Available online at www.sciencedirect.com

Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321www.nanomedjournal.com

Neurology Nanomedicine

Reknitting the injured spinal cord by self-assemblingpeptide nanofiber scaffold

Jiasong Guo, PhD,a,b Huanxing Su, MD,a Yuanshan Zeng, MD, PhD,c

Yu-Xiang Liang, PhD,a,d Wai Man Wong, MS,a Rutledge G. Ellis-Behnke, PhD,a,d,e

Kwok-Fai So, PhD,a,d,f Wutian Wu, MD, PhDa,d,g,⁎aDepartment of Anatomy, The University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong SAR, China

bDepartment of Histology and Embryology, Southern Medical University, Guangzhou, ChinacDepartment of Histology and Embryology, Zhongshan Medical College, Sun Yat-sen University, Guangzhou, China

dState Key Laboratory for Brain and Cognitive Sciences, The University of Hong Kong Li Ka Shing Faculty of Medicine, Pokfulam, Hong Kong SAR, ChinaeDepartment of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

fResearch Centre of Heart, Brain, Hormone and Healthy Aging, The University of Hong Kong Li Ka Shing Faculty of Medicine,Pokfulam, Hong Kong SAR, China

gResearch Center of Reproduction, Development and Growth, The University of Hong Kong Li Ka Shing Faculty of Medicine,Pokfulam, Hong Kong SAR, China

Received 1 July 2007; accepted 20 September 2007

Abstract In traumatic spinal cord injury, loss of neurological function is due to the inability of damaged axons

R.G.E.-B. has a finMatrix, manufacturer

⁎ Corresponding aKong Li Ka Shing Fac

E-mail address: w

1549-9634/$ – see frodoi:10.1016/j.nano.20

to regenerate across large, cystic cavities. It has recently been demonstrated that a self-assemblednanofiber scaffold (SAPNS) could repair the injured optical pathway and restore visual function. Todemonstrate the possibility of using it to repair spinal cord injury, transplanted neural progenitor cellsand Schwann cells were isolated from green fluorescent protein–transgenic rats, cultured withinSAPNS, and then transplanted into the transected dorsal column of spinal cord of rats. Here wereport the use of SAPNS to bridge the injured spinal cord of rats, demonstrating robust migration ofhost cells, growth of blood vessels, and axons into the scaffolds, indicating that SAPNS provides atrue three-dimensional environment for the migration of living cells.© 2007 Elsevier Inc. All rights reserved.

Key words: Self-assembling peptide; Nanofiber scaffold; Spinal cord injury; Schwann cell; Neural progenitor cell

Traumatic spinal cord injury results in a loss of nervoustissue characterized by the formation of large cystic cavitiesand a partial or complete loss of neurological functions [1-6].For the restoration of function following spinal cord injury,damaged axons must regenerate across and beyond the siteof injury. One of the major efforts to repair spinal cord injuryhas been to construct a bridge through the injured area overwhich the axons can grow. To do that, many potentialstrategies have been applied, including the transplantation of

ancial interest in Clear Nano Solutions, Inc. and in 3Dof the material distributed by BD Biosciences.uthor. Department of Anatomy, University of Hongulty of Medicine, Pokfulam, Hong Kong SAR, China.twu@hkucc.hku.hk (W. Wu).

nt matter © 2007 Elsevier Inc. All rights reserved.07.09.003

(1) fetal spinal cord tissue [7], (2) peripheral nerves [8], (3)Schwann cells (SCs) [9], (4) olfactory ensheathing cells [10],(5) embryonic stem cells [11], (6) marrow stromal cells [12],(7) neural stem cells (NSCs) [13], and (8) geneticallymodified cells [14,15].

Because of the limited source of fetal tissue andautologous nerve grafts, using cells amplified in vitro is amore viable option for transplantation. In addition, injectingthe cells into the host can be problematic because of the largecavities that form, necessitating a scaffold to contain thesecells and bridge the gap [5].

The need for tissue repair has encouraged the develop-ment of biomaterials scaffolds that can be used to fill thecavities that develop as a result of an injury in the spinal

312 J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

cord. There are primarily two kinds of biomaterials currentlybeing used in spinal cord injury: (1) naturally derived animalproducts such as matrices of collagen and (2) syntheticmacromolecular polymers such as poly lactic acid, and polylactic-co-glycolic acid [16,17]. Both can present potentialproblems; first, naturally derived animal products, such asmatrices of collagen, can carry dangerous pathogens [18,19].Second, some of the problems associated with chemicallysynthetic macromolecular polymers, such as poly lactic acidand poly lactic-co-glycolic acid [16,17] are (1) degradation[20,21], (2) damage caused by the degradation byproducts[16], (3) the limited biocompatibility of the environmentprovided by these materials, and (4) the occasional rigidity ofthe structures with small pores, restricting both the migrationof cells in and out of the material as well as the growth ofaxons [22-24].

Recently it was shown that a self-assembled nanofiberscaffold (SAPNS) could repair an injured optical pathwayand restore visual function [25], demonstrating the possibi-lity of using SAPNS for the effective treatment of centralnervous system injury.

