Evaluation of Sterilisation Techniques for Regenerative...

Research ArticleEvaluation of Sterilisation Techniques for RegenerativeMedicine Scaffolds Fabricated with PolyurethaneNonbiodegradable and Bioabsorbable Nanocomposite Materials

Michelle Griffin 123 Naghmeh Naderi1345 DeepakM Kalaskar1

EdwardMalins6 Remzi Becer6 Catherine A Thornton4 Iain S Whitaker45

Ash Mosahebi3 Peter E M Butler123 and Alexander M Seifalian 7

1UCL Centre for Nanotechnology amp Regenerative Medicine University College London Royal Free London NHS Foundation TrustPond Street London NW3 2QG UK

2The Charles Wolfson Center for Reconstructive Surgery Royal Free London NHS Foundation Trust Hospital London UK3Department of Plastic Surgery Royal Free London NHS Foundation Trust Pond Street London NW3 2QG UK4Reconstructive Surgery amp Regenerative Medicine Group Institute of Life Science Swansea University Medical School Singleton ParkSwansea SA2 8PP UK

5Welsh Centre for Burns amp Plastic Surgery ABMU Health Board Heol Maes Egwlys Swansea SA6 6NL UK6Polymer Chemistry Laboratory School of Engineering and Materials Science Queen Mary University of London Mile End RoadLondon E1 4NS UK

7DirectorProfessor Nanotechnology amp Regenerative Medicine NanoRegMed Ltd The London BioScience Innovation CentreLondon NW1 0NH UK

Correspondence should be addressed to Michelle Griffin 12michellegriffingmailcom

Received 13 March 2018 Revised 18 June 2018 Accepted 9 August 2018 Published 3 October 2018

Academic Editor Rosalind Labow

Copyright copy 2018 MichelleGriffin et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

An effective sterilisation technique thatmaintains structure integritymechanical properties and biocompatibility is essential for thetranslation of new biomaterials to the clinical setting We aimed to establish an effective sterilisation technique for a biodegradable(POSS-PCL) and nonbiodegradable (POSS-PCU) nanocomposite scaffold that maintains stem cell biocompatibility Scaffolds weresterilised using 70 ethanol ultraviolet radiation bleach antibioticantimycotic ethylene oxide gamma irradiation argon plasmaor autoclaving Samples were immersed in tryptone soya broth and thioglycollate medium and inspected for signs of microbialgrowth Scaffold surface and mechanical and molecular weight properties were investigated AlamarBlue viability assay of adiposederived stem cells (ADSC) seeded on scaffolds was performed to investigate metabolic activity Confocal imaging of rhodaminephalloidin and DAPI stained ADSCs was performed to evaluate morphology Ethylene oxide gamma irradiation argon plasmaautoclaving 70 ethanol and bleach were effective in sterilising the scaffolds Autoclaving gamma irradiation and ethylene oxideled to a significant change in the molecular weight distribution of POSS-PCL and gamma irradiation and ethylene oxide to that ofPOSS-PCU (plt005) UV ethanol gamma irradiation and ethylene oxide caused significant changes in the mechanical propertiesof POSS-PCL (plt005) Argon was associated with significantly higher surface wettability and ADSC metabolic activity (plt005)In this study argon plasma was an effective sterilisation technique for both nonbiodegradable and biodegradable nanocompositescaffolds Argon plasma should be further investigated as a potential sterilisation technique for medical devices

1 Introduction

Synthetic biomaterials are being used to replace the extracel-lular matrix to restore damaged and failing tissues and organs

[1] Amongst biomaterials polymeric scaffolds have gainedsignificant popularity due to their ease of fabrication andversatility [1] Polymeric scaffolds for tissue engineering areeither manufactured aseptically or sterilised after processing

HindawiInternational Journal of BiomaterialsVolume 2018 Article ID 6565783 14 pageshttpsdoiorg10115520186565783

