evidence of innervation following extracellular matrix scaffold-mediated remodelling of muscular...

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE R E S E A R C H A R T I C L EJ Tissue Eng Regen Med 2009; 3: 590–600.Published online 21 August 2009 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.200

Evidence of innervation following extracellularmatrix scaffold-mediated remodelling of musculartissues

Vineet Agrawal1,2,4, Bryan N. Brown1,2, Allison J. Beattie1,3, Thomas W. Gilbert1,2,3

and Stephen F. Badylak1,2,3*1McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA2Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA3Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA4Medical Scientist Training Program, University of Pittsburgh, Pittsburgh, PA, USA

Abstract

Naturally occurring porcine-derived extracellular matrix (ECM) has successfully been used asa biological scaffold material for site-specific reconstruction of a wide variety of tissues. Thesite-specific remodelling process includes rapid degradation of the scaffold, with concomitantrecruitment of mononuclear, endothelial and bone marrow-derived cells, and can lead to theformation of functional skeletal and smooth muscle tissue. However, the temporal and spatialpatterns of innervation of the remodelling scaffold material in muscular tissues are not wellunderstood. A retrospective study was conducted to investigate the presence of nervous tissue ina rat model of abdominal wall reconstruction and a canine model of oesophageal reconstructionin which ECM scaffolds were used as inductive scaffolds. Evidence of mature nerve, immaturenerve and Schwann cells was found within the remodelled ECM at 28 days in the rat body wallmodel, and at 91 days post surgery in a canine model of oesophageal repair. Additionally, amicroscopic and morphological study that investigated the response of primary cultured neuronsseeded upon an ECM scaffold showed that neuronal survival and outgrowth were supported by theECM substrate. Finally, matricryptic peptides resulting from rapid degradation of the ECM scaffoldinduced migration of terminal Schwann cells in a concentration-dependent fashion in vitro. Thefindings of this study suggest that the reconstruction of tissues in which innervation is an importantfunctional component is possible with the use of biological scaffolds composed of extracellularmatrix. Copyright 2009 John Wiley & Sons, Ltd.

Received 5 January 2009; Revised 15 May 2009; Accepted 1 July 2009

Keywords extracellular matrix (ECM); innervation; regenerative medicine; tissue remodelling; biologicalscaffolds

1. Introduction

Innervation is implicit to the success of tissue engineeringand regenerative medicine strategies with an endgoal of functional tissue restoration. Numerous in vitrostudies have investigated the ability of naturally derived

*Correspondence to: Stephen F. Badylak, McGowan Institutefor Regenerative Medicine, University of Pittsburgh, 100Technology Drive, Suite 200, Pittsburgh, PA 15219, USA.E-mail: [email protected]

biomaterials (Hadlock et al., 2001; Smith et al., 2004;Phillips et al., 2005; Donzelli et al., 2006; Su et al.,2007; Chunzheng et al., 2008) and artificially synthesizedconstructs (Johansson et al., 2006; Recknor et al., 2006;Gomez et al., 2007; Schnell et al., 2007; Sorensen et al.,2007) to support neuronal outgrowth and differentiation.However, relatively few studies have investigated theprocess of innervation following in vivo placement of suchbiomaterials or tissue-engineered constructs (Pope et al.,1997; Patrick et al., 2001; Caione et al., 2006; Caissieet al., 2006; Lopes et al., 2006; Ueno et al., 2007).

Copyright 2009 John Wiley & Sons, Ltd.

Innervation following ECM scaffold-mediated remodelling of muscular tissues 591

Biological scaffold materials composed of extracellularmatrix (ECM) have been used in preclinical animal studiesand human clinical applications to facilitate constructiveremodelling of a variety of muscular tissues, includingoesophagus (Badylak et al., 2000, 2005; Lopes et al.,2006), myocardium (Kochupura et al., 2005; Robinsonet al., 2005; Badylak et al., 2006; Ota et al., 2007),blood vessels (Badylak et al., 1989; Lantz et al., 1990,1992), urinary bladder (Kropp et al., 1995, 1996; Popeet al., 1997; Record et al., 2001) and skeletal muscle(Prevel et al., 1995; Clarke et al., 1996; Badylak et al.,2001, 2002). These studies have shown that the hostresponse to ECM scaffold implantation is characterizedby rapid degradation of the scaffold, with concomitantreplacement by organized, site-appropriate, functionalhost tissue (Allman et al., 2001, 2002; Record et al., 2001;Gilbert et al., 2007B).

The mechanisms responsible for the remodellingresponse following placement of an ECM scaffold arenot fully understood, but are thought to include bioactivematricryptic peptides generated following rapid scaffolddegradation (Record et al., 2001; Li et al., 2004; Brennanet al., 2006; Gilbert et al., 2007A, 2007B), recruitment ofboth differentiated and multi-potential cells to the site ofremodelling (Badylak et al., 2002; Valentin et al., 2006;Zantop et al., 2006), angiogenesis (Badylak et al., 2000;Li et al., 2004) and deposition of site-appropriate tissuein response to appropriate mechanical cues (Gilbert et al.,2006; Gilbert et al., 2007C). However, the contributionof nervous tissue to the remodelling process has not beeninvestigated.

Recent studies compared the in vivo remodelling ofcommercially available ECM scaffold devices in a ratmodel of abdominal wall repair (Valentin et al., 2006;Brown et al., 2009). Islands of morphologically normalskeletal muscle replaced portions of acellular ECMscaffolds that had not been chemically cross-linked withinthe first 28 days of the remodelling process. However,it was not reported whether these islands of musclewere innervated and functional. An earlier study of ECM-mediated oesophageal reconstruction showed peristalsisin positive-contrast oesophagrams and spontaneouscontraction immediately after euthanasia (Badylak et al.,2005). ECM scaffold remodelling in the urinary bladder(Pope et al., 1997; Caione et al., 2006) and stomach(Ueno et al., 2007) has shown innervation of new muscletissue. It is logical that morphologically normal-appearingskeletal or smooth muscle formed during remodelling ofan ECM scaffold would be accompanied by a motor nervesupply.

The objective of the present study was to determinethe temporal and spatial patterns of innervation duringthe process of ECM scaffold remodelling through aretrospective analysis of animal models of oesophagealand body wall reconstruction (Badylak et al., 2005;Brown et al., 2009). Additionally, in vitro studies wereperformed to determine the ability of an ECM scaffoldto support attachment and survival of a neuronalcell population and the ability of ECM matricryptic

peptides to recruit cells known to support neuronalregeneration.

