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Introducing the Concept of the 3Rsinto Tissue Engineering ResearchClaudia Eder', Erwin Falkner':', Stefan Nehrer', Udo M. Losen? and Harald Schoffi'lBioMed - Society for the Promotion of Biomedical and Medical Technological Research in Upper Austria, Linz, Austria;2Core Unit for'Biomedical Research, Medical University Vienna, Austria; 3Dept. of Orthopaedic Surgery, Medical UniversityVienna, Austria; "zet - Centre for Alternative and Complementary Methods to Animal Testing, Linz, Austria

SummaryTissue engineering, defined as using a combination of culturedcells and biodegradable scaffolds to repair tissue damaged byinjury or disease, represents a booming sector of biomedicalresearch. Animal experimentation is routinely performed priorto clinical trials. The presented study tries to translate theaspect of the 3Rs to tissue engineering research: Cell cultureprotocols were adapted to antibiotic free and serum free condi-tions. Biomaterials (Bio-Gide® and a collagen sponge proto-type) were pre-tested using the HET-CAM assay. CAM-testingsuggested a protocol change for application of the Bio-Gide®scaffold and demonstrated unsuitable material properties ofthecollagen sponge. Application of 3R compliant protocols fortissue engineering research led to increased cell proliferation,higher synthesis of extracellular matrix molecules, reduceddedifferentiation and more information about the biomaterialsat an early experimental stage. Tissue engineering research cantherefore profit from the increased efforts to validate in vitroalternatives and supplements to animal testing.

Zusammenfassung: Biomedizinische Forschung aus dem Blick-winkel der 3R betrachtetDie Technik des .Jissue Engineering" repräsentiert einenboomenden Zweig der biomedizinischen Forschung. Menschli-che Zellen werden isoliert, expandiert und auf eine Matrixaufgebracht, um geschädigtes Gewebe zu regenerieren. Diehierfür eingesetzten Tierversuchsprotokolle wurden am Beispieleines Experiments zur Meniskusreparatur im Sinne der 3Rüberarbeitet: Antibiotika- und serumfreie Zellkulturtechnikenkamen zum Einsatz, und die als Matrix dienenden Biomateria-lien (Bio-Gide® und ein Kollagenschwamm-Prototyp) wurdenmit Hilfe des HET-CAM Tests evaluiert. Die HET-CAM Ergeb-nisse führten zu einer Änderung des Protokolls für die in vivoEvaluierung der Bio-Gide® Matrix und ergaben unbefriedigen-de Materialeigenschaften für den Kollagenschwamm. Die 3R-konforme Überarbeitung der Versuchspläne führte zu verbes-serter Zellvermehrung, gesteigerter Synthese extrazellulärerMatrixmoleküle und verringerter Dedifferenzlerung der Zellen.Durch den Einsatz des HET-CAM Tests konnte zusätzlicheInformation über die Biokompatibilität der Implantate gewon-nen werden. Dies zeigt, dass die Bestrebungen zur Validierungvon Ersatz- und Ergänzungsmethoden zu Tierversuchen auchin der biomedizinische Forschung Anwendung finden.

Keywords: tissue engineering, the 3Rs, serumfree cell culture, antibioticfree cell culture, HET-CAM

1 Introduction

Tissue Engineering has emerged as anew field in the biomedical sciences.Twenty-five years ago scientists believedthat human tissue could only be replacedeither by transplantation of other tissuefrom a donor or with fully artificial ma-terials. Today, ambitions have movedfrom simple replacement towards recon-struction of the original tissue. Tissueengineering is defined as "the applicationof knowledge and expertise from a

multidisciplinary field to develop andmanufacture therapeutic products thatutilise a combination of matrix scaffoldswith viable human cell systems, or cellresponsive biomolecules derived fromsuch cells, for the repair, restoration orregeneration of cells or tissue damagedby injury or disease" (European Com-mission, Health & Consumer ProtectionDirectorate, 2001).

