Study of biocompatibility of small intestinal submucosa (SIS) with Schwann cells in vitro

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  • ag,

    A R T I C L E I N F O

    Article history:Accepted 17 January 2007Available online 13 February 2007

    seeded with SCs has potential to be an alternate candidate of autografting for repairing long

    2007 Elsevier B.V. All rights reserved.

    of autologous nerve grafts by means of tissue engineering

    B R A I N R E S E A R C H 1 1 4 5 ( 2 0 0 7 ) 4 1 4 7

    ava i l ab l e a t www.sc i enced i rec t . com

    mTreatment of the injured peripheral nerve with a long defectremains one of the most difficult problems in nervereconstructive surgery. Transplantation of autologous nerveis the widely utilized method when direct anastomosis isdifficult. However, this technique is limited when the sourceof donor nerve is insufficient, in particular when considering

    (Bellamkonda, 2006; Fansa and Keilhoff, 2004; Rochkind et al.,2004).

    Schwann cells (SCs) are known to play an obligatory role inperipheral nerve regeneration by providing bioactive sub-strates on which axons migrate and by releasing moleculesthat regulate axonal outgrowth (Fawcett and Keynes, 1990;1. Introduction environment for axonal regeneration, to overcome drawbacksthe morbidity in the donor site and atime (Lundborg and Malm, 2000; TeTherefore, efforts have been made tonerve conduits, which are creating a

    Corresponding author. Fax: +86 21 64369181E-mail address: yansu2004@yahoo.com.cn

    0006-8993/$ see front matter 2007 Elsevidoi:10.1016/j.brainres.2007.01.138peripheral nerve defects.A B S T R A C T

    No satisfactory method currently exists for repairing long peripheral nerve defects. Effortshave been made to fabricate bioactive artificial nerve conduits, comprised of a biomaterialpre-seeded with Schwann cells (SCs), which creating a favorable micro-environment foraxonal regeneration, to be an alternative to autografting by means of tissue engineering.Small intestinal submucosa (SIS) possesses special biological characteristics and iscomprehensively researched for tissue repairing at varied tissues and organs. This studyinvestigated the biocompatibility of SISwith SCs in vitro. Cultured rat SCswere seededonSIS.Cell morphology was observed by light microscopy, scanning electron microscopy andtransmission electron microscope. The viability of SCs was measured by MTT assay.Secretion of NGF- and BDNF was quantitatively assessed by ELISA, and NGF-mRNA andBDNF mRNA were semi-quantitatively assessed by RTPCR. The results indicated that SCscould adhere,migrate and proliferate on the surface of SIS in good condition with productivefunction of secreting growth factors. SIS has a good biocompatibility with SCs and SIS pre-Keywords:Schwann cellBiocompatibilitySmall intestinal submucosaPeripheral nerve regenerationDepartment of Orthopaedics, The Sixth Affiliated People's Hospital, Shanghai Jiaotong University, 600 Yishan Road, Shanghai 200233, ChinaResearch Report

    Study of biocompatibility of smwith Schwann cells in vitro

    Yan Su, Bing-Fang Zeng, Chang-Qing Zhan

    www.e l sev i e r. codditional operationrzis et al., 1997).fabricate artificialfavorable micro-

    .(B.-F. Zeng).

    er B.V. All rights reservedll intestinal submucosa (SIS)

    Kai-Gang Zhang, Xue-Tao Xie

    / l oca te /b ra in resThompson and Buettner, 2004). Thus, they are commonlyused in nerve tissue engineering as seed cells. Meanwhile,searching for an appropriate kind of material served asscaffold is critical. A variety of materials, including synthetic

    .

  • 2. Results

    2.1. Light microscopy

    At 24 h after isolation by enzyme digestion, a large amount ofcells adhered and grew in spherical and short olivary shape.After 2 days, the adherent SCs appeared a bi- or tripolarmorphology with oval nuclei and connected to each other. At6 days, the identification of SCs was verified using theimmunocytochemistry for S-100 and the purity of SCsexceeded 95% (Fig. 1).

    At 5 days after seeding on SIS, SCs were intensive aroundSIS and adhered on the edge of SIS. SCs grew in long olivaryshape with obvious cell body processes (Fig. 2).

    2.2. SEM

    1 1 4 5 ( 2 0 0 7 ) 4 1 4 7and natural materials have attempted to be employed tobridge nerve gaps (Evans et al., 2002; Keilhoff et al., 2003;Mackmmon and Dellon, 1990; Smith et al., 2004; Hudson et al.,1999). Yet despite some advances in the field of tissueengineering, axonal regeneration using neither synthetic nornatural materials has reached to the equivalent level of theuse of autografts. Thus, further investigation to searchalternative nerve scaffold is demanded.