2 International Journal of Biomaterials

[2 3] For economical and practical reasons the latter strategyhas been employed with polymeric scaffolds intended forin vivo use and is considered a more realistic approach toachieve sterile implantable scaffolds [1 2] Nevertheless thechallenge remains to determine an efficient and nondestruc-tive sterilisation procedure for polymer scaffolds that pre-serves their structure and surface properties [3] Sterilisationtechniques may influence a materialrsquos structural chemicaland biological properties thus it is important to ensure themodality implemented does not affect biocompatibility [3]Sterilisation of biomaterials accepted by the FDA for medicaldevices includes ethylene oxide autoclaving and gammasterilisation [4] The success of an implant for sterilisationis dependent not only on the implant remaining sterilebut also on achieving sterility without adversely affectingthe materialrsquos properties Different sterilisation agents haveshown that they can attack polymers causing hydrolysismelting or depolymerisation [5 6]

Our group have developed and patented two familiesof nanocomposite polymers for the development of organsand tissues [7ndash9] The nonbiodegradable polymer incorpo-rates POSS nanoparticles into polycarbonate-based urea-urethane (POSS-PCU UCL-Nano) Its biodegradable coun-terpart modifies poly(caprolactone urea-urethane) POSS-PCL Understanding an appropriate sterilisation techniquefor POSS-PCU and POSS-PCL is crucial for translation toclinical practice A brief previous study compared threetechniques of sterilising POSS-PCU and POSS-PCL scaffoldsincluding autoclaving gamma irradiation and ethanol [7]Autoclaving was found to be effective in maintaining steril-isation of the scaffolds without degrading the material Thefirst authors of this paper have also demonstrated that bleachmay be useful for sterilising POSS-PCL scaffolds comparedto ethanol and autoclaving sterilisation [8] Adipose derivedstem cells (ADSCs) were shown to adhere to the POSS-PCL scaffolds following ethanol and bleach sterilisation [8]Lastly a study comparing the effects of autoclave microwaveantibiotics and 70 ethanol sterilisation on POSS-PCLscaffolds found ethanol to be a suitable sterilisation techniquewith maintained fibroblast attachment [9] The aim of thisstudy was to compare all available sterilisation techniquesfor POSS-PCU and POSS-PCL in a single study includinga new method using argon plasma sterilisation buildingon previous studies to understand the optimal sterilisationtechnique for nanocomposite scaffolds

An increasingly popular method of modifying the sur-face functionality to enhance cell behaviour on scaffolds isplasma modification [10 11] Plasma consists of electronsions energy rich neutrals molecules fragments atomsand photons It can be under low atmospheric or highpressureThe distinct behaviour of gases led to the suggestionthat plasma is the ldquofourth state of matterrdquo [12] Plasmamodification (PM) is an easy reliable clean way of creatingreactive functional groups on the surfaces of biomaterials andfor creating anchoring sites for further chemical reactions[12]

This study compared ethylene oxide argon plasmableach antibioticantimycotic ethanol ultraviolet radiationautoclaving and gamma irradiation sterilisation procedures

for morphological alteration chemical damage effects onpolymer degradation and biocompatibility The viability ofADSCs was assessed following the sterilisation of POSS-PCUand POSS-PCL scaffolds to assess biocompatibility of thesterilisation techniques

2 Materials and Methods

21 Polymer Synthesis

211 POSS-PCU The nanocomposite scaffolds were man-ufactured as previously described [7 13] In brief polycar-bonate polyol 2000mwt and trans-cyclohexanechloroydrin-isobutyl-silsesquioxane (Hybrid Plastics Inc) was heated to135∘C and then cooled to 70∘C Flakes of 441015840-methylenebis(phenyl isocyanate) (MDI) were then added to the mixtureat 75ndash85∘C for 90minutes to form the prepolymerThenNN-dimethylacetamide (DMAc) was to form a solution Furtherchain extension was completed by the drop-wise additionof ethylenediamine and diethylamine in DMAc This thencreated the POSS-modified polycarbonate urea-urethane inDMAc All chemicals and reagents were purchased fromAldrich Limited Gillingham UK

