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Biomaterials 27 (2006) 3560–3569

www.elsevier.com/locate/biomaterials

The promotion of oriented axonal regrowth in the injured spinal cord byalginate-based anisotropic capillary hydrogels

Peter Pranga, Rainer Mullerb, Ahmed Eljaouharib, Klaus Heckmannb, Werner Kunzb,Thomas Weberc, Cornelius Faberc, Maurice Vroemena, Ulrich Bogdahna, Norbert Weidnera,�

aDepartment of Neurology, University of Regensburg, Universitatsstr. 84, 93053 Regensburg, GermanybInstitute of Physical and Theoretical Chemistry, University of Regensburg, Universitatsstr. 31, 93053 Regensburg, Germany

cDepartment of Physics, EP5 (Biophysics), University of Wurzburg, Am Hubland, 97074 Wurzburg, Germany

Received 25 October 2005; accepted 30 January 2006

Available online 28 February 2006

Abstract

Appropriate target reinnervation and functional recovery after spinal cord injury depend on longitudinally directed regrowth of

transected axons. To assess the capacity to promote directed axon regeneration, alginate-based highly anisotropic capillary hydrogels

(ACH) were introduced into an axon outgrowth assay in vitro and adult rat spinal cord lesions in vivo. In an entorhino-hippocampal

slice culture model, alginate-based scaffolds elicit highly oriented linear axon regrowth and appropriate target neuron reinnervation.

Coating of alginate-based ACH with the extracellular matrix components collagen, fibronectin, poly L-ornithine and laminin did not

alter the axon regrowth response as compared to uncoated alginate-based ACH. After implantation into acute cervical spinal cord

lesions in adult rats, alginate-based ACH integrate into the spinal cord parenchyma without major inflammatory responses, maintain

their anisotropic structure and in parallel to findings in vitro induce directed axon regeneration across the artificial scaffold.

Furthermore, adult neural progenitor cells (NPC), which have been shown to promote cell-contact-mediated axon regeneration, can be

seeded into alginate-based ACH as a prerequisite to further improve the regenerative capacity of these artificial growth supportive

matrices. Thus, alginate-based ACH represent a promising strategy to induce directed nerve regrowth following spinal cord injury.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Self-assembly; Stem cell; Nerve regeneration; Nerve tissue engineering; Hydrogel; Alginate

1. Introduction

Approximately 10,000 mostly young individuals eachyear suffer acute spinal cord injury in the United States [1],resulting in permanent and severe disability. Currently nosufficient regenerative therapeutic strategy exists for thisdisease. The injured spinal cord lacks the intrinsic capacityto replace organotypic tissue, which, besides expression ofgrowth inhibitory factors and lack of growth promotingfactors, represents the major factor contributing to thefailure of central nervous system (CNS) axons to regener-ate [2,3]. Substantial progress in promoting axonalregeneration has been achieved through cell transplanta-

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.01.053

ing author. Tel.: +49 941 941 3031; fax: +49 941 941 3005.

ess: norbert.weidner@klinik.uni-regensburg.de

tion strategies. Specific primary cell populations replacelost spinal cord parenchyma [4–6]. In particular, adultneural progenitor cells (NPC) allow organotypic tissuereplacement and build a cellular scaffold for injured axonsto regenerate on [7–9]. However, in all of these strategiesaxon regeneration does not occur in a directed rostro-caudal fashion, preventing reconnecting of disrupted axonpathways with their target neurons located caudal to thespinal cord lesion site. Therefore, the injured spinal cordrequires a regrowth directing structured scaffold, whichshould also allow the integration of growth promoting cellpopulations.Numerous natural (e.g. collagen, agarose, alginates) and

synthetic polymers (e.g. poly a-hydroxy acids, polyvi-nylchloride) are candidates for nerve repair strategies[10,11]. However, most of these substrates do not permitlongitudinally oriented axon regrowth, since they are

ARTICLE IN PRESSP. Prang et al. / Biomaterials 27 (2006) 3560–3569 3561

characterized by a more or less amorphous and irregularthree-dimensional (3D) structure.

Alginic acid is a chain-forming polysaccharide, whichcan be cross-linked by complexation of its carboxylicgroups with many multivalent cations such as Cu2+, Ca2+

or Al3+, thus producing mechanically stable hydrogels.Calcium alginates represent biocompatible and non-im-munogenic polymers, which have been used as scaffoldmaterial for tissue engineering and transplantation of cells[12]. Studies analyzing non-structured alginate-based sub-strates under cell culture conditions or after transplanta-tion into the mammalian CNS demonstrated that alginatesare biodegradable without causing allergic/inflammatoryreactions and promote limited axonal regeneration [13,14].However, axonal regrowth rarely occurs in a longitudinallyoriented fashion, thus preventing reconnection with thecaudal spinal cord.