    Small intestinal submucosa (SIS) is a multilaminar portionof the small intestine and has been applied as a biomaterialscaffold for tissue engineering applications to artery (Badylaket al., 1989), the lower urinary tract (Campodonico et al., 2004),bone (Suckow et al., 1999), and abdominal wall (Zhang et al.,2003). Encouraging results with appropriate tissue regenera-tion and functional recovery have been reported in each ofthese applications. Therefore, SIS appears to have potential

    Fig. 1 SCs were positive for S-100 staining using theimmunocytochemistry method (fluorescence microscope,100).

    42 B R A I N R E S E A R C Hto facilitate host tissue regeneration without concurrentimmunologic rejection or alteration (Badylak et al., 1989;Campodonico et al., 2004; Suckow et al., 1999; Zhang et al.,2003). Rat SIS was also applied to bridge a sciatic nerve gap inrats and the results demonstrated the feasible use of SC-seeded SIS as nerve guidance channel that was comparableto nerve autografts. But rat SIS was not firm enough toprevent lamellae from collapsing onto one another, therebypreventing further axonal regeneration (Hadlock et al., 2001).Porcine SIS is thicker and has greater mechanical strengththan rat SIS and may be more suitable to be applied asscaffold in peripheral nerve tissue engineering.

    Good biocompatibility with SCs is essential for the bioma-terials used in peripheral nerve tissue engineering. However,the biocompatibility of porcine SIS with SCs has not beenevaluated in vitro previously andwe are not sure if porcine SISis suitable for SCs adherence and growth. In this study, wecultured rat SCs on porcine SIS in vitro and examinedadhesion, migration and proliferation of SCs. Moreover,secretion of growth factors by SCs on SIS was also evaluated.The study aimed to investigate the biocompatibility of porcineSIS with SCs in vitro and determine the feasibility of potentialapplication in nerve tissue engineering.At 1 day, SCs scattered and adhered on the surface of SIS,exhibiting a round cell body with short cell body processes(Fig. 3A). Then the density of SCs increased with prolonged co-culture time. At 5 days, SCs were observed to grow inmultilayer fashion on SIS. They migrated and proliferatedactively along the SIS surface in three-dimensional fashion,demonstrating long olivary, triangular or long fusiform shape.Furthermore, SCs arranged in bundles or took on radiatingvortex end to end arrangement (Figs. 3B, C). Protein granulessecreted on cellular surface were also shown. At 7 days, SCsgrew intensively and reached confluency (Fig. 3D).

    2.3. TEM

    SCs grew and adhered to the surface of SIS in good condition.The cell body of SCs stretched out and demonstrated longolivary with extending processes, having microvilli on theirsurface in abundance. A considerable number of chondrio-somes and ribosomes were seen in cytoplasm, and an ovalnuclear located at one side. At the interface of SCs and SIS,plenty of the minute foot plates were observed to attach SIStightly (Fig. 4).

    Fig. 2 SCs grew and adhered on the edge of SIS in good

    condition (phase contrast microscope, 100). The arrowindicates the interface of SCs and SIS.

  • Fig. 3 SEM of Schwann cells cultured on SIS. SCs exhibited a roSCs migrated and proliferated actively along the SIS surface in thradiating vortex end to end arrangement at 5 days (B, C). SCs gre

    Fig. 4 TEM of Schwann cells attached and adhered to thesurface of SIS at 7 days. At the interface of SCs and SIS, plentyof the minute foot plates were observed to attach SIS tightly.The arrow indicates the interface of SCs and SIS (bar=2 m).SC: Schwann cell; N: nucleus of Schwann cell; SIS: smallintestinal submucosa.

    B R A I N R E S E A R C H 1 1 434 5 ( 2 0 0 7 ) 4 1 4 72.4. MTT assay

    The results are given in Fig. 5. The SCs' viability of SIS groupwas significantly higher than that of the control group at 3, 7and 10 days.