2. Methods

2.1. Overview

A retrospective analysis was conducted to determinethe temporal appearance and spatial distribution ofinnervation at the site of ECM remodelling in two separatestudies of rat abdominal wall reconstruction (Brownet al., In press) and canine oesophageal repair (Badylaket al., 2005) (Table 1). The biological scaffold materialused in both studies consisted of ECM derived from theporcine urinary bladder (UBM). Immunohistochemicalmethods were used to identify the presence of maturenerve (neurofilament), growing nerves (GAP-43) (Goslinet al., 1990) and the presence of Schwann cells [markedby glial fibrillary acidic protein (GFAP)] (Triolo et al.,2006) in the tissues of interest. GFAP was chosen asthe marker for Schwann cells because previous studieshave demonstrated that Schwann cells retain expressionof GFAP following dedifferentiation, while losing theexpression of other mature markers such as S-100(Jessen and Mirsky, 1991; Garavito et al., 2000; Trioloet al., 2006). In a separate prospective study, primaryspinal cord neurons were isolated from embryonic ratspinal cords, cultured upon UBM and examined for theexpression of neuron and glial specific markers (β-tubulin-III and GFAP) by immunofluorescent labelling techniques.Finally, the migration of Schwann cells in response tomatricryptic peptides from UBM was investigated in aBoyden chamber assay.

All methods were approved by the Institutional AnimalCare and Use Committee at the University of Pittsburghand performed in compliance with NIH Guidelines for theCare and Use of Laboratory Animals.

Table 1. Surgical groups for canine oesophagus and rat abdomi-nal wall studies∗

Group No. Surgical procedure

1 Segmental resection of full-thickness canineoesophagus, replacement with ECM

2 Segmental removal of full-circumference canineendomucosa, muscularis externa left intact, noECM scaffold

3 Segmental removal of full circumference canineendomucosa plus 70% of muscularis externa. ECMscaffold used to replace endomucosa

4 Segmental removal of full circumference canineendomucosa. ECM scaffold used to replaceendomucosa

5 Partial thickness resection of rat abdominal wall,ECM scaffold used to replace external and internaloblique muscle

∗Badylak et al. (2005); Brown et al. (2009).

Copyright 2009 John Wiley & Sons, Ltd. J Tissue Eng Regen Med 2009; 3: 590–600.DOI: 10.1002/term

592 V. Agrawal et al.

2.2. Preparation of urinary bladder matrix

The urinary bladder matrix was prepared using previouslydescribed methods (Freytes et al., 2004; Robinson et al.,2005). Briefly, porcine urinary bladders were harvestedfrom market weight pigs (approximately 110–130 kg)immediately after sacrifice. Residual external connectivetissues, including adipose tissue, were trimmed and allresidual urine was removed by repeated washes withtap water. The urothelial layer was removed by soakingof the material in 1 N saline. The tunica serosa, tunicamuscularis externa, tunica submucosa and most of themuscularis mucosa were mechanically delaminated fromthe bladder tissue. The remaining basement membraneof the tunica epithelialis mucosa and the subjacent tunicapropria were collectively termed UBM. The UBM wasdecellularized and disinfected by immersion in 0.1% v/vperacetic acid (PAA; σ ), 4% v/v ethanol and 96% v/vdeionized water for 2 h. The material was then washedtwice for 15 min with phosphate-buffered saline (PBS),pH = 7.4, and twice for 15 min with deionized water.

2.3. Rat abdominal wall model

2.3.1. Preparation of four-layer body wall ECMdevice

Multilaminate (four-layer) sheets were constructed aspreviously described (Freytes et al., 2004). In brief, fourhydrated sheets of UBM were stacked on top of oneanother, each at a 90◦ orientation to the adjacent layers.The constructs were then placed into plastic pouches andattached to a vacuum pump (Model D4B, Leybold, Export,PA, USA) with a condensate trap inline. The constructswere subjected to a vacuum of 710–740 mmHg for10–12 h to remove the water and form a tightly coupledmultilaminate construct. The devices were terminallysterilized with ethylene oxide.

2.3.2. Surgical procedure

The surgical procedure of the partial-thickness rat abdom-inal wall reconstruction has been previous described(Badylak et al., 2002; Brown et al., In press). A ven-tral midline incision was made and the adjacent subcutisdissected to expose the ventral lateral abdominal wall.A 1 cm × 1 cm partial thickness defect was then cre-ated in the exposed musculature, leaving the underlyingperitoneum, transversalis fascia, transversus abdominisand the overlying skin intact. The muscular defects werereconstructed with a UBM test article. The test article wassutured to the adjacent abdominal wall with 4-0 Prolenenon-resorbable suture at each corner to secure the testarticle and to allow for identification of the device bound-aries at the time of euthanasia and explantation. The skinwas closed using an resorbable 4-0 Vicryl suture. The ani-mals were recovered from anaesthesia on a heating padand allowed normal activity and diet for the remainder

of the study period. The animals were euthanized at timepoints of 3, 7, 14 and 28 days and the defect site wasexcised with a small portion of the surrounding nativetissue.

2.4. Canine oesophageal resection study

2.4.1. Device preparation

Multilayer tubes were created by wrapping hydratedsheets of UBM around a 30 mm perforated tube/mandrelthat was covered with umbilical tape for a total of fourcomplete revolutions (i.e. a four layer tube)(Badylaket al., 2005). The constructs were then placed into plasticpouches and attached to a vacuum pump (Leybold) witha condensate trap inline. The constructs were subjected toa vacuum of 710–740 mmHg for 10–12 h to remove thewater and form a tightly coupled multilaminate construct.After removal of each device from the mandrel, they wereterminally sterilized with ethylene oxide.