Thiele described already in 1967 that anisotropiccapillary hydrogels (ACH) are formed when an aqueoussolution of sodium alginate and a solution containingmultivalent cations are superimposed in layers under theassumption that convection is avoided [15]. After forma-tion of a membrane-like boundary between the two liquids,which is consisting of non-soluble, ionically cross-linkedalginate, oriented diffusion of the electrolyte ions into thesolution of the polymer follows, which causes continuousgel formation. A dissipative convective process resultingfrom opposing diffusion gradients and friction of thepolyelectrolyte chains is the reason that the ongoingprecipitation results in an almost hexagonally structuredanisotropic capillary gel [16]. The walls of the capillariesconsist of the precipitated metal alginate and their luminacontain the water extruded during precipitation.

In the current study, we assessed the capacity of ACH topromote directed axonal regrowth in the injured mamma-lian CNS. The entorhino-hippocampal slice culture wasemployed first (a) to analyze the capacity of ACH topromote oriented CNS axon regrowth in vitro and (b) toscreen the optimal preparation of ACH (introduction ofvarious extracellular matrix molecules (ECM)) for subse-quent in vivo experiments. Based on these in vitro results,ACH implants were investigated in an established ratmodel of spinal cord injury (wire knife dorsal columntransection). To determine whether ACH can be colonizedwith growth permissive cells, NPC were seeded intoalginate gels.

2. Material and methods

2.1. Animals

For all in vivo experiments, adult female Fischer 344 rats (160–180 g)

were used. All experiments were carried out in accordance with the

institutional guidelines for animal care. All efforts were made to minimize

the number of animals used. Animals had ad libidum access to food and

water throughout the study. All surgical procedures were performed under

anesthesia with a combination of ketamine (62.5mg/kg body weight;

WDT, Germany), xylazine (3.175mg/kg body weight; WDT) and

acepromazine (0.625mg/kg body weight, Sanofi-Ceva, Germany) in sterile

0.9% saline solution.

2.2. Preparation of alginate-based ACH

Alginic acid sodium salt (Manugels DJX; Kelco Int. Ltd., England)

with an average molecular weight of 100,000 g/mol was used. The content

of guluronic acid moieties was about 70%, polyguluronic acid sequences

were 50–55%, polymanuronic acid sequences were 10–14%, and alternat-

ing sequences were 31–40%. The sodium alginate was dissolved in

deionized water, which was purified by ultrafiltration (Millipore, France)

at a concentration of 2% and subsequently filtered through a 0.2mm pore

size filter (Sartorius, Germany). Sixty millimeters of the sodium alginate

solution were placed in a cylindrical aluminum mold (5 cm in diameter)

and carefully superimposed with 20ml of a 1mol/l copper nitrate solution

(VWR International, Germany) using pump spray bottles (VWR). The

filled mold was covered with a vaulted glass bowl and the gel was allowed

to form for 24 h at room temperature avoiding concussion of the mold.

The obtained gel bodies were cut perpendicular to the longitudinal axis of

the capillaries using a custom-made cutting machine. Gel bodies were

mounted onto a slide, which was adjustable in relation to the blade. After

removing the non-structured top layer of the gel (2mm) two 1 cm thick

slices were prepared. For chemical cross-linking, gel slices were dehydrated

in pure acetone several times and then soaked in a 10% hexamethylene

diisocyanate solution (VWR) in pure acetone for 12 h at room

temperature. After removing excess cross-linking solution, the gel slices

were heated for 20min in 70 1C water. Fixed gel slices were rinsed

in a hydrochloric acid solution (1mol/l) several times to exchange copper

ions for protons. The metal free state was determined by emission

spectroscopy.

Gels were cut into defined dimensions (average width�height� length,

3� 0.3� 0.5mm for entorhino-hippocampal slice cultures; 0.5� 0.5�

1mm for spinal cord implants) on a vibratome (Leica). ACH were

sterilized by incubation in 70% alcohol for 5min and finally kept in sterile

0.1M phosphate-buffered saline (PBS; pH 7.4). Adsorption of ECM on the

capillary walls was achieved by immersing the cross-linked alginate-based

scaffolds in solutions of laminin (5mg/ml; Sigma), collagen (50 mg/ml; Pan

Biotech, Germany), fibronectin (10mg/ml; Sigma), and poly L-ornithine

(250mg/ml; Sigma) for 12 h.

2.3. Preparation of adult NPC

For the isolation of NPC, adult green fluorescent protein (GFP)

transgenic Sprague–Dawley rats [17] were deeply anesthetized as described

above and killed by decapitation. NPC were isolated from the cervical

spinal cord as described before [8]. Briefly, the cervical spinal cord was

dissected out, minced and washed in sterile Dulbecco’s PBS/D-glucose

(PAA Laboratories, Austria) followed by digestion in a solution of papain

(0.01%; Worthington Biochemicals, USA), neutral protease (0.1%;

