Effect of sterilization on structural and material properties of 3-D silkfibroin scaffolds

Sandra Hofmann a,⇑, Kathryn S. Stok a, Thomas Kohler a, Anne J. Meinel b, Ralph Müller a

a Institute for Biomechanics, ETH Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerlandb Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerland

a r t i c l e i n f o

Article history:Received 1 May 2013Received in revised form 15 August 2013Accepted 26 August 2013Available online 4 September 2013

Keywords:Silk fibroinSterilizationScaffoldTissue engineering

a b s t r a c t

The development of porous scaffolds for tissue engineering applications requires the careful choice ofproperties, as these influence cell adhesion, proliferation and differentiation. Sterilization of scaffolds isa prerequisite for in vitro culture as well as for subsequent in vivo implantation. The variety of methodsused to provide sterility is as diverse as the possible effects they can have on the structural and materialproperties of the three-dimensional (3-D) porous structure, especially in polymeric or proteinous scaffoldmaterials. Silk fibroin (SF) has previously been demonstrated to offer exceptional benefits over conven-tional synthetic and natural biomaterials in generating scaffolds for tissue replacements. This studysought to determine the effect of sterilization methods, such as autoclaving, heat-, ethylene oxide-,ethanol- or antibiotic–antimycotic treatment, on porous 3-D SF scaffolds. In terms of scaffold morphol-ogy, topography, crystallinity and short-term cell viability, the different sterilization methods showedonly few effects. Nevertheless, mechanical properties were significantly decreased by a factor of twoby all methods except for dry autoclaving, which seemed not to affect mechanical properties comparedto the native control group. These data suggest that SF scaffolds are in general highly resistant to varioussterilization treatments. Nevertheless, care should be taken if initial mechanical properties are of interest.

! 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

The development of sophisticated porous scaffolds for use intissue engineering applications requires the careful choice of scaf-fold properties, as the requirements for the materials are explicitfor the specific tissue to be regenerated. Scaffold morphology andtopology, crystallinity and scaffold stiffness are amongst theparameters that have been shown to influence the ability of cellsto attach, produce extracellular matrix and form a complex tissueconstruct [1]. Such porous three-dimensional (3-D) scaffolds usedin tissue engineering must also be safe for clinical use. Thus a ster-ilization process is used to inactivate potentially present microbialcontaminants. As this is usually achieved through denaturing con-ditions, disruption of the microorganisms’ enzyme system or thedestruction of their nucleic acid bases, sterilization methods areknown to cause alterations in biomolecules and may have seriouseffects on their performance [2]. This is even more important forscaffolds that are susceptible to degradation and/or morphologicaldegeneration by high temperature and pressure, leaving only a fewsterilization techniques suitable [3].

Harsh sterilization techniques have been shown to significantlyaffect polymeric and proteinous materials, especially those withlow melting points, complex architectures and hydrolytic degrada-tion mechanisms [4]. Polylactic acid, polyglycolic acid (PGA)and their copolymers, for example, have been shown to undergodeformation/degradation from attack by water when steamsterilized, and melting and softening after sterilization with dryheat [5]. Sterilization effects have also been thoroughly investi-gated for 3-D poly(lactide-co-glycolide) (PLGA) scaffolds [6]. Whilegamma-irradiation has been shown to be efficient in sterilizing andpreserving the 3-D morphology, it decreased the polymer molecu-lar weight by 54% and accelerated degradation over time [6]. Inpolycaprolactone foams, the elasticity of the scaffolds was shownto be a function of the irradiation dose [7]. Ethylene oxide (EO)treatment, on the other hand, was less damaging in terms ofmolecular weight loss, but had a profound effect on the scaffold’smorphology [6]. For collagen-based scaffolds, manifold steriliza-tion effects have been described, such as denaturation after steamsterilization, partial denaturation and crosslinking of collagenchains after sterilization with dry heat, modification of thesecondary structure of collagen through chemical inactivators,and crosslinking and chain scission processes or collapsed porosityafter beta- or gamma-irradiation [2,8,9].

Three-dimensional porous scaffolds fabricated from silk fibroin(SF) have previously been demonstrated to offer exceptional

1742-7061/$ - see front matter ! 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.08.035

⇑ Corresponding author. Present address: Department of Biomedical Engineering,Eindhoven University of Technology, GEM-Z 4.124, P.O. Box 513, 5600 MBEindhoven, The Netherlands. Tel.: +31 40 247 3494; fax: +31 40 247 3744.

E-mail addresses: sahofmann@ethz.ch, S.Hofmann.Boss@tue.nl (S. Hofmann).

Acta Biomaterialia 10 (2014) 308–317

Contents lists available at ScienceDirect

Acta Biomaterialia

journal homepage: www.elsevier .com/locate /ac tabiomat

benefits over conventional synthetic and natural biomaterials invarious tissue-engineering studies [10–18]. The effect of steriliza-tion was not investigated in all of these studies, and often theapplied sterilization method was not even described. To ourknowledge, there has been no systematic study to investigatehow sterilization methods affect porous 3-D SF scaffold properties,although they are widely used for tissue engineering applications.SF has been reported to be an environmentally stable protein incomparison to globular proteins after it has been conformationallytransformed with methanol from a predominantly random coilstructure into a water-insoluble b-sheet structure [19–22]. How-ever, virgin silk ophthalmic sutures are not recommended for reuseas they are weakened by thermal methods of sterilization and dis-infection, and appear to be strengthened by immersion in ethanolor glutaraldehyde [23].