2.4.2. Surgical procedure

Surgical procedures were completed as reported (Badylaket al., 2005). Briefly, adult female mongrel dogs weighing17–24 kg were divided into four groups and subjectedto surgical resection of 5 cm of the full circumferenceof the oesophageal endomucosa (epithelium, basementmembrane, lamina propria, muscularis mucosa andtunica submucosa), with removal of varying amountsof the muscularis externa. Four surgical groups wereinvestigated in the canine study, as listed in Table 1.In group 1, the full thickness of the 5 cm oesophagealsegment was removed and the defect was repairedwith a tubular UBM scaffold. In group 2, all layers ofthe oesophagus except for the muscularis externa wereremoved, and the defect was untreated to serve as acontrol. In group 3, 70% of the circumference of the 5 cmsegment of oesophagus was subjected to full thicknessresection and the defects were repaired with a tubularUBM scaffold. Finally, group 4 was the same as group 2,with the exception that the resected endomucosal tissuewas replaced with a tubular UBM scaffold. In groups 3 and4, the UBM was sutured to the native endomucosa using5-0 Prolene sutures and the muscle and subcutaneoustissues were closed with 4-0 Vicryl sutures. Followingsurgery, the animals were recovered from anaesthesiaand allowed normal ambulation. The animals were givensoft food from a raised bowl for 2 weeks or until they couldtolerate a normal diet. At the time of sacrifice, the defectsite was excised with a small portion of the adjacent nativetissue. For the present study, sections of the remodelledscaffold from three animals from group 3 that had beensacrificed at 91, 101 and 104 days after surgery wereexamined for markers of nervous tissue. Sections fromthe animal in group 2 that showed a scar tissue responsewith stricture served as a negative control. Sections froman animal in group 4 that had normal intact muscle servedas a positive control for immunohistochemical studies.



2.5. Immunohistochemistry of animal sections

Paraffin-embedded specimens from both studies were cutinto 6 µm thick sections and mounted onto glass slides.The specimens were deparaffinized by treatment withxylene and exposure to a graded series of ethanol solutions(100–70%). Following deparaffinization, the slides wereplaced in citrate antigen retrieval buffer (10 mM citric acidmonohydrate, pH 6.0; C1285, Spectrum, New Brunswick,NJ, USA) at 95–100 ◦C for 20 min. The buffer was allowedto cool to room temperature on ice and the slides werewashed twice in Tris-buffered saline/Tween 20 (TrizmaBase, T6066; and Tween 20, P7949; Sigma, St. Louis, MO,USA) solution, pH 7.4, and twice in PBS. The sections werethen incubated in 2% normal horse serum (S-2000, VectorLabs, Burlingame, CA, USA) for 1 h at room temperaturein a humidified chamber to inhibit non-specific binding ofthe primary antibody. Following incubation in blockingserum, the sections were incubated in primary antibodyin a humidified chamber at 4 ◦C overnight. Each tissuespecimen was exposed to antibodies to the light chain ofneurofilament (Anti-neurofilament; M0762, Dako USA,Carpinteria, CA, USA), to a marker that localizes tothe growth cones of regenerating neurons (Anti-GAP43;NB600, Novus Biologicals, Littleton, CO, USA) or to amarker that is known to localize to Schwann cells in theperipheral nervous system following injury (Anti-GFAP;M0761, DakoUSA). Following overnight incubation, theslides were washed three times in PBS. The sections werethen incubated in a solution of 3% H2O2 in methanol for30 min at room temperature to quench any endogenousperoxidase activity. Following H2O2 treatment, the slideswere washed three times in PBS and incubated insecondary antibody for 30 min in a humidified chamberat room temperature prior to three more washes in PBS.The sections were then incubated in Vectastain ABC (EliteABC Kit, PK6200, Vector Labs) reagent for 30 min in ahumidified 37 ◦C chamber, rinsed three times in PBS andincubated in 4% diaminobenzadine substrate solution(SK4100, Vector Labs) at room temperature until theslides showed the desired staining affinity. Finally, theslides were rinsed in water to stop the developmentof the diaminobenzadine substrate and counterstainedusing Harris’s haematoxylin stain (Thermo Electron Corp.,Shandon, Pittsburgh, PA, USA). The slides were thendehydrated using the reverse of the deparaffinizationtreatment described above and then coverslipped. EachPBS rinse in the protocol was for 3 minutes at roomtemperature, with occasional agitation.

Anti-neurofilament antibody was used at a concentra-tion of 1 : 100. Anti-GAP43 was used at a concentrationof 1 : 250. Anti-GFAP antibody was used at a concen-tration of 1 : 500. Corresponding secondary antibodieswere used at a concentration of 1 : 100 for biotinylatedanti-mouse IgG (BA-2000, Vector Labs) and 1 : 200 forbiotinylated anti-rabbit IgG (PK-4001, Vector Labs). Allantibodies were diluted in filtered PBS, pH 7.4. Nativecanine oesophagus and rat abdominal wall were used aspositive control tissue samples for staining.

2.6. Culture of neurons on UBM

2.6.1. Isolation of embryonic spinal cordneurons

Spinal cord neurons were isolated from embryonic day 14Sprague–Dawley rat pups. Spinal cords were collected incold Hanks’ buffered salt solution without Ca2+ and Mg2+(14 170-112, Gibco, Carlsbad, CA, USA), minced intopieces approximately 0.5 mm2 in size and enzymaticallydissociated in 2 ml 0.25% trypsin solution containing0.05% collagenase L1 (MP Biomedicals, Solon, OH, USA)at 37 ◦C for 20 min. Cell digestion was inhibited by adding2 ml SBTI-DNAse solution (0.52 mg/ml soybean trypsininhibitor, T-9003; 3.0 mg/ml BSA, A-2153; 0.04 mg/mlbovine pancreas DNAse, D-4263; Sigma). The cellsuspension was gently triturated with a Pasteur pipetteand centrifuged at 800 × g for 5 min. The resulting pelletwas then resuspended in plating medium and gentlytriturated. The plating medium consisted of 20% horseserum (16 050-130, Gibco), 2 mM L-glutamine (25 030-081, Gibco), 5 ml HBSS without Ca2+ and Mg2+ (14 170-112, Gibco) and 9.8 ml Dulbecco’s modified Eagle’smedium (DMEM; D5648, Sigma). All non-dispersed tissuewas allowed to settle before being discarded.

2.6.2. Cell seeding

Single sheets of UBM were cut with a 1.5 cm diametercircular punch to create discs for use in cell cultureexperiments. Using sterile stainless steel rings to anchorthe UBM discs, spinal cord neurons were seeded onthe abluminal surface of the discs in plating mediumat a plating density of 3 × 105 cells/cm2 and allowedto adhere for 4 h in culture conditions of 5% CO2

and 37 ◦C in a humidified environment. After 4 h, themedium was exchanged with 1 ml serum-free culturemedium containing Neurobasal-A (NBA, 10 888-022,Gibco), 1× B27 supplement and 1 mM Glutamax (35 050-061, Gibco). The cells were maintained in a humidified5% CO2 atmosphere at 37 ◦C, with 50% of the culturemedium changed every 4 days. Cells were seeded atthe same concentration on poly-L-lysine-coated coverslips(12.5 µg/ml in H2O; P1274, Sigma) as a control.