Tissue engineered products are usuallybased on autologous cells - cells derivedfrom the same individual's body - ex-

Received 7 November 2005; received in final form and accepted for publication 31 December 2005

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panded in vitro and, once new tissue ofthe desired phenotype has been formed,re-implanted into the patient. Traditionalanimal models for tissue engineering re-search include rats, mice, guinea pigs,hamsters and rabbits. For most surgicalapplications, large animal models areused for their anatomical and physiolog-ical similarities to man. In 1986 theEuropean Community passed a directiveon the protection of animals used for ex-perimental and other scientific purposes(Directive 86/609/EEC; Louhimies,2002) stating that the comrnission andthe member states should actively sup-

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port the development, validation and ac-ceptance of methods which could reduce,refine or replace the use of laboratory an-imals. The concept of replacement/re-ductionJrefinement (Russel and Burch,1959, reprinted 1992) should lead to areplacement of experimental anirnals bynonsentiest organisms, areduction of thetotal number of experimental animalsneeded and refined testing protocolsleading to less distressing experimentalprocedures.Total replacement of animal experi-

mentation for tissue engineering researchseems impossible at the moment. Yet,there are several possibilities for reduc-tion or at least refinement: Standard cellexpansion in vitro is performed usingculture media supplemented with foetalcalf serum (FCS). Besides ethical con-cerns regarding the harvesting of FCS(Shah, 1999; Jochems et al., 2002; vander Valk et al., 2004), traces of serum inthe transplanted cells may cause an im-mune reaction in the host, leading to an-tibody formation. The use of serum freeculture conditions might pose an alterna-tive. Also, the use of growth factors topromote cell proliferation for tissue engi-neering applications is controversiallydiscussed (van Tienhoven et al., 2001).The package insert of Carticel® culturedautologous chondrocytes states that in43% out of 81 treated patients hyper-trophic tissue was noted in the follow-uparthroscopy (package insert of Carti-cel®). Finally, aserum free cell cultureprotocol not requiring the supplementa-tion of growth factors might be anadvantage. The routine antibiotic supple-mentation of culture media may masklatent infections (Kuhiman, 1993) andeventually lead to transplantation of anunsterile product. Allergies to antibioticsare a potential risk for the recipient oftissue engineered products, and they arenot restricted to man (Ndiritu and Enos,1977; Gauchia et al., 1996). Antibioticsupplementation of cell culture mediashould therefore be discontinued priorto implantation of a tissue engineeredproduct.Animal experirnentation for the evalu-

ation of safety and efficiency of biomed-ical devices in Austria is regulated by theNational Federal Law concerning exper-imentation on living animals (1989).

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Although most research institutions per-form numerous in vitro tests to ensurebiocompatibility prior to animal testing,the implantation of biomedical deviceswithout obtaining preliminary data ispossible under the current legislation.The HET-CAM (Hen Egg Test -Chorioallantoic Membrane) test, origi-nally validated for toxicity and irritationstudies (Kalweit et al., 1987; Spielmann,1995), is gaining increasing attention forbiomaterial evaluation (Borges et al.,2003; Jux et al., 2003). Cells and bioma-terials selected for animal experimenta-tion could be studied using theHET-CAM model first to exc1ude unsuit-able setups and to optimise experimentalprocedures prior to animal testing(Falkner et al., 2004; Eder et al., 2005).The aim of the presented study was totranslate 3R aspects to tissue engineeringresearch and to test the feasibility of 3Rcompliant procedures in biomedicalapplication development. The experi-mental setup for a meniscus regenerationexperiment planned in a sheep modelwas evaluated according to the principlesof the 3Rs.

2 Material and methods

2.1 Cell cultureSheep primary meniscus cells were iso-lated from cadaver material accruing atthe university's animal experimentationfacility. The menisci were digested incollagenase type II (Invitrogen, Lofer,Austria), 0.15% (w/v), for 8 hours. Thecell suspension was filtered through a40 f111l cell strainer (Becton DickinsonLabware, NJ, USA), washed in phos-phate buffered saline (PBS; Invitrogen)and seeded into 25 cm" culture fiasks.Culture flasks were coated with 2%bovine skin gelatine (Sigma-Aldrich,Vienna, Austria) for serum free cell cul-tures. Cells were cultured in Dulbecco'sModified Eagle's Medium (DMEM;Invitrogen) supplemented with 0.05mg/mi ascorbic acid (Sigma-Aldrich)and either 10% foetal calf serum (FCS;PAA Laboratories, Pasching, Austria) or2% (v/v) Insulin/TransferrinJSeleniummixture (ITS; Sigma-Aldrich) for serumfree cell culture. Antibiotic supplementa-tion was performed using 100 U/ml peni-

cillin and 100 ug/ml streptomycin.Serum free cultures were split at subcon-fiuency at a ratio of 1:2 using 0.25%Trypsin-EDTA (Invitrogen Gibco, Carls-bad, USA) for cell detachment. Trypsinwas incubated for 1 minute at room tem-perature and incubation was terminatedby addition of pre-cooled soybeantrypsin inhibitor (Sigma-Aldrich) at aratio of 1:1, followed by centrifugation at1000 rpm for 5 minutes.