Three-dimensional scaffolds need to be sterilized in a reproduc-ible and safe way. This study systematically determines for the firsttime the effects of various SF scaffold sterilization methods, com-prising autoclaving (of scaffolds in dry (autodry), humid (autohu-mid) or wet state (autowet)), sterilization with dry heat (dryheat), EO treatment (EO) or sterilization with chemicals such as70% ethanol (EtOH 70%) or antibiotic–antimycotic solution(P/S/F), in comparison to non-sterilized (not sterile) or asepticallyprepared (aseptic) scaffolds. The effect of these treatments on scaf-fold 3-D morphology, surface topography, crystallinity, mechanicalproperties and cell metabolic activity is investigated. The purposeof the study is to find an effective method for sterilizing porous3-D silk fibroin structures derived from a 1,1,1,3,3,3-hexafluoro-propan-2-ol solution by the sodium chloride salt leaching methodfor use as in vitro scaffolds for tissue engineering applications.

2. Materials and methods

2.1. Materials

Silkworm cocoons from Bombyx mori L. were kindly provided byTrudel Inc. (Zurich, Switzerland). Fetal bovine serum, Dulbecco’smodified Eagle’s medium (DMEM), basic fibroblast growth factor,antibiotic–antimycotic (consisting of 10,000 units ml!1 penicillin,10,000 units ml!1 streptomycin and 25 lg ml!1 amphotericin B),nonessential amino acids (consisting of 890 mg l!1L-alanine,1320 mg l!1L-asparagine, 1330 mg l!1L-aspartic acid, 1470 mg l!1

L-glutamic acid, 750 mg l!1 glycine, 1150 mg l!1L-proline,1050 mg l!1L-serine) and trypsin (0.05% with EDTA⁄4NA) werepurchased from Invitrogen (Basel, Switzerland). All other sub-stances were of analytical or pharmaceutical grade and obtainedfrom Sigma-Aldrich (Buchs, Switzerland).

2.2. Preparation of SF scaffolds

Three-dimensional SF scaffolds were prepared as described pre-viously using the salt leaching method [17,21,24]. In brief, cocoonswere boiled twice for 1 h in an aqueous solution of 0.02 M Na2CO3

solution and rinsed with water to remove sericin. Purified SF wasdissolved in an aqueous 9 M LiBr solution at 55 "C and dialyzedagainst water for 2.5 days (Pierce, MWCO 3500 g mol!1, obtainedfrom Fisher Scientific AG, Wohlen, Switzerland). SF was frozen at!80 "C overnight, lyophilized and redissolved in 1,1,1,3,3,3-hexa-fluoropropan-2-ol (HFIP; ABCR-Chemicals, Karlsruhe, Germany).Sodium chloride crystals with diameters in the range of 300–400 lm were weighed in a Teflon container and SF/HFIP solutionwas added at a ratio of 20:1 (NaCl:SF). HFIP was allowed to evap-orate and the blocks were treated in a 90% methanol solution for30 min to induce a conformational transition to b-sheet domains[17]. Blocks were allowed to dry in a chemical cabinet at room

temperature for at least 3 days before sodium chloride was re-moved by leaching out in deionized water. Disk-shaped scaffolds5 mm in diameter and 1–2 mm in thickness were prepared usinga razor blade and dermal punch.

2.3. Sterilization methods

Scaffolds were left untreated, prepared aseptically in a laminarflow cabinet after the methanol treatment step or sterilizedaccording to established protocols. An overview of the differentsterilization methods used can be found in Table 1.

2.3.1. Steam autoclaveScaffolds were packed in glass Petri dishes in either a dry state

(autodry) or after being humidified in water for 10 min (autohu-mid). A third batch of scaffolds was put in Schott glass bottles filledwith 25 ml of phosphate-buffered saline (PBS; autowet). Steamsterilization was performed at 121 "C and 1 bar for 15 min in aVarioklav 65T (H+P Labortechnik AG, Oberschleissheim, Germany).

2.3.2. Sterilization in dry heatDry scaffolds were placed in a glass Petri dish and heat steril-

ized at 180 "C for 30 min in a drying oven (Binder, Tuttlingen,Germany).

2.3.3. Sterilization with EODry scaffolds were sterilized with EO for 4 h at 55 "C followed

by overnight aeration to remove residual EO. Before seeding withcells, scaffolds were extensively rinsed in PBS.

2.3.4. Sterilization by immersion in disinfecting reagentsIn a laminar flow cabinet, scaffolds were incubated in either 70%

ethanol (EtOH 70%) or a solution of 1 vol.% P/S/F (10,000 units ml!1

penicillin G, 10,000 units ml!1 streptomycin sulphate and25 lg ml!1 amphotericin B, obtained from Invitrogen, Basel,Switzerland) in PBS for 30 min at room temperature. Samples werethen rinsed three times with PBS before further use.

2.4. Test for sterility

Following sterilization, samples (n = 5 per method) were testedfor sterility. In a sterile environment, scaffolds of each group wereimmersed into 30 ml of soybean casein digest broth for cultivationof fastidious microorganisms and were incubated at 37 "C for

Table 1Overview of the various sterilization methods.

Group Sterilization method Abbreviation Settings

A. Scaffold beforesterilization

Not sterile –

B. Aseptic preparation Aseptic –C. Autoclaving in a dry

stateAutodry Dry scaffolds, 121 "C,

1 bar, 15 minD. Autoclaving in a humid

stateAutohumid Humid scaffolds, 121 "C,

1 bar, 15 minE. Autoclaving in a wet

stateAutowet Scaffolds immersed in

H2O, 121 "C, 1 bar, 15 minF. Dry heat Dry heat 180 "C, 30 minG. Ethylene oxide EO 55 "C, 4 h, then overnight

aerationH. Ethanol 70% EtOH 30 min, three rinsings in

PBSI. Antibiotic–antimycotic P/S/F 30 min, three rinsings in

PBSJ. SF before conversion to

water insoluble form– Raw silk fibroin, no

scaffold

S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317 309

5 days. Samples were checked periodically for signs of infection, aswould be indicated by increased opacity of the medium. Mediawithout scaffolds and non-sterilized scaffolds were used ascontrols.