Roche, Germany), DNase I (0.01%; Worthington Biochemicals) and

12.4mM MgSO4, dissolved in Hank’s balanced salt solution (HBSS; PAA

Laboratories). The resulting cell suspension was plated into culture dishes

in serum-free growth medium consisting of Neurobasal medium with B27

supplement (Gibco) and 20 ng/ml recombinant human FGF-2 (R&D

System, Germany). Cells were grown as neurospheres for 2–3 passages in

uncoated culture flasks. Thereafter, the medium containing the neuro-

spheres was centrifuged and resuspended in 1ml of Accutase (Innovative

Cell Tech, USA). The resulting single cell suspensions were seeded at a

density of 3000 cells/cm2 in fresh growth medium. For the consecutive 2–3

passages, NPC were grown as monolayers in culture flasks coated with

poly L-ornithine (20mg/cm2) and laminin (0.4mg/cm2). Monolayer

cultures were detached by incubation with 40 ml/cm2 Accutase, centrifuged

and resuspended in fresh growth medium.

A sample of single cell suspensions was stained with Trypan Blue

(Sigma) and counted using a Neubauer hemocytometer. The remaining

single cell suspension was washed twice and resuspended in PBS to yield a

final concentration of 1� 105 cells/ml. For seeding, a volume of 1 ml cell

ARTICLE IN PRESSP. Prang et al. / Biomaterials 27 (2006) 3560–35693562

suspension was applied onto ACH (capillaries oriented vertically), which

were placed on Millicell-CM membranes (Millipore, France). NPC-seeded

ACH were incubated in a humidified atmosphere with 5% CO2 at 37 1C in

serum-free growth medium (as described above). After 7 days of

incubation, NPC-seeded ACH were inserted into lesioned entorhino-

hippocampal slice cultures.

2.4. Entorhino-hippocampal slice cultures

Slice cultures were prepared from postnatal day 2–4 Fischer rat brain

according to previously published protocols [18,19]. In brief, after

decapitation of the animals, hippocampi with the attached entorhinal

cortex were dissected in ice-cold preparation medium (minimal essential

medium, MEM; Gibco) containing 2mM glutamax adjusted to pH 7.3.

Transverse slices of 400mm were cut using a tissue cutter (McIllwain).

Each postnatal rat brain yielded between 6 and 8 entorhino-hippocampal

slices, which were evenly distributed as individual samples across the

different experimental conditions (see below). Entorhino-hippocampal

slices were completely transected from the rhinal fissure to the

hippocampal fissure using a sterile scalpel blade as described [20,21] and

transferred onto Millicell-CM membranes. Defined blocks of ACH were

inserted in between the entorhinal cortex and the hippocampus followed

by incubation for 7 days in a humidified atmosphere with 5% CO2 at

37 1C. The medium contained 50% MEM, 25% basal medium eagle, 25%

heat-inactivated normal horse serum, 2.5% HEPES buffer solution,

0.65% glucose, and 2mM glutamax (all Gibco) adjusted to pH 7.2.

The entorhino-hippocampal projections were specifically labeled with

the anterogradely transported fluorophor coupled dextran Microruby

(Molecular Probes, Netherlands). Using a glass pipette, a few crystals of

Microruby were placed onto the entorhinal cortex. The slices were

incubated again for 6–7 days before processing.

2.5. Morphological analysis of slice cultures

For analysis of entorhino-hippocampal slice cultures, slices were fixed

in 4% paraformaldehyde in PBS for 2 h at room temperature and

incubated in blocking solution containing Tris-buffered saline (TBS; pH

7.4), 0.5% Triton X-100 (Sigma), 1% bovine serum albumin (Biomol) and

0.2% fish skin gelatin (Sigma). Double/triple labeling immunofluorescence

techniques were performed to assess axon regrowth into ACH, target

reinnervation, identification of seeded adult NPC and their interaction

with transected hippocampal projections. The following primary anti-

bodies were used: mouse anti-GAP-43 (for regenerating axons; Chemicon,

USA; at 1/100), rabbit anti-GFP (for seeded GFP-transgenic NPC;

Molecular Probes; at 1/1000) and mouse anti-NeuN (for neurons;

Chemicon; at 1/500). Sections were incubated in TBS+3% donkey

serum+0.1% Triton-X for 1 h, then transferred into the first primary

antibody and incubated overnight at 4 1C. The following day, sections

were rinsed with TBS and incubated with Alexa Fluor 488 donkey anti-

mouse (Molecular Probes; at 1/1000) in TBS+0.1% Triton X for 2 h to

visualize the primary antibodies. Slices were counterstained with ToPRO-

3 nuclear staining (Molecular Probes; at 1/2000). To enhance the signal of

the anterograde tracer Microruby, slices were incubated with peroxidase-

conjugated avidin–biotin complex (Vectors Laboratories, USA; at 1/50) in

50mM TBS, 0.1% Triton X-100, for 2 h at room temperature. Finally,

sections were incubated with Texas-Red-conjugated streptavidin (Mole-

cular Probes; at 1/1000). Samples were coverslipped with ProLong

Antifade Kit (Molecular Probes).