The benefits of SAPNS over current biomaterials are:(1) a minimized risk of carrying biological pathogens orcontaminants that are typically present in animal-derivedbiomaterials [19,26]; (2) a true three-dimensional (3D)environment for cell growth and migration, similar to thenative extracellular matrix [22,27-29]; (3) excellent physio-logical compatibility as well as minimal cytotoxicity due toits composition of naturally occurring amino acids [25,30];and (4) no apparent immune response or tissue inflammationwhen introduced into animals [25,27].

We proposed that the successful reconnection of dis-connected parts observed in the optic tract regenerationexperiments [25] might also be effective in treating spinalcord injury. The present study was designed and implemen-ted to determine (1) if the SAPNS can provide a friendly 3Denvironment for neural progenitor cell (NPC) survival,migration, and differentiation both in vitro and in vivo; (2) ifthe SAPNS can integrate into, as well as bridge, the injuredspinal cord; and (3) if the SAPNS will elicit axonregeneration alone, or with the addition of Schwann cells(SCs) or NPCs to it.

Materials and methods

We used four materials to test axon regeneration:(1) SAPNS [31,32] alone, (2) SAPNS pretreated to adjustthe pH, (3) SAPNS cultured with SCs, and (4) SAPNScultured with NPCs.

Pretreatment of SAPNS

In the present study RADA16-I peptide (BD Biosciences,Cambridge, MA), which is a type I SAPNS, was used fortransplantation. The RADA16-I peptide is an amphiphilicmolecule in water containing a hydrophobic face and ahydrophilic face. When ionic strength is increased or pH

values are raised to neutrality (e.g., physiological saltconcentrations, culture media, buffers), the peptides sponta-neously self-assemble into nanofibers about 10 nm indiameter. The nanofibers further form a hydrogel withgreater than 99% water content [22,29,33,34].

Because the untreated SAPNS has a very low pH (about3–4) and our preliminary study showed that untreatedSAPNS would damage the host tissue either in dorsalcolumn–transected or right-hemisected spinal cord (data notshown), we neutralized the SAPNS in culture medium beforetransplantation. Briefly, 1% RADA16-I peptide was gentlyand quickly plated to dish in DMEM/F12 with 10% fetalbovine serum (FBS; Gibco, Grand Island, NY). The self-assembly occurred as soon as the material contacted with themedium. The medium was changed twice at 1, 10, and 30minutes after plating, and once every 3 days during thefollowing days. On day 7, the medium-treated SAPNSmaterial was used for transplantation.

Isolation and culture of SCs and NPCs

Schwann cellsThe SCs were isolated as described elsewhere [35,36].

Briefly, the sciatic nerves from adult green fluorescentprotein (GFP)–transgenic Sprague-Dawley rats [“green ratCZ-004” SD TgN (act-EGFP) OsbCZ-004] were cut into1-mm3 explants and placed in culture dishes with DMEM/F12 (Gibco) supplemented with 10% FBS (Gibco). Whenthe outgrowth of migratory cells (predominantly fibroblasts)reached a near-confluent monolayer around the explants(about 7 days), the explants were transferred to new culturedishes with fresh medium. After three to five such passages(3–5 weeks) the cells that emerged from the explants wereprimarily SCs. The explants were then transferred to a35-mm dish containing 1.25 U/mL dispase, 0.05% collage-nase, and 15% FBS in DMEM/F12 for incubation overnightat 37°C in 5% CO2. On the following day the explants weredissociated and the cells were plated onto poly-l-lysine(0.01%; Sigma, St. Louis, MO)-coated dishes in DMEM/F12with 10% FBS. Later the cultures were re-fed with the samemedium supplemented with 20 μg/mL pituitary extract(Sigma) and 2 μM forskolin (Sigma) for dividing. Whenthe SCs reached confluence they were rinsed in Ca2+- andMg2+-free Hanks balanced salt solute ion (CMF-HBSS;Gibco) and briefly treated with 0.05% trypsin (Gibco)and 0.02% EDTA (Gibco) in CMF-HBSS. Cells werewashed twice in DMEM/F12 with 10% FBS and transferredinto new dishes at a density of 2 × 106 cells per 100-mm dish.When the cells reached confluence again they were collectedfor transplantation.

NPCsThe generation and characterization of embryonic NPC

cultures have been described [37,38]. The hippocampi ofembryonic day 16 (E16) embryos of GFP-transgenicSprague-Dawley rats were dissected in cooled CMF-HBSSand dissociated mechanically. The cells were collected by

313J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

centrifugation and resuspended in DMEM/F12 supplemen-ted with B27 supplement (2%, Gibco) N2 (1%, Gibco),epidermal growth factor (EGF, 20 ng/mL, Gibco), basicfibroblast growth factor (bFGF, 20 ng/mL, Sigma), penicillin(100 U/mL), and streptomycin (100 μg/mL). The cells wereadjusted to 1 × 105 cells/mL and planted into culture flasks.The medium was replaced by one-half every 3 days.Typically the cells grew in suspending neurospheres,which were mechanically dissociated approximately onceeach week and reseeded at approximately 1 × 105 cells/mL.In this study the second neurospheres were used fortransplantation. To assess the purification of the NPCs,some of the neurospheres were dissociated and seeded ontopoly-l-lysine-coated plates with the same medium as above.