2.5. Characterization of scaffolds

2.5.1. Scaffold morphologyScaffold morphology before and after the different sterilization

procedures was assessed by a microcomputed tomography (lCT)imaging system (lCT40, Scanco Medical AG, Brüttisellen,Switzerland). Samples were scanned dry in air at an energy of40 kVp, an intensity of 180 lA and an integration time of 200 ms,with a nominal (isotropic) resolution of 6 lm. Four times frameaveraging was applied. The measured data was filtered using a3-D constrained Gaussian filter with finite filter support (1 voxel)and filter width (r = 0.8). A global threshold was applied at 4.5%of the maximum grayscale value to separate the scaffold frombackground and thus to binarize the image. Disconnected partswere removed by a component labeling algorithm. The resulting

3-D volumes were evaluated morphometrically for scaffoldporosity, scaffold surface density and pore size distribution, asdescribed previously [25,26].

2.5.2. Scaffold topographyDry scaffolds were sputter coated with platinum and their sur-

face topography was assessed with scanning electron microscopy(SEM; Zeiss Leo Gemini 1530, Cambridge, UK).

2.5.3. SF conformationApproximately 5 mg of each scaffold was ground with a mortar

and pestle, mixed with 300 mg of KBr and pressed into a pellet.Spectra from 1800 to 800 cm!1 were collected under a vacuumusing a Bruker IFS 66 V spectrometer (Bruker Optik GmbH,Ettlingen, Germany). Background measurements were taken twicewith an empty cell and subtracted from the sample readings.

2.5.4. Mechanical propertiesScaffolds were prewetted in PBS at room temperature for at

least 2 h, placed in a custom-built, confined, sample holder and

A. not sterile B. aseptic C. autodry

F. dry heat E. autowet D. autohumid

I. P/S/F H. EtOH 70% G. EO

0 m 550 m

Fig. 1. Typical lCT images of SF scaffolds for various sterilization methods. The top image displays the scaffold, the bottom images show a color-coded visualization of thepore diameters of each respective scaffold. (A) Before sterilization, (B) aseptically prepared, (C) autoclaved in a dry state, (D) autoclaved in a humid state, (E) autoclaved in awet state, (F) sterilized with dry heat, (G) sterilized with ethylene oxide, (H) sterilized by immersion in 70% EtOH and (I) sterilized by immersion in P/S/F.

310 S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317

covered with PBS. Testing was performed according to a previouslypublished, modified protocol [27] on a commercial materials test-ing machine (Zwick Z005, Ulm, Germany) equipped with a 10 Nload cell and a cylindrical, plane-ended, stainless steel indentertip with a diameter of 0.9 mm. A small preload of 1–5 mN was ap-plied to determine sample thickness. Stepwise stress–relaxationindentation of the measured specimen thickness was carried outin five strain steps (of 5, 10, 15, 20 and 25%). After each indentationstep, the relaxation response to equilibrium was measured for60 min. The equilibrium modulus, instantaneous modulus andmaximum stress were determined from the resulting data.

2.5.5. Cell cultureSterilized scaffolds were immersed in PBS for 2 h to allow com-

plete humidification. The scaffolds were then removed from thePBS and distributed into the wells of a 24-well culture plate. Next,1 " 106 human mesenchymal stem cells (hMSCS) suspended in20 ll of culture medium (DMEM supplemented with 10% fetal bo-vine serum and 1% antibiotic/antimycotic solution) were pipettedon top of each scaffold and inoculated at 37 "C and 5% CO2. After15 min, an additional 20 ll drop of culture medium was added inorder to keep the cells wet. After 30 min, scaffold–cell constructswere placed in new wells of a 24-well culture plate and 2 ml of cul-ture medium was added to each well. This culture medium was re-placed every other day. Cell metabolic activity was assessed after24 h or 7 days (alamarBlue# Assay, Invitrogen, Basel, Switzerland),according to the manufacturer’s protocols.

2.6. Statistical analysis

Statistical analysis was performed using R statistical software(www.r-project.org). A nonparametric Kruskal–Wallis test was ap-plied to investigate whether the sterilization method had an influ-ence on the scaffold morphological parameters, the mechanicalproperties or the cell metabolic activity. p-values < 0.05 were con-sidered significant. If significant, pairwise comparisons were madeusing Mann–Whitney tests with Bonferroni correction.

3. Results

3.1. Sterilization

All of the applied methods resulted in sterility of the 3-D scaf-folds since all the applied media stayed clear. No growth of fungior bacteria could be observed either in the test for sterility or after1 week of culture with seeded hMSCs (data not shown).

3.2. Scaffold morphology and topography

lCT has previously been shown to be a powerful tool for char-acterizing scaffold 3-D architecture, comparing scaffold productiontechniques and assessing the effects of production parameters. Noobvious differences in scaffold morphology could be detected afterscaffold sterilization (Fig. 1). All scaffolds showed a highly irregularpore system, with a macroporosity of >80%. Pore size distributionplots (Fig. 2) showed that the average pore diameter was close to200 lm and therefore much smaller than the porogen diameter(300–400 lm). As these parameters were assessed in the scaffold’sdry state, this difference could be attributed to scaffold shrinkageduring the drying process. Only scaffolds autoclaved in a humidstate (Fig. 2E) seemed to be influenced by the sterilization process,indicated by a non-significant broadening of the pore size distribu-tion and a shift towards a smaller average pore diameter. AKruskal–Wallis test confirmed that scaffold porosity was influ-enced by the sterilization method (p < 0.05). Pairwise post hoc

comparison with the Mann–Whitney test revealed that scaffoldsautoclaved in a humid state showed significantly decreased poros-ity as compared to unsterilized scaffolds (p < 0.05) or any othersterilization method (p < 0.05 for each comparison) except sterili-zation with P/S/F (p = 0.15) (Table 2). The mean scaffold pore diam-eter and scaffold surface density showed no significant differencesbefore or after sterilization (Table 2). In general, most sterilizationmethods showed a tendency towards an increased variance com-pared to unsterilized scaffolds. This trend was most prominent in

A. controlB. asepticC. autodryD. autohumid

I. P/S/FH. EtOH

F. dry heatG. EO

E. autowet

0 600500400300200100

Pore diameter [µm]

Freq

uenc

y of

occ

uren

ce

Fig. 2. Average pore size distribution of SF scaffolds for various sterilizationmethods (n = 5). The y-axis displays arbitrary units, the x-axis shows the scaffoldpore diameter (in lm). (A) Before sterilization, (B) aseptically prepared, (C)autoclaved in a dry state, (D) autoclaved in a humid state, (E) autoclaved in a wetstate, (F) sterilized with dry heat, (G) sterilized with ethylene oxide, (H) sterilizedby immersion in 70% EtOH and (I) sterilized by immersion in P/S/F.