2.6.3. Immunofluorescent labelling

Cells seeded on control coverslips were maintained inculture for 5 days. They were then washed three timeswith PBS warmed to 37 ◦C, fixed with 4% formaldehydeat 37 ◦C for 15 min and incubated in permeablizationbuffer (0.5% Triton X) for 15 min at room temperature.All antibodies were diluted to working concentration ina modified PBS solution (0.3 M NaCl PBS, 0.3% TritonX). All primary antibodies were diluted to a workingdilution of 1 : 100 and incubated overnight at 4 ◦C. Allsecondary antibodies, also at a working dilution of 1 : 100,were incubated for 1 h at 37 ◦C. After each stage ofantibody incubation, the samples were gently washed



three times for 5 min in PBS. Samples were examinedusing antibodies against β-tubulin III (Sigma) and GFAP(Dako) and secondary antibodies, FITC-conjugated goatanti-mouse IgG2b and TRITC-conjugated goat anti-rabbit(Cambridge Biosciences, Cambridge, UK), respectively.All samples were mounted in Vectashield mountingmedium containing DAPI (Vector Labs).

The UBM cell-seeded discs were harvested after 5 daysand fixed with 10% neutral buffered formalin for 24 h.Sections of the discs were trimmed, embedded in paraffinand then cut in cross-sections into 5 µm thick sections. Allsections were first deparaffinized before being immersedin permeablization buffer (0.5% Triton X) for 15 minat room temperature. Sections were triple-stained asdescribed above, with and without the primary antibodies.

2.7. Preparation of samples for SEM analysis

All samples examined by SEM were fixed for 2 h with2.5% gluteraldehyde. After three 15 min washes in PBS,the samples were dehydrated through a graded series ofethanol washes, followed by chemical drying with 100%hexamethyldisilazane (HMDS) overnight. Samples weresputter-coated with a 4 nm thick layer of gold palladium(Cressington 108 Sputter Coater, Cressington, UK) andthen examined using a JEM 6335F field emission gun SEM(JEOL, Peabody, MA) with an excitation voltage of 4 kV.

2.8. Migration and survival of Schwann cellsin response to ECM degradation

2.8.1. Formation of peptide fragments of UBM

The preparation of UBM matricryptic peptides has beendescribed previously (Reing et al., 2008). UBM wasdigested with pepsin by mixing 1 g lyophilized, powderedUBM with 100 mg pepsin (Sigma-Aldrich, St. Louis, MO,USA) in 100 ml 0.01 M HCl. The pepsin was allowed todigest the UBM for 48 h under constant stirring at 4 ◦C.The pepsin was inactivated by bringing the mixture to a pHof 7.4 using PBS, and the resulting peptide fragments wereused in migration and survival assays at concentrations of50, 100 and 200 µg/ml of dry weight protein.

2.8.2. Cell migration assay

Schwann cells (RT4-D6P2T cell line) were obtainedfrom the ATCC and cultured in medium containingDMEM, 10% FBS, 100 U/ml penicillin and 100 µg/mlstreptomycin. Passages 2–4 were utilized for the presentstudy. Cells at 70–80% confluence were starved in serum-free DMEM for 16 h. After starvation, the cells weretrypsinized with 0.25% trypsin and 0.53 mM EDTA for2 min, and resuspended in culture medium. The cellswere centrifuged at 1200 r.p.m. (Sorvall Legend RT,Heraeus 6445 Rotor) for 5 min and the pellet wasresuspended in serum-free DMEM and incubated in a

humidified environment at 37 ◦C in 5% CO2 for 1 h.Polycarbonate PFB filters (NeuroProbe, Gaithersburg, MD,USA) with pore size of 8 µm were coated on both sideswith 0.05 mg/ml collagen type I (BD Biosciences, SanJose, CA, USA). Peptide fragments of UBM were placedin the bottom chamber of 48-well migration chambers(NeuroProbe) and the filter was then placed over thebottom chamber. The upper chamber was then assembledonto the lower chamber and sealed. Approximately 30 000cells were placed into each well of the top chamber, andthe entire system was placed in a humidified environmentat 37 ◦C in 5% CO2 for 3 h, after which cells facingthe upper chamber on the filter were scraped off andcells facing the bottom filter were fixed in methanol andstained with DiffQuick (Dade AG, Liederbach, Germany).Each concentration of peptide fragments was run inquadruplicate, and the results of each condition werecompared to the results of a buffer control that containedall components at the same concentration except for thepeptide fragments. The total number of migrated cellsin each well was then counted, and a mean numberof migrated cells were calculated from the quadruplicatessample. The experiment was repeated three times, and thevalues for the chemotactic activity obtained for the peptidefragments and control were compared via Student’s t-test(two-tailed, unequal variance).

2.8.3. Survival assay

Schwann cells at 70–80% confluence were starved inserum-free DMEM for 16 h. After starvation, the cellswere trypsinized with 0.25% trypsin and 0.53 mM EDTAfor 2 min and then resuspended in culture medium.The cells were centrifuged at 1200 r.p.m. for 5 min,resuspended in serum-free DMEM and incubated in ahumidified environment at 37 ◦C in 5% CO2 for 1 h.In each well of a 96-well plate, 8000 cells were platedin serum-free DMEM. Varying concentrations of peptidefragments or corresponding buffer controls were addedto the wells. Culture medium was used in positive controlwells. After 48 h, the supernatants were removed andthe samples were extensively washed in PBS, lysed, and

Figure 1. (Badylak et al. 2005, Brown et al. 2009). Timeline forevaluation of innervation in the rat abdominal wall model andthe canine oesophageal model.



cell number was assayed using the Cambrex ViaLight

Plus Cell Proliferation Kit (Cambrex Corp.) and a M2SpectraMax plate reader. Each condition was testedin triplicate, and the assay was repeated three times.Differences were assessed via Student’s t-test (two-tailed,unequal variance).

3. Results

3.1. Rat abdominal wall model

Following abdominal wall repair in the rodent model, theUBM scaffold showed progressive remodelling with time.Histological examination showed a mixture of skeletalmuscle cells and organized collagenous connective tissueat the site of UBM placement at day 28. No positivestaining for neurons or Schwann cells was present at days3, 7 and 14. At day 28, positive staining for neuronswas observed in the remodelled UBM scaffold adjacentto the leading edge of growth of the adjacent nativeexternal oblique muscle. Nerves were localized to regions

of neo-muscular growth. Positive staining for GAP-43confirmed that these nerves were actively growing (Skeneand Willard, 1981; Hoffman, 1989; Goslin et al., 1990).Positive stain for Schwann cells co-localized with positivestain for nerves within the pockets of neo-muscular growth(Figure 2). The specificity of neurofilament, GFAP andGAP-43 stains was confirmed via staining of nerve innative rat abdominal wall muscle.

