lntracerebral Implantation ofHydrogel-Coupled …downloads.hindawi.com/journals/np/1995/854291.pdf(ATR-FTIR) Infrared (i.r.) spectra were recorded with a NicoletMagna-550Fouriertransforminfrared

(C) Freund Publishing House Ltd., 1995

lntracerebral Implantation of Hydrogel-Coupled Adhesion Peptides:Tissue Reaction

S. Woerly1, G. Laroche1, R. Marchand2, J. Pato3, V. Subr4 and K. Ulbrich4

Qubec Biomaterials Insitute, H6pital Saint-Franqois d’Assise, Quebec, CanadaCentre de Recherche en Neurobiologie, H6pital de l’Enfant-Jdsus, Qud.bee, Canada

Central Research Instituteror Chemistry, Budapest, HungaryaM Insttute ofMacromolecular Chemistry, Cech Academy ofSciences, Prague, Cech RepubBc

SUMMARY KEY WORDS

Arg-Gly-Asp peptides (RGD) were synthe-sized and chemically coupled to the bulk of N-(2-hydroxypropyl) methacrylamide-based polymerhydrogels. Fourier Transform Infrared Spectro-scopy (FFIR) and amino acid analysis confirmedthe peptide coupling to the polymer. Activatedand control (unmodified) polymer matrices werestereotaxicaHy implanted in the striata of ratbrains, and two months later the brains wereprocessed for immunohistochemistry using anti-bodies for glial acidic fibrillary protein (GFAP),laminin and neurof’daments. RGD-containingpolymer matrices promoted stronger adhesion tothe host tissue than the unmodified polymermatrices. In addition, the RGD-grafted polymerimplants promoted and supported the growthand spread of GFAP-positive gliai tissue ontoand into the hydrogels. Neurof’dament-positivefibers were also seen running along the surfaceof the polymer and, in some instances, pene-trating the matrix. These findings are discussedin the context of using bioactive polymers as anew approach for promoting tissue repair andaxonal regeneration of damaged structures ofthe central nervous system.

Reprint address:Dr. S. WoerlyInstitut des Biomat6riauxHfpital Saint-Franqois d’Assise10 rue de l’EspinayQu6bec G1L 3L5, Canada

hydrogels, peptides,regeneration, plasticity

intracerebral implantation,

INTRODUCTION

Fetal neural gratis, when transplanted intodamaged regions of the central nervous system(CNS), can compensate for tissue damage and pro-mote reorganization of the neural circuit. Alterna-tively, a synthetic polymer scaffold may provide anextracellular matrix (ECM) that assists the course oftissue healing so that new tissue can be constitutedwith the CNS’s own tissue constituents, enablingaxonal reg.eneration to occur/27/. The combinationofthese two approaches, namely entrapment of fetalgrafts within a polymer scaffold, is attractivebecause it may allow us to produce cell-based poly-mer hybrid devices that associate properties ofbiocompatible polymers with those of physiolo-gically active cells for replacement of tissue massand ftmction /29/. However, one critical issue in thedesign ofpolymer matrices for brain tissue implant-ation is the properties of the polymer surface thatinterfaces with the host tissue, as these determinethe type of tissue response and the fate of the poly-mer matrix in the host. The polymer surface can betailored with biomolecular cues that may optimizethe ftmction of the implanted polymer matrices bypromoting a specific response from host cells, suchas selective binding and axonal regeneration. In thisrespect, short peptides containing the Arg-Gly-Asp(RGD) sequences found in many proteins of ECMand which are related to cell-binding and cell-ECMrecognition processes have incited a great deal ofinterest because they provide a unique model for the

VOLUME 5, NO. 4, 1995 245

246 S. WOERLY ET AL.

study of cell receptor-ligand interactions in relationto biological activities. Such sequences can besynthesized and immobilized on a polymer surface,thereby reproducing the chemical environment ofECM. Thus, polymer substrates grafted withbioactive peptides have been shown to supportattachment and spreading of a variety of cells/4/and neurite outgrowth was shown to occur onlaminin oligopeptide immobilized surfaces/3/.