Table 2Quantitative 3-D evaluation of scaffold morphological parameters for varioussterilization methods (n = 5 per group).

Sterilizationmethod

Scaffold porosity(%)

Mean porediameter (lm)

Scaffold surfacedensity (mm!1)

Average SD(CV%)

Average SD(CV%)

Average SD(CV%)

A. Not sterile 90 1 (0.8) 176.8 4 (2.2) 130.1 11(8.0)

B. Aseptic 89 1 (1.3) 179.4 12(6.9)

122.1 12(10.0)

C. Autodry 89 2 (2.2) 184.6 15(7.9)

126.9 22(17.0)

D. Autohumid 84* 1 (0.6) 139.6 18(12.6)

124.0 15(12.0)

E. Autowet 90 1 (0.7) 171.8 13(7.3)

138.6 9 (6.0)

F. Dry heat 89 3 (3.5) 169.2 17(10.0)

137.8 36(26.0)

G. EO 90 0.5(0.5)

158.6 20(12.8)

151.9 17(11.0)

H. EtOH 90 2 (1.7) 165.8 20(11.9)

150.9 19(13.0)

I. P/S/F 88 4 (4.7) 172.8 36(20.8)

128.9 40(31.0)

Data are expressed as average, standard deviation (SD) and coefficient of variation(CV%).* The porosity of scaffolds autoclaved in a humid environment was significantlylower than the porosity of scaffolds sterilized by any other method (p < 0.05) exceptsterilization with P/S/F.

S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317 311

samples sterilized through dry heat and P/S/F, but EtOH treatmentand autoclaving in a dry state also appeared to influence variancefor at least two of the assessed parameters.

The surface topography of the scaffolds before and after the dif-ferent sterilization procedures was assessed by SEM (Fig. 3A–I).The images confirmed the high porosity and pore interconnectivityof the scaffolds. A detailed view of the surface topography showedthat the surface was not smooth but rather patchy, with many cav-ities of diameters between 1 and 10 lm. No influence from thesterilization methods was obvious.

3.3. SF conformation

To investigate whether the various sterilization methods in-duced a conformational change in SF, Fourier-transform infraredspectroscopy (FTIR) analysis was performed (Fig. 4). It has beenshown that MeOH treatment results in a conformational shift ofthe SF that renders it water-insoluble. This water-insolubility isachieved through the transition from a predominantly random coilor silk I structure into a water-insoluble b-sheet-enriched structure[28]. All MeOH-treated scaffolds showed the typical intensity shiftin the amide II region from #1540 to #1520 cm!1, indicating anN–H bending vibration bond. The peaks and shoulders in theunsterilized sample (Fig. 4A) seemed to be slightly more pro-nounced than in all of the treated samples, indicating that, what-ever sterilization treatment was used, only small further changesare induced. As expected, the amide I intensity peak shift from#1660 to #1630 cm!1 after MeOH treatment was most prominenton the heat-treated samples (Fig. 4C–F) when compared with thosesamples that had not been sterilized with heat.

3.4. Mechanical properties

Stepwise stress–relaxation indentation testing was performedto determine whether the sterilization process had an influenceon the mechanical properties of the scaffolds under a compressiveload (Fig. 5). Specimens reached equilibrium during the relaxationphase of the test setup (data not shown). The sterilization method

had a significant influence on the equilibrium modulus (p = 0.009,Fig. 5A), instantaneous modulus (p = 0.032, Fig. 5B) and maximumstress (p = 0.007, Fig. 5C). For the equilibrium and instantaneousmoduli, most sterilization methods resulted in a non-significantreduction of these parameters compared to the non-sterilized con-trol, the significant effect mainly being caused by the difference be-tween some groups compared to samples autoclaved in a dry state.Scaffolds autoclaved in a wet state and prepared asepticallyshowed a significant reduction in maximum stress in comparisonto the controls. Notably, except for the autoclaving of scaffolds ina dry state, all methods resulted in a decrease in sample variationcompared to the control. As expected, scaffold thickness was notsignificantly influenced by the sterilization method (Fig. 5D).

3.5. Cell metabolic viability

Cell metabolic activity was not significantly influenced by thesterilization method (Fig. 6). The total cell metabolic activity in-creased in all groups from 1 to 7 days after seeding, which mightbe attributed to a higher cell number or higher cell metabolic activ-ity. Scaffolds sterilized with EO, EtOH or P/S/F, where potentialtoxic remnants might have impaired cell metabolic activity,showed the highest variances after 7 days of culture – a potentialindicator that the washing steps applied may have beeninsufficient.

4. Discussion

Biodegradable porous scaffolds are widely used in tissue engi-neering to provide a structural template for cell seeding and extra-cellular matrix formation. These scaffolds must often possesssufficient structural integrity to temporarily withstand functionalloading or cell traction forces in vitro and in vivo. Physical featuresof a substrate that may affect cell responses include the crystallin-ity, morphology and compliance of the material, whereas chemicalproperties are based on surface energy and specific protein motifs.Topographical cues are also imparted by the size features and

B. aseptic C. autodry

G. EO H. EtOH 70% I. P/S/F

D. autohumid E. autowet F. dry heat

A. not sterile100 µm

10 µm

Fig. 3. SEM images of SF scaffolds for various sterilization methods. (A) Before sterilization, (B) aseptically prepared, (C) autoclaved in a dry state, (D) autoclaved in a humidstate, (E) autoclaved in a wet state, (F) sterilized with dry heat, (G) sterilized with ethylene oxide, (H) sterilized by immersion in 70% EtOH and (I) sterilized by immersion inP/S/F. Scale bar: 100 lm; scale bar insets: 10 lm.