    2.5. ELISA

    The results are given in Figs. 6 and 7. After seeding on SIS, theamount of NGF- and BDNF which were secreted by SCsincreased with the prolonged time. At 5 days, each groupreached a high level relatively. Compared to the control group,the level of both growth factors of the SIS group wassignificantly higher at 3, 5 and 7 days (P

  • are suitable for SCs attachment, proliferation and secretion of

    Fig. 5 The results of MTT assay (n=6). Error bars represent

    Fig. 7 The concentration of BDNF in culture supernatantmeasured by ELISA (n=6). Error bars represent means

    44 B R A I N R E S E A R C H 1 1 4 5 ( 2 0 0 7 ) 4 1 4 7growth factors.SIS is essentially acellular extracellular matrix (ECM), with

    about 40% of the dry weight being composed of fibrillarcollagen. Studies have documented that SIS contains glyco-be significantly higher than that of the control group (P1 cm in a rat model or 3 cm in human subjects (Lundborg,1988;Machinnon andDellon, 1990). Hence, interests have beendirected to introduce cultured SCs into scaffold to constructbioactive nerve conduits for promoting nerve regeneration.Although the ideal material for a nerve guide has not beenidentified, successful materials must be biocompatible which

    meansSE. *P

  • adhesion, survival, migration and proliferation on its surface.

    1 1Observation of the ultrastructure of SCs by TEM demon-strated that SCs adhered tightly and grew productively on thesurface of SIS. MTT assay also showed that SIS had nocytotoxicity for SCs. Quantitative analysis of NGF- and BDNFby ELISA, as well as semi-quantitative analysis of NGF-mRNA and BDNF mRNA by RTPCR, showed that SCs seededon SIS had more productive function of secretion than thenormal cultured SCs. NGF- and BDNF are the main growthfactors secreted by SCs, which are known to have neuro-trophic effects on nerve regeneration (Schicho et al., 1999;Serpe et al., 2005).

    SIS-contained glycoprotein and cellular factors may playimportant roles in supporting SC growth. The proteoglycancompositions in SIS binds with specific receptors on cellsurfaces and links ECM components, such as collagen typeIV, which facilitates SC adherence and migration. Further-more, extractable growth factors from SIS include FGF-2 andTGF- (Voytik-Harbin et al., 1997). Studies have shown thatFGF-2 is mitogenic for cultured SCs in vitro (Mauritz et al.,2004), and TGF- can promote proliferation and differentia-tion of SCs (Sulaiman and Gordon, 2002). In addition, as akind of extracellular matrix-derived scaffolds, the naturalthree-dimensional ultrastructure of SIS may also be suitablefor cells adherence and growth (Badylak et al., 1999). Takentogether, these factors create a proper microenvironment toenhance axonal regeneration.

    As a xenogenic graft, host immunological response shouldalways be concerned. Allman has reported that porcine SISelicits an immunological response just restricted to the Th2pathway, which is consistent with a remodeling reactionrather than rejection (Allman et al., 2001). As a ureteralallograft, despite the presence of chronic inflammatoryprocess, porcine SIS behaves as a biological tissue withoutacute rejective reaction (Greca et al., 2004). Absence of anacute rejection following porcine SIS xeno-implantationappears to be related to its characteristics of acellular extra-cellular matrix.

    This study evaluated the biocompatibility of SIS with SCs invitro. It demonstrated that SCs could adhere and grow well onthe surface of SIS with productive function of secreting growthfactors. In conclusion, as a natural biomaterial, SIS hasexcellent neuroglial cell affinity. SIS pre-seeded with SCs haspotential to be an alternate candidate for autografting forrepairing long peripheral nerve defects. Further studies ofconstructing artificial nerve conduit using SIS pre-seeded withSCs to promote axonal regeneration will be followed in thefuture.

    4. Experimental procedures

    4.1. SIS preparationBadylak, 2001). In the present study, the biocompatibility ofSIS with SCs was further investigated in vitro. The resultsindicated SIS was useful for growth of nerve cells. When co-cultured with SCs, SIS showed good ability to support SCs

    B R A I N R E S E A R C HPreparation of porcine SIS was followed previous descriptionby others (Abraham et al., 2000). Briefly, a segment of freshjejunum was harvested from healthy swine (weighting over200 kg) from a closed herd (Qi Xing Farm, Shanghai). Aftergently cleaning in water, the segment was everted and thetunica mucosa was abraded using longitudinal wipingmotions with a scalpel handle wrapped with moistenedgauze. The treated jejunal segment was everted again andthe serosa and tunica muscularis were then gently removedusing the same procedure. After mechanical cleaning, theintestine was slit longitudinally and cut into 15 cm sectionsthat were processed through a series of chemical cleaningsteps. The v/v ratio of each chemical cleaning solution totissue was 100:1, and the incubations all were carried out atambient room temperature. The tissue was first incubatedfor about 16 h in a solution of 100 mM ethylenediaminete-traacetic acid (EDTA) in 10 mM sodium hydroxide (NAOH)with a pH of 1112. The second incubation in 1 M hydro-chloric acid (HCl) in 1 M sodium chloride (NaCl) at pH 01was carried out for 68 h. This was followed by incubation in1 M sodium chloride and 10 mM phosphate-buffered saline(PBS) at pH 77.4 for 16 h and then 2-h incubation in 10 mMPBS at pH 77.4. Finally, the tissue was rinsed in sterile waterat pH 5.87.0 for at least 2 h.