In the present study synthetic hydrogels weremodified by covalently coupling the tripeptide Arg-Gly-Asp to the polymer network of synthetic hydro-gels. RGD peptide sequences are specifically recog-nized by integrin cell surface receptors/20,30/andconstitute a recognition system which is involved incell adhesion, migration and growth during tissueremodelling and regeneration /20/. RGD-adhesionreceptor systems have been shown to be expressedby neural and glial cells/3,13,16,26/, and it was ex-pected that RGD-coupled polymer matrices wouldbe able to induce a different tissue reaction to thatwhich follows injury by promoting tissue adhesionand cell growth, and thus form a solid integration ofthe polymer matrix to the host tissue. RGD peptideswere chemically attached to the bulk of N-(2-hy-droxypropyl) methacrylamide-based hydrogels usingglycylglycine spacers. Such bioactive polymer hy-drogels were implanted into the striata of adult ratbrains and the tissue reaction was examined twomonths later using standard immunocytochemicalmethods. The potential of bioactive polymer ma-trices as brain tissue implants is discussed in thecontext of the emerging field of neural tissue en-gineering as a new method of repairing structuralbrain damage and promoting axonal regeneration.

MATERIAL AND METHODS

RGD peptide synthesis

The protected amino acids, Asp dibenzylester,Boc-Gly, and carbobenzyloxy nitro-arginine wereprepared according to standard methods. The pep-tide coupling (BocGlyAspOBzl, ZNO2ArgGlyAspOBzl) was carried out using the standard solutionphase method with isobutyl chloroformate (ZNO2ArgGlyAspOBzl). Removing the Z and benzylprotecting groups of fully protected peptides was

achieved by catalytic hydrogenation and the Bocgroup was removed by HC1 in ethylacetate. The freepeptide was purified on a Sephadex G-25 column.The RGD fraction was collected and freeze-dried.

Synthesis ofHPMA copolymers

N-(2-hydroxypropyl) methacrylamide [1] andmethacryloylglycylglycine p-nitrophenyl ester (Ma-GlyGlyONp) [2] were prepared according topreviously described procedures /16,22/. Othermonomers- 2-hydroxyethyl methacrylate [3] andtriethyleneglycol dimethacrylate [4] from Rohmand Haase (Germany), were purified by vacuumdistillation. The synthesis of the coupling reactionsand the synthesis of the activated gels are shown inFig. 1.

Preparation of methacryloylglycylglycylargynylglycyl-aspartic acid [5]

The synthesis of [5] involved the aminolysis of[2] with the aliphatic NH2 group of Arg /18/.MaGlyGlyONp (0.04 g, 1.2 x 10 mol)wasdissolved in dry DMSO (0.5 ml) and the solutionwas purged of oxygen with nitrogen. Undercontinuous stirring, RGD (0.05 g, 1.4 x 10-4 mol)was added to [2]. Afterwards 1 h triethylamine(0.023 g, 2.3 x 10"4 mol) was added to the reactionmixture in equimolar ratio to ONp ester. Thereaction was carried out overnight at roomtemperature. After the reaction octylpyrocatechin asa polymerization inhibitor was added and DMSOwas removed using a rotatory vacuum evaporator at40C. The oil residue was triturated with drydiethylether (6 times 5 ml). The white precipitatewas filtered, washed with diethylether and driedunder vacuum. The crude product was used forpolymerization without further purification.

Synthesis ofhydrogel containingRGD

Hydrogels were synthesized by radical cross-linking polymerization in DMSO at 60C for 24 husing AIBN as an initiator (0.6% wt/wt). The poly-merization mixture was purged with nitrogen for 3min. The reaction was carried out between twoTeflon plates connected by silicone rubber sealingand thermostated. The concentration of monomers[1], [3], [4] was 14.8: 22.2:1.4 wt% and the

JOURNAL OF NEURAL TRANSPLANTATION & PLASTICITY

NEURAL TISSUE INTERACTION WITH RGD PEPTIDES 247

CH3

H2C C- CO- (NHCH2 CO)2- O -(-NO2

NH2C=NH

NH COOH

(CH2) CH2

H2N CH CO Nil CH2 CO NH CH COOH

[2] [RGD]