312 S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317

periodicity of the substratum [29]. In order to be used clinically,sterility is required for every material or device, and the efficacyof the sterilization procedure must be confirmed [30]. Sterilizationmethods may adversely affect material properties, and any suchproperties must be fully characterized and accounted for in themanufacturing process if scaffolds cannot be fabricated aseptically.Such an effect must not only be negative, if its magnitude is knownin detail and predictable, but could also be used to tailor scaffoldproperties for a specific need. Knowledge and quantification of ascaffold’s structure–function relationships is critical for simulatingand predicting its mechanical and biological performance.

As expected, all methods applied in this study resulted in scaf-fold sterilization. The local microarchitecture was quantified withlCT, as shown previously [25,31,32], as it affects both the mechan-ical and biological properties of porous 3-D scaffolds [33]. Scaffoldsdisplayed a high porosity of between 84 and 90% for all scaffoldsand a large pore diameter distribution with mean values smallerthan the used porogen. The second point might be attributed totwo effects: (i) shrinking during the drying process as the scaffolds

had to be measured in their dry state in air in order to provide en-ough contrast and (ii) limited solubilization of the surface of thesodium chloride crystals during supersaturation of the SF solutionprior to solidification [34]. The morphometric parameters wereequal in all groups after all sterilization methods, with the excep-tion of scaffolds that had been sterilized in a humid environment,which showed a slightly reduced porosity (Table 2) and a tendencytowards a smaller pore size distribution (Fig. 2).

Musculoskeletal cell interaction with the substratum topogra-phy appears to be a multifaceted, multiscale response [35,36].Nanometer-sized pits and islands have been shown to affect cellmorphology, proliferation and alignment [36]. Micron-sizedgrooves on substrate surfaces have also been studied and shownto affect musculoskeletal cell behavior. In contrast to other com-monly used scaffold materials such as Tecoflex# SG-80A polyure-thane [37] and PLGA [4], SF scaffolds showed no signs of changein surface topography, such as reduced porosity or increased sur-face wrinkling, or other signs of deformation after sterilizing byany of the applied methods.

Similarly to surface topography, it is suggested that surfacechemistry and energy play important roles in musculoskeletal cellmechanosensing, differentiation and phenotype maintenance [5].The production of SF scaffolds includes methanol treatment thatrenders the silk insoluble in aqueous solution [21,28]. Through thisprocess, b-sheets are rearranged so that the methyl groups andhydrogen groups of opposing sheets interact to form the inter-sheet stacking in the crystals [38]. The SF crystalline structureand degree of crystallinity were not affected by the sterilizationprocess in any of the applied sterilization methods. Therefore itcan be expected that there was no additional effect on surfacechemistry. Control over the rate of degradation is an important fea-ture of functional tissue design, such that the rate of scaffold deg-radation matches the rate of tissue growth [39]. As the in vitrodegradability of SF biomaterials has been related to the mode ofprocessing and the corresponding content of b-sheets [19,38], itcan be deduced that this parameter was not highly affected bythe sterilization method.

The sterilization processes had an influence on the maximumstress and the equilibrium modulus, as well as on the instanta-neous modulus, with a tendency for all sterilization methods ex-cept for autoclaving in a dry state to reduce the mechanicalproperties when compared to the control.

A particular physical environment, such as the scaffold’s 3-Dstructure, its surface or mechanical properties, can be cell instruc-tive as well as potentially contribute towards the physical functionof the tissue [1,33,40,41]. Short-term in vitro assessment of theability of hMSCs to attach and remain metabolically active on SFscaffolds showed that all the sterilization methods applied enabledcell attachment and survival over 7 days in culture and confirmedthe minimal effects of the sterilization methods, as seen with theother parameters investigated in this study (Fig. 6).

An interesting finding was that the decrease in mechanicalproperties compared to the unsterilized control corresponded toa decrease in sample variability (Fig. 5), a trend which was some-what inverted in the scaffold morphological parameters, wheremethods including treatment with heat and sterilization withchemicals (EtOH and P/S/F) showed a tendency towards highervariability than the unsterilized control (Table 2). Different molec-ular weight distributions of the SF molecules before and after ster-ilization might be a possible explanation for this phenomenon.

This study has a number of limitations. A number of steriliza-tion techniques were not investigated in this paper, includinggamma-irradiation, which has been shown to cause substantialdegradation of polyester chains with increasing dosages of radia-tion. For example, at the standard 2.5 Mrad sterilization dose,considerable damage was observed in PGA sutures [42].

B. aseptic

1800 1600 1400 1200 8001000

C. autodry

D. autohumid

E. autowet

F. dry heat

H. EtOH 70%

I. P/S/F

J. Silk fibroin

G. ethylenoxid

A. not sterile

Wavenumber [cm-1]

Amide I Amide IIAmide III

Fig. 4. FTIR spectra of SF scaffolds for various sterilization methods. (A) Beforesterilization, (B) aseptically prepared, (C) autoclaved in a dry state, (D) autoclaved ina humid state, (E) autoclaved in a wet state, (F) sterilized with dry heat, (G)sterilized with ethylene oxide, (H) sterilized by immersion in 70% EtOH, (I)sterilized by immersion in P/S/F and (J) SF without MeOH treatment.

