and atrophy of neuronal cell bodies could be observed. The inflammatory reaction was moderate. The interface formed between the brain tissue and the implant showed a close apposition of the tissue with the polymer surfaces and points of adhesion (Figure 5a). A summary of the histopathological reaction of host neural tissue 3 month after implantation of the hydrogels is given in Tab/e 3. The main features of the reactivity of the host tissue were the astrocytosis and the proliferation of mesenchymal cells, probably of meningeal and vascular origin, that invaded the gels. Proliferation of capillary and axonal sprouts were also

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199

Neural tissue interaction with synthetic hydrogels: S. Woerly et al.

1OOr

80 70

2.0

5 .vl

60

: e 5

40

50

cxc 30

2

1.5

0.1

E E 2 ._ u E i E b

10.0

1.0

100,o

200.0

Diameter (pm) R

1.0

Z

10.0

1.0

Diameter

100.0

___ _

zoo.0

(urn)

Figure 4 Mercuryporosimetry ofpoly(glyceryimethacryiate) (pGMA) andpGMA-glycidylmethacrylate (GdMA), showing the fractional porosity (inset) and the distribution of pore sizes of hydrogels. The GMA and the GdMA increase the pore size but have an opposite effect on the fractional porosity. 0, pGMA20; A. pGMA30: 0, pGMAPO-Gd2; n , pGMA20-Gd6.

Table 2

Values of pore parameters of pGMA and pGMA-GdMA

Hydrogel

pGMA20 pGMA30 pGMA20-Gd2 pGMA206d6 pGMA-GdMA,

hydrogels

Fractional porosity(%)

(rm)

Average pore size (pm)

65 60 60 90

6 10 2 8

7 13 2 10.5

Hyperporous

poly(glyceryl methacrylate-glycidyl methacrylate).

seen in the vicinity of the implant and at the brain-implant interface. Newly formed collagen showing polarization features different from that of the collagen introduced in the matrices was detected as early as 2 wk in the bioimplants by polarization and light microscopy, after staining with the picrosirius red (Figure 56). This collagen displayed a bright birefringence and variable polarization colours whilst the introduced collagen was red and weakly birefringent. On silver-impregnated sections, extracellular materials showing strong argyrophilic properties were deposited into the matrices and could correspond to reticularfibres and/or type III collagen fibress (e.g. Figure 6~). Tab/e 3 shows that the neural tissue reacted variably to the bioimplants and that the polymers were variably penetrated by the host tissue. Maximal tissue ingrowth was found for neutral and anionic gels based on pGMA30. Effect ofporeparamaters. The effect of pore structure on the response of the host tissue was best studied with the gels pGMA20, pGMA30, pGMA20-Gd2 and pGMA20-Gd6 for

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Biomaterials

199 1, Vol 12 March

which increasing concentrations of GMA or GdMA affect the structure of the gel (Table 2). The formation of a fibrous capsule around the implants occurred for matrices having a microporous structure, i.e. hyperporous below 10 pm (Tab/e 2). The capsule was of variable thickness, composed of newly formed and densely aggregated collagen and glial elements. Penetration of tissue into the matrices was variable according to their structures. TheGMA/GdMAcopolymers were penetrated marginally by a few cells only. The gels pGMA20 and pGMA30 were penetrated by host tissue progressively with time forming, 3 month after implantation, gradients of cellularization. The best biological response was obtained with the homopolymer pGMA30, although the pGMA30 copolymers also displayed excellent biological properties. Indeed, for this series of gels there was no or little encapsulation and tissue penetration was found maximal compared with the other implants. Glial cells and processes, cells of heterogeneous origin and capillary sprouts infiltrated deeply into the interspaces of polymeric matrices (Figure 6a). In addition, the series of pGMA30 polymers displayed good structural stability and the patterns of cell infiltration were seen to superimpose the microgeometryof the polymer network. The implant pGMA20 did not show the same stability. The infiltration of host tissue caused extreme deformations of its structure with large fractures through the polymer network. Nerve fibres were seen growing into the pGMA30 gels at variable degrees ( vide infra ) . Effects of polar groups. Comparison of the tissue reaction after implantation of gels pGMA30, pGMA30 + and