DMSO, Et3N

NH2

C NH

NH COOH

CH3 (CH2)3 CH2- CH2 C CO (Nil CH2 CO)2 NH CH CO NH CH2 CO NH CH COOH

[5]

CHCH C +

C=O

O

crt2CH

0

0

12--0

CH C

CH

[4]

CH

CH C +

C=O

CH2

CHOH

CH

Ill

CH3 CH

CH2 C + CH2 C

C=O C--O

oNil

CH2CH2

CH2OHC=O

[31

CH2

C=O

IICH-- (CH2)-- Nil--C--NH2

C=O

CH

CO

CH CH2- COOH

COOH

[51

DMSO, AIBN

60C, 24h.

CH3 CH CH CHi

C=O C=O C=O C--O

CH

CH

cnCi-l

0

cn0

12-0

CH

Gly NH O

CHGly CH2

CH2OHrg I CHOH[;ly CH3

Fig. 1: Reaction schemes for the synthesis of RGD-grafted hydrogels.

VOLUME 5, NO. 4, 1995


concentration of [5] was 1.7 wt% (gel 1) and 1.18wt% (gel 2). After polymerization the gels werewashed in ethanol and swollen in ethanol/water (1:1v/v). The thickness ofthe gels was 1.5 mm.

Amino acid analysis

Amino acid analysis was carried out on anAmino Acid Analyser (LDC Analytical, USA) witha reverse phase column C18 (250 x 4 mm) using o-phthaldialdehyde (OPA) precolumn derivatization.A fluorescence detector Fluoro-Monitor 4100(LDC Analytical) was used for detection (Ex 330nm, Em 450 nm). The mobile phase consisted of amixture ofbuffer A (0.05M sodium acetate pH 6.5)and buffer B (methanol: 0.1M sodium acetate; 1:1)with a gradient of concentration increasing from 0%B to 100% B within 45 min at a flow rate of 0.5

The hydrogel samples (free of low molecularweight impurities) were hydrolyzed with 200tl 6NHC1 at 115C for 16 h. The hydrolyzate was driedin vacuo over NaOH, dissolved in distilled waterand, after OPA dedvatization, 20 tl was used forinjection.

Attenuated total reflection-Fourier transform infrared(ATR-FTIR)

Infrared (i.r.) spectra were recorded with aNicolet Magna-550 Fourier transform infrared spec-trometer with a DTGS detector and a germaniumcoated KBr beamsplitter. Two hundred and fiftyscans were acquired with an optical retardation of0.25 cm, triangularly apodized and Fourier trans-formed to yield a 4 cm1 resolution. The attenuatedtotal reflectance (ATR) mode was used for re-cording the infrared spectra ofthe hydrogel sampleswith a Split Pea attachment (Harrick ScientificCorporation) equipped with a Si hemispherical, 3mm diameter internal reflection element (IRE). TheIRE was beveled on the edge of its flat surface toprovide a sampling area slightly larger than the 150-200 tm diameter hot spot on the crystal.

Implantation and tissue processing

Twelve young Sprague-Dawley rats (250 g)were used as recipients. Rats were anesthesized byan intramuscular injection (1 mg/kg) of a solution

comaining ketamine hydrocholoride (87 mg/ml) andxylazine (13 mg/ml), and placed in a stereotaxichead frame. Under a surgical microscope, a smallcraniotomy was made over the let parietal cortex.Hydrogels were cut into 2 x 1.5 mm size pieceswhich were implanted into the striatum of the ratbrain using the following coordinates: bregma -0.8;lateral 3.6; vertical 5.2 (Paxinos G. and Watson C.).First, a channel was created through the cortex upto the stereotaxic point using a capillary glass tubewith an opening of 1.5 mm. The polymer implantwas then slowly inserted into the preformed channelusing the glass tube. Of the host rats, two receivedunmodified HPMA polymer and two were shamoperated.