212 POSS-PCL POSS-PCL nanocomposites solution wasmanufactured as described previously [7] In brief polyca-prolactone diol (2000 gmol) and trans-cyclohexanechloro-hydrinisobutyl-polyhedral oligomeric silsesquioxane (POSS)were mixed and heated to 135∘C Then 94 g of 441015840-methylenebis(cyclohexylisocyanate) was added to form aprepolymer Following this 100 g of DMAC was added tothe prepolymer Chain extension was performed by drop-wise addition of 1 g of ethylenediamine in 80 g of dry DMACFollowing this 2 g of 1-butanol in 5 g of DMAC was addedto form the nanocomposite All chemicals and reagents werepurchased from Aldrich Limited Gillingham UK

213 Sample Preparation Polymers were fabricated as 3Dscaffolds using the phase separationparticulate-leachingtechnique as described previously [13] Firstly NaCL (200-250120583m) was dissolved in POSS-PCL and POSS-PCU inDMAc containing Tween-20 surfactant (11 weight ratio)Thesolution was dispersed and degassed in a Thinky AER 250mixer (Intertonics Kidlington UK) The polymer mixturewas spread onto steel moulds The moulds were then washedin deionised water to dissolve the solvent and DMAC for 7days Following washing polymer sheets with 700-800120583mthicknessweremanufactured For cell culture analysis 16 mmpolymer disks were cut from the sheets using a steel manualshape cutter

22 Sterilisation

221 Gamma Irradiation Scaffolds were irradiated with adose of 25 kGy at room temperature using a 60Co gamma-ray source (Synergy Health Swindon UK) Scaffolds wereexposed on a continuous path for 10 hours as describedpreviously [7]

International Journal of Biomaterials 3

222 Autoclaving The scaffolds were exposed to steam at121∘C for 15 minutes at pressures of 115 kPa as previouslydescribed [7]

223 Ethanol Polymer scaffolds were submerged in 70(vv) ethanol on a roller mixer for 30 minutes as previouslydescribed [8] Following alcohol sterilisation scaffolds arewashed in sterile deionised water on a roller for 15 minuteswhich is then repeated five times

224 Plasma Scaffolds are placed in 24-well plates fortreatment by LF (radio frequency) argon plasma generatoroperating at 40 KHz at 100 W Scaffolds then enter thechamber of the electrode-less glow discharge apparatuswhich is purged 3 times with argon gas (9999purity BOCUK) for 2minutes The chamber is then evacuated to 10 TorrPlasma is then ignited by a radio frequency excitation sourceand was maintained at 100 W for 5 minutes The scaffoldswere treated with plasma and immediately seeded with cellsfor in vitro analysis to prevent hydrophobic recovery of thescaffolds

225 Ethylene Oxide The ethylene oxide sterilisation is ini-tiated with a preconditioning of the samples which is carriedout at 41∘C for 13 hours at 42 humidityThe sterilisation stepis thenperformed by 100 ethylene oxide atmosphere at 49∘Cfor 2 12 hours Scaffold are then in air for 9 hours at 43∘C

226 Ultraviolet Irradiation Scaffolds were UV irradiatedby placement in a UV decontamination device (40 wattwavelength 254nm mean density 15 kJcm2) for 3 hours aspreviously described [8]

227 Antibiotic Antimycotic Treatment Scaffoldswere placedin a 1 (vv) antibiotic antimycotic solution (10 000 UmLpenicillin G 10mgmL streptomycin sulphate and 25mgmLamphotericin B diluted in sterile phosphate buffered saline(PBS)) for 24 hours at 4∘C Following sterilisation thescaffolds were washed five times with sterile deionised wateron a roller for 15 minutes each time

228 Bleach (SDIC) The scaffolds were submersed in1000ppm slow chlorine releasing compound sodium dichlo-roisocyanurate dihydrate (SDIC) at room temperature for 20minutes as described previously [8] The scaffolds are thenwashed in sterile deionisedwater daily for 7 daysThe removalof remaining SDIC was confirmed by pH testing