2.2 Cell characterisationPrimary cells were seeded on sterileglass slides at a concentration of 105cellsper slide. Evaluation was performed onday one, three and five of either serumsupplemented or serum free monolayerculture. Slides were rinsed in PBS andfixed with acetone for 10 minutes. Slideswere then washed in distilled water andstained with HemalaunJEosin (HIE) tomonitor dedifferentiation, and Azan,Alcian Blue, Safranine Ü and withspecific antibodies against collagen typeland 11 for characterisation of the prima-ry cultures. All staining protocols wereperformed according to Romeis (1989).Immunohistochemistry was performedusing goat anti-human collagen type Iand type II antibodies (Abcam, Cam-bridge, UK). Slides were washed in PBS,incubated in 0.3% H2ü2 to block endo-genous peroxidase reactions followingincubation in 2% (w/v) bovine serum al-bumin (BSA; Sigma-Aldrich) and 10%(v/v) horse serum (PAA) to block non-specific reactions. The specific antibod-ies were diluted 1:100 in PBS containing2% (w/v) BSA and incubated over night.Sampies were washed in PBS and incu-bated with biotinylated horse anti-goatIgG (Abcam; Cambridge/UK) for 30minutes, followed by washing in PBS,incubation in ABC-Reagent (Vectas-tain®, Vector Inc., Peterborough, UK)and antibody detection using di-aminobenzidine (Sigma-Aldrich) dilutedto 0.06% (w/v) in PBS containing 0.5%Triton X-100 (Sigma-Aldrich). 10 J1l30% H2ü2 were added per 100 J1l di-aminobenzidine solution.For three-dimensional cell culture,

5xlOs cells of passage 2 were pelleted bycentrifugation at 1200 rpm for 10 min-utes. The pellets were cultured for twoweeks prior to histological analysis.

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2.3 Antibiotic free cell cultureAntibiotic free cell culture was per-formed in a standard research laboratory.Cells were maintained in aseparate in-cubator. Laminar air flow, water bath andincubator were washed and sterilisedmonthly. The humidity-sensor waschanged every#lther month. The deviceswere scrubbed with S&M LaboratoryGranulate (Schülke & Mayr, Austria),rinsed with distilled water and dried. Allparts were sprayed with Biotenside®(Arkana, Austria) and dried. The laminarair fiow was soaked in Biotenside® overnight and left to air dry. The water bathwas treated the same way. The incubatorwas ventilated and sterilised using thedevice's self-sterilising programme. Theeffects of antibiotic free cell culture weremonitored via growth curves and histo-logical characterisation. To evaluate cellproliferation, cells were plated in 24 wellplates (1.9 cnr' per well) at a density of104 cells per well. Assessment of cellcounts was performed daily in triplicatesby detaching the cells using Trypsin-EDTA 0.25% (Gibco Invitrogen), stain-ing with Trypan Blue 0.4% (Sigma-Aldrich) and counting the viable cells ina Neubauer haemocytometer. Routinemycoplasma testing was performed us-

ing in situ DNA fiuorescence with 4'6-diamidino-2-phenylindole (DAPI; Sig-ma-Aldrich, ViennaJAustria) accordingto the supplier's protoco1.