Neural

Figure

5

The

brain-bioimplant

interface,

a, phosphotungstic

acid haematoxylin-eosin

zones

of the neural

of adhesion

methacrylic

acid

projections

(large

the

(pGMA-MAAJ arrows)

bioimplant

delimited

materials

section

stained

formed

collagen,

deposited

with

in

accumulation

Table

3

bend

(weakly

stained) red

IS strongly

of collageneoos

features

hydrogels,

Hydrogel

have

fibrino-granular

6, Microphotograph bright

field.

the picrosirius,

with

of a Newly

has

Note

the

the neural

tissue

been slight (nt).

pertaining

3 month

to the reaction

after

of the neural

tissue

implantation

Astrocyte

AXO”

ingrowth

pro]ectlo”s

penetration

-++

+

+

n.d.

pCiMA20-Gd2

+

+marg.

+

n.d.

6.

pGMA20-Gd6

+

+marg.

+

n.d.

Astrocytic

pGMA30

-+

++>

++

pGMA30+

0

t

++

;a”d

$

consrituting

pGMA30-

0

+++>

+++

lland

5

nssue.

discontmuity

of the capsule;

f.

Capsule

Figure

6

Examples

ofhydrogels. ingrowth

pGMA20

++.

0, noencapsulatlon;

thick

Tissue

Axon

adhesion

+, moderate;

prolectlons:

$, axons

+, few;

+ +, Important; ++,

numerous;

n.d., not detected; projecting

into the

1, axonal matrix.

>, onentated; +++,

of

marg.,

formation

sprouts

at the

marginal.

of glial matrix. brain-implant

the

neural

an

tissue

(nt)

to

haematoxylm-eosm grown

different and

show from

and grown

cortical

up to 200

of the

variable

corner

of the microphotograph.

refractive

certainly

Bodian’s

index

correspond

pm.

Other

of the

gel

the

the

axons -

to reticubn

project

arrows]

with

fuzzy

the

or fibrillar

host

hydrogel

entered

be traced

zones

show

a

at the interface

have

cannot

of

hydrogel,

c, pGMA-MAA

sprouts

The arrowheads

section.

(pGMA-M/IA)

/long

Note

bioimplant.

interspaces

is m continuity

axonal fibres

of

(PTAHJ-stained into

of the biotmplant.

6). Numerous

regeneratmg

of tissue

in this case].

surface

sod fhat

gradients

(pGMA30

extensively

framework

the limits

after implantation

month showrng

the

have

Interlaced

The arrows

could

into the polymer

methacrylate)-methacryhc

(arrow)

that

sect/on

acid

processes

(sample

tissue.

of the neural3

blue-stained

deeply

phosphotungsoc

poly(glyceryl

thin capsule;

of the react/on

a, Coomassie

that extended

broimplant

projections:

interface:

-,

the

capsule.

Ingrowth:

Astrocyte

et a/.

to astrocytic

Tissue

Capsule:

S. Wow/y

showing

processes

hydrogel.

scar at the interface

hydrogels:

methacrylate)-

and

under

with

a pGMA30

synthetic

showed differences in the reaction of the elements of the neural tissue. Compared with hydrogel, charged hydrogels had a different reactivity of the host tissue. Although the gliosis

Histological

dffferent

stamed

of

These

viewed

with

of the gel or infiltrate

arrows)

accumulate. and

interaction

implantation.

section

corresponding

stained). fsmall

picrosirios

after

a poly(glyceryyl

(MJ and

cells

interspaces

month

on to the surface

weakly

which

which

the

pGMA30histological the neutral effect on the

to the

into

with

matrix

that

(arrowheads.

cavities

extracellular

tissue

3

(PTAHJ-stained

tissue

on the

the

because left

argyrophilic collagen.

upper

material nt, neural

method.