Two months after implantation, the animals weredeeply anesthetized and perfused with PBScontaining heparin, followed by 4% paraformal-dehyde in PBS. Brains were post-fixed overnight,the implant area blocked out and transferred in 30%buffered sucrose. Coronal brain sections (40 tm)were cut with a cryostat and processed usingstandard procedures for indirect immunohisto-chemistry. The following antibodies were used: glialfibrillary acidic protein (GFAP, Dako), 200-kDaneurofilaments (NF, Sigma) and laminin (LN,Sigma). Sections were preincubated for 30 minuteswith 10% normal goat serum in DPBS (Dulbecco)and then with the above antibodies diluted in DPBScontaining 0.1% bovine serum albumin (BSA,Sigma) and 0.2% Triton at the followingconcentrations: GFAP (1:200), LN (1:200) and NF(1:64). GFAP and LN were incubated with thetissue overnight at room temperature and underagitation, while NF was incubated for 48 h at 4C.After washing in DPBS (3x10 minutes), sectionswere incubated for 2 hours with a sheep anti-mouseFITC (Sigma) or with RITC (Dako), at a dilution of1:100 in 0.1% BSA/DPBS/0.2% Triton. Sectionswere then washed in DPBS for 3x10 minutes,mounted onto slides, and coverslipped in a mixtureof glycerol-water. They were then examined on aNikon fluorescent microscope. Additional sectionswere stained with hematoxylin-eosin (H&E). Tovisualize the gel implants, sections were observedon an Olympus inverted microscope equipped witha Hoffman modulation contrast system.


NELrRAL TISSUE INTERACTION WITH RGD PEPTIDES 249

RESULTS

Amino acid analysis

Amino acid analysis showed that RGD wassuccessfully incorporated in the bulk of hydrogelsvia methacryloylglycylglycyl spacers. The amotmt ofArg and Asp in the hydrolyzate was used to cal-culate the amount of RGD incorporated into thehydrogels. Calibration curves (concentration versusarea of the peak) obtained for arginine and tyrosinestandards were used to calculate the amount ofbothamino acids in the hydrolyzate. The amount ofpeptides was 0.5 tool% of MA-GlyGly-ArgGlyAsp(1.2% RGD) and 0.36 tool% (0.86% RGD) for gel1 and gel 2 respectively.

ATR-FT.IR spectroscopy

Figure 2 shows the FTIR spectra ofthe polymergel containing RGD (a) and an unmodified controlpolymer (b). Spectrum (a) is valid for gel 1 and gel2 since both polymer gels show spectra with verysimilar characteristics. This Figure shows that gel aand gel b exhibit very similar infrared features.However, the analysis of the infrared spectra of theindividual components ofthe hydrogels (not shown)demonstrates that the 747 cm1 feature is due to theHEMA spectral contribution. Thus, the RGDpeptide in the hydrogel should be detected bysubtracting the infrared spectra of the control poly-mer b from that ofthe polymer a, using the 747 cmpeak as an internal intensity standard. As a result,the subtraction spectrum (c) clearly exhibits (despitea poor signal to noise ratio due to the very lowconcentration of the peptide) features characteristicof RGD such as the carbonyl (C=O) and C-Ostretching modes vibration ofthe acidic fimctions ofArg at 1721 and 1160 cm respectively, and theamide I and amide II bands characteristic of thepeptide bonds at 1676 and 1577 cm, respectively.It may be noted that both spectra show very similarcharacteristics. Even though these data do notdemonstrate that the RGD is covalently bound tothe polymer gel, it is clear that the peptide issuccessfully coupled to the HEMA-HPMA copoly-mer.

747m1

AmideC=O

ooo doo ’oo .’oo ’oo ooo oo

Frequency (cml)ATR-FTIR spectra of: (a) polymer gel containingRGD peptide sequence; (b) unmodified polymer geland (c) subtraction spectrum of (a) minus (b) (seeresult for details).

Findings at autopsy

All animals that received polymer gel implantssurvived to the schedule date of sacrifice. Neitherneurological deficits nor abnormal behaviors wereobserved during the two-month survival period.