SCs and NPCs in 3D culture within the SAPNSBefore transplantation the SCs and NPCs were respec-

tively cultured within SAPNS scaffold. Briefly, the SCs andNPCs were collected and finally adjusted to 5 × 105 cells/μL.Then, 1 μL of the cell suspension was mixed with 9 μL ofRADA16-I peptide; themixturewas gently and quickly platedto dish in DMEM/F12 with 10% FBS. The medium waschanged twice at 1, 10, and 30 minutes after plating, and onceevery 3 days during the following days. On day 7 the cultureswere used for transplantation. Some of the cultures weremaintained for 4 weeks, and the images of living cells weretaken by two-photon confocal microscope (Zeiss LSM510META, Jena, Germany). Four weeks later, the cultures werefixed with 4% paraformaldehyde for immunostaining.

Immunocytochemistry

For immunostaining the SCs and dissociated culturedNPCs were directly fixed with 4% paraformaldehyde. Theneurospheres of the NPCs were plated on poly-l-lysine-coated coverslips and grown in culture medium. After theneurospheres were attached on the coverslips (about 1 hour)they were fixed with 4% paraformaldehyde. The fixedcultures of SAPNS with SCs or NPCs were first cryopro-tected in 30% sucrose, and then cryostat sections (10 μm)were cut and mounted on to gelatin-coated slides.

Before exposure to antibodies, all samples were blockedwith 1% bovine serum albumin (BSA), 10% normal goatserum, and 0.3% Triton X-100 in phosphate-buffered saline(PBS) for 1 hour at room temperature (25°C), and then thefollowing primary antibodies were applied: (1) rabbit anti-p75 (1:200; Promega, Madison, WI) for identifying SCs; (2)mouse anti-nestin (1:2000; BD Biosciences, Cambridge,MA) for NPCs; (3) rabbit anti-glial fibrillary acidic protein(anti-GFAP, 1:1000; Chemicon, Temecula, CA) for astro-cytes; (4) mouse anti-Rip (1:50; gift of Dr. Xu XM, KentuckySpinal Cord Injury Research Center, Louisville, Kentucky)for oligodendrocytes; and (5) mouse anti-β-tubulin type III(1:1000; Sigma) for neurons. The sections were incubatedwith the primary antibody in PBS plus 1% BSA and 0.3%Triton X-100 overnight at 4°C, and treated with fluorescentAlexa 568 goat anti-mouse or anti-rabbit secondary antibody

(1:400; Molecular Probes, Eugene, OR), respectively, for2 hours at room temperature. Finally the sections weremounted with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) to counterstain the nuclei.

Surgical procedures

Thirty adult female wild-type Sprague-Dawley rats(220–250 g) and three adult female GFP-transgenic Sprague-Dawley rats (220–250 g) were used in this project. The ratswere divided to five groups: (1) saline control group,(2) uncultured SAPNS group, (3) precultured SAPNS-alonegroup, (4) SAPNS seeded with NPCs group, and (5) SAPNSseeded with SCs group. Each group had six wild-type rats;three GFP-transgenic rats were added to the preculturedSAPNS-alone group. The surgical procedures carried out inthe project were approved by the Committee for the Use ofLive Animals in Teaching and Research at the Universityof Hong Kong. Rats were anesthetized with ketamine(80 mg/kg) and xylazine (10 mg/kg). A dorsal laminectomyon the sixth and seventh cervical vertebrae was performed.The dura was incised longitudinally and pulled laterally.A spinal cord dorsal column transection was made betweenC6 and C7, followed by the removal of 1 mm dorsal columntissue. With the assistance of a micro-scoop, 5 μL ofprecultured SAPNS scaffold, or an equal volume of thecultured mixture of SAPNS and SCs or NPCs, respectively,was transferred from the culture dish into the lesion cavity. Ascontrols, 5 μL of uncultured SAPNS or saline was placed inthe lesion cavity. After transplantation the dura was closed bysuturing with an 11-0 suture. The muscle layers and skin werealso closed with a suture. Manual bladder expression wasperformed twice daily until recovery of the bladder reflex.

Immunohistochemistry on tissue sections

Animals were perfused transcardially with 4% parafor-maldehyde in 0.1 M PBS (pH 7.4) after an overdose ofpentobarbital anesthesia 6 weeks after transplantation. Thespinal cords were subsequently postfixed overnight in theperfusing solution plus 10% sucrose at 4°C. Then the tissueswere cryoprotected in 30% sucrose in PBS for 12 hoursat 4°C. A 1-cm length of the spinal cord centered at theinjury site was separated and embedded in optimum cuttingtemperature compound. Horizontal cryostat sections (30 μm)were cut and mounted onto gelatin-subbed slides and storedat –20°C. For immunostaining the frozen slides were air-dried at room temperature for 30 minutes and washed withPBS for 10 minutes. Then they were blocked with 1% BSA,10% normal goat serum, and 0.3% Triton X-100 in PBS for1 hour at room temperature. Afterward, the following primaryantibodies were applied overnight at 4°C: mouse anti-nestin(1:2000; BD Biosciences, Cambridge, MA) for NPCs;rabbit anti-GFAP (1:1000; Chemicon) for astrocytes; mouseanti-Rip (1:50, gift from Dr Xu XM, Kentucky Spinal CordInjury Research Center, Louisville, Kentucky) for oligoden-drocytes; mouse anti-β-tubulin III (1:500; Sigma, St. Louis,MO) for neurons; mouse anti-NF200 (1:400; Sigma, St.