S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317 313

Gamma-irradiation also showed certain limitations in sterilizingdemineralized bone matrices, such as impaired stem cell andosteoprecursor proliferation rates, and is only suggested if thereis no alternative available [43]. Additionally, this method is notas easily accessible in laboratories as the herein described meth-ods. Sterilization with ultraviolet light is another method, but, asit is only a surface treatment, it is more suitable for electrospunscaffolds than for large, 3-D volumes [30]. Another limitation con-cerns the characterization of the bulk material. One batch of SFwas used to manufacture all of the scaffolds used in this study –therefore the results do not take potential batch-to-batch varia-

tions into account. Moreover, Yamada and colleagues [44] haveshown that even simple changes in SF processing, such as varyingthe boiling time, can degrade the fibroin molecule, resulting in pro-tein fractions of lower molecular weight. Such degradation mightbe an explanation for observed differences such as those in themechanical properties. It is therefore also important to note thatour study can only show a general trend, and variations may occurby using a different batch of SF or changing the parameters of theprocessing (including sterilization) protocol. This becomes evenmore important as changes in the surface chemistry were notassessed, though they may well influence cellular behavior. Such

Fig. 5. Equilibrium modulus (A), instantaneous modulus (B), maximum stress (C) and scaffold thickness (D) of SF scaffolds for various sterilization methods. Sterilizationmethods applied: unsterilized control, aseptical preparation, autoclaving in a dry state, autoclaving in a humid state, autoclaving in a wet state, sterilization with dry heat,sterilization with EO or sterilization by immersion in either 70% EtOH or P/S/F.

314 S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317

effects are highly dependent on the cell type used and the culturingconditions, and therefore need to be assessed for each plannedapplication individually; they could not be systematically checkedhere. Cell studies have been limited to short-term cell metabolicactivity assessment, as all other investigations depend highly onthe intended application and the cell type used. It should also benoted that all the conclusions in this article are valid only forHFIP-derived SF scaffolds prepared by the sodium chloride saltleaching method described herein; scaffolds prepared by othermethods might well show different results.

For any tissue engineering application with SF scaffolds,researchers need to decide a priori on the appropriate sterilization.Aseptic preparation is tedious and time consuming, and offers noobvious advantage over the other sterilization methods. Chemicalsterilization by EO gas is a widely used method in which neitherhigh heat nor moisture is used, and very little degradation occursin a variety of degradable polymers [6,45,46]. However, as with li-quid chemical sterilization, the process can leave residues in harm-ful quantities on the surface or within the scaffold, and additionallycan alkylate proteins. Ethanol at concentrations of 60–80 vol.% hasbecome a favorite and widely used method for in vitro studies, dueto its ease of use and apparent effectiveness [6]. However, it is con-sidered to be a chemical disinfectant rather than a sterilizationagent because of its inability to destroy hydrophilic viruses orbacterial spores, so it cannot be used for in vivo applications ofbiomedical devices. While ethanol treatment is an inappropriatesterilization technique with respect to biocompatibility, it affectedneither the scaffold’s morphology nor the conformational ormechanical properties, and could serve as a control against whichthe other sterilization techniques can be compared. Similarly,antibiotic–antimycotic treatment is not a standard sterilizationprocedure and no official treatment duration has been reported.In our study, a duration of 30 min was enough to provide sterilityof up to 40 mm3 scaffolds, while other studies recommend dura-tions of 6–24 h [4]. Shearer et al. [4] even recomend this treatmentfor the sterilization of hollow fiber PLGA scaffolds, as it had theleast effect on the scaffold structure and mechanical properties ofthe techniques they investigated. As it has been shown that

gentamycin negatively affects human mesenchymal stem cell pro-liferation and differentiation, the influence of potential remnantsof antibiotic–antimycotic solution in the scaffolds would also needfurther investigation [47]. It is well known that aliphatic polyestersare sensitive to both moisture and heat, which makes it difficult touse dry heat sterilization and steam sterilization. In both cases, be-sides the degrading effects, the porous structures were destroyedas a consequence of plastic deformation from the heat treatment[48]. A recent study on recombinantly produced miniature spidersilk protein fibers suggested that the known stability of silk mightbe an advantage in this regard too [49]. Our study confirms that3-D scaffolds made of SF from regenerated B. mori L. cocoons canstructurally withstand the heat treatment experienced with heatsterilization or autoclaving. The final selection of a suitable sterili-zation method depends on the scaffold material, the scaffoldingtechnique and the final intended function. For musculoskeletal tis-sue engineering, we recommend autoclaving of dry SF scaffolds, asthis is a simple, reliable sterilization method that is likely to haveminimal detrimental effects on the scaffold. This was the onlygroup that kept the natural variance of the samples that stemsfrom the production process of the scaffolds. All other techniquesreduced this variability, though at the cost of much lower mechan-ical properties. Cell studies are usually influenced by many param-eters and produce results with large variations, leading to evenworse reproducibility. In the future it would be advisable to createnative samples with lower variation in order to have more repro-ducible results.

The importance of the chemical and physical features of a scaf-fold on the determination of cell fate is well known [1,35,41].Knowledge of the effects of sterilization allows incorporation ofthese effects into future scaffold design and should be consideredwhen choosing a certain scaffold material [4]. Whilst these altera-tions are generally viewed as being detrimental to the final productin terms of bulk and/or surface properties, they may also give riseto potentially beneficial changes if the quantitative extent of theeffect is known in advance. The small changes in structure andproperties by treatment with the different sterilization methodsapplied in this study will probably have little effect on the

A B Cell metabolic activity 1 day after seeding Cell metabolic activity 7 days after seeding Fl

uore

scen

ce

Fluo

resc

ence

0

5000

10000

15000

0

5000

10000

15000

Fig. 6. Cell metabolic activity at (A) 1 or (B) 7 days after seeding hMSCs on sterilized SF scaffolds. Sterilization methods applied: aseptical preparation, autoclaving in a drystate, autoclaving in a humid state, autoclaving in a wet state, sterilization with dry heat, sterilization with EO or sterilization by immersion in either 70% EtOH or P/S/F.