3.2. Canine oesophageal model

Histological examination showed the presence of a welldefined muscularis externa, tunica submucosa and tunicamucosa with complete epithelium. The remodelled tissuediffered from normal oesophagus in that the remodelledoesophagus lacked rugal folds, mature rete pegs in theepithelium and glandular structures in the submucosa(Badylak et al., 2005). Muscular tissue subjacent to thesubmucosa was examined for the presence of neuraltissue. Schwann cells and regenerating neurons wereobserved adjacent to mature skeletal muscle cells in allthree specimens at 91, 101 and 104 days post-surgery

Figure 2. Histological images of remodelled UBM 28 days after repair of a 1 cm × 1 cm defect in the rat abdominal wall, withspecific staining for (A) neurofilament, (B) GAP43 and (C) GFAP. Specificity of staining was confirmed by primary delete negativecontrols for (D) neurofilament, (E) GAP43 and (F) GFAP. Positive control staining was confirmed in native rat abdominal wallmuscle for (G) neurofilament, (H) GAP43 and (I) GFAP. All scale bars are 75 µm.



(Figure 3). In a region of neo-muscular growth, positivestaining for Schwann cells and GAP-43 was present in theabsence of positive staining for neurofilament (Figure 4).Positive staining was confirmed in the myenteric plexusof samples from group 4, where the muscularis externawas left intact (Figure 5). In samples from group2 (scar tissue with stricture), there was a lack oforganized muscularis mucosa and externa and incompleteepithelialization. No evidence of nerve was present inthese samples.

3.3. Neuronal survival and axonal outgrowthon UBM

Approximately 90% of cells isolated from the spinalcord tissue of embryonic Sprague–Dawley rat pups andgrown on control coverslips were positively labelled forthe neuron-specific cytoskeletal marker β-tubulin-III andwere therefore classified as neurons (Figure 6A). Toconfirm that the cells and corresponding cell processesattached to the abluminal scaffold surface were neurons,

Figure 3. Histological images of remodelled UBM 91 days after repair of a 5 cm long canine oesophageal endomucosal resectionof with removal of 70% of the muscularis externa. Specific staining is shown for (A) neurofilament, (B) GAP43 and (C) GFAP.Specificity of staining was confirmed by primary delete negative controls for (D) neurofilament, (E) GAP43 and (F) GFAP. All scalebars are 75 µm.

Figure 4. Histological images of remodelled UBM 104 days after repair of a 5 cm long canine oesophageal endomucosal resectionof with removal of 70% of the muscularis externa. Specific staining is shown for (A) neurofilament, (B) GAP43 and (C) GFAP.Specificity of staining was confirmed by primary delete negative controls for (D) neurofilament, (E) GAP43 and (F) GFAP.



Figure 5. Histological images of native oesophagus displaying the myenteric plexus of nerves, with specific staining for(A) neurofilament, (B) GAP43 and (C) GFAP.

Figure 6. β-tubulin III (green), glial fibrillary acidic protein (GFAP) (red) and DAPI (blue). (A) Immunofluorescent staining of cellsseeded on poly-L-lysine control coverslips after 5 days in culture. (B) Immunofluorescent images of cells seeded on the abluminalsurface of the UBM scaffolds. (C) Immunofluorescent images of seeded sections stained with secondary antibodies only. All scalebars are 50 µm.

sections were labelled for β-tubulin-III and glial markerGFAP. Cells seeded on the scaffold surface stainedpositive for both cytoskeletal markers (β-tubulin-III andGFAP; Figure 6B, C). SEM analysis showed that seededcells displayed long cellular processes that spanned theinter-fibril spaces of the abluminal surface structure(Figures 7A, 7B). At high magnification it was evidentthat cellular processes were interacting with the surfacestructure (Figure 7C).

3.4. Peptide fragments following degradationof UBM scaffold recruit Schwann cells

A concentration-dependent increase in migration ofSchwann cells towards UBM peptide fragments wasnoted, and migration at all concentrations was increasedcompared to buffer controls at each concentrationand positive control fibronectin (Figure 8A). The onlyconcentration for which an increase in cell survivalcompared to the negative control was noted was at aconcentration of 50 µg/ml (Figure 8B).

4. Discussion

This retrospective study showed the presence of nervetissue within sites of ECM scaffold remodelling, firstobserved at 28 days in the rat model and at 91 daysin the canine model. While sites of positive staining didnot morphologically match the appearance of normal

nerve fibres in muscle, these results are in accordancewith the findings that ECM-reconstructed oesophagusshowed positive-contrast oesophagrams and spontaneouscontraction during dissection at necropsy (Badylak et al.,2005). Because remodelling as a result of implantation ofa biological scaffold did not result in complete reformationof structures normally found in histological sections ofthe oesophagus and abdominal wall, it is possible thatnerve fibres also repopulated the site of remodelling anddid not resemble what is seen in normal sections. Theinitial appearance of neuronal axons in both models wasspatially and temporally associated with the presenceof new muscle tissue. In addition, the present studydemonstrated that the surface topology of the ECMscaffold itself supports in vitro neuronal outgrowth andsurvival. Finally, the present study shows that degradationof the ECM scaffold can produce peptide fragments thatrecruit Schwann cells. Overall, the findings of the presentstudy support the hypothesis that functional remodellingof ECM scaffolds is associated with the presence of matureand regenerating nerve.

The present findings are consistent with previous stud-ies that have shown innervation of the urinary bladderand oesophagus after repair with an ECM scaffold com-posed of porcine small intestinal submucosa (SIS–ECM)(Pope et al., 1997; Caione et al., 2006; Lopes et al., 2006).Although it is not known whether ECM scaffolds derivedfrom other tissue and organs (i.e. liver, skin and peri-cardium) can also support innervation during the processof in vivo remodelling, it is likely that innervation is arequisite component of any functional tissue remodelling



Figure 7. Scanning electron micrographs of primary spinal cord neurons seeded on the abluminal surface of the UBM scaffold(A–C).