Fig 1. Immunocytochemical identification of cultured SCs and NPCs from GFP-transgenic rats. Almost all GFP cells (A) were p75-positive (B). (C) Mergedimage of A and B, with DAPI staining. GFP NPC neurospheres (D) were nestin-positive (E). (F) Merged image of D and E. After dissociation and plating onpoly-l-lysine-coated dishes, the majority of GFP NPCs (G) attached to the dish, extended varying processes, and were nestin-positive (H). (I) Merged imageshows G and H, with DAPI staining. Scale bars: A–F = 50 μm; G–I = 20 μm.

314 J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

Louis, MO) for axons; rabbit anti-5HT (serotonin, 1:200;Sigma, St. Louis, MO) for raphespinal axons; rabbit anti-calcitonin gene-related peptide (CGRP, 1:200; Sigma, St.Louis, MO) for primary sensory axons; mouse anti-ED1(1:1000; Serotec, Raleigh, NC) for macrophages; rabbit anti-p75 (1:200; Promega) for SCs; and mouse anti-myelin basicprotein (anti-MBP, Ipswich, MA, 1:1000) for myelin. Theslides were washed in PBS three times and incubated withfluorescent Alexa 568 goat anti-mouse or anti-rabbitsecondary antibody (1:400; Molecular Probes) for 2 hoursat room temperature. The slides were coverslipped withmounting medium (Dako, Carpinteria, CA) containing DAPIto counterstain the nuclei. The images were taken using aconfocal microscope (Zeiss LSM510 META).

Hematoxylin and eosin (H&E) staining and alkalinephosphatase (AP) histochemical staining

To show the morphology and the angioregenerationaround the grafts the H&E and native endothelial alkalinephosphatase (AP) were used as described elsewhere [39].

Quantification of axons

Quantification of NF-200-, CGRP-, and 5HT-positiveaxons that regenerated within the graft was performed usingmethods described elsewhere [40,41]. Briefly, axons in thegraft were quantified on immunostaining sections by using a

fluorescent microscope. Three lines at intervals of 0.5 mmwere superimposed onto the graft perpendicularly to thelongitudinal axis with the middle one through the center ofthe graft. Axons intercepted with the superimposed lines werecounted. The mean number of axons obtained from fivesections of each animal was defined as the number of axons.Statistical comparisons were made by analysis of variancewith repeated measures or by Student's paired t-test forpaired observations. P values of .05 or less were consideredsignificant. Where applicable, data are shown as mean ±standard error.

Results

Characterization of the cultured SCs and NPCs

SCsAfter 6 weeks of primary culture and 7 days passaged

culture, the cellular outgrowth of sciatic nerve explants fromGFP-transgenic rats consisted almost entirely of SCs. All ofthe cells were positive for green fluorescence, and morethan 95% of the cells in the culture were positive for p75(Figure 1, A–C).

NPCsIn the presence of EGF and bFGF the embryonic

hippocampus-derived NPCs formed floating neurospheres

Fig 2. Images of live SCs and NPCs that survived and migrated three-dimensionally within the SAPNS. All images are merged stacks of multiple confocal opticalsections taken by a two-photon microscope 4 weeks after the cells were cultured within SAPNS in vitro. (A, B) SCs growing three-dimensionally in SAPNS.(A) Horizontal stacked image; (B) vertical stacked image. (C, D) NPCs growing three-dimensionally in SAPNS. (C) Horizontal stacked image; (D) verticalstacked image. Scale bar = 25 μm.

Fig 3. Immunocytochemical staining of cultured GFP SCs (A) showing p75-positive SCs within the SAPNS (B). (C) Merged image of A and B with DAPIstaining. Immunocytochemical staining of transplanted GFP SCs (D and G) showing p75-positive (E) and MBP-positive (H) cells in the transplant. (F) and(I) Merged images of (D, E) and (G, H), respectively, with DAPI staining. Scale bar: A–F = 30 μm; G–I = 10 μm.

315J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

Fig 4. Immunocytochemical staining showing the differentiation patterns of NPCs cultured within SAPNS in vitro and after transplantation into the injured spinalcord. (A–L) Four weeks after NPCs were cultured in vitro. (M–X) Six weeks after cultured NPCs were transplanted into the injured spinal cord. Most of theNPCs remained undifferentiated (nestin-positive) in vitro (A–C) and in vivo (M–O). β-tubulin III –positive cells were found (arrows) both in vitro (D–F) and invivo (P–R); Rip-positive cells were found (arrows) both in vitro (G–I) and in vivo (S–U); and GFAP-positive cells were found (arrows) both in vitro (J–L) andin vivo (V–X). Scale bars = 25 μm.

316 J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

in the uncoated flasks (Figure 1, D–F). When the neuro-spheres were dissociated and plated on poly-l-lysine-coateddishes, the cells attached to the dish and extended varyingprocesses (Figure 1, G–I). All of the cells showed greenfluorescence, and the majority of them (∼95%) were positivefor nestin (Figure 1, D–I).

SAPNS produced a good 3D environment for cellularsurvival, migration, and differentiation

The SAPNS assembled into a clear fibrous gel veryquickly (about 1 or 2 seconds) after it was transferred into thedish with culture medium. The SCs and NPCs were able tosurvive and migrate within the SAPNS, in a 3D manner invitro, after being introduced into it (Figure 2).