S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317 315

suitability of porous SF scaffolds as scaffolds for tissue engineering,but most of them lower the scaffold’s mechanical properties, aparameter usually stressed as one of the biggest advantages ofSF. A big challenge will be to find protocols to produce SF scaffoldswith more precise geometries and increased reproducibility, in or-der to draw conclusions on the effects of morphological parame-ters on cell behavior in future tissue engineering studies.

5. Conclusion and outlook

The objective of this study was to determine the optimal steril-ization technique causing minimum damage to the delicate 3-D SFscaffold morphology, surface topology, crystallinity and mechani-cal properties, each of them being factors that have been shownto be critical for successful tissue engineering. It is hoped that iden-tification of the mechanisms governing cell interactions with theseparameters will allow rule-based design of biomaterial scaffolds toengineer specific cellular responses for a desired application in thefuture. While the presented data show that most sterilizationmethods have a slight effect on some of the parameters investi-gated, hMSCs viability was not significantly affected and no steril-ization method was clearly inappropriate. Especially when thescaffold’s mechanical properties are of importance, we recommendautoclaving of the scaffolds in a dry state as this method is conve-nient and does not affect or even lower the scaffold’s initialmechanical properties. The effect of the sterilization method oncell differentiation and extracellular matrix production needs tobe determined separately for every cell type and tissueinvestigation.

5. Disclosures

The authors confirm that there are no known conflicts of inter-est associated with this publication and there has been no signifi-cant financial support for this work that could have influenced itsoutcome.

Acknowledgements

The authors gratefully acknowledge financial support from theRMS Foundation (Bettlach, Switzerland) and the provision of SFcocoons from Trudel Silk Inc. (Zurich, Switzerland). They thankAlexandra Krause and Colin Firminger for assistance in theexperimental work.

Appendix A. Figures with essential color discrimination

Certain figures in this article, particularly Fig. 1 is difficult tointerpret in black and white. The full color images can befound in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013.08.035.

References

[1] Place ES, Evans ND, Stevens MM. Complexity in biomaterials for tissueengineering. Nat Mater 2009;8:457–70.

[2] Wiegand C, Abel M, Ruth P, Wilhelms T, Schulze D, Norgauer J, et al. Effect ofthe sterilization method on the performance of collagen type I on chronicwound parameters in vitro. J Biomed Mater Res B Appl Biomater2009;90:710–9.

[3] Holy CE, Fialkov JA, Davies JE, Shoichet MS. Use of a biomimetic strategy toengineer bone. J Biomed Mater Res 2003;65A:447–53.

[4] Shearer H, Ellis MJ, Perera SP, Chaudhuri JB. Effects of common sterilizationmethods on the structure and properties of poly(D, L lactic-co-glycolic acid)scaffolds. Tissue Eng 2006;12:2717–27.

[5] Athanasiou KA, Niederauer GG, Agrawal CM. Sterilization, toxicity,biocompatibility and clinical applications of polylactic acid/polyglycolic acidcopolymers. Biomaterials 1996;17:93–102.

[6] Holy CE, Cheng C, Davies JE, Shoichet MS. Optimizing the sterilization of PLGAscaffolds for use in tissue engineering. Biomaterials 2001;22:25–31.

[7] Olah L, Filipczak K, Czvikovszky T, Czigany T, Borbas L. Changes of porouspoly(epsilon-caprolactone) bone grafts resulted from e-beam sterilizationprocess. Radiat Phys Chem 2007;76:1430–4.

[8] Noah EM, Chen J, Jiao X, Heschel I, Pallua N. Impact of sterilization on theporous design and cell behavior in collagen sponges prepared for tissueengineering. Biomaterials 2002;23:2855–61.

[9] Doillon CJ, Drouin R, Cote MF, Dallaire N, Pageau JF, Laroche G. Chemicalinactivators as sterilization agents for bovine collagen materials. J BiomedMater Res 1997;37:212–21.

[10] Altman GH, Horan RL, Lu HH, Moreau J, Martin I, Richmond JC, et al. Silk matrixfor tissue engineered anterior cruciate ligaments. Biomaterials2002;23:4131–41.

[11] Karageorgiou V, Meinel L, Hofmann S, Malhotra A, Volloch V, Kaplan D. Bonemorphogenetic protein-2 decorated silk fibroin films induce osteogenicdifferentiation of human bone marrow stromal cells. J Biomed Mater Res A2004;71:528–37.

[12] Kim HJ, Kim UJ, Leisk GG, Bayan C, Georgakoudi I, Kaplan DL. Boneregeneration on macroporous aqueous-derived silk 3-D scaffolds. MacromolBiosci 2007;7:643–55.

[13] Kim KH, Jeong L, Park HN, Shin SY, Park WH, Lee SC, et al. Biological efficacy ofsilk fibroin nanofiber membranes for guided bone regeneration. J Biotechnol2005;120:327–39.

[14] Meinel L, Hofmann S, Betz O, Fajardo R, Merkle HP, Langer R, et al.Osteogenesis by human mesenchymal stem cells cultured on silkbiomaterials: comparison of adenovirus mediated gene transfer and proteindelivery of BMP-2. Biomaterials 2006;27:4993–5002.

[15] Meinel L, Karageorgiou V, Hofmann S, Fajardo R, Snyder B, Li C, et al.Engineering bone-like tissue in vitro using human bone marrow stem cells andsilk scaffolds. J Biomed Mater Res A 2004;71:25–34.

[16] Perez-Rigueiro J, Viney C, Llorca J, Elices M. Silkworm silk as an engineeringmaterial. J Appl Polym Sci 1998;70:2439–77.

[17] Sofia S, McCarthy MB, Gronowicz G, Kaplan DL. Functionalized silk-basedbiomaterials for bone formation. J Biomed Mater Res 2001;54:139–48.

[18] Wang Y, Kim HJ, Vunjak-Novakovic G, Kaplan DL. Stem cell-based tissueengineering with silk biomaterials. Biomaterials 2006;27:6064–82.