Figure 8. (A) Migration of Schwann cells towards matricryptic peptides resulting from rapid degradation of UBM or correspondingcontrol. (B) Survival of Schwann cells in the presence of matricryptic peptides resulting from rapid degradation of UBM orcorresponding control. ∗p < 0.05 compared to control; ∗∗∗p < 0.005 compared to control.

that occurs, regardless of the tissue source of the ECMscaffold used. ECM scaffolds may have an advantage oversynthetic scaffolds in this regard, since they are secretedby the resident cells of each tissue and the matrix is likelyoptimized to support the maintenance of cell phenotypesnative to that tissue (Sellaro et al., 2007).

A number of studies have shown that soluble factorsreleased from ECM and the degradation productsthemselves are capable of recruiting both differentiatedcells and progenitor cells to the site of remodelling(Li et al., 2004; Reing et al., 2008). The presentstudy demonstrated that peptides resulting from rapiddegradation of ECM scaffolds are chemoattractant forSchwann cells. Following injury and denervation, theterminal Schwann cells covering the neuromuscularjunction are induced to proliferate and guide the neuronalaxon back to the muscle (Sanes, 1983, 1989; Grinnell,1995; La Fleur et al., 1996; Son et al., 1996; Auld andRobitaille, 2003; Reddy et al., 2003). Thus, the directedmigration of the RT4-D6P2T cell line, a model for terminalSchwann cells (Bansal and Pfeiffer, 1987; Ponomarevaet al., 2006), suggests that terminal Schwann cells maybe at least partially responsible for the ECM-mediatedneurite outgrowth into the site of remodelling.

The temporal and spatial association between nerveand muscle in the current and previous studies(Pope et al., 1997) suggests that either these cell

types or their precursors all respond to the samestimulus or, more likely, participate in paracrineinteraction or direct cell–cell communication thatsupports their simultaneous growth and development.Previous developmental studies show that nerves cancreate a myotrophic environment to promote therecruitment of muscle (Brockes, 1984; Dietz, 1989;Ashby et al., 1993; Suzuki et al., 2005). The reciprocalneurotrophic effect of muscle formation is well known inboth developmental and regenerative models (McCaig,1986; Grinnell, 1995; Carlson, 2005; Kingham andTerenghi, 2006). The dual neurotrophic and angiogeniceffects of blood vessels and nerves, respectively, have alsobeen extensively studied (Carmeliet, 2003), and nervemay in fact guide the growth of blood vessels in normaldevelopment in vivo (Mukouyama et al., 2002).

The interactions between these cell types are notwell understood in the context of biological scaffoldsand regenerative medicine. The ability of ECM scaffoldsto recruit and support endothelial cells (Stupack andCheresh, 2002; Li et al., 2004; Sellaro et al., 2007) andmuscle cells (Prevel et al., 1995; Clarke et al., 1996;Kropp et al., 1996; Pope et al., 1997; Badylak et al., 2000,2002, 2005; Caione et al., 2006; Ueno et al., 2007) iswell known. It is not clear whether neuronal axonsfirst promote the recruitment of endothelial cells andmuscle cells, or whether endothelial cells and muscle cells



promote the directed outgrowth of neurons at the site ofinjury.

While the present study has demonstrated that nervefibres and associated Schwann cells repopulate the remod-elled tissue as a result of implantation of a biologicalscaffold composed of ECM, it is not clear whether thenervous tissue is functional. Although the present studydid not assess functionality, it is plausible that a numberof the nerve fibres observed in the present study couldeventually reform functional connections. Because tissueremodelling was likely still active within the tissues inves-tigated (Gilbert et al., 2007B), the process of functionalinnervation may not have reached equilibrium at the timeof sacrifice. Thus, it would be expected that any functionalinnervation of muscle was not complete at that time.

Additionally, the present study cannot distinguishbetween various subsets of neurons, such as motor, gen-eral sensory and nociceptive sensory neurons, in the siteof remodelling. However, retrospective immunhistochem-ical analysis to distinguish between various subsets ofneurons is not reliable in a regenerative setting, becauseexpression of subset-specific markers and genes is alteredfollowing injury to peripheral nerves (Navarro et al.,2007). Future studies that prospectively assess the func-tionality and phenotype of nerves in vivo and ex vivo willbe required to adequately understand the nature of theregenerated neuronal axons.

In summary, the collective findings of this retrospectivestudy and previous studies (Pope et al., 1997; Caioneet al., 2006; Lopes et al., 2006; Ueno et al., 2007)demonstrate that mature and regenerating nerve tissue ispresent in the site of remodelling following placement ofan ECM scaffold. Further investigation is needed of themechanisms underlying the process of innervation duringin vivo ECM scaffold remodelling. By understanding themechanisms that promote innervation, it may be possibleto control the spatio-temporal patterns of innervationand ultimately promote faster remodelling of functional,site-appropriate tissue.

5. Conclusions

The findings of this study demonstrate the temporaland spatial association of innervation during the in vivoremodelling of the ECM scaffold used for reconstruction oftwo muscular tissues: the oesophagus and muscular bodywall. Further investigation in well-designed prospectivestudies is warranted and needed.

Acknowledgements

The authors would like to thank Jennifer DeBarr for herassistance in the preparation of histological sections. The authorswould also like to thank the Center for Biological Imaging CoreFacility at the University of Pittsburgh for the use of theirequipment for imaging. This work was supported by NIH GrantNo. 5R01 AR05494003.

References

Allman AJ, McPherson TB, Badylak SF, et al. 2001; Xenogeneicextracellular matrix grafts elicit a TH2-restricted immuneresponse. Transplantation 71(11): 1631–1640.

Allman AJ, McPherson TB, Merrill LC, et al. 2002; The Th2-restrictedimmune response to xenogeneic small intestinal submucosa doesnot influence systemic protective immunity to viral and bacterialpathogens. Tissue Eng 8(1): 53–62.

Ashby PR, Wilson SJ, Harris AJ. 1993; Formation of primary andsecondary myotubes in aneural muscles in the mouse mutantperoneal muscular atrophy. Dev Biol 156(2): 519–528.

Auld DS, Robitaille R. 2003; Perisynaptic Schwann cells atthe neuromuscular junction: nerve- and activity-dependentcontributions to synaptic efficacy, plasticity, and reinnervation.Neuroscientist 9(2): 144–157.

Badylak S, Kokini K, Tullius B, et al. 2002; Morphologic study ofsmall intestinal submucosa as a body wall repair device. J SurgRes 103(2): 190–202.

Badylak S, Kokini K, Tullius B, et al. 2001; Strength over time of aresorbable bioscaffold for body wall repair in a dog model. J SurgRes 99(2): 282–287.