23 Material Characterisation

231 Tensiometry Tensiometry to evaluate the mechanicalproperties of the scaffolds following sterilisation was per-formed as described previously [13] In brief dumbbell-shaped scaffolds (dimensions of 10 times 2mm) were tensileloaded at a loading speed of 100mmmin (n = 6) using anInstron 5565 (HighWycombe Bucks UK) Youngrsquos modulusof elasticity at the 0-25 portion of the curve maximum

tensile strength and elongation at break were calculatedStatistical differences in the tensile properties between ster-ilisation techniques were evaluated using two-way ANOVAwith post hoc Turkey test

232 Attenuated Total Reflectance Fourier Transform InfraredSpectroscopy (ATR-FTIR) FTIR spectrophotometer wasused to analyse changes in the surface chemistry of thescaffolds treated with the different sterilisation techniquesas previously described [8] Chemical groups were detectedusing attenuated total reflectance (ATR)-FTIR mode (JascoFTIR 4200 Spectrometer (JASCO Inc USA)) (n=6)FTIR testing parameters were recorded at 20 scans at a4 cm-1 resolution with a wavenumber range of 600 cm-1 to4000 cm-1

233 Gel Permeation Chromatography (GPC) Molecularweight averages and polymer dispersity were determined byGPC as previously described [8] (n=6) In brief samples wereprepared to a 1mgmL concentration and passed througha 022120583m nylon filter GPC analysis was conducted on theAgilent 1260 infinity system using 2 PLgel 5 120583m mixed-Dcolumns (300 times 75mm) a PLgel 5mm guard column (50times 75mm) a differential refractive index (DRI) and variablewavelength detector (VWD) Statistical differences in theGPC analysis between sterilisation techniques were evaluatedusing two-way ANOVA with post hoc Turkey test

234 Water Contact Angle Measurements The static watercontact angle of the scaffolds was performed as describedpreviously [13] In brief the water contact angle was analysedusing the sessile drop method (DSA 100 instrument (KRUSSGermany)) A 5 120583l volume of deionised water was used in allexperiments Measurement of a single drop was performedon six independent scaffolds (n=6)The average contact anglewas calculated using the KRUSS drop shape software (version190014)

235 Scanning Electron Microscopy (SEM) The surface ofthe scaffolds treated with the different sterilisation tech-niques was analysed using SEM as described previously [8](n=6) Scaffolds were dehydrated in acetone prior to dryingovernight The scaffolds were then mounted on aluminiumpin stubs using sticky carbon tape After coating the scaffoldswith a thin layer of AuPd (approximately 2 nm thick) using aGatan ion beam coater the surface of the scaffolds was imagedwith a Carl Zeiss LS15 Evo HD SEM

24 Cytotoxicity

241 ADSC Isolation and Seeding ADSCswere isolated fromadipose tissue according to the method described by Zuk etal with modifications as previously described [8 14 15] Inbrief following removal of fibrous tissue adipose tissue wascut into small pieces of lt 3mm3 The tissue was then furtherdigested in Dulbeccorsquos Modified Eaglersquos MediumNutrientMixture F-12 Ham (DMEMF12) containing 300UmL crudecollagenase I (Invitrogen Life Technologies Ltd Paisley UK)


for 30 minutes in an incubator (37∘C 5 CO2) Following

filtration through 70120583m Cell Strainers (BD BiosciencesOxford UK) the samples underwent centrifugation (290timesG5min) Then the ADSC-rich cell preparation formed a pelletat the bottom of the tube The ADSC cells were cultured forup to 2 passages When the ADSCs reached approximately80 confluence subculture was performed through trypsin-isation For cell cultures analysis 15 X 104 ADSCs at passage2 were seeded on each polymer disk following sterilisationWritten consent was taken from all patient donors in thestudy and was approved by the North Scotland ethical reviewboard reference number 10S080220

242 AlamarBlue AlamarBlue viability assay was per-formed as described previously [8 13] Briefly followingincubation of scaffolds with complete medium for 24 hoursscaffolds were seeded with 15 X 104 ADSC per well At 1 37 and 14 days medium was removed and 10 AlamarBlueprepared in fresh media was added for 3 hours Followingincubation AlamarBlue fluorescence was quantified at therespective excitation and emission wavelength of 540 and595nm The mean fluorescent units for the six replicatecultures of three individual experiments were calculated foreach exposure treatment and the mean blank value wassubtracted from these