[19] Minoura N, Tsukada M, Nagura M. Physico-chemical properties of silk fibroinmembrane as a biomaterial. Biomaterials 1990;11:430–4.

[20] Jin HJ, Chen J, Karageorgiou V, Altman GH, Kaplan DL. Human bone marrowstromal cell responses on electrospun silk fibroin mats. Biomaterials2004;25:1039–47.

[21] Nazarov R, Jin HJ, Kaplan DL. Porous 3-D scaffolds from regenerated silkfibroin. Biomacromolecules 2004;5:718–26.

[22] Altman GH, Diaz F, Jakuba C, Calabro T, Horan RL, Chen J, et al. Silk-basedbiomaterials. Biomaterials 2003;24:401–16.

[23] Shuttleworth GN, Vaughn LF, Hoh HB. Material properties of ophthalmicsutures after sterilization and disinfection. J Cataract Refract Surg1999;25:1270–4.

[24] Hofmann S, Hagenmüller H, Koch AM, Müller R, Vunjak-Novakovic G, KaplanDL, et al. Control of in vitro tissue-engineered bone-like structures usinghuman mesenchymal stem cells and porous silk scaffolds. Biomaterials2007;28:1152–62.

[25] Hildebrand T, Laib A, Müller R, Dequeker J, Rüegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone:microstructural data from spine, femur, iliac crest, and calcaneus. J BoneMiner Res 1999;14:1167–74.

[26] van Lenthe GH, Hagenmuller H, Bohner M, Hollister SJ, Meinel L, Muller R.Nondestructive micro-computed tomography for biological imaging andquantification of scaffold-bone interaction in vivo. Biomaterials2007;28:2479–90.

[27] Stok KS, Lisignoli G, Cristino S, Facchini A, Müller R. Mechano-functionalassessment of human mesenchymal stem cells grown in three-dimensionalhyaluronan-based scaffolds for cartilage tissue engineering. J Biomed MaterRes A 2010;93:37–45.

[28] Wilson D, Valluzzi R, Kaplan D. Conformational transitions in model silkpeptides. Biophys J 2000;78:2690–701.

[29] McCullen SD, Haslauer CM, Loboa EG. Musculoskeletal mechanobiology:interpretation by external force and engineered substratum. J Biomech2010;43:119–27.

[30] Rainer A, Centola M, Spadaccio C, Gherardi G, Genovese JA, Licoccia S, et al.Comparative study of different techniques for the sterilization of poly-L-lactideelectrospun microfibers: effectiveness vs. material degradation. Int J ArtifOrgans 2010;33:76–85.

[31] Müller R, Van Campenhout H, Van Damme B, Van Der Perre G, Dequeker J,Hildebrand T, et al. Morphometric analysis of human bone biopsies: aquantitative structural comparison of histological sections and micro-computed tomography. Bone 1998;23:59–66.

[32] Rüegsegger P, Koller B, Müller R. A microtomographic system for thenondestructive evaluation of bone architecture. Calcif Tissue Int1996;58:24–9.

[33] Lin AS, Barrows TH, Cartmell SH, Guldberg RE. Microarchitectural andmechanical characterization of oriented porous polymer scaffolds.Biomaterials 2003;24:481–9.

[34] Kim UJ, Park J, Kim HJ, Wada M, Kaplan DL. Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin. Biomaterials 2005;26:2775–85.

316 S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317

[35] Curtis AS, Dalby M, Gadegaard N. Cell signaling arising from nanotopography:implications for nanomedical devices. Nanomedicine (Lond) 2006;1:67–72.

[36] Stevens MM, George JH. Exploring and engineering the cell surface interface.Science 2005;310:1135–8.

[37] Andrews KD, Hunt JA, Black RA. Effects of sterilisation method on surfacetopography and in vitro cell behaviour of electrostatically spun scaffolds.Biomaterials 2007;28:1014–26.

[38] Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci 2007;32:991–1007.[39] Langer R, Vacanti JP, Vacanti CA, Atala A, Freed LE, Vunjak-Novakovic G. Tissue

engineering: biomedical applications. Tissue Eng 1995;1:151–61.[40] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage.

Biomaterials 2000;21:2529–43.[41] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell

lineage specification. Cell 2006;126:677–89.[42] Chu CC, Williams DF. The effect of gamma irradiation on the enzymatic

degradation of polyglycolic acid absorbable sutures. J Biomed Mater Res1983;17:1029–40.

[43] Han B, Yang Z, Nimni M. Effects of gamma irradiation on osteoinductionassociated with demineralized bone matrix. J Orthop Res 2008;26:75–82.

[44] Yamada H, Nakao H, Takasu Y, Tsubouchi K. Preparation of undegraded nativemolecular fibroin solution from silkworm cocoons. Mater Sci Eng C2001;14:41–6.

[45] Hooper KA, Cox JD, Kohn J. Comparison of the effect of ethylene oxide andgamma-irradiation on selected tyrosine-derived polycarbonates and poly(L-lactic acid). J Appl Polym Sci 1997;63:1499–510.

[46] Weir NA, Buchanan FJ, Orr JF, Farrar DF. Influence of processing andsterilisation on properties of poly-epsilon-caprolactone. Plast RubberCompos 2003;32:265–70.

[47] Chang Y, Goldberg VM, Caplan AI. Toxic effects of gentamicin on marrow-derived human mesenchymal stem cells. Clin Orthop Relat Res2006;452:242–9.

[48] Plikk P, Odelius K, Hakkarainen M, Albertsson AC. Finalizing the properties ofporous scaffolds of aliphatic polyesters through radiation sterilization.Biomaterials 2006;27:5335–47.

[49] Hedhammar M, Bramfeldt H, Baris T, Widhe M, Askarieh G, Nordling K, et al.Sterilized recombinant spider silk fibers of low pyrogenicity.Biomacromolecules 2010;11:953–9.

S. Hofmann et al. / Acta Biomaterialia 10 (2014) 308–317 317