Only ACH blocks, in which (i) most part of the entorhinal cortex was

stained by a dense network of Microruby-traced neurons with their fibers

and (ii) no tracer was spilled on the hippocampal part of the culture were

used for further microscopical analysis. For individual ECM coating

conditions the sample sizes were as follows: n ¼ 19 for laminin, n ¼ 18 for

collagen, n ¼ 23 for poly L-ornithine, n ¼ 17 for fibronectin coated and

n ¼ 42 for uncoated ACH. For the quantification of the axon density, the

three capillaries of each ACH block containing most Microruby-labeled

axonal profiles were chosen. A single scan was obtained from these

capillaries by confocal immunofluorescence microscopy (LEICA TCS-

NT) choosing a level in the z-axis of the slice culture containing most

Microruby labeled axonal profiles. Using NIH Image 1.62 software, the

area of Microruby labeled axon profiles was highlighted with a digital pen

and the number of pixels representing the marked area was assessed. To

determine the maximum distance of entorhinal axon regeneration into the

ACH (axon length) the longest distance from the entorhinal cortex-ACH

interface to the longest axon ending was measured using NIH Image 1.62.

An observer blinded to group identity rated the degree of axonal

regeneration of each sample.

2.6. Statistical analysis

All data are expressed as mean7standard deviation (SD). All values

from experimental slice culture–alginate complexes were compared to

control culture–alginate complexes prepared in the same batch. The

statistical significance of differences (Po0:05) in parameters was assessed

by parametric one-way ANOVA.

2.7. Surgical procedures

Spinal cord lesions were performed as previously described [8,22].

Briefly, the dorsal columns containing the dorsal corticospinal tract and

ascending proprioceptive projections were transected at cervical level C3

using a tungsten wire knife (David Kopf Instruments, USA). Immediately

after the injury, a dura incision was made following one half of the

previous wire knife transection. ACH gel blocks with standardized

dimensions were inserted into the transection site (n ¼ 4). The orientation

of the ACH capillaries followed the longitudinal axis of the spinal cord.

The lesion/implantation site was covered with gel foam (Braun, Germany)

and antibiotic powder before readapting muscular layers and stapling the

skin above the lesion.

2.8. Morphological analysis of spinal cord tissue

At 6 weeks post-lesioning/transplantation, animals were transcardially

perfused with a 0.9% saline solution in 0.1M PBS followed by 4%

paraformaldehyde in 0.1M PBS. Sagittal 35mm cryostat sections were

processed for immunohistochemistry. Every seventh section was mounted

for Nissl staining to determine the lesion size. Double labeling immuno-

fluorescence techniques were performed with free-floating sections to

assess interaction of implanted ACH with injured axons. The following

primary antibodies were used: rabbit anti-GFAP (for astroglial cells;

Molecular Probes; at 1/1000), rabbit anti-200 kD neurofilament (for

axonal profiles; Chemicon; at 1/500), mouse anti-GAP-43 (for regenerat-

ing axons; Chemicon; at 1/100), mouse anti-ED1 (for macrophages,

activated microglia; Chemicon; at 1/1,000), rabbit anti-tyrosine hydro-

xylase (for coerulospinal axons; Chemicon; at 1/500). Immunolabeling was

visualized using fluorescein-linked donkey secondary antibodies (Mole-

cular Probes; at 1/1000). Sections were rinsed in 50mM TBS, incubated in

TBS+1% donkey serum+0.1% Triton-X for 1 h, then incubated with the

primary antibody overnight at 4 1C on a rotating platform. The following

day, sections were incubated with fluorophor-conjugated (Alexa488,

Alexa568) secondary donkey antibodies (Molecular Probes; at 1/1000)

for 2 h. Finally, sections were mounted onto glass slides and cover-slipped

with ProLong Antifade Kit (Molecular Probes). For brightfield immuno-

histochemical analysis mouse anti-ED1 (Chemicon; at 1/1000), rabbit anti-

GFAP (Molecular Probes; at 1/1000) primary antibodies were used. After

overnight incubation with the respective primary antibodies, sections were

transferred to biotinylated donkey IgG (Dianova, Germany; 1/1000)

followed by incubation with avidin biotinylated peroxidase complex

(Vector Laboratories; at 1/1000) for 1 h each. Immunolabeling was

visualized with a 0.05% solution of 3,30-diaminobenzidine, 0.01% H2O2,

and 0.04% nickel chloride in TBS. Tissue sections were mounted onto

gelatin-coated glass slides, air dried, dehydrated, and cover-slipped with

Neo Mount (Merck).

ARTICLE IN PRESSP. Prang et al. / Biomaterials 27 (2006) 3560–3569 3563

2.9. In vivo magnetic resonance imaging

All magnetic resonance imaging (MRI) experiments were conducted on

a vertical Bruker 750 wide bore magnet system (Bruker Biospin, Germany)

at 17.6T with a bore size of 89mm and a custom-built probehead/surface

coil as described [23]. Rats were anesthetized by inhalation of 4%

isoflurane followed by 2% isoflurane throughout the imaging time. Body

temperature was maintained by heating the gradient cooling unit to

3772 1C. An animal gradient system with 57mm inner diameter and

200mT/m was used. A multislice 2D spoiled gradient echo sequence was

used with a TE/TR ¼ 3.5/600ms. Up to 16 slices were acquired per scan.