243 Immunofluorescence Rhodamine Phalloidin and DAPITo study adhesion and morphology of the ADSC onto thescaffolds immunocytochemistry morphology staining wasperformed as described previously [8 13] At 24 hoursthe media was removed and the cells were washed withPBS three times Following this cells were fixed with 4(wv) paraformaldehyde for 15 minutes The scaffolds werethen washed thrice in PBS01 Tween-20 and washed in01 tritonX100 to improve permeability for 5 minutes Thescaffolds were then stained with rhodamine phalloidin dye(Molecular Probes Life Technologies Paisly UK) in theratio 140 (dissolved in 1mL of methanol) in PBS for 40minutes Following washing the nuclei was stained withDAPI (Molecular Probes Life Technologies Paisley UK)The ADSCs on the scaffolds were visualised using confocalmicroscopy Zeiss LSM 710 (Zeiss Jena Germany) ImageJ (National Institute of Health NIH) software was used todetermine circularity of the ADSCs on the scaffolds

25 Sterility Testing All samples were tested for the effec-tiveness of sterilisation as previously described [8] In briefscaffolds were immersed in tryptone soya broth (TSB)and fluid thioglycollate medium (THY) for cultivation ofmicroorganisms (Wickham Laboratories Hampshire) for 7days Sterile broth was considered the negative control andunsterilised samples as the positive control Both broths weremacroscopically observed every 1ndash3 days for clouding asan indicative of contamination and ineffective sterilisationA clear broth was considered to have no infection and aneffective sterilisation of the samples (n = 9)

26 Statistical Analysis All statistical analyses were per-formed using Prism software (GraphPad Inc La Jolla USA)

Means and standard deviations were calculated from numer-ical data In figures bar graphs represent means whereaserror bars represent 1 standard deviation (SD) A 119901 value ofle 005 was defined as significant The exact statistical analysisperformed for each dataset is described in the figure legend

3 Results


311 Visual Inspection after Sterilisation All samples with-stood treatment with UV antibioticantimycotic bleach andplasma treatment Although the POSS-PCU samples wereunaffected by the autoclaving process the POSS-PCL sampleswere destroyed therefore it was not possible to examinethe autoclaved POSS-PCL samples further Both the POSS-PCU and POSS-PCL samples held up well against gammairradiation with slight discolouringyellowing of the POSS-PCU samples being observed Ethylene oxide gas causedslight yellow discolouring and ethanol visible deformation ofPOSS-PCL samples

312 Tensiometry Quantitative values of mechanical prop-erties of POSS-PCL and POSS-PCU samples subjected toethanol bleach (SDIC) plasma ethylene oxide gas UVradiation antibioticantibiotic treatment gamma irradiationautoclaving (only POSS-PCU) and unsterilised controls foreach sterilisation method are presented in Table 1 No sig-nificant difference in elongation at break Youngrsquos modulusor maximum stress was seen between any of the POSS-PCUsamples The ultimate tensile stress for POSS-PCL increasedfrom056plusmn008MPa in control samples to 171plusmn019 MPa afterethylene oxide gas treatment to 145plusmn004 after UV radiationand to 117plusmn015 after gamma irradiation (p lt 005) Thisincrease in tensile stress of ethylene oxide treated POSS-PCL translated itself to Youngrsquos modulus which was alsosignificantly increased compared to control (040plusmn011 versus018plusmn00 p lt 005) Compared to control POSS-PCL UVradiated POSS-PCL and ethanol had a significantly shorterelongation at break (p lt 005)

313 Water Contact Angle Measurements The difference insurface hydrophilicity of POSS-PCL and POSS-PCU samplesafter each sterilisation technique was assessed by measuringthe water contact angle (Figure 1) Plasma treated POSS-PCLand POSS-PCU samples had statistically significant lowercontact angles when compared to untreated controls andother sterilisation methods (p lt 0001) Ethanol treatmentof POSS-PCL was associated with a significant decreasein contact angles when compared to controls (p lt 005)Amongst the treated POSS-PCU samples UV radiation wasassociated with a statistically significant decrease in contactangle measurements compared to controls (p lt 005)