Biomatenals

199 1. Vol

12 March

201

Neural tissue j~taractjo~ with synthetic hydmgels: S. Woarly et al.

that developed in the vicinity of the implants seemed influenced by both charged hydrogels, it was obvious that pGMA30- had the strongest effect on reactive astrocytes. The hydrogel was extensively penetrated by a network of astrocytic processes so that it resembled a glial matrix that superimposed the microgeometry of the polymer network (Figure 6L)j. Interestingly, the astrocytic processes seemed to have extended upon newly formed collagen. On sections passing near to the brain-implant interface, polarization of astrocytic processes could be observed. For this series of hydrogels, axonal sprouts most often arranged in fascicules and showing swollen tips, were detected at the interface using Bodian’s method. The sprouts projected towards the marginal zone of the bioimplant. Although for both ionic hydrogels, cortical nerve fibres either packed or individual were seen entering the gel and growing inside the polymer network, axons projecting into the hydrogel were more numerous with negatively charged polymer (Figure 6~). However, it was difficult to evaluatethe exact extent to which they penetrated the matrix because of the variable refractive index of the gel.

DISCUSSION Preparation of hydraaels and effect of GdfUlA The method used in this work for the preparation of p(GMA)collagen gels yields the formation of an interpenetrating network composed of two cross-linked polymer networks, i.e. ~l~ethac~ate and collagen, topologicaliy independent. This material has therefore the m~hanicai stability of the synthetic polymeric matrix with the adhesivity and the biological acceptability of collagen. In contrast to EGDMA and GMA which have only a slight influence on the structure of hydrogels”,GdMAaffects the pore structure significantly. Moreover, tripling the concentration of GdMA increases dramatically the fractional porosity of pGMA and shifts the pore size distribution towards macroporous domains. This effect is most likely related to a decrease of the cross-linking density of pGMA, as has been shown in a previous study5 by hindering the formation of interchain bounds by the epoxide groups.

related to collapsed pores or non-permeable surface of the matrix.

pores at the

Effect of polar groups Compared with the reference matrix pGMA30, it is clear that charged hydrogels belonging to this series, i.e. pGMA + and pGMA_, have a potent effect on the reactivity of the neural tissue and on cellular organization. These copolymeric matrices acquire their charges by ionization of carboxylic acid groups and amino groups in contact with polar media, i.e. the introduced collagen solution and the interstitial fluids of the surgical lesion (Figure 7). However, 96% of carboxyl groups are ionized while less than 1% of amino groups become charged at physiological pH (values calculated from the constant of dissociation of a dilute solution of MAA and DMAEMA of known pH). Different mechanisms may operate for substrates with negative charges and can explain the strong reactivity of the glial cells. First, since the ionic conductivity of the gels is related to the level of charges, pGMA30is the material that has the highest ionic conductivity. Ionic currents generated in pGMA30and carried by highly mobile ions like H+, might have favoured migration and growth of cells towards the jmmobilized negative charges. Indeed, it is known that ionic currents generated by electrical field influence cell motility during morph~enesis and tissue regeneration”, ’ ‘. A second mechanism could be the buffering by Ca*’ that is released after cell injury. Since it is known that Ca2+ forms a stable complex with acid functions, the pGMA-Ma polymers can have reduced the amount of free CaZf and consequently reduced the extent of depolymerization of the cell cytoskeleton. Finally, the high level of charges in pGMABO_ might have two other consequences that could mediate tissue kinetics: the increase of the degree of swelling and the adsorption of blood proteins. The conversion of MAA to its carboxylate salt increases the swelling capacity of the gel12, making more pores open and available for tissue ingrowth. Adsorption of polymer surfaces with plasma proteins (e.g. fibronectin, thrombospondin,von Willebrand factor) might haveconferred bioadhesive properties on polymers. Moreover, the presence of MAA in hydrogels causes a great increase of protein adsorption13.