Each slice was averaged eight times to increase the signal-to-noise ratio.

The spatial resolution was 156� 137mm in-plane with a slice thickness of

300mm.

3. Results

3.1. Fabrication of stable highly anisotropic alginate-based

capillary gels

Oriented diffusion of copper ions into sodium alginatesols caused self-formation of almost hexagonally struc-tured anisotropic capillary gels (Fig. 1a). The prepared gelbodies were of cylindrical shape and had diameters ofabout 5 cm and heights of about 2 cm. After removing thenon-structured top layers of the gels, the whole gel bodieswere traversed by capillaries, which were circular in cross-section and aligned parallel to each other in longitudinalsection (Fig. 1b–d). After exchanging the copper ions forprotons the structure of the capillary gels remainedunchanged, since protonated alginic acid is not soluble inwater.

Within the present study several gels were prepared,which displayed very similar structural properties: The gelsexhibited capillary diameters of 2774 mm and the numberof capillaries per cross-section was 530/mm2 with 30% ofthe cross-section covered by the water-filled capillarylumen (Fig. 1d). As soon as the gels got in contact withbiological medium, the alginic acid dissolved, becausemonovalent cations, which were present in cell culturemedium in high amounts, displaced the protons of thepolyelectrolyte and formed water-soluble salts [24]. There-

Fig. 1. Ultrastructure of alginate-based anisotropic capillary gels (ACH). (a) Il

Macroscopic appearance of ACH bodies. (c) ACH in cross- and (d) longitudi

fore, the anisotropic structure of the alginate gels wasfurther stabilized by chemically cross-linking the hydroxylgroups of the carbohydrate chains via hexamethylenediisocyanate yielding a still soft and flexible gel.

3.2. Anisotropic alginate-based gels elicit highly oriented

axon regeneration and appropriate target reinnervation in

vitro

To evaluate the permissiveness and the ability of ACH tosupport directed CNS axonal regrowth, defined size blocksof stabilized ACH were introduced into rat entorhino-hippocampal slice cultures (Fig. 2a,b) [20,21,25]. The ACHcapillaries were oriented in such a way to allow directtransition of axons from the entorhinal cortex to thehippocampus (Fig. 2b). ACH maintained their 3Dstructure under in vitro conditions for at least 14 days.Lesioned entorhinal axons, either identified by specificanterograde labeling with Microruby (Fig. 2c) or bydetection of GAP-43 immunoreactivity (Fig. 2d), robustlyregenerated into ACH mostly as dense fascicles containingnumerous axons. Axons regrew in a longitudinally orientedfashion, frequently crossing the complete distance of thecapillaries (500 mm). Moreover, anterogradely labeledentorhinal axons were found to terminate along neuronsin the outer molecular layer of the dentate gyrus, theappropriate target site of entorhinal projections (Fig. 2e).As soon as the orientation of the alginate gel was turned901 (the direction of the capillaries was now perpendicularto the direct axon regeneration path) very few axonscrossed the alginate-based scaffold following a moretortuous path below or above the alginate gel, whereasthe majority of lesioned axons still followed the changeddirection of the capillaries (Fig. 2f). Taken together,alginate-based ACH direct CNS axon regeneration, whichultimately leads to appropriate target reinnervation in an invitro model of CNS axonal injury.We thought to further enhance the regenerative capacity

of ACH by introducing specific ECM components, whichare known to support axon regeneration or which might

lustration of the different phases of anisotropic capillary gel formation. (b)

nal sections. Scale bars (b) 1 cm; (c) and (d) 100mm.

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Fig. 2. Axon regrowth and target reinnervation in vitro. (a,b) Entorhino-hippocampal slice culture model: the entorhino-dentate pathway originates in the

entorhinal cortex (EC) and terminates within the outer molecular layer of the dentate gyrus (DG) of the hippocampus (CA1, CA3). (b) ACH capillaries are

oriented to allow direct transition of regenerating axons across the lesion gap. (c–f). The orientation of all confocal fluorescence micrographs except (e) is

in analogy to the illustration in (a, b): the EC ends at the right margin, the hippocampus starts at the left margin of the micrograph. Arrows delineate the

alginate–slice culture transition. (c) Microruby labeled axon bundles (red; arrowheads) within the ACH. (d) GAP-43 immunoreactive (green) regenerating

axons in the same section. (e) Microruby-labeled entorhinal axon projections (red) terminate specifically along neurons (NeuN; green) in the outer

molecular layer (OML). Arrows delineate the ACH–hippocampus transition, shown at lower magnification in the boxed micrograph. (f) After a 901

rotation of ACH in the horizontal plane, GAP-43 immunoreactive axons (green) follow the path of the misaligned ACH. Scale bars (c–f) 50mm.