2.4 HET-CAMassayFertilised, specific pathogen free WhiteLeghorn (Gallus domesticus) eggs weresupplied by Baxter Vaccine AG (Orth a.d. Donau, Austria) on incubation day 4.The egg shells were disinfected and 2 mlalbumen was removed as previouslydescribed (Falkner et al. , 2004). Theegg shell was opened and the amnioticmembranes were excised to expose thedeveloping CAM in a way accessible totreatment. The egg shell was closed withtin foil and the eggs were incubated in astandard cell culture incubator set to37°C, 5% CO2 and 95% humidity. Acommercially available collagen typeIIIII scaffold for tissue regeneration(Biogide®; Geistlich, Wolhusen,Switzerland) and a collagen sponge pro-totype (Geistlich) intended for meniscusrestoration were tested using theHET-CAM assay. The scaffolds wereaseptically cut into 25 rnrrr' pieces andmoistened in either FCS supplementedcell culture medium or PBS prior totransplantation onto the CAM.

Fig. 1: Serum supplemented and serum free primary cultures (Passage 0).a) FeS supplemented culture on the first day after isolation, demonstrating round as weilas fibroblast-like cells b) same culture after 3 days c) after 5 days in culture only thefibroblast-like phenotype prevails d)-f) serum free cultures at the same time points, alwaysshowing a round, chondrocyte-like appearance. Staining: H/E (The coloured figures of thisarticle are available on www.altex.ch).

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All HET-CAM experiments were per-formed in triplicate and repeated on twoseparate occasions. Sampies were ap-plied on incubation day 7 and maintainedin ovo for three days. Embryo viabilitywas controlled hourly for the first 12hours after sample application and at12 hour intervals afterwards. Digitaldocumentation was performed every24 hours. The embryos were killed afterspecimen explantation according toanimal welfare procedures by openingall major blood vessels and freezing at-20°C (Falkner et al., 2004). The CAMarea carrying the implant was complete-ly processed for histological analysis:CAM specimens were fixed in 4% (w/v)neutral buffered paraformaldehyde at4°C and embedded in Paraplast (Shan-don, Runcorn, UK). 5 um serial sectionswere prepared and stained in HemalaunlEosin according to Romeis (1989).

3 Results

3.1 Serum free cell cultureSheep meniscus primary cells main-tained under serum free conditions start-ed to attach to culture fiasks after 24hours and were securely attached after 48hours. Proliferation was slower than inFCS supplemented cultures. Populationdoubling times of the primary cultureswere 30.5 hours for FCS supplementedand 57 hours for serum free cultures. Themorphologie appearance of the cells wasdifferent under the different culture con-ditions: FCS supplemented meniscuscultures demonstrated three phenotypesafter cell isolation: Round cells, polygo-nal cells and spindie shaped cells werepresent on day 1 after isolation. After fivedays in vitro, only the polygonal fibro-blast-like phenotype could be observed.Serum free primary cultures demonstrat-ed a round phenotype, which was stillpresent after 5 days in vitro (Fig. 1). De-differentiation towards the fibroblast-likephenotype was observed after approxi-mately 10 days in culture.Histological characterisation of FCS

supplemented primary cultures demon-strated negative Alcian Blue and Safra-nine 0 staining, indicating the absence ofglycosaminoglycan synthesis. Azanstaining for non-specific collagens was

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slightly positive while immunohisto-chemistry demonstrated slightly positiveimmunostaining of collagen type landintensive staining of collagen type 11(Fig. 2 a-e). Native sheep meniscidemonstrate both collagen type landtype 11 positive staining at comparablelevels. )lheep meniscus fibrochondro-cytes maintained in serum free monolay-er culture demonstrated positive AlcianBlue and Safranine 0 staining andmarkedly higher levels of collagen type Isynthesis (Fig. 2 f-j).Three dimensional cell culture in-

creased collagen type land 11synthesisin FCS supplemented cultures, so thatculture results were comparable to thoseof serum free cultures regarding collagentype I synthesis. Collagen type 11synthe-sis was lower than in serum free culturesand glycosaminoglycan staining re-mained negative (Fig. 3 a-f).

3.2 Antibiotic free cell cultureNo bacterial or fungal contamination oc-curred during more than 12 months ofculture. Routine mycoplasma testing wasnegative. Proliferation of serum free cul-tures was markedly improved by theomission of antibiotic supplementation(Fig. 4). Population doubling time wasshortened to 25.6 hours. Generationtimes were 0.79 for FCS and antibioticsupplemented cultures, 0.42 for serumfree/antibiotic supplemented culturesand 0.94 for serum free/antibiotic freecultures. The collagen content was in-creased and dedifferentiation - indicatedby loss of collagen synthesis at higherpassages - was reduced (Fig. 5).