Badylak S, Meurling S, Chen M, et al. 2000; Resorbable bioscaffoldfor esophageal repair in a dog model. J Pediatr Surg 35(7):1097–1103.

Badylak SF, Kochupura PV, Cohen IS, et al. 2006; The use ofextracellular matrix as an inductive scaffold for the partialreplacement of functional myocardium. Cell Transplant 15: (suppl1): S29–40.

Badylak SF, Lantz GC, Coffey A, et al. 1989; Small intestinalsubmucosa as a large diameter vascular graft in the dog. J SurgRes 47(1): 74–80.

Badylak SF, Vorp DA, Spievack AR, et al. 2005; Esophagealreconstruction with ECM and muscle tissue in a dog model. J SurgRes 128(1): 87–97.

Bansal R, Pfeiffer SE. 1987; Regulated galactolipid synthesis andcell surface expression in Schwann cell line D6P2T. J Neurochem49(6): 1902–1911.

Brennan EP, Reing J, Chew D, et al. 2006; Antibacterial activitywithin degradation products of biological scaffolds composed ofextracellular matrix. Tissue Eng 12(10): 2949–2955.

Brockes JP. 1984; Mitogenic growth factors and nerve dependenceof limb regeneration. Science 225(4668): 1280–1287.

Brown BN, Valentin JE, Stewart-Akers AM, et al. 2009; Biologicscaffold remodeling and macrophage polarization in a rat bodywall model. Biomaterials 30(8): 1482–1491.

Caione P, Capozza N, Zavaglia D, et al. 2006; In vivo bladderregeneration using small intestinal submucosa: experimentalstudy. Pediatr Surg Int 22(7): 593–599.

Caissie R, Gingras M, Champigny MF, et al. 2006; In vivoenhancement of sensory perception recovery in a tissue-engineeredskin enriched with laminin. Biomaterials 27(15): 2988–2993.

Carlson BM. 2005; Some principles of regeneration in mammaliansystems. Anat Rec B New Anat 287(1): 4–13.

Carmeliet P. 2003; Blood vessels and nerves: common signals,pathways and diseases. Nat Rev Genet 4(9): 710–720.

Chunzheng G, Shengzhong M, Yinglian J, et al. 2008; Siatic nerveregeneration in rats stimulated by fibrin glue containing nervegrowth factor: an experimental study. Injury 39(12): 1414–1420.

Clarke KM, Lantz GC, Salisbury SK, et al. 1996; Intestine submucosaand polypropylene mesh for abdominal wall repair in dogs. J SurgRes 60(1): 107–114.

Dietz FR. 1989; Effect of denervation on limb growth. J Orthop Res7(2): 292–303.

Donzelli R, Maiuri F, Piscopo GA, et al. 2006; Role of extracellularmatrix components in facial nerve regeneration: an experimentalstudy. Neurol Res 28(8): 794–801.

Freytes DO, Badylak SF, Webster TJ, et al. 2004; Biaxial strengthof multilaminated extracellular matrix scaffolds. Biomaterials25(12): 2353–2361.

Garavito ZV, Sutachan JJ, Muneton VC, et al. 2000; Is S-100 proteina suitable marker for adult Schwann cells? In vitro Cell Dev BiolAnim 36(5): 281–283.

Gilbert TW, Sacks MS, Grashow JS, et al. 2006; Fiber kinematics ofsmall intestinal submucosa under biaxial and uniaxial stretch.J Biomech Eng 128(6): 890–898.



Gilbert TW, Stewart-Akers AM, Badylak SF. 2007a; A quantitativemethod for evaluating the degradation of biologic scaffoldmaterials. Biomaterials 28(2): 147–150.

Gilbert TW, Stewart-Akers AM, Simmons-Byrd A, et al. 2007b;Degradation and remodeling of small intestinal submucosa incanine Achilles tendon repair. J Bone Joint Surg Am 89(3):621–630.

Gilbert TW, Stewart-Akers AM, Sydeski J, et al. 2007c; Geneexpression by fibroblasts seeded on small intestinal submucosaand subjected to cyclic stretching. Tissue Eng 13(6): 1313–1323.

Gomez N, Lu Y, Chen S, et al. 2007; Immobilized nerve growth factorand microtopography have distinct effects on polarization versusaxon elongation in hippocampal cells in culture. Biomaterials28(2): 271–284.

Goslin K, Schreyer DJ, Skene JH, et al. 1990; Changes in thedistribution of GAP-43 during the development of neuronalpolarity. J Neurosci 10(2): 588–602.

Grinnell AD. 1995; Dynamics of nerve–muscle interaction indeveloping and mature neuromuscular junctions. Physiol Rev75(4): 789–834.

Hadlock TA, Sundback CA, Hunter DA, et al. 2001; A newartificial nerve graft containing rolled Schwann cell monolayers.Microsurgery 21(3): 96–101.

Hoffman PN. 1989; Expression of GAP-43, a rapidly transportedgrowth-associated protein, and class II β-tubulin, a slowlytransported cytoskeletal protein, are coordinated in regeneratingneurons. J Neurosci 9(3): 893–897.

Jessen KR, Mirsky R. 1991; Schwann cell precursors and theirdevelopment. Glia 4(2): 185–194.

Johansson F, Carlberg P, Danielsen N, et al. 2006; Axonal outgrowthon nano-imprinted patterns. Biomaterials 27(8): 1251–1258.

Kingham PJ, Terenghi G. 2006; Bioengineered nerve regenerationand muscle reinnervation. J Anat 209(4): 511–526.

Kochupura PV, Azeloglu EU, Kelly DJ, et al. 2005; Tissue-engineeredmyocardial patch derived from extracellular matrix providesregional mechanical function. Circulation 112: (suppl 9):I144–149.

Kropp BP, Eppley BL, Prevel CD, et al. 1995; Experimentalassessment of small intestinal submucosa as a bladder wallsubstitute. Urology 46(3): 396–400.

Kropp BP, Rippy MK, Badylak SF, et al. 1996; Regenerativeurinary bladder augmentation using small intestinal submucosa:urodynamic and histopathologic assessment in long-term caninebladder augmentations. J Urol 155(6): 2098–2104.

La Fleur M, Underwood JL, Rappolee DA, et al. 1996; Basementmembrane and repair of injury to peripheral nerve: defininga potential role for macrophages, matrix metalloproteinases,and tissue inhibitor of metalloproteinases-1. J Exp Med 184(6):2311–2326.