The SCs within SAPNS were p75-positive, whethercultured in vitro (Figure 3, A–C) or after being transplantedinto the injured spinal cord (Figure 3, D–F). Moreover,6 weeks after being transplanted to the injured spinal cordsome of the SCs had matured with tubelike processes andwere shown to be MBP-positive (Figure 3, G–I).

The NPCs within SAPNS had multiple processes, someof them differentiating into neurons (β-tubulin III-positive),astrocytes (GFAP-positive), and oligodendrocytes (Rip-positive); but most of them remained nestin-positive. The

differentiation of the NPCs was similar both in vitro(Figure 4, A–L) and in vivo (Figure 4, M–X).

Pretreated SAPNS integrated well with injured spinal cord

The spinal cord injury created a large cavity in the injurysite, and multitudinous macrophages (ED1-positive cells)surrounded the cavity (Figure 5, A). The SAPNS directlytransplanted to the injured spinal cord without beingpretreated with the culture medium did not integrate wellwith the host and caused serious inflammation. There wereobvious gaps and cysts between the implanted and hosttissue (Figure 5, B).

In contrast, when the SAPNSwas pretreated with a culturemedium before transplantation it was able to properly bridgethe lesion area. The implants integrated very well with thehost tissue with no obvious cavities or gaps between theimplanted material and host (Figure 5, C). In addition, therewas much less inflammation (as shown by the ED1-positivecells) surrounding the lesion site (Figure 5, C). Moreover,DAPI staining (Figure 5, C) showed large numbers of hostcells that had migrated into the implants. In the cases of thepretreated SAPNS, which was transplanted into the injuredspinal cord of GFP-transgenic rats, we found GFP-positive

Fig 5. Integration of implants within the injured spinal cord. (A) Control. Lesion cavity filled only with saline during surgical procedures. Six weeks after dorsalcolumn transection the site of injury showed inflammation with many ED1-positive cells (macrophages, red) surrounding the cavity (star). Nuclei (blue) arelabeled with DAPI. (B) Uncultured SAPNS implant resulted in serious inflammation showing gaps and cysts (arrows) between the implant and host tissue withmany ED1-positive cells (red) in the lesion area. Nuclei (blue) are labeled with DAPI. (C–G) SAPNS pretreated with culture medium before transplantation.(C) SAPNS implants integrated very well with host tissue, with no obvious cavities or gaps and only slight inflammation, compared with the uncultured SAPNS.Many host cells migrated into the implants, shown by DAPI staining. (D) Implantation of precultured SAPNS into the injured spinal cord of GFP-transgenic ratsresulted in many GFP-positive cells migrating from the host into the implant. (E) Implantation of precultured SAPNS with GFP NPCs. (F) Implantation ofprecultured SAPNS with GFP SCs. Both transplanted NPCs and SCs were found to migrate into the host tissue. (G) H&E staining showed a high level ofintegration between the implants and host, although in most cases a few small cysts were found near the implants. (H) AP histochemistry staining showed(arrows) that blood vessels grew into the implants. Scale bar = 500 μm.

317J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

cells that had moved from host to implant (Figure 5,D). Afterthe SAPNS with GFP-positive SCs (Figure 5, E) or NPCs(Figure 5, F) had been transplanted into the spinal cord ofwild-type rats, the grafted cells survived very well in theimplants with some of them migrating into the host tissue.

H&E staining showed that the most of the interfacebetween the implants (either pretreated SAPNS alone orseeded with grafted cells) and host integrated very well(Figure 5, G). Another important finding was that therewere many blood vessels in the implants; some vessels

Fig 6. Axons regenerated into SAPNS 6 weeks after transplantation. (A) Transplantation of SAPNS alone. No GFP-positive cells were transplanted in thelesioned spinal cord. (B) NF-positive axons (arrows) are visible in the implant. (C) Merged image of A and B, with DAPI staining. (D) Transplantation ofSAPNS seeded with NPCs. (E) NF-positive axons (arrows) are visible in the implant. (F) Merged image of D and E, with DAPI staining. (G) Transplantationof SAPNS seeded with SCs. (H) NF-positive axons (arrows) are visible in the implant. (I) Merged image ofG andH, with DAPI staining. (J) Transplantation ofSAPNS seeded with SCs. (K) 5HT-positive axons (arrows) are visible in the implant. (L) Merged image of J and K, with DAPI staining. (M) Transplantationof SAPNS seeded with SCs showing CGRP-positive axons (arrows, N). (O) Merged image of M and N, with DAPI staining. Scale bar = 20 μm.

318 J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

Fig 7. Quantification of axonal regeneration into the implants. Afterimmunostaining, the numbers of NF-, CGRP-, or 5HT-positive axons werequantified and compared among the SAPNS implant alone, SAPNS withNPCs, and SAPNS with SCs groups. The data are expressed as mean ±SEM, P N .05, *P b .05, **P b .01.

319J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

were very large, especially at the edges of the implants(Figure 5, H).

Axons regenerated into the implants

Anti-neurofilament (NF) immunolabeling revealed thataxons grew into the implants whether the SAPNS was usedalone or seeded with SCs or NPCs. However, the numberand distribution of the axons in the implants varied in thedifferent groups. Very few NF axons were observed in theSAPNS-alone transplant animals (Figure 6, A–C).