    The preparation of porcine SIS was rinsed extensively with0.1% peracetic acid for 2 h, vacuum-sealed into hermeticpackaging, and terminally sterilized by gamma irradiation(2535 kGy).

    4.2. Isolation and culture of SCs

    The bilateral sciatic nerves and brachial plexuses of 10neonatal Sprague-Dawley rats (Animals Center of AcademiaSinica, Shanghai) were harvested and washed 3 times withPBS (pH 7.4). The epineurium was separated and nerves weredissected into discrete fascicles (less than 1 mm), which wereenzymatically dissociated using 0.03% type I collagenase(Sigma, USA) and 0.25% trypsin in 10 ml PBS for 30 min at37 C. The resulting cell suspension was centrifuged andplaced onto poly-L-lysine- (Sigma, USA) coated cell culturedishes in Dulbecco's modified Eagle's medium (DMEM)(Gibco, USA) with 10% fetal bovine serum (Hyclone, USA),incubated in a 37 C humidified incubator with 5% CO2. After12 h, the medium containing 5 M cytosine arabinoside(Sigma, USA) was added to deplete rapidly proliferatingfibroblasts for 48 h. The cells were then fed with DMEMsupplemented with 1% antibiotic (10,000 U/ml penicillin Gand 10 mg/ml streptomycin), 2 mM glutamine, 10% fetalbovine serum and 40 g/ml bovine pituitary extract (Upstate,USA). The medium was changed every 3 days. After 67 days,the cells were passaged with 0.25% trypsin/EDTA afterconfluent monolayer was obtained. The whole surgicalprocedure was performed in laminar flow hood under asepticconditions following the guidelines of the institute's ethicalcommittee.

    4.3. Seeding SCs on SIS

    The prepared SIS was cut into pieces of 1 cm1 cm andattached on each well of a 24-well tissue culture plate, after

    454 5 ( 2 0 0 7 ) 4 1 4 7which they were rinsed extensively with PBS. 2 ml SCs wereseeded onto SIS pieces at density of 5105 cell/cm2. Wells

  • different groups, and statistical significance was accepted atthe 0.05 confidence level.

    Acknowledgments

    We wish to thank Professor Wang Yang of Department ofAnatomy and HistologyEmbryology, Shanghai MedicalCollege, Fudan University for her kind help in cell culture.

    BDNF Forward 5: AAACCATAAGGACGCGGACTReverse 5: GATTGGGTAGTTCGGCATTG

    1 1embedded with poly-L-lysine-coated coverslips were seededwith SCs as the control group. Wells attached with SIS alonewithout SCs seeding served as the blank control group. Allwells were incubated in DMEM containing 1% antibiotic(10,000 U/ml penicillin G and 10 mg/ml streptomycin), 2 mMglutamine, 10% fetal bovine serum and 40 g/ml bovinepituitary extract, in a 37 C humidified incubator with 5% CO2.

    4.4. Evaluation of biocompatibility

    4.4.1. Light microscopyThe growth of SCs surrounding SIS was observed under aninverted microscope (Olympus, Tokyo, Japan) every day.Photographs were taken at regular intervals.

    4.4.2. Scanning electron microscopy (SEM)On days 1, 3, 5, and 7 after SCs were seeded onto SIS, thesamples were washedwith PBS and fixed in 2% glutaraldehydesolution. The samples were then postfixed with 1% osmiumtetroxide, dehydrated in a graded series of ethanol solutions,dried at critical point drier (Hitachi, Tokyo, Japan) and gold-coated.All sampleswere observedusingSEM (PhilipsQUANTA-200, Holland).

    4.4.3. Transmission electron microscope (TEM)On day 7 after SCs were seeded onto SIS, the samples werewashed with PBS and fixed in 2% glutaraldehyde solution.The samples were then postfixed with 1% osmium tetroxide,dehydrated in a graded series of ethanol solutions andembedded in Epon 812 epoxy resin. Ultrathin sections (7090 nm) were prepared, stained with lead citrate and uranylacetate and observed using TEM (Hitachi H-600, Tokyo,Japan).