Effect of pore st~~ture Our findings show that the composition of polymers affect the reactivity of the neural tissue. The best biological properties were obtained with neutral and ionic pGMA30 gels for which we observed extensive tissue ingrowth and axonal regeneration. In accordance with other in viva studies involving non-neural tissue’, encapsulation and tissue ingrowth were found dependent upon the porosity of the polymer network. However, our study shows also that the pore size distribution of the inner structure of matrices is critical in initiating tissue ingrowth. For instance, the best tissue response was observed for matrices pGMA30 which have 50% of their volume occupied by pores larger than 10 orm. This conclusion is apparently inconsistent with the pGMA20-66 matrix which has a pore distribution similar to that of pGMA30, but for which tissue penetration remains limited. One possible explanation is that the lowering of the cross-linking density of the polymer network by GdMA certainly diminished the polymer surface area available for cell growth. However, the degree of tissue penetration can also be

202

Biomaterials

199 1, Voi 12 March

0 0 MAA

M%@J +

MAA

GM/j

N(CH&H +

I

DMAEMA

DMAEMA

MAA

Gj$A I

I

N(CH~~H+ @ DMAEMA

@ mobile ions @

fixed charges

Figure 7 Schematic representation of cherged hydrogets. The polymers acquire their charges by ionization of carboxy! and amino groups.

Neurai tissue in?eractfon with synthetic hydrogefs: S. Wuerly et al.

Because of the low level of charges, pGMA + behaves as a neutral polymer with a lower ionic conductivity and this can explain the weak penetration of tissue into this matrix. Nevertheless, some growth of tissue occurred and this could be modulated by different mechanisms: mechanical guidance or the adsorption of amphoteric molecules that might create a bridge between the polymer surfaces and the negatively charged cell surface. However, in all cases, the co-network of collagen introduced into pGMA polymers was essential in initiating tissue ingrowth as shown previously4, and this is consistent with in vitro studies which have shown that the adhesion of cells to synthetic substrates is promoted by preadsorption of surfaces with bioadhesive proteins14. It is most likely that the growth of nerve fibres is a consequence of the growth of astrocytes into the matrix and not the result of a direct effect of implanted synthetic substrates. Astrocytic processes, together with newly deposited ECM macromoi~ules, may have provided appropriate pathways for the growth of axons. Supporting this, is the importance of neuroglial interactions in determining the outcome of neuroregenerative processes in vivo ’ 5,” and in vitro ’ ’ . In conclusion, among pGMA hydrogels tested, pGMA30 is the most bioactive material. Furthermore, the introduction of negative charges potentiates tissue interactions and the importance of anionic groups in adhesion mechanisms has already been reported’*.

Synthetic polymeric matrix as artificial

ECIW

Studies point to the importance of an orderly cellular microenvironment in the lesion for axonal regeneration’6,‘g. 4xonal regeneration in lower vertebrates is a natural successful process but is dependent upon the establishment of cellular-extracellular substructures at the site of injury*‘. In mammals, regeneration of severed axons is not naturally successful but experimentally can be promoted by the availability of glial substrates*‘, **. In this context, introducing synthetic porous matrices into surgical lesions of the CNS tissue aims at diverting the kinetics of wound healing in a direction that promotes migration and growth of cells into the lesion and the spatial organization of collagen. As a result, the matrix is progressively transformed (figure 81, and a three-dimensional cellular-extracellular matrix establishes in the implantation site. However, an important process, essential for the adhesion and growth of cells onto synthetic substrates, is the interface conversion23 by which the polymer surface acquires physicochemical properties that promote the initial attachment of cells. This involves the adsorption of plasma proteins, the reinforcement of bioadhesive properties by the deposition of newly formed extracellular matrix compounds and, in the case of charged surfaces, the conversion of acid or basic residues to their respective salts.

ACKNOWLEDGEMENTS

--+

REFERENCES 1

2

3

4

8 9

10

11 12

13

14

15

16

17

18

19

20

We would like to express our thanks to Dr R. Guidoin of the laboratory of biomaterials for the use of the scanning electron syntheticpolymeric matrix(protomatrix)

microscope. We thank MS Louise Bertrand and Nicole Massicotte for their technical assistance. This work was supported by the Medical Research Council of Canada and the Fonds de la recherche en Sante du Quebec.

reorganization of the gliosis and scaning

-+

tissue matrix

Figure 8 The fate of synthetic polymeric matrices in conracr wirh the neural rlssue.

21

22

23

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