SAPNS implants with either the NPCs (Figure 6, D–F) orSCs (Figure 6, G–I) showed more axons than the implants ofSAPNS alone. SAPNS transplanted with SCs showed manymore axons than the SAPNS plus NPCs (Figure 7).

To assess whether raphespinal axons and primary sensoryaxons grew in the implants, 5HT and CGRP immunohis-tochemistry was performed. Similar to the NF-positive axons,more 5HT-positive (Figure 6, J–L) and CGRP-positive(Figure 6, M–O) axons could be found in the SAPNS plusSCs transplant group than other groups (Figure 7).

Discussion

Two-way migration of cells

We found that cells migrated in both directions: first,DAPI staining showed large amounts of host cells that hadmigrated into the implant of pretreated SAPNS. Second,where pretreated SAPNS was transplanted into the injuredspinal cord of GFP-transgenic rats, GFP-positive cells werealso found migrating from host to implant. Finally, thegrafted cells of SAPNS with GFP-positive SCs or NPCs,transplanted into the spinal cord of wild-type rats, survivedvery well in the implants, with some of them even migratinginto the host tissue.

In another important finding we observed many bloodvessels in the implants, indicating the presence of a bloodsupply to allow healing and regeneration. Some vessels werevery large, especially at the edges of the implants.

Integration with surrounding tissue

Many kinds of cells, tissues, or biomaterials have beenused for transplantation in an attempt to repair the spinalcord's conductive function [2,16,17,42] and until now, mosthave not integrated with surrounding tissue [23,24,43,44], asa result of the difficulty of filling and holding the injectedcells in the lesion cavities [5]. These cavities, or cysts,formed in the injured spinal cord are one of the mostimportant physical obstacles for axon regeneration, structurerebuilding, and return of function.

We learned that to fill the cavity and build a bridge it isimportant and necessary to pretreat SAPNS in culturemedium before transplantation, so as to buffer the pH. Weobserved that untreated SAPNS elicited serious inflamma-tion in the host tissue, creating distinct gaps and cystssurrounding the implants, and believe the primary reason isdue to low pH. It appeared that there was insufficientbuffering of the low pH in the lesion site, thus leading to aninflammatory response. After SAPNS was pretreated inculture medium, boosting the pH to a level similar to that ofblood, as in a previous study of hemostasis [45], it was ableto acclimatize to the microenvironment of the host.

It is interesting to note that implantation of SAPNS alonewithout pretreatment could repair an injured optical pathwayand restore visual function [25]. However, the present studyshows that SAPNS, without pretreatment, had little reparativeeffect on the injured spinal cord. Mechanisms underlying thisdifference between the brain and spinal cord injuries areunknown. One possibility is that there is more cerebrospinalfluid in the lesioned area of the brain than spinal cord, whichhas a better buffering effect for the untreated SAPNS.

Pre-treated SAPNS transplants have good properties inspinal cord repair, acting like a biomaterial to bridge thecavities caused by acute trauma. The pretreated SAPNSshowed significantly improved biocompatibility with thehost tissue, reducing the inflammation, filling the cysts andbuilding a bridge across which axons were able to grow intothe implants. It appeared to be completely biocompatibleboth in vivo and in vitro, integrating very well with the hostspinal cord in vivo and reducing inflammation caused by thetrauma. In vitro, merged stacks of multiple confocal opticalsections taken by a two-photon microscope 4 weeks after thecells were cultured within SAPNS, show images of live SCsand NPCs that have survived and migrated three-dimension-ally 4 weeks after the cells were cultured within SAPNS.

Increased axonal regeneration

The therapeutic potential of NPCs and SCs is wellestablished [9,46,47]: NPCs promote axonal regenerationeither by reconstituting a “bridge” through a lesion site capableof supporting axonal attachment and growth, or by secreting

320 J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

diffusible molecules, such as growth factors, to attract injuredaxons [4,47,48]. SCs are successful in spinal cord repair,expressing a variety of neurotrophic factors that support thegrowth of axons, axonal growth-supporting cell adhesionmolecules and axonal growth-promoting substrates, such aslaminin and fibronectin. In addition, SCs have also been shownto remyelinate regenerated axons [9]. Pretreated SAPNStransplants can fill and bridge the cavities caused by acutetrauma, which suggests that grafted biomaterials in combina-tion with SCs or NPCs may be the best strategy to bridge theinjured spinal cord and a potential solution for repair.

Conclusion

Artificial 3D scaffolds that store or attract cells arecritically important in regenerative medicine [49]. TheSAPNS scaffold has been shown to support the attachmentof a variety of cells in culture [27], but until now the survivaland migration of cells in that scaffold, after transplantationinto the spinal cord, has not been reported. Our study showsthat both NPCs and SCs can survive, migrate, anddifferentiate within the SAPNS scaffolds three-dimension-ally, not only in vitro but in vivo. Furthermore, there is bothrobust migration of host cells and growth of blood vesselsinto the scaffolds. These results indicate that the SAPNS is apotential material to repair damaged tissue and to bridge theinjured axons cross the lesion site after spinal cord injury.

Acknowledgments

Funding support was provided by the University of HongKong and Hong Kong Research Grants Council to W.T. Wu.

References

[1] Schwab ME, Bartholdi D. Degeneration and regeneration of axons inthe lesioned spinal cord. Physiol Rev 1996;76:319-70.