P. Prang et al. / Biomaterials 27 (2006) 3560–35693564

facilitate the colonization with growth supportive cellpopulations such as adult NPC. ACH were coated eitherwith laminin, collagen, poly L-ornithine or fibronectin andwere placed in between transected slices. Neither thedensity (P ¼ 0:22) of Microruby-labeled axon profileswithin the ACH (Fig. 3a) nor the maximum length ofaxon ingrowth (P ¼ 0:48) into the capillaries (Fig. 3b) wassignificantly different in any of the ECM coating condi-tions compared to uncoated ACH. Axon density was70471248 pixels with laminin, 7107978 pixels withcollagen, 136171441 pixels with poly L-ornithine,4687424 pixels with fibronectin coating and 83071454pixels with uncoated ACH. Coating with laminin induced amaximum axon outgrowth of 1037125 mm, with collagen84789 mm, with poly L-ornithine 134769 mm and withfibronectin 95780 mm. In comparison, uncoated ACHelicited a maximum axon outgrowth of 109793 mm.

3.3. Anisotropic alginate-based gels promote directed axon

regrowth following spinal cord injury

Four weeks after transplantation of uncoated definedsize ACH into cervical dorsal funiculus transection sites,

we employed 17.6 T MRI based in vivo structural analysis,which allows to analyze implant integration and hostresponses (edema, cyst formation) in situ, thus gettingaround artifacts, which arise from postmortem tissuealterations caused by histological processing. ACH wereplaced correctly in the lesion site (Fig. 4a). There were nosignal changes indicating significant inflammatory re-sponses or cyst formation adjacent to ACH implants. Sixweeks postoperatively, postmortem histological evaluation(Nissl staining) showed that ACH scaffolds remainedstable and maintained their microstructure (Fig. 4b).Cyst-like spaces surrounding the ACH more likelyrepresent artifacts due to tissue shrinkage in the course oftissue processing (fixation, staining procedure). This notionis supported by the fact that MRI did not revealhyperintense signal changes as a correlate of cyst formationin the vicinity of the ACH. Specific macrophage/microglialabeling revealed that the inflammatory reaction in thesurrounding spinal cord parenchyma was restricted toareas of Wallerian degeneration in the dorsal columns(Fig. 4c), which is typically observed in transection spinalcord injuries (N. Weidner, unpublished observation).GFAP immunoreactive glial processes were identified,

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Fig. 3. Effects of extracellular matrix coating. (a) Axon density and (b) the length of axon ingrowth in the different ECM-coated ACH conditions. Bars

represent standard deviation (SD).

P. Prang et al. / Biomaterials 27 (2006) 3560–3569 3565

which entered the ACH implant indicating a continuoustransition from the spinal cord into the implanted matrix(Fig. 4d). The overall GFAP immunoreactivity around theimplant was increased, however, not beyond levelsobserved after spinal cord lesions without therapeuticinterventions.

Axonal profiles immunoreactive for neurofilament,tyrosine hydroxylase indicating ingrowth of coerulospinalprojections and GAP-43 identifying regenerating axons,were found to robustly grow into alginate-based capillarygels in a longitudinally oriented fashion (Fig. 5). Moreover,neurofilament positive axons were aligned along GFAPpositive processes within the capillaries (Fig. 5b), suggest-ing that host astroglial cells provide a growth permissiverather than a growth-inhibitory milieu. Thus, ACH elicithighly oriented robust axonal regeneration following spinalcord injury in adult rats, which parallels data obtained withthe entorhino-hippocampal slice culture.

3.4. Alginate scaffolds serve as platform for adult NPC

The intention is to combine a structurally guidingsubstrate (ACH) with a cell-based growth promotingstructure. Therefore, we first investigated the feasibility tointroduce NPC, which have been shown to promote cell-contact-mediated axon regrowth [7], into ACH in vitro.Two weeks after inserting GFP-transgenic NPC-seededACH into the slice culture system, cells were identifiable

within ACH frequently extending processes along thehydrogel capillary walls (Fig. 6a). Nuclear counterstainingindicated that counting just GFP expressing NPC withinACH underestimated the total number of NPC seeded intothe alginate scaffolds. This is attributable to the fact thatdifferentiating NPC rapidly downregulate their transgene,which has been shown recently [9]. Transected entorhinalaxons grew into NPC-seeded ACH and were frequentlyaligned along GFP-expressing NPC (Fig. 6c–e). These cellsdisplayed a bipolar morphology, paralleling the appear-ance of NPC graft-derived glial cells, which have beenshown to promote cell-contact-mediated axon regenerationin the injured spinal cord [7–9]. Taken together, NPC canbe introduced into ACH and support axon regeneration invitro as a prerequisite for the combined use of directingscaffolds and growth permissive cell populations for nerverepair following spinal cord injury.