314 Attenuated Total Reflectance Fourier Transform InfraredSpectroscopy (ATR-FTIR) Figure 2 shows the peak assign-ment in the FTIR spectra of unsterilised control POSS-PCLand POSS-PCU and POSS-PCL and POSS-PCU samples


Table1Mecha

nicalP

rope

rtieso

fPOSS

-PCUan

dPO

SS-PCLaft

ersterilisatio

nprocessesYo

ungrsquos

mod

ulusm

axim

umstr

esselon

gatio

natbreakandthickn

esso

fcon

trolP

OSS-PCL

andPO

SS-PCU

andsamples

subjectedto

ethano

lbleach

(SDIC)plasmaethylene

oxideUVradiation

antib

iotic

antim

ycotictre

atmentandgammairradiatio

n

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Gam

ma

Ethy

lene

Oxide

UV

Antibiotic

Antim

ycotic

Plasma

Autoclaving

Youn

grsquosMod

ulus

(MPa)PO

SS-PCL

018plusmn00

029plusmn002

020plusmn002

032plusmn004

040plusmn011

035plusmn002

025plusmn003

030plusmn005

NA

POSS-PCU

055plusmn004

057plusmn002

056plusmn002

056plusmn004

055plusmn002

055plusmn002

053plusmn001

055plusmn003

054plusmn002

Maxim

umStress(M

Pa)

POSS-PCL

056plusmn008

061plusmn010

031plusmn011

117plusmn015

171plusmn

019

145plusmn004

110plusmn014

080plusmn026

NA

POSS-PCU

083plusmn003

082plusmn006

085plusmn003

083plusmn001

084plusmn001

083plusmn001

081plusmn012

083plusmn002

082plusmn004

Elon

gatio

natbreak(

)PO

SS-PCL

5896plusmn262

5497plusmn68

2930plusmn61

4622plusmn16

4716plusmn39

3875plusmn22

4695plusmn11

5001plusmn

30NA

POSS-PCU

2833plusmn95

72736plusmn1001

2792plusmn174

2880plusmn305

2886plusmn14

62693plusmn855

2738plusmn90

12811plusmn412

2805plusmn78

8

Thickn

ess(mm)

POSS-PCL

20plusmn0

143plusmn015

1525plusmn013

16plusmn022

12plusmn025

138plusmn005

155plusmn017

2433plusmn

012

NA

POSS-PCU

075plusmn004

077plusmn002

084plusmn002

077plusmn005

076plusmn002

077plusmn002

075plusmn003

076plusmn002

074plusmn006


POSS-PCL POSS-PCU

lowastlowastlowast

0

50

100

150

Con

tact

Ang

le (

∘)

lowast

UVAntibioticAntimycotic

ControlBleach (SDIC)EthanolGammaEthylene Oxide

PlasmaAutoclaving

lowastlowastlowast

lowast

Figure 1Contact angles of POSS-PCL and POSS-PCU samplesmeasured after different sterilisation techniquesOne-wayANOVA andTurkeyrsquosmultiple comparison testwas used to show statistical significancelowast indicates statistically significant differences (lowastplt 005 andlowastlowastlowastplt 0001)


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Abs

Wavenumber (cm -1)

04

03

02

01

0

(a) POSS-PCL FTIR spectra

Autoclaving


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm -1)

07

06

04

02

0

minus01

Abs

(b) POSS-PCU FTIR spectra

Figure 2 POSS-PCL and POSS-PCU FTIR spectra after different sterilisation techniques [a] SDIC treatment led to a slight decreasein the peaks at 1634 cmminus1 and 1557 cmminus1 and an increase in the peak at 1520 cmminus1 compared to untreated control POSS-PCL [b] No majorchanges were noted in the FTIR spectra of POSS-PCU samples after different sterilisation methods

after different sterilisation methods Bleach (SDIC) treatmentled to a slight decrease in the peaks at 1634 cmminus1 and1557 cmminus1 and an increase in the peak at 1520 cmminus1 POