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A Customizable Instrument for Measuring the Mechanical Properties of Thin Biomedical Membranes

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A Customizable Instrument for Measuring the Mechanical Properties of Thin Biomedical Membranes
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  A Customizable Instrument for Measuring theMechanical Properties of Thin Biomedical Membranes Gracia´ n Trivin˜ o, * 1  Marı´ a Concepcio´ n Serrano, 2  Raffaella Pagani, 2  Marı´ a Teresa Portole´ s 2 1 Department of Photonics Technology, Universidad Polite´cnica de Madrid, Campus de Montegancedo, 28710-Madrid, SpainFax: 34-91-336 73 72; E-mail: gtrivino@fi.upm.es 2 Department of Biochemistry and Molecular Biology I, Faculty of Chemistry, Universidad Complutense, 28040 Madrid, SpainReceived: July 6, 2005; Accepted: August 5, 2005; DOI:10.1002/mame.200500243 Keywords:  degradation; mechanical properties; polycaprolactone; stress-strain curve;thin elastic membrane; tissue engineering Introduction In recent years, the need for replacements for diseasedand damaged biological structures has resulted in tissueengineering becoming a promising research field. Thisrequires a new interdisciplinary approach that applies theprinciples of engineering and life sciences in the develo-pment of biological substitutes with prominent applica-tions. [1] Depending on the case, bioresorbable or non-bioresorbable polymers with suitable physico-mechanical,chemical and biochemical properties have been used forthese purposes. The mechanical properties of the polymerselected for tissue engineering applications can affect themorphology, proliferation and differentiation of the cellscultured on its surface [2] and, moreover, the compliance of the implant. An ideal scaffold must have the appropriatemechanical properties to match those of the natural tissuesat the site ofimplantation. [3] Structural integrity is requiredbefore new tissue can be formed, [4] so the rate of elasticityorstiffnessandtheresistanceorthestraintofailuremustbechosen for the implant depending on its future application.In the case of vascular replacements, properties such aselasticity, mechanical breakdown or fatigue acquire theutmost importance.Nowadays,severaldevicesarecommerciallyavailabletomechanically characterizethe scaffoldsobtained.Theclas-sicinstrumentsconsistofatestingsystemthatmeasuresthestrain after producing a uniaxial deformation on the sam-ple. [5,6] However, these tests can be inadequate due to themechanical context the scaffold will have to support afterimplantation in vivo. Thus, a biaxial test is often required.Instron [7] and EnduraTEC [8] are prestigious commercialproviders of instruments that are capable of performingeitheruniaxialorevenbiaxialmechanicaltesting.Themain Summary:  A customized instrument has been developed aspart of multidisciplinary research work relating to the devel-opmentofabiodegradablevascularscaffold.Thisinstrumentaims to measure the mechanical properties of elastic andviscoelastic thin membranes with tissue engineering appli-cations. Uniformand omni-directional pressure isapplied onthe whole membrane which is uniformly clamped and sub-merged into a liquid medium. The mechanical testing des-cribed in this study is focused on the stress-strain curves of polycaprolactone (PCL) films after different treatments. Theinfluence of Dulbecco’s modified Eagle’s culture medium,L929 fibroblast culture, NaOH treatment and film thicknessonthemechanicalpropertiesofPCLfilmswasevaluatedafterdifferent times.These studiesshow thatthe PCL degradationprocess is influenced by immersion in the culture medium,inducing an increment in the slope of the pressure-dilationcurve which is indicative of an increase in the polymer stiff-ness. On the other hand, long NaOH treatments make PCLfilms have more flexible behavior.A computerized version of the instrument: (1) Electricalcompressor; (2) Filter; (3) Voltage-pressure converter; (5)Pressure sensor;(6)Differentialpressuresensor;(7–8)Mainandauxiliarypipettes;(9)Printedcircuitboard;(10)Personalcomputer.  Macromol. Mater. Eng.  2005 ,  290 , 953–960 DOI:10.1002/mame.200500243    2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Full Paper  953  drawbacks of these commercial tools are their relativelyhigh cost and difficulties with clamping the sample ade-quatelytoperformthetestinthedesiredway.Specificmodi-fications in these kinds of systems have been developed inorder to adapt the devices to different samples. [4,9–11] Recently, several approaches have been taken to designcustomized instruments aimed at measure the mechanicalproperties of a thin elastic membrane. Some of their char-acteristics could be considered to be precedents for theinstrument presented in this paper. Ju et al. have describedan instrument based on the use of a video enhanced micro-scope to provide the capability to measure both the appliedforce and the resultant displacement of the biologicalmembrane under a central point force simultaneously. [12] Roeder et al. have designed a device based on the idea of inflating a recipient made of small intestine submucose. [13] By maintaining this graft under a liquid medium, it waspossible to obtain the relation between the applied pressureand the increment of volume. As will be explained below,we have used the same basic principle to measure themembrane deformation with our instrument. Mackin et al.performed a mechanical fatigue test by applying forcewithasphericalindenterwhileusinganinfraredsystemtodetectthe appearance of cracks. [14] More recently, Ju et al. havedescribed the use of the weight of a ball left alone on amembrane to measure the progressive deformation with avision system. [15] The data obtained in this way refer to theviscoelastic properties of the membranes. Recently, Khooet al. have presented a new instrument that allows themeasurement of the mechanical characteristics of a mem-brane that support a very small deformation produced by acentral indenter. [16] Intheframeworkofamultidisciplinaryprojectaimingtodesign a tissue-engineered scaffold, the need to measuringthe mechanical characteristics of handled membranesarose.Afteradetailedanalysisofpresentandfutureprojectneeds,itwasdeterminedthatitwasnecessarytoprovideaninstrument defined by the following list of requirements.1. The assays must be omni-directional due to theheterogeneityinthickness,compositionandphysicalstruc-ture that the sample membranes could show.2. Clamping must be as hard and uniform as possible toavoid slipping. The use of local claws should be avoidedbecause of the risk of tearing the membranes.3. Different sizes of samples must be allowed.4. It should be possible to perform the test while themembrane is submerged in a liquid medium. The mechan-ical properties of both biological and synthetic samplescouldchangeiftheyaremaintaineddry,particularlyinlongtime tests.5. Force must be applied as a uniform pressure in orderto reproduce the membrane working conditions.6. The instrument must provide enough resources toperform tests concerning mechanical parameters such asstress-strain, breakdown, fatigue and viscoelasticity.7. It would be desirable to have optical access to themembrane deformation process. This could be useful toperform tests based on computer vision measurements.8. It would be desirable to obtain a low cost instrument.Thispaperdescribesacustomizeddevicedevelopedwiththe aim of measuring the mechanical properties of elasticand viscoelastic thin membranes with tissue engineeringapplications. These membranes are either artificial bio-resorbable substrates or biological tissues. The first resultsconcerning to the mechanical behaviour of polycaprolac-tone membranes during degradation are included. Experimental Part The Chamber  The mechanical principles of the instrument are described asfollows. The pressurewas applied in a controlled manner via aliquidatonesideofthesample.Then,themembranebulgewasobtained by measuring the displacement of the liquid on theothersideofthesample.Thecoreoftheinstrumentiscalledthechamber (Figure 1) and contained the sample, which wasclampedinitsedges.Thisisanadequaterecipientfortheliquidat both sides of the membrane. The diagram in Figure 1 showshow the membrane was placed and deformed after pressurewas applied. The chamber had four pipes, two on each side.These were an input for pressurized liquid, an output for purg-ing and plugging a pressure sensor, an output for the displace-ment sensor and an output for purging. Note that the internalpipes in the outputs were dedicated to making bubbleextraction easy. Figure 2 represents the chamber breakdown.The central piecewasa cylindrical pipe threaded at both sides.There were also two other cylindrical pipes with diameterssuitable for clamping the membrane between them and twoscrew-tops which provided the necessary strength between theinner cylinders to clamp the membrane (which is convenientlycircular) when threaded on (1). All the pieces were made of poly(methylmethacrylate) (PMMA),atransparentandeasytoshape material.Two versions of the instrument are presented here, but thechamberissimilarinboth.Thefirstonewaslowcostandeasierto make. The second one was a computerized version withelectronic sensors that allowed test precision and reproduci-bility to be improved, increasing the reliability of the data.Moreover, this automated version provided the necessaryresources to perform viscoelasticity and fatigue analysis. Figure 1. A diagram and a photo of the chamber of theinstrument: (1) Membrane; (2–5) Pipes to access to the chamber. 954  G. Trivin˜o, M. C. Serrano, R. Pagani, M. T. Portole´s  Macromol. Mater. Eng.  2005 ,  290 , 953–960 www.mme-journal.de    2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim   Manual Version Themanualversionoftheinstrumentusedasyringetoprovidethe necessary pressure. A mechanical manometer (0–2.5 bar;NuovaFima) measured the pressure at the left side of thechamber.Tomeasuretheliquiddisplacementattherightside,a10mlpipettewasused.Figure3showstheinstrumentreadytouse. Two people could easily perform a stress-strain test,applying force in a manual but controlled manner. Computerized Version This version had several advantages over the manual one.Firstly, the maximum level of applied pressure could be incre-ased considerably. Moreover, the measure resolution wasimproved because human participation was substituted byelectronic sensors and the data could be stored in real time.However, the main advantage consisted of the possibility of applying pressure with a compressor at an automatic andcontrolled speed. Thanks to this feature, it was possible to testthe membrane viscoelastic properties and to perform fatiguetesting.Figure 4 shows the main components of this version of theinstrument. Pressure was obtained from an electrical 8 barcompressor (Vento Model, ABAC) (1). This model was main-tenance free, of small size and had more than enough volumecapability. The flow of pressurized air passed through a filter(2) and then was introduced into a voltage-pressure converter(5354R0400R Model, Watson Smith) (3). This provided anoutputbetween2and120psiforvoltagesintherange1.0–10V.ThepressureintheleftsideofthechamberwasmeasuredusingaMotorolaMP4500pressure sensor(5).Thevolumedisplace-ment was measured using a differential pressure sensor(164PC01D76 Model, Honeywell) (6). This sensor was ableto measure very low differences in pressure, thus detecting amillimeter of liquid level of difference between the mainpipette (7) and the auxiliary one (8). The electronic signalsprovided by both pressure sensors and the one required tocontrol the voltage-pressure converter were handled using acustomized electronic circuit. The microcontroller (16F876model, Microchip), the sensors, the connexion for thevoltage-pressure converter and a serial interface RS232 were includedinaprintedcircuitboard(9).Thiselectroniccircuitwasusedasan interface between the mechanical components and apersonal computer (10). This electronic circuit had a reducedcomplexity and it was used by authors as a didactical resourcefor teaching Electronic Instrumentation Development. [17] Thepersonal computer ran a program developed using the Micro-soft Visual C þþ Programming Language. The computationalpower of this computer and the programming language usedprovide a very flexible tool for developing different kinds of testing in accordance with the project requirements. Figure 5showsthecomputerizedversionoftheinstrumentreadytouse. Figure 2. A diagram of the chamber breakdown: (1) Maincylinder; (2) Internal cylinders; (3) Tops.Figure 3. Manual version of the instrument ready to use.Figure 4. Computerizedversionoftheinstrument:(1)Electricalcompressor;(2)Filter;(3)Voltage-pressureconverter;(5)Pressuresensor; (6) Differential pressure sensor; (7–8) Main and auxiliarypipettes, respectively; (9) Printed circuit board including themicrocontroller, the sensors, the connexion for the voltage-pressure converter and a serial interface RS232; (10) Personalcomputer.Figure 5. Computerized version of the instrument ready to use. A Customizable Instrument for Measuring the Mechanical Properties of Thin Biomedical Membranes  955  Macromol. Mater. Eng.  2005 ,  290 , 953–960 www.mme-journal.de    2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  Stress-Strain Tests The classical stress-strain test consists of obtaining the figureof the material deformation versus the applied force. Theinstrument presented provided this type of curve directly bymeasuring the displaced volume versus the applied pressure.Notethatthemeasuredvolumeisdirectlyrelatedtothedilationof the membrane. The deflection can be obtained easily con-sidering that, for a limited bulgevalue, the membrane deformsfollowingthe surface of an ellipsoid (Figure 6).Thevolumeof this semi-ellipsoid is: V   ¼ 23 p r  2  f  ;  f   ¼  3 V  2 p r  2  ð 1 Þ Notethatthe deflectionis proportionalto thevolumedisplace-ment. For homogeneous and elastic materials, this relation islinear and the ratio of stress-strain corresponds to the Young’sModulus. A formula to obtain the Young’s Modulus has beendeduced for clamped circular plates under uniform pressurewith a small deflection: [18]  f   ¼  q*r  4 64 *D  where  D ¼  E*h 3 12 * ð 1  n 2 Þ ð 2 Þ where q istheappliedpressure(kg  cm  2 ), r  istheradiusofthemembrane(cm), E  istheYoung’smodulus, h isthethicknessof the membrane (cm),  u  is Poisson’s ratio ( e 0  /  e  which isconsidered to be equal to 0.5 for elastic and incompressiblematerials) and  f   is the deflection of the membrane (cm).Therefore: E  ¼  PV r  6 3 : 39 h 3  ð 3 Þ This parameter is enough to represent the mechanicalbehavior ofmaterialswiththementioned restrictions.Figure7shows a stress-strain curve obtained with the instrument. Thiscurve corresponds to a polycaprolactone sample. In the firstphase, the membrane needs to be aligned since the applicationof the load, which is indicative of the damping process of thesample (volume displacement of less than 0.3 ml). The secondphase corresponded to the elastic zone where a characteristicslope is shown. Finally, the curve ends show plastic behavior.Note that the polymer shows quasi-crystalline behavior underthe experimental conditions. Unfortunately, the type of mate-rials handled in bioengineering cannot be characterized bysimple elastic behavior. They do not usually have a homoge-neous structure or uniform thickness. In the context of thisproject,ourcurrentchallengeistobecapableofcomparingtheevolution of the mechanical properties of heterogeneous andviscoelastic membranes.The analyses havebeen performed by comparing the curvesobtained from sets of at least three samples with similarcharacteristicsandwhichweretreatedundersimilarconditionsin each case. Other Tests Mechanical BreakdownThistestconsistsofincreasingthepressureuntilthemembranebreaks. This situation is detectable because of the abruptchange in the membrane resistance.ViscoelasticityUsing the computerized version, a test has been developed tomeasure the viscoelastic properties of a material (time depen-dence). The instrument was programmed to maintain a fixedpressure while measuring the volume increment. Then themembrane deformation versus time was reported.FatigueUsing the computerized version, it was possible to obtain thestress-strain curve before and after applying a sequence of anumber of pulses of moderate pressure to membrane samples.Polymeric SamplesPCL was directly used as purchased (  M  w ¼ 65000 g  mol  1 ,Sigma-Aldrich Corporation, St. Louis, USA). The PCL filmswere prepared by hot pressing under 20 tons at 100 8 C for twominutes and were characterized as previously described inref. [19] The film thickness was controlled to obtain homo-geneous groups of samples. PCL films were cut into circularpieces (3.1 cm diameter) and sterilized with 30 min UVirradiation. Some membranes were submerged in a 2  N  NaOHsolution for two hours to increase their hydrophilicity. Thistreatment improves the cell-polymer interaction and has beenused in previous biocompatibility studies. [20] The effects of both culture medium and cell culture on the mechanical beha-vior of untreated and NaOH treated PCL films were studied inorder to evaluate the possible degradation of these membranesunderconditionssimilartoabiologicalenvironment.Withthispurpose in mind, untreated and NaOH treated films wereimmersed in Dulbecco’s modified Eagle’s medium (DMEM, Figure 6. The membrane deformation follows the surface of anellipsoid.Figure 7. A pressure-dilation curve of a PCL film obtained withthe instrument. 