Histology

The hydrogels were readily recognized in thedorsal striatum on H&E-stained brain sections ob-served at low magnification. The gel implants wereencased in the host tissue without damage to theadjacent tissue (see Fig. 4B). In a few instances thepolymer gel penetrated the corpus callosum and the

VOLUME 5, NO. 4, 1995

250 S. W()ERLY ET AL.

adjacent layers of the cerebral cortex. In all casesthe implants were surrounded by a mild-to-densecelhflar reaction, mainly of glial origin as demon-strated by immunohistochemistry (vide infra). Highmagnification showed that cell spreading occurredin the marginal structures of the RGD-containingpolymer gel (Fig. 3), but cell spreading could alsohave extended deeper in the gel along the surface ofdefects as seen by immunocytochemistry (see Fig.5C). The unmodified polymer gels did not showtissue ingrowth and remained acellular.

Differences in tissue adhesiveness were foundbetween polymer implants containing RGD andunmodified polymer. Polymer gel containing pep-tides showed stronger adhesion to the host tissuethan the unmodified polymer as there was no orlittle partition between the gels and the host tissue.Control polymer gels had either lost contact at thepolymer-tissue interface or showed a discontinuousinterface. No evidence of polymer degradation ortoxicity was found, and the architecture of thestriatum in all cases appeared normal.

Immunocytochemistry

Immunocytochemical analysis of GFAP-stainedtissue sections revealed that both control andmodified gels were associated with low-gradegliosis in the surrounding host tissue when com-pared to control lesions. The gliosis had developed

P

Photomicrograph taken with Hoffman modulationoptics (HMO) of an H&E stained brain sectionafter implantation of a RGD-grafted polymer gel(P) showing the infiltration of the polymer gel byhost tissue cells (arrows). Bar: 100 pm

at and around the polymer-striatum interface andwas characterized by tightly packed GFAP-positiveastrocytes and processes. Control lesions and thewound created by the implantation needle were alsoassociated with intensely GFAP-positive astrocytes(e.g., Fig. 4A). Furthermore, the intensity of GFAP-immunostaining decreased away from the implant-ation site. However, there were differences in theorganization ofthe gliosis between the two types ofgel implants. The unmodified polymer gels showedmarked GFAP-immunostaining along the edgestructure while the bulk of the gels did not showany visible staining (Figs. 4A-4B). In contrast, pep-tide-grafted polymer implants were positivelyimmunostained for GFAP, but immunostaining wasseen only in localized regions of a gel section andcould not be seen through consecutive serialsections. Immtmolabelled cellular patterns could beobserved at the surface and within the gel byfocusing throughout the 40 pm-thick gel slice.GFAP immunofluorescence was comparable in in-tensity of the reaction to that of the astrocytes ofthe scar in the surrounding tissue or to that whichoccurred in control lesions. Reactive astrocyteslocated immediately adjacent to the polymer implantextended intensely stained GFAP-positive processesalong the surface and within the polymer implant(Fig. 5A), or formed a discrete tissue bridge incontact with the polymer gel from both sides of thehost tissue (Fig. 5B). Although the extent of im-munostaining was usually limited to the marginallayer of the gel, it reached the deeper layers of theimplant in instances where processes migrated alongthe surface of gel defects or cracks (Figs. 5C-5D).Results were similar within this group of animals,and there were no significant differences betweenthe gels containing different amounts ofpeptides.

Examination of brain sections immunostainedwith antibodies to neurofilaments revealed the ab-sence of fluorescence signal at the surface and with-in the control gel implants; positively immuno-stained neurofilaments were seen only along theedge of the gel implant and in the host striatum,which showed a typical patchy organization. Gelimplants containing RGD peptides were immuno-positive after treatment with neurofilament anti-bodies. Intensely immunostained nerve fibers ofstriatal origin and, when the polymer gel contacted

J()IJRNAL ()F NIURAL TRANSPLANTATI()N & PLASTICITY

NEIJRAI TISS[JE INTERACTION WITtt RGD PEPTIDES 251

Fig. 4: Brain section containing an unmodified control polymer gel after immunostaining with GFAP antibodies (A) and underHMO (B). (A) Low-magnification photomicrograph showing a marked zone of GFAP-positive astrocytes along thetranscortical implantation track (arrows) and at the dorsal aspect of the polymer implant (arrowheads), while theremainder of the gel implant is surrounded by a thin zone of reactive astrocytes. Note the absence of immunostaining ofthe gel. (B) High-magnification photomicrograph of the area indicated in (A), showing the close apposition between thepolymer gel (P) and the host striatum (St). Bars: A, 500/am; B, 10 lttm