4. Discussion

Self-assembling anisotropic alginate-based capillarygels, which were stabilized by chemical cross-linking,represent a unique biopolymer, which combine severalkey features required for axonal regeneration in theCNS. They promote highly oriented axon regenerationin vitro and in vivo in the injured mammalian CNS lead-ing to appropriate target reinnervation in vitro. Further-more, alginate-based scaffolds provide a platform, which

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Fig. 5. ACH regrowth in vivo. (a) Oriented neurofilament immunoreactive axons (green) within ACH (arrows). (b) Neurofilament expressing axons

(green) co-localized (arrow) with GFAP expressing astroglial processes (red) within ACH capillaries. (c) Tyrosine hydroxylase immunoreactive

coerulospinal axons (arrow; green) within ACH capillaries. (d) Dense GAP-43 immunoreactive axon bundles within ACH capillaries. Orientation in all

confocal fluorescence micrographs: rostral is to the left, dorsal to the top. Scale bars (a–c) 25mm; (d) 40mm.

Fig. 4. Host responses after ACH implantation. (a) In vivo MRI at 17.6T detects the ACH block (arrow) within the injured spinal cord at cervical level

C3. Sagittal scan, rostral to the left, dorsal to the top. (b) Nissl-stained sagittal spinal cord section with the ACH located directly within the lesion site. (c)

ED-1 immunoreactive macrophages/microglial cells after implantation of ACH. (d) GFAP immunoreactive astroglial processes extend towards and

sometimes into the ACH (arrow, shown in boxed micrograph at higher magnification). Orientation of all micrographs: rostral to the left, dorsal to the top.

Scale bars (a) 1mm, (b–d) 0.5mm.

P. Prang et al. / Biomaterials 27 (2006) 3560–35693566

ARTICLE IN PRESS

Fig. 6. Adult neural progenitor cell survival within ACH. (a, b) Adult GFP-expressing NPC (green). (b) Overlay of GFP-expressing NPC and nuclear

counterstain with ToPro3 (blue). (c–e) Axonal regeneration into NPC-seeded ACH. (c) Microruby labeled entorhinal axons (red). (d) GFP-expressing

NPC (green). (e) Overlay of (c) and (d) combined with nuclear counterstain (ToPro3; blue). Arrows highlight axons co-localized with GFP positive cellular

processes. Confocal immunofluorescence micrographs. Scale bars (a–e) 10mm.

P. Prang et al. / Biomaterials 27 (2006) 3560–3569 3567

supports survival of adult NPC as a prerequisite for acombined cell- and artificial scaffold-based regenerativetherapy.

Although alginate gels have been employed in the pastfor a number of biomedical applications (e.g. impressionmaterial in dentistry, wound dressings, encapsulationmaterial for cells), alginates have not been used to produceanisotropic structured capillary gels for biomedical pur-poses thus far. Several studies continued the work of Thiele[15] concentrating on the theoretical aspects of dissipativestructure formation [16,26] and on the kinetics of sol–geltransformation [27]. Based on Thiele’s description, algi-nate-based capillary hydrogels were used as templates forceramic materials [28]. ACH contain self-assembled linearcapillaries of almost equal dimensions, which are parallelto each other and extend uniaxially through the full lengthof the hydrogel. The small pore size—in this case adjustedaround 27 mm—allows physical guidance of regeneratingaxons by restricting the directions of regrowth. However, asystematic analysis investigating the degree of directedaxon regrowth in relation to the pore size of ACH remainsto be done. The observed physical stability of the ACHscaffolds for at least 6 weeks after implantation is aprerequisite to promote CNS axon regeneration, sinceaxonal regeneration within the injured CNS is likely to takemonths. A major concern using artificial nerve guides is thebiocompatibility. Alginate is known to be immunologicallyinert and is not digested by mammalian cells [10]. Theintroduction of a chemical cross-linking agent into alginategels in this study did not affect biocompatibility, sinceinflammatory responses or other signs of tissue damagebeyond changes occurring following spinal cord injurywithout therapeutic intervention were not observed.

Of all natural and synthetic biopolymer implants (forreview see [10,11]) investigated regarding their regenerativecapacity in the injured spinal cord, only agarose-based

scaffolds have been described to promote linear axongrowth across the lesion gap to an extent comparable toACH [29]. Overall, ACH have properties similar to theagarose-based scaffold used by Stokols. Like agarose,ACH do not evoke immune or inflammatory responses andcan be processed to yield soft and flexible scaffoldsmatching physical properties of spinal cord parenchyma.In contrast to the highly anisotropic structure ofACH, freeze-dry processing produced uniaxial agarose-based channels with a much higher variability in respectto size and shape of individual channels. The averagecross-sectional diameter (125 versus 27 mm) is much highercompared to ACH. Whether the almost identical shape/size and the smaller cross-sectional capillary diameter ofACH improve axon regrowth beyond the regenerativeresponse observed with agarose scaffolds remains to bedetermined.The main rationale for the application of artificial

growth supporting matrices is to guide axon regrowth insuch a way that it would allow topographically appropriatereentry of regenerating axons into the spinal cord distal tothe lesion site and ultimately appropriate reinnervation ofprevious target neurons. In vitro we were able todemonstrate that ACH are capable to guide growing axonsin a longitudinal fashion and to promote reinnervation oforiginal target neurons in the dentate gyrus of thehippocampus. Future experiments need to evaluatewhether ACH elicit target reinnervation in vivo afterspinal cord injury and restore function. However, thisregenerative approach alone is unlikely to be sufficient topromote substantial reentry into the host spinal cord andtarget reinnervation, since other factors known to inhibitaxon regeneration in the injured adult mammalian CNS,namely the upregulation of growth-inhibitory moleculesaround the ACH [30] and the lack of growth-promotingmolecules [31], need to be addressed at the same time.