956  G. Trivin˜o, M. C. Serrano, R. Pagani, M. T. Portole´s  Macromol. Mater. Eng.  2005 ,  290 , 953–960 www.mme-journal.de    2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim  Sigma Chemical Company, St. Louis, USA) supplementedwith10%fetalbovineserum(FBS,Gibco,BRL),1  10  3 M L- glutamine (BioWhittaker Europe, Belgium), penicillin(20000 U  ml  1 , BioWhittaker Europe, Belgium) and strep-tomycin (20000  m g  ml  1 , BioWhittaker Europe, Belgium)(culture medium) for progressive times. On the other hand,L929 mouse fibroblasts were seeded on PCL films, untreatedandNaOHtreated,in6wellcultureplatesinaculturemedium,under a CO 2  (5%) atmosphere and at 37 8 C for different times.Finally, to see the effect of long NaOH treatments on themechanical properties of PCL membranes, some films weresubmerged into 2  N  NaOH solution for one, two or four weeks.Organic SamplesSegments of inferior cava vein (5 cm length) were obtainedfrompigsasbiologicalmembranesformechanicaltesting.Thesamples were washed with phosphate buffered saline (PBS)and opened lengthways with a scalpel. Statistics In all cases, each point in the curves was expressed as themean  standard deviation of the results from more than threesamples. Statistical analysis was performed using the Statis-tical Package for the Social Sciences (SPSS) version 11.5software. Statistical comparisons were made using theStudent’s t-test or univariate analysis of variance when appro-priate. The film thickness was fitted as a covariable in order toeliminate its potential effect on the mechanical parameters. Inall statistical evaluations,  p < 0.05 was considered as statisti-cally significant. Results and Discussion  Mechanical Testing of PCL Films During Degradation: Effect of Short NaOH Treatment  Polycaprolactone (PCL), a semi-crystalline linear resorb-able aliphatic polyester, exists in a rubbery state at roomtemperature. The mechanical properties of solid PCL havebeen previously reported. [5] In vitro studies indicate thatPCL is a suitable, biodegradable material for use as a scaf-fold for vasculargraftdevelopment. Ashort treatment(2 h)ofthe PCL films with NaOH 2 N  improvesthe adhesionandproliferation of vascular cells on these membranes. [20] Thispolymer is subjected to biodegradation because of thesusceptibility of its aliphatic ester linkage to hydrolysis. [21] The mechanism by which these polyesters are degraded isnot completely understood. The first phase of their biode-gradation is consistent with a mechanism which involvesrandom chain scission by the hydrolytic cleavage of estergroups,excludingenzymeparticipation.Asecondmechan-ism of weight loss evidently becomes important when theparticle size of the implant is decreased. [22] In order to study how the mechanical properties of PCLfilms are modified by the environmental conditions andthe degradation processes, both untreated and NaOHtreated PCL films were submerged into culture mediumfor 34 weeks (Figure 8(A) and 8(B), respectively). The un-treatedPCLfilms showed asignificantincreaseinthe slopeof the stress-strain curves after immersion in the culturemedium, which is indicative of PCL becoming stiffer withtime. According to this result, Tan et al. found that thedegree of PCL crystallinity would increase with storagetime and thus it affected the mechanical properties mea-sured. [4] As shown in Figure 8(B), short NaOH treatmentinduces an increment in PCL stiffness at time zero. How-ever,afterimmersionintheculturemedium,theslopeofthecurve decreased and the initial NaOH treatment effectdisappeared. After this first phase, NaOH treated filmsshowed similar behavior to untreated ones, so PCL becamemore rigid with longer immersion times.Toanalyzethe influence thatcellculturecouldhaveoverthe mechanical parameters of PCL films, L929 mousefibroblasts were seeded on both untreated and NaOH treat-ed PCL membranes for different times. As shown inFigure 9(A) and 9(B), respectively, cell culture seemed notto have a relevant influence over the PCL pressure-dilationcurves.Nosignificantdifferenceswerefoundasawholeforeither untreated or treated films. The significant influenceon mechanical parameters observed during immersionin the culture medium was mitigated by cell culture. Figure 8. Pressure-dilation curves of PCL films after progres-sive immersion times in culture medium at 37 8 C: (A) UntreatedPCL films; (B) NaOH treated PCL films. Significant differ-ences were found as a whole. A Customizable Instrument for Measuring the Mechanical Properties of Thin Biomedical Membranes  957  Macromol. Mater. Eng.  2005 ,  290 , 953–960 www.mme-journal.de    2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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