Fluorescence photomicrographs of three different brain sections containing RGD-grafted polymer gels after stainingwith GFAP antibodies (A-C). (A) is the polymer implant/brain interface (arrowheads) showing spreading of reactiveastrocytes and their processes from the striatum (St) onto the polymer surface. (B) GFAP-positive astrocytic fibers havegrown in contact with the polymer surface (P) to form a bridge between the two interfaces of the host. (C) GFAP-positiveastrocytic fibers have spread along the surface of a crack in the polymer gel (P). (D) is identical to (C) under HMOshowing the defect in the structure of the polymer gel. Bars: 25 pm

VOLIJME 5, NO. 4, 1995


the adjacent cortex, of cortical origin were seenclearly growing onto and into the polymer gelsections. Figure 6 shows two examples of the hosttissue reaction a/ter neurofilament immunostaining.In one case, regeneration of long individual axonswas seen on a tissue section that included the entiresurface of the gel and showed the growth of nervefibers originating from the adjacent host tissue ontothe gd surface (Figs. 6A-6B). In other cases, thegels showed a punctuated fluorescence resemblingdensely associated axonal sprouts (Figs. 6C-6D).Nerve fibers could also be seen on the upper andlower surfaces of the gel section as they hadpenetrated the gel implant.

Laminin immunoreactivity was associated withproliferating blood vessels and was moderate in thevicinity of the implantation site while it was intensealong the wound of the needle track (Figs. 7A-7B).For the same survival period, the control lesionsthat included the cortex down to the dorsal striatumwere the site of dense scar tissue positively im-munostained for GFAP without evidence of axonalregeneration through the wound.

DISCUSSION

The main findings ofthis study are: (i) that RGDpeptides were successfully immobilized in HPMA-based polymer matrices via glycylglycine spacerarms. Amino acid analysis and ATR-FTIR con-firmed that the coupling of the peptide wasachieved; (ii) that RGD-grafted polymer substratesmediate a host tissue reaction involving glialproliferation and axonal regeneration.

The RGD peptide, a cell-adhesion sequencefound in a large number of extracellular matrixproteins/30/, is specifically recognized by integrincell surface receptors/5/. This recognition system isinvolved in cell-matrix and cell-cell attachments,spreading and growth/20/, and has been demon-strated to be expressed by astrocytes/26/ and byneuronal cells/19/. Therefore, in comparison withthe unmodified gel implants, it is reasonable to thinkthat the observed behavior of regenerating axonsand astrocytes on RGD-grafted polymer wasinfluenced by the immobilized peptides on the poly-mer surface. Interaction of reactive astrocytes withthe modified polymer implants may involve focal

contacts through the expression of integrin recep-tors, as has been shown in vitro /26/. Likewise,axonal regeneration on the surface of the substratemay occur through the expression of specific inte-grin receptors, which have been shown to beoverexpressed following injury /8/. Such inter-actions are highly specific; exogenous RGD peptideis able to inhibit neuronal attachment and growthwhen neuronal cells are plated onto RGD-containing protein fragments/13/. Although surfaceadsorption of plasma proteins (e.g., fibronectin,albumin) onto the polymers may favor cell attach-ment and growth, it is unlikely that this occurredhere, since the unmodified polymer gel did not sup-port tissue growth and spreading.

The observation that spreading of glial cells andaxonal growth occurred only in very localizedregions of implants suggests that the chemical andphysical characteristics of the polymer hydrogdsmay also be important in the interactions ofthe hostcells with the polymer. For instance, the limitedgrowth of cells and processes onto the polymer mayresult from the dependency that cells with RGD-grafted polymer have on some complex factors suchas peptide surface density, expression of the RGDrecognition integrin receptors, spatial conformationand accessibility of the peptide. Thus, we canspeculate that the density of immobilized RGDpeptides was non-uniform throughout the gel ma-trix, while interactions resulting in successful tissueproliferation would be dependent on a thresholdvalue of RGD concentration, as has been shownwith other cell-type cultures /6,11/. Also, thestructure of the gels may have influenced tissuegrowth; a micro- or non-porous surface induces theformation of a tissue scar that further impedes thegrowth of nerve fibers, while a porous structurefacilitates tissue ingrowth and organization/27/.