3.3 HET-CAMassay3.3.1 Bio-Gide® scaffoldThe scaffold was securely attached to theCAM after three days in ova and no ob-vious adverse effects such as bleeding,vessel malformation or vessel thrombo-sis were observed. Scaffolds moistenedin culture medium containing 10% FCSprior to application onto the CAMdemonstrated a significant angiogenic ef-fect, which was not observed in scaffoldsmoistened in PBS only (Fig. 6 a,b). His-tological analysis demonstrated good tis-sue integration and blood vessel forma-tion in the surrounding of the implant(Fig. 6c).

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Fig. 2: Improvement of histological characteristics by serum free cell culture(Passage 0).a)-e) FCS supplemented culture: a) negative Alcian Blue staining, b) slightly positive Azanstaining, c) negative Safranine 0 staining, d) positive collagen type I staining, e) positivecollagen type 11staining. f)-j) Serum free cultured cells: f) positive Alcian Blue staining, g)positive Azan staining, h) positive Safranine 0 staining, i) collagen type I, j) collagen type 11.

Fig. 3: Spheroid culture of sheep meniscus cells.al-cl FCS supplemented culture: a) negative Alcian Blue staining, b) collagen type I, c)collagen type 11.d)-f) Serum free culture: d) positive Alcian Blue staining, e) collagen type I,f) collagen type 11.

~~10000~--~~~~~~::==::!:::====~~~~~~--------~oGio

day 1 day 3 day 5-+serum free/antibiotic free -11-- serum free/antibiotic supplemented....•.. FC S /antibiotic supplemented

Fig. 4: Proliferation of FCS/antibiotic supplemented, serum free/antibiotic free andserum free/antibiotic supplemented primary cultures of sheep meniscus cells(Passage 0), n=3.

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3.3.2 Collagen spongeThe collagen sponge prototype showedextremely rapid degradation patterns inova and could not be manipulated withtweezers for sample explantation. Spon-taneous CAM bleeding and a retractionof blood vessels from the implantationsite were obsewed (Fig. 6 d,e). Histolog-ical analysis demonstrated failed implantattachment and a foreign body tissue re-sponse (Fig. 6 f). The material was there-fore considered unsuitable for meniscustissue engineering and was not includedin further experimental plans.

4 Discussion

The presented study attempted to transferaspects of the 3Rs to tissue engineeringresearch. Routine cell culture for tissueengineering is performed using FCS as asupplement to promote attachment andproliferation of mammalian cells. Serumis sourced as a by-product of the beefindustry. The high protein diet of dairycows may consist of meat and bone meal,which is a scientifically accepted causeof Bovine Spongiform Encephalopathy(BSE; Falkner et al., 2003). Serum usedfor cell cultivation in vitro is a majorsource of viral contaminants (Jennings,1999), which often do not produce cyto-pathic effects or morphological changes.A great number of viruses have the po-tential to infect cell cultures, but only alimited number is likely to be detected inquality controlled sera (Rhangan et al. ,1979; Kappeler, 1996; van der Noordaaet al., 1999).

Traces of bovine pro teins derivingfrom FCS supplementation of cell cul-tures may cause allergie reactions in therecipient, as tissue engineering experi-ments are usually not performed usingbovine animal models. FCS supplement-ed "autologous" cell cultures shouldtherefore be regarded as xenografts.Looking at data obtained from humanclinical trials, several studies have beenpublished reporting allergic reactions toFCS components used for cell expansionin vitro (Mackensen et al., 2000; Tschuonget al., 2002; Spees et al., 2004).

The benefits of serum free culturemedium are easily deducible: Serum freecell culture promotes chemically defined

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and controlled culture conditions(Gstraunthaler, 2003), leads towardselimination of a potential source ofmicrobial contamination (Froud, 1999;Merten, 1999), allows the selection ofspecific cell types and delays dedifferen-tiation phenomena (Brown and Schnei-der, 2002; Chaipinyo, 2002; Yokoyama,2004). Regarding serum free culture of

meniscus fibrochondrocytes, only fewprotocols are published. Tumia and John-stone used DMEM supplemented with50 ug/ml ascorbic acid and various con-centrations of bFGF for serum free cul-ture of sheep meniscus cells (Tumia andJohnstone, 2004). The benefit of ITSsupplementation in combination withvarious growth factors was demonstrated

Fig. 5: Cells cultured in medium containing 10% FCS with and without antibioticsupplementation.Passage 5 cells maintained in antibiotic free medium stain positive for collagen type I (a)while corresponding cells cultured in antibiotic supplemented medium demonstratenegative staining results (b).

Fig. 6: HET-CAM testing results of the Bio-Gide® scaffold and the collagen spongeprototype.a) Bio-Gide®, angiogenic response caused by traces of culture medium used for scaffoldmoistening after 3 days in ovo b) Bio-Gide®, absent angiogenic response after scaffoldmoistening in PBS after 3 days in ovo c) Bio-Gide®, histological analysis demonstratinggood tissue integration and the formation of blood vessels in the surrounding of thescaffold (arrows); staining: H/E. d) Collagen sponge on day 1 after sam pie application;stars marking blood vessels present on day 1 but absent on day 3 after sam pieapplication; e) collagen sponge after 3 days in ovo: vessel bleeding and anti-angiogeniceffect; f) histology demonstrates failed tissue integration and a foreign body tissueresponse; staining: H/E.

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for chondrocytes by various authors(Chua et al., 2005; Morgan et al., 2005).The presented study tested serum freeprimary culture of meniscus cells with-out addition of specific growth factors.Serum supplemented primary cultures

isolated from meniscus fibrocartilagecontained a large number of cells with aphenotype different from fibrochondro-cytes. After 5 days in vitro, these culturesdemonstrated a unique, fibroblast-likeappearance. Under serum free condi-tions, cultures demonstrated a round phe-notype for the first 10 days of culture aswell as synthesis of extracellular matrixproteins typical for chondrocyte-likecells. As meniscus cells derive from apoorly vascularised tissue, serum freeculture conditions might refiect a morephysiological environment.Although antibiotic supplementation is

standard practice in cell culture routineand clinical applications, the uncriticaluse of antibiotics carries several disad-vantages: Besides induction of resistantbacterial strains by insufficient dosage orincorrect waste disposal and the possibil-ity of side effects or allergic reactions incase of cell transplantation, the antipro-liferative effect on allliving cells shouldbe taken into account (Kuhlmann, 1993).As a routine research laboratory is hard-ly comparable to an automated GMPcompliant tissue engineering facility, an-tibiotic free cell culture was performedunder non-GMP conditions. The resultsdemonstrated that antibiotic free cell cul-ture was not only possible under non-GMP conditions, but carried several ben-efits for the cell cultures: Populationdoubling time of serum free cultures wasdecreased and dedifferentiation phenom-ena were reduced.The HET-CAM test offers a cheap,

simple and 3R compliant borderline invitro/in vivo model, allowing the pre-evaluation of cells and scaffolds prior toactual animal experimentation (Falkneret al., 2004; Eder et al., 2005). Using theHET-CAM test for pre-evaluation of bio-materials designed for tissue engineer-ing, the Bio-Gide® scaffold demonstrat-ed impressive angiogenic propertieswhen applied after pre-moistening in cul-ture medium. Applying scaffolds soakedin PBS as control indicated that the an-giogenic effect was due to the FCS in the

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culture medium and not to material prop-erties. A change in protocol was there-fore suggested for later animal testing ofthe Bio-Gide® scaffold to avoid traces ofculture medium infiuencing experimentalresults. The collagen sponge prototypedemonstrated rapid in ovo degradation,failed tissue integration and it elicited aforeign body tissue response. The bioma-terial was therefore considered inappro-priate and excluded from further experi-mental procedures. Considering thefinancial, personnel and time require-ments of animal experimentation, bioma-terial pre-testing using the HET-CAMbioassay is not only desirable in terms ofthe 3Rs, but also beneficial for the re-searcher.The presented study tried to transfer

aspects of the 3Rs to tissue engineeringresearch. All starting points tested turnedout to be not only favourable in terms ofthe 3Rs, leading to refinement and reduc-tion of animal experiments, but alsofavourable in terms of the scientific ex-periment, leading to better cell prolifera-tion, higher synthesis of extracellularmatrix molecules, reduced dedifferentia-tion phenomena and adding additionalinformation about biocompatibility at anearly experimental stage. Tissue engi-neering research can therefore profitfrom the increased efforts to standardisecell culture technologies and in vitro re-search and to validate in vitro alternativesand supplements to animal testing.

ReferencesAustrian Federal Law 1989 regulatingexperimental Procedures on livinganimals, 27 September 1989.http://www.bmbwk.gv.at/forschung/recht/tierversuche/tvg.xml?style=print(last accessed 5.11.2005).

Borges, 1.,Tegtrneier, F. T., Padron, N. T.et al. (2003). Chorioallantoic mem-brane angiogenesis model for tissueengineering: a new twist on a classicmodel. Tissue Engineering 9(3), 441-450.

Brown, L. D. and Schneider, M. F.(2002). Delayed dedifferentiation andretention of properties in dissociatedadult skeletal muscle fibres in vitro. InVitro Cell Dev. Biol. Anim. 38(7),411-422.

Carticel package insert http://www.carti-

cel.com/pdfs/carticelproductIabel.pdf (last accessed 29.12.2005).

Chaipinyo, K., Oakes, B. W. and vanDamme, M. P. (2002). Effects ofgrowth factors on cell proliferationand matrix synthesis of low-density,primary bovine chondrocytes culturedin collagen I gels. J. Orthop. Res.20(5), 1070-1078.

Chua, K. H., Aminuddin, B. S., Fuzina,N. H. and Ruszymah, B. H. I. (2005).Insulin- Transferrin-Selenium preventhuman chondrocyte dedifferentiationand promote the formation of highquality tissue engineered human hya-line cartilage. European Cells and Ma-terials 9,58-67.

Eder, C., Falkner, E., Mickel, M. et al.(2005). A Modified HET-CAM Ap-proach for Biocompatibility TestingOf Medical Devices. Animal Welfare14(4),297-302.

European commission, Health & Con-sumer Protection Directorate (2001).Opinion on the state of the artconcerning tissue engineering,europa.eu.int/commlfood/fs/sc/scmp/out37_en.pdf (last accessed 5.11.2005).

Falkner, E., Schöffl, H., Appl, H. et al.(2003). Replacement of Sera for CellCulture Purposes: A Survey, ISBN 3-900 l31-00-7, Linz, Austria.

Falkner, E., Eder, C. and Kapeller, B. etal. (2004). The mandatory CAM test-ing of cells and scaffolds for tissueengineering: Benefits for the 3Rs ofcooperation with the vaccine industry.ATLA 32, 573-580.

Froud, S. J. (1999). The developmentbenefits and disadvantages of serumfree media. Dev. Bio. Stand. 99, 157-166

Gauchia, R., Rodriguez-Serna, M., Sil-vestre.T, F. et al. (1996). Allergie con-tact dermatitis from streptomycin in acattle breed. Contact Dermatitis 35(6),374-375.

Gstraunthaler, G. (2003). Alternatives tothe use of fetal bovine serum: serum-free cell culture. ALTEX 20(4), 275-281.

Jennings, A. (1999). Detecting viruses insera: methods used and their merits.Dev. Biol. Stand. 99, 51-59.

Jochems, C. E. A., van der Valk, J. B.,Stafieu, F. R. and Baumans, V. (2002).The use of fetal bovine serum: ethical

ALTEX 23, 1/06

~ EDER ETAL.-~--------------

or scientific problem? ATLA 30, 219-227.

Jux c., Wohlsein, P., Bruegmann, M. etal. (2003). A new biological matrix forseptal occlusion. Journal of Interven-tional Cardiology 6(2), 149-152.

Kalweit, S., Gerner, 1. and Spielmann, H.(1987). Valieation project of alterna-tives for the Draize eye test. MolecularToxicology I, 597-603.

Kappeler, A., Lutz-Wallace, C., Sapp, T.and Sidhu, M. (1996). Detection ofbovine polyomavirus contamination infetal bovine sera and modified liveviral vaccines using polymerase chainreaction. Biologieals 24(2),131-135.

Kuhiman, 1. (1993). Problems of theprophylactic use of antibiotics in cellculture. ALTEX 10(2),27-49.

Louhimies, S. (2002). Directive86/609IEEC on the protection of ani-mals used for experimental and otherscientific purposes. ATLA 30, Suppl. 2,217-219.

Mackensen, A., Drager, R., Schlesier, M.et al. (2000). Presence of IgE anti-bodies to bovine serum albumin in apatient developing anaphylaxis aftervaccination with human peptide-pulsed dendritic cells. CancerImmunol. Immunother. 49(3), 152-156.

Merten, O. W. (1999). Safety issues ofanimal products used in serum freemedia. Dev. Bio. Stand. 99, 167-180.

Morgan, T. J., Xu, X., Rowan, A. D. andCawston, T. E. (2005). Evaluation ofchondrocyte micromass culture for thestudy of cartilage degradation. Arthri-tis Research & Therapy 7 (Suppl 1),58.

Ndiritu, C. G. and Enos, L. R. (1977).Adverse reactions to drugs in a veteri-nary hospital. 1. Am. VetoMed. Assoc.171(4),335-339.

Rhangan, S. R. and Gallagher, R. E.

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(1979). Tumors and viruses in non-human primates. Adv. Virus Res. 24,1-123.

Romeis, B. (1989). MikroskopischeTechnik (Title translation: Microscopictechnology). Munich, Germany: Urban& Schwarzenberg.

Russel, W. M. S. and Burch, R. L. (1959,reprinted 1992). The principles 01humane experimental technique.Wheathampstead, UK: UniversitiesFederation for Animal Welfare.http://wwwJorschung3r.ch/de/publica-tions/bu7.htrnl (last accessed 5.11.2005).

Shah, G. (1999). Why do we still useserum in the production of biopharma-ceuticals? Dev. Biol. Stand. 99,17-22.

Spees, J. L., Gregory, C. A., Singh, H. etal. (2004). Internalised antigens mustbe removed to prepare hypoimmuno-genic mesenchymal stern cells for celland gene therapy. Mol. Ther. 9(5),747-756.

Spielmann, H. (1995). HET-CAM test.Methods in Molecular Biology 43,199-204.

Tschuong L., Soenen, S. L., Blaese, R.M. et al. (2002). Immune response tofetal calf serum by two adenosinedeaminase-deficient patients after Tcell gene therapy. Rum. Gene Ther.13(13),1605-1610.

Tumia, N. S. and Johnstone, J. (2004).Promoting the Proliferative and Syn-thetic Activity of Knee Meniscal Fi-brochondrocytes Using Basic Fibro-blast Growth Factor In Vitro. TheAmerican Journal 01Sports Medicine32,915-920.

van der Noordaa, J., Sol, C. J. and Schu-urman, R. (1999). Bovine poly omavirus, a frequent contaminant of calfsera. Dev. Biol. Stand. 99,45-47.

van der Valk J., Mellor, D., Brands, R. etal. (2004). The humane collection of

fetal bovine serum and possibilitiesfor serum free cell and tissue culture.Toxicol. in vitro 18(1), 1-12.

van Tienhoven, E. A. E, Geertsma, R. E.,Wassenaar, C. and Jong, W. H. (2001).Pre-clinical safety assessment of Tis-sue Engineered Medical Products(TEMPs) - An investigation on assaysand guidelines for biocompatibilitytesting http://www.rivm.nl/biblio-theek/rapporten/640080002.html (lastaccessed 29.12.2005).

Yokoyama, A. (2004). Microglia, a po-tential source of neurons, astrocytes,and oligodendrocytes. Glia 45(1), 96-104.

AcknowledgementsThe presented study was embedded inthe European Comrnission's 5th Frame-work Programme "Innovative materialsand technologies for a bio-engineeredmeniscus substitute" (Project No GRD 1-2001-40401, Contract No G5RD-CT-2002-00703). Parts of the work weresupported by the Pollux Private Trust.The authors would like to acknowledgeBaxter Vaccine AG, Egg ProcessingUnit, Orth a. d. Donau, Austria for sup-plying the SPF chicken eggs from sur-plus.

Parts of the work presented wereawarded with the Viennese Animal Wel-fare Award 2005.

Correspondence toDr. Claudia EderBiomed - Society for the Promotion ofBiomedical and Medical TechnologicalResearch in Upper AustriaP.O. Box 210A-4021 LinzAustriae-mail: claudia.eder@biomed.or.at

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