    4.4.4. MTT assay3-(4,5-Dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bro-mide (MTT, Sigma, USA) was prepared as 0.5 mg/ml stocksolution in PBS. On days 1, 3, 7, and 10 after SCs were seeded ineach group, the viability was assessed. Briefly, the culturemedium of each well was replaced with 100 l DMEM. 25 lMTT in PBS (0.1 M, pH 7.2) was added to each well. After 4 h ofincubation at 37 C, 100 l lysis buffer (20% SDS in 50% N,N-dimethylformamide, pH 4.7) was added. After 20 h of incuba-tion at 37 C, the supernatants of all wells were respectivelyaspirated out to be measured photometrically with an EIX-800Microelisa reader (Bio-Tek Inc., USA) at 570 nm.

    4.4.5. Enzyme-linked immunosorbent assay (ELISA)On days 1, 3, 5, and 7 after SCs were seeded in each group,cellular supernatant was analyzed by ELISA kits (Boster,China) for nerve growth factor- (NGF-) and brain-derivedneurotrophic factor (BDNF) according to the manufacturer'sdirections. Briefly, the samples were added to each well of96-well plates pre-coated with specific antibody and incu-bated for 90 min at 37 C. The plates were washed andincubated with biotinylated rabbit anti-rat isotype-specificantibody for 90 min at 37 C. The plates were washed; avidinbiotin complex (ABC) working dilutions was added and

    46 B R A I N R E S E A R C Hincubated for 30 min at 37 C. The plates were washed againand TMB solution was added and incubated for 25 min at37 C. Finally, stop solution was added and absorbance wasread at 450 nm on a microplate reader (Bio-Tek Instruments,Winooski, VT).

    4.4.6. Reverse transcriptasepolymerase chain reaction(RTPCR)On day 7 after SCs were seeded in each group, the total RNAwas extracted using the RNeasy extraction kit (Invitrogen,USA) and genomic DNAwas removed by DNAse I, according tothemanufacturer's protocol. RNAwas reverse transcribed in a20 l final volume using 10 l of total RNA, 0.5 g oligodT,200 M of each deoxyribonucleoside triphosphate (dNTP),20 units RNasin and 200 units M-MLV Reverse Transcriptase(Promega, USA), according to the protocol and with the bufferssupplied by the manufacturer. Two separate negative controlreactions, either without RNA or without M-MLV, alsoaccompanied each reaction.

    PCR was performed using 2 l of synthesized cDNA with1.25 U Taq polymerase (Tiangen Biotech, Germany), 200 MdNTPs, 0.4 M of each primer, 1.5 mM MgCl2, the buffersupplied by the company, and deionized distilled water in a50 l total reaction volume. All common components wereadded into a master mix and then aliquotted into tubes. Thecycling conditions were as follows: Initial denaturation at95 C for 3 min followed by 30 cycles of: 95 C for 30 s, 60 C for30 s, 72 C for 45 s, and a final extension of 72 C for 5 min. Theproducts were analysed on a 1.5% agarose (Invitrogen) gelcomplemented with 0.025 ethidium bromide, where aDL2000 DNA-ladder (TaKaRa, Japan) was run in parallel tothe samples. -Actin was used as the loading control. The gelswere pictured with a digital camera (Sony DSC-S75 Cybershot)and a Kodak Digital Science 1D analysing system (KodakScientific Imaging Systems, New Haven, CT, USA) was used todetermine the densities of NGF- and BDNF bands for both SISand control groups.

    Primers were designed in Table 1.

    4.4.7. Statistical analysisA one-way ANOVA was used to compare the means of

    Length of product (bp) 393Table 1 Nucleotide sequence and size of the expand PCRproducts for oligonucleotide primers for RTPCR

    NGF- Forward 5: GGCCACTCTGAGGTGCATAGReverse 5: CATGGGCCTGGAAGTCTAAALength of product (bp) 349

    4 5 ( 2 0 0 7 ) 4 1 4 7This work was supported by National Natural ScienceFoundation of China (Grant No. 303714444).

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    Study of biocompatibility of small intestinal submucosa (SIS) with Schwann cells in vitroIntroductionResultsLight microscopySEMTEMMTT assayELISARTPCR

    DiscussionExperimental proceduresSIS preparationIsolation and culture of SCsSeeding SCs on SISEvaluation of biocompatibilityLight microscopyScanning electron microscopy (SEM)Transmission electron microscope (TEM)MTT assayEnzyme-linked immunosorbent assay (ELISA)Reverse transcriptasepolymerase chain reaction (RTPCR)Statistical analysis

    AcknowledgmentsReferences

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