ARTICLE IN PRESSP. Prang et al. / Biomaterials 27 (2006) 3560–35693568

The rationale to introduce artificial matrices into theinjured spinal cord in the context of cell transplantation istwofold: to build a platform for transplanted cells toadhere to and to support the longitudinal orientation ofgrafted cells as a prerequisite for directed axon regrowth[7–9]. From the perspective of biomaterial science, therationale to introduce cells into artificial matrices is tocreate a milieu within the substrate, which is moreconducive and stimulating for regenerating axons thanthe matrix alone. Several previous studies have alreadycombined the administration of artificial growth scaffoldswith cell therapy. Most scaffolds were hollow single-channel tubes, which only allow a very limited degree ofguidance [32–34]. Few data are available regardingmicrostructured guidance channels combined with celltherapy. The combination of freeze-dried poly (D,L-lacticacid) (PLA) [35], alginate [36] or poly (lactic-co glycolicacid) (PLGA) [37] with neural stem cells or Schwann cellsallowed cell survival within the scaffold, promoted modestfunctional recovery or induced directed axon regrowth tosome modest degree. However, a clear correlation betweencell administration, directed axon growth and functionalrecovery has yet to be demonstrated. Our in vitro datashow that adult NPC survive within ACH and appear topromote not only cell-contact-mediated axon regrowth,recapitulating previous observations with NPC/fibroblastco-grafts in vivo [7]. Moreover, in combination with ACHaxon regeneration occurs in a longitudinally orientedfashion. This finding has to be verified in vivo andcompared to ACH application without cell administrationbefore further conclusions regarding the regenerativecapacity of ACH combined with NPC can be drawn.

Diisocyanate cross-linked ACH promote axonal re-growth in vitro and in vivo independent of surface coatingwith various ECM components (laminin, fibronectin,collagen, poly L-ornithine). This is in line with previousreports, which have demonstrated axon regrowth followingspinal cord injury in young rats into unmodified freeze-dried alginate gels [13,38–40]. However, evidence fromother studies suggests that alginate gels without surfacemodification are not sufficient to promote neurite extension[41]. In the light of these conflicting data, severalexplanations for our findings are conceivable: (1) thecoating process with ECM was not sufficient to ensure theiradsorption within the ACH, since alginate hydrogels areknown to promote minimal protein adsorption [42]. Wehave no data confirming that ECM were adsorbed to theACH, since immunohistochemistry failed to detect thesecomponents within ACH (data not shown). On the otherhand, a standard coating procedure of alginate gels withlaminin comparable to ours resulted in an enhanced celladhesion effect implying that ECM become adsorbed toalginate gels [43]. (2) In case if ECM are adsorbed, theybecome inactivated. Coating alginate gels with laminin hasbeen described to enhance cell adhesion, however, does notpromote neurite extension, which has been attributed tosteric hindrance at the active site or to interaction of

cations present in the hydrogel with laminin [43]. (3) Thechosen ECM, even though they are properly adsorbed andactive, do not alter axon regrowth in vitro and in vivo,because of differential axon growth responses. Forexample, inhibition of collagen synthesis has been shownto promote axon regrowth after postcommissural fornixtransection in the adult rat brain [44]. In contrast, theidentical treatment in the spinal cord does not induceregrowth of transected corticospinal axons [22].

5. Conclusion

A yet novel kind of alginate-based biopolymers contain-ing linear capillaries of almost equal dimensions, whichextend through the full length of the hydrogel with adefined small pore size, promote axonal regeneration in theinjured CNS in a highly oriented fashion. In vitro findingssuggest that the regenerative capacity of ACH can befurther improved by the introduction of growth promotingcells such as adult NPC. Future studies need to evaluate,whether anisotropic ACH either alone or in combinationwith adult NPC elicit axon regrowth beyond the implant,proper target reinnervation and functional recovery in arelevant animal model of spinal cord injury. Of course,directed axon regrowth promoted by anisotropic hydrogelsis just one piece of the puzzle. Considering matriximplantation as a clinically relevant regenerative strategy,other strategies including the application of growth factorsand the neutralization of growth inhibitory molecules needto be addressed in order to elicit substantial functionalrecovery in humans suffering from spinal cord injury.

Acknowledgements

This work was supported by the ReForM-Program ofthe University of Regensburg, School of Medicine to P.P.We thank Massimiano Caioni for excellent technicalassistance.

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