There are several lines of evidence that suggestthat inhibition of axonal regeneration in the centralnervous system may be the result of the effect ofdifferent factors: absence of appropriate substrate-bound neuroregenerative molecular cues/9/, form-ation of a glial scar/17/and presence of growth-inhibitory molecules from central myelin /23/ orfrom extracellular matrix/1,12/. Our results showthe importance ofgrowth-promoting molecular cuesfor sustained axonal regeneration and, at the same


NEIJRAL TISSUE INTI’;RACTION WITtt RGI) PEPTII)ES 253

Fluorescence photographs of brain sections including RGD-grafted polymer gels after immunostaining with anti-neurofilament antibodies. (A) Numerous regenerating axons of striatal origin are seen coursing onto the surface of thegel, covering almost the entire surface, while other axons seem to have penetrated the polymer matrix (arrows). (B) Aview of the same section under HMO; the limit of the gel implant (arrowheads) can be seen to merge with the braintissue. (C) High-magnification photomicrograph showing punctuated immunostaining of axons at the interface with thepolymer gel. This view corresponds to the region depicted by the square in (D), at lower magnification under HMOwhich shows the polymer gel (P) in contact with the striatum (St). Bars: A and B, 50 lam; C, 15 lam: D, 75 lam

Fig. 7: (A) Brain tissue section through the striatum (St) under HMO showing structure details of the interface with the polymerimplant (P). (B) Same view after laminin immunostaining showing numerous stained blood vessels and theimmunopositive interface (arrowheads). Bars: 20

VOLUME 5, NO. 4, 1995


time, suggest that if such cues are available at thesite of injury, reactive gliosis which occurs at theinterface might not impede axonal regeneration.This hypothesis is in agreement with the currentcontroversy concerning the traditional view of theglial scar as a mechanical barrier to axonal growthand a more modem view of astrocyte’s modulatoryresponse to injury /17/. On the other hand, it isknown that reactive astrocytes do not supportneuronal attachment or growth ofneurites/21/.

Therefore, regeneration in our model is due topositive interactions of growing nerve fibers withthe RGD-bound substrate, and this suggests thatimmobilized RGD peptides provide a strong enoughstimulatory effect to overcome the inhibitoryinfluence of the astrocyte scar and related mole-cules. We propose that regeneration of centralaxons can be induced either by suppressing inhi-bitory factors to regeneration as previously demon-strated/22/or, as demonstrated here, by providinga substrate with neuroregeneration molecular cues.

This study has shown that it is feasible to modifybrain healing, particularly the astrocytic reaction,and to promote axonal regeneration by means ofpolymer matrices into which bioactive peptides havebeen integrated. Although this study has shown thatsubstrate-bound RGD favors axonal regeneration,other peptides (natural or synthetic) have beenidentified as interacting specifically with neuronalcells to promote regeneration. Thus, laminin penta-peptides of various sizes have been shown to sup-port cell attachment and axonal growth /7,10,15,25/. Taken together, these results allow us toenvisage the possibility of engineering healing in theCNS in a direction that would lead to tissuerestructuring at the wound site and axonal regen-eration. This could be achieved by providing a well-controlled molecular environment that mediates thecellular and molecular mechanisms that are requiredfor tissue regeneration and organization/28/. How-ever, if we consider the diversity of receptors in-volved in cellular interactions with substrate-boundmolecules and the various modes ofthese molecularinteractions that occur during regeneration anddevelopment, engineering brain wound healing andregeneration will probably imply the binding ofseveral peptides of various sequences and sizes toelicit specific cell interactions/2/.

ACKNOWLEDGEMENTS

This work was supported by the MedicalResearch Council of Canada and the Fond de laRecherce en Sant du Quebec.

10.

11.

12.

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