Lantz GC, Badylak SF, Coffey AC, et al. 1990; Small intestinalsubmucosa as a small-diameter arterial graft in the dog. J InvestSurg 3(3): 217–227.

Lantz GC, Badylak SF, Coffey AC, et al. 1992; Small intestinalsubmucosa as a superior vena cava graft in the dog. J SurgRes 53(2): 175–181.

Li F, Li W, Johnson S, et al. 2004; Low-molecular-weight peptidesderived from extracellular matrix as chemoattractants for primaryendothelial cells. Endothelium 11(3–4): 199–206.

Lopes MF, Cabrita A, Ilharco J, et al. 2006; Esophageal replacementin rat using porcine intestinal submucosa as a patch or a tube-shaped graft. Dis Esophagus 19(4): 254–259.

McCaig CD. 1986; Myoblasts and myoblast-conditioned mediumattract the earliest spinal neurites from frog embryos. J Physiol375: 39–54.

Mukouyama YS, Shin D, Britsch S, et al. 2002; Sensory nervesdetermine the pattern of arterial differentiation and blood vesselbranching in the skin. Cell 109(6): 693–705.

Navarro X, Vivo M, Valero-Cabre A. 2007; Neural plasticity afterperipheral nerve injury and regeneration. Prog Neurobiol 82(4):163–201.

Ota T, Gilbert TW, Badylak SF, et al. 2007; Electromechanicalcharacterization of a tissue-engineered myocardial patch derivedfrom extracellular matrix. J Thorac Cardiovasc Surg 133(4):979–985.

Patrick CW, Zheng B, Schmidt M, et al. 2001; Dermal fibroblastsgenetically engineered to release nerve growth factor. Ann PlastSurg 47(6): 660–665.

Phillips JB, Bunting SC, Hall SM, et al. 2005; Neural tissueengineering: a self-organizing collagen guidance conduit. TissueEng 11(9–10): 1611–1617.

Ponomareva ON, Fischer TM, Lai C, et al. 2006; Schwann cell-derived neuregulin-2α can function as a cell-attached activator ofmuscle acetylcholine receptor expression. Glia 54(6): 630–637.

Pope JC, Davis MM, Smith ER Jr, et al. 1997; The ontogeny of caninesmall intestinal submucosa regenerated bladder. J Urol 158(3 Pt2): 1105–1110.

Prevel CD, Eppley BL, Summerlin DJ, et al. 1995; Small intestinalsubmucosa: utilization for repair of rodent abdominal wall defects.Ann Plast Surg 35(4): 374–380.

Recknor JB, Sakaguchi DS, Mallapragada SK. 2006; Directedgrowth and selective differentiation of neural progenitor cellson micropatterned polymer substrates. Biomaterials 27(22):4098–4108.

Record RD, Hillegonds D, Simmons C, et al. 2001; In vivodegradation of 14C-labeled small intestinal submucosa (SIS) whenused for urinary bladder repair. Biomaterials 22(19): 2653–2659.

Reddy LV, Koirala S, Sugiura Y, et al. 2003; Glial cells maintainsynaptic structure and function and promote development of theneuromuscular junction in vivo. Neuron 40(3): 563–580.

Reing JE, Zhang L, Myers-Irvin J, et al. 2008; Degradation productsof extracellular matrix affect cell migration and proliferation.Tissue Eng Part A 15(3): 605–614.

Robinson KA, Li J, Mathison M, et al. 2005; Extracellular matrixscaffold for cardiac repair. Circulation 112: (suppl 9): I135–43.

Sanes JR. 1983; Roles of extracellular matrix in neural development.Annu Rev Physiol 45: 581–600.

Sanes JR. 1989; Extracellular matrix molecules that influence neuraldevelopment. Annu Rev Neurosci 12: 491–516.

Schnell E, Klinkhammer K, Balzer S, et al. 2007; Guidance of glialcell migration and axonal growth on electrospun nanofibers ofpoly-ε-caprolactone and a collagen/poly-ε-caprolactone blend.Biomaterials 28(19): 3012–3025.

Sellaro TL, Ravindra AK, Stolz DB, et al. 2007; Maintenance ofhepatic sinusoidal endothelial cell phenotype in vitro usingorgan-specific extracellular matrix scaffolds. Tissue Eng 13(9):2301–2310.

Skene JH, Willard M. 1981; Axonally transported proteins associatedwith axon growth in rabbit central and peripheral nervous systems.J Cell Biol 89(1): 96–103.

Smith RM, Wiedl C, Chubb P, et al. 2004; Role of small intestinesubmucosa (SIS) as a nerve conduit: preliminary report. J InvestSurg 17(6): 339–344.

Son YJ, Trachtenberg JT, Thompson WJ. 1996; Schwann cellsinduce and guide sprouting and reinnervation of neuromuscularjunctions. Trends Neurosci 19(7): 280–285.

Sorensen A, Alekseeva T, Katechia K, et al. 2007; Long-term neuriteorientation on astrocyte monolayers aligned by microtopography.Biomaterials 28(36): 5498–5508.

Stupack DG, Cheresh DA. 2002; ECM remodeling regulatesangiogenesis: endothelial integrins look for new ligands. Sci Signal2002(119): PE7.

Su Y, Zeng BF, Zhang CQ, et al. 2007; Study of biocompatibility ofsmall intestinal submucosa (SIS) with Schwann cells in vitro. BrainRes 1145: 41–47.

Suzuki M, Satoh A, Ide H, et al. 2005; Nerve-dependent and -independent events in blastema formation during Xenopus frogletlimb regeneration. Dev Biol 286(1): 361–375.

Triolo D, Dina G, Lorenzetti I, et al. 2006; Loss of glial fibrillaryacidic protein (GFAP) impairs Schwann cell proliferation anddelays nerve regeneration after damage. J Cell Sci 119(Pt 19):3981–3993.

Ueno T, de la Fuente SG, Abdel-Wahab OI, et al. 2007; Functionalevaluation of the grafted wall with porcine-derived small intestinalsubmucosa (SIS) to a stomach defect in rats. Surgery 142(3):376–383.

Valentin JE, Badylak JS, McCabe GP, et al. 2006; Extracellularmatrix bioscaffolds for orthopaedic applications. A comparativehistologic study. J Bone Joint Surg Am 88(12): 2673–2686.

Zantop T, Gilbert TW, Yoder MC, et al. 2006; Extracellular matrixscaffolds are repopulated by bone marrow-derived cells in amouse model of Achilles tendon reconstruction. J Orthop Res24(6): 1299–1309.


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