[2] Dusart I, Schwab ME. Secondary cell death and the inflammatoryreaction after dorsal hemisection of the rat spinal cord. Eur J Neurosci1994;6:712-24.

[3] Hulsebosch C. Recent advances in pathophysiology and treatment ofspinal cord injury. Adv Physiol Educ 2002;26:238-55.

[4] Lu P, Jones L, Snyder E, TuszynskyM. Neural stem cells constitutivelysecrete neurotrophic factors and promote extensive host axonal growthafter spinal cord injury. Exp Neurol 2003;181:115-29.

[5] Vroemen M, Aigner L, Winkler J, Weidner N. Adult neural progenitorcells grafts survival after acute spinal cord injury and integrate alongaxonal pathways. Eur J Neurosci 2003;18:743-51.

[6] Deumens R, Koopmans GC, Honig WM, Maquet V, Jerome R,Steinbusch HW, et al. Chronically injured corticospinal axons do notcross large spinal lesion gaps after a multifactorial transplantationstrategy using olfactory ensheathing cell/olfactory nerve fibroblast-biomatrix bridges. J Neurosci Res 2006;83:811-20.

[7] Reier PJ, Stokes BT, Thompson FJ, Anderson DK. Fetal cell grafts intoresection and contusion/compression injuries of the rat and cat spinalcord. Exp Neurol 1992;115:177-88.

[8] David S, Aguayo A. Axonal elongation into peripheral nervous system“bridges” after central nervous system injury in adult rats. Science1981;215:931-3.

[9] Oudega M, Xu X. SC transplantation for repair of the adult spinal cord.J Neurotrauma 2006;23:453-67.

[10] Li Y, Decherchi P, Raisman G. Transplantation of olfactoryensheathing cells into spinal cord lesions restores breathing andclimbing. J Neurosci 2003;23:727-31.

[11] Mcdonald JW, Liu XZ, Qu Y, Liu S, Mickey SK, Turetsky D,et al. Transplanted embryonic stem cells survive, differentiate andpromote recovery in injured rat spinal cord. Nat Med 1999;5:1410-2.

[12] Hofstetter CP, Schwarz EJ, Hess D, Widenfalk J, El Manira A, ProckopDJ, et al. Marrow stromal cells form guiding strands in the injuredspinal cord and promote recovery. Proc Natl Acad Sci U S A 2002;99:2199-204.

[13] Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead C, FehlingsM. Delayed transplantation of adult neural precursor cells promotesremyelination and functional neurological recovery after spinal cordinjury. J Neurosci 2006;26:3377-89.

[14] Guo JS, Zeng YS, Li H, Huang WL, Liu RY, Li XB, et al. Co-transplant of neural stem cells and NT-3 gene modified Schwann cellspromote the recovery of transected spinal cord injury. Spinal Cord2007;45:15-24.

[15] Mitsui T, Fischer I, Shumsky JS, Murray M. Transplants of fibroblastsexpressing BDNF and NT-3 promote recovery of bladder and hindlimbfunction following spinal contusion injury in rats. Exp Neurol 2005;194:410-31.

[16] Schmidt C, Leach J. Neural tissue engineering: strategies for repair andregeneration. Ann Rev Eng 2003;5:295-347.

[17] Geller H, Fawcett J. Building a bridge: engineering spinal cord repair.Exp Neurol 2002;174:125-36.

[18] Scott M,Will R, Ironside J, Nguyen H, Tremblay P, DeArmond S, et al.Compelling transgenetic evidence for transmission of bovine spongi-form encephalopathy prions to humans. Proc Natl Acad Sci U S A1999;96:15137-42.

[19] Holmes T. Novel peptide-based biomaterial scaffolds for tissueengineering. Trends Biotechnol 2002;20:16-21.

[20] Nomura H, Tator C, Shoichet M. Bioengineered strategies for spinalcord repair. J Neurotrauma 2006;23:496-507.

[21] Moore M, Friedman J, Lewellyn E, Mantila S, Krych A, AmeenuddinS, et al. Multiple-channel scaffolds to promote spinal cord axonregeneration. Biomaterials 2006;27:419-29.

[22] Zhang S, Gelain F, Zhao X. Designer self-assembling peptidenanofiber scaffolds for 3D tissue cell cultures. Semin Cancer Biol2005;15:413-20.

[23] Stokols S, Tuszynski M. Freeze-dried agarose scaffolds with uniaxialchannels stimulate and guide linear axonal growth following spinalcord injury. Biomaterials 2006;27:443-51.

[24] Prang P, Muller R, Eljaouhari A, Heckmann K, KunzW,Weber T, et al.The promotion of oriented axonal regrowth in the injured spinal cordby alginate-based anisotropic capillary hydrogels. Biomaterials2006;27:3560-9.

[25] Ellis-Behnke RG, Liang YX, You SW, Tay DK, Zhang S, So K, et al.Nano neuro knitting: peptide nanofiber scaffold for brain repair andaxon regeneration with functional return of vision. Proc Natl Acad SciU S A 2006;103:5054-9.

[26] Lupi O. Prions in dermatology. J Am Acad Dermatol 2002;46:790-3.[27] Holmes T, Lacalle S, Su X, Liu G, Rich A, Zhang S. Extensive neurite

outgrowth and active synapse formation on self-assembling peptidescaffolds. Proc Natl Acad Sci U S A 2000;97:6728-33.

[28] Zhang S. Fabrication of novel biomaterials through molecular self-assembly. Nat Biotechnol 2003;21:1171-8.

[29] Garreta E, Genove E, Borros S, Semino C. Osteogenic differentiationof mouse embryonic stem cells and mouse embryonic fibroblasts in athree-dimensional self-assembling peptide scaffold. Tissue Eng2006;12:2215-27.

[30] Davis M, Hsieh P, Takahashi T, Song Q, Zhang S, Kamm R,et al. Local myocardial insulin-like growth factor 1 (IGF-1)delivery with biotinylated peptide nanofibers improves cell therapyfor myocardial infarction. Proc Natl Acad Sci U S A 2006;103:8055-60.

321J. Guo et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 3 (2007) 311–321

[31] Zhang S, Holmes T, Lockshin C, Rich A. Spontaneous assembly of aself-complementary oligopeptide to form a stable macroscopicmembrane. Proc Natl Acad Sci U S A 1993;90:3334-8.

[32] Zhang S, Holmes T, DiPersio C, Hynes R, Su X, Rich A. Self-complementary oligopeptide matrices support mammalian cell attach-ment. Biomaterials 1995;16:1385-93.

[33] Caplan M, Moore P, Zhang S, Kamm R, Lauffenburger D. Self-assembly of a beta-sheet oligopeptide is governed by electrostaticrepulsion. Biomacromolecules 2000;1:627-31.

[34] Marini D, Hwang W, Lauffenburger D, Zhang S, Kamm D. Left-handed helical ribbon intermediates in the self-assembly of a β-sheetpeptide. Nano Lett 2002;2:295-9.

[35] Morrissey TK, Kleitman N, Bunge RP. Isolation and functionalcharacterization of Schwann cells derived from adult peripheral nerve.J Neurosci 1991;11:2433-42.

[36] Xu XM, Guénard V, Kleitman N, Bunge M. Axonal regeneration intoSC-seeded guidance channels grafted into transected adult rat spinalcord. J Comp Neurol 1995;351:145-60.

[37] Johe KK, Hazel TG, Muller T, Dugich-Djordjevic MM, Mckay RD.Single factors direct the differentiation of stem cells from the fetal andadult central nervous system. Genes Dev 1996;10:3129-40.

[38] Enomoto M, Shinomiya K, Okabe S. Migration and differentiationof neural progenitor cells from two different regions of embryoniccentral nervous system after transplantation into the intact spinal cord.Eur J Neurosci 2003;17:1223-32.

[39] Challa VR, Moody DM, Brown WR, Zagzag D. A morphologic studyof the vasculature of malignant gliomas using thick celloidin sectionsand alkaline phosphatase stain. Clin Neuropathol 2004;23:167-72.

[40] Hsu JY, Xu XM. Early profiles of axonal growth and astroglialresponse after spinal cord hemisection and implantation of Schwanncell-seeded guidance channels in adult rats. J Neurosci Res 2005;82:472-83.

[41] Kamada T, Koda M, Dezawa M, Yoshinaga K, Hashimoto M,Koshizuka S, et al. Transplantation of bone marrow stromal cell–derivedSchwann cells promotes axonal regeneration and functional recoveryafter complete transection of adult rat spinal cord. J NeuropatholExp Neurol 2005;64:37-45.

[42] Burge M, Pearse D. Transplantation strategies to promote repair of theinjured spinal cord. J Rehabil Res Dev 2003;40:55-62.

[43] Teng Y, Lavik E, Qu X, Park K, Ourednik J, Zurakowski D, et al.Functional recovery following traumatic spinal cord injury mediatedby a unique polymer scaffold seeded with neural stem cells. Proc NatlAcad Sci U S A 2002;99:3024-9.

[44] Patista C, Mulder M, Gautier S, Maquet V, Jerome R, Oudega M.Freeze-dried poly(d,l-lactic acid) macroporous guidance scaffoldsimpregnated with brain-derived neurotrophic factor in the transectedadult rat thoracic spinal cord. Biomaterials 2004;25:1569-82.

[45] Ellis-Behnke RG, Liang YX, Tay DK, Kau PF, Schneider GE, ZhangS, et al. Nano hemostat solution: immediate hemostasis at thenanoscale. Nanomedicine 2006;2:207-15.

[46] Zeng YS, Ding Y, Wu LZ, Guo JS, Li HB, Wong WM, et al. Co-transplantation of SC promotes the survival and differentiation ofneural stem cells transplanted into the injured spinal cord. DevNeurosci 2005;27:20-6.

[47] Enzmann G, Benton R, Talbott J, Cao Q, Whittemore S. Functionalconsiderations of stem cell transplantation therapy for spinal cordrepair. J Neurotrauma 2006;23:479-95.

[48] Toda H, Tsuji M, Nakano I, Kobuke K, Hayashi T, Kasahara H, et al.Stem cell–derived neural stem/progenitor cell supporting factor is anautocrine/paracrine survival factor for adult neural stem/progenitorcells. J Biol Chem 2003;278:35491-500.

[49] Silva G, Czeisler C, Niece K, Beniash E, Harrington D, Kessler J, et al.Selective differentiation of neural progenitor cells by high-epitopedensity nanofibers. Science 2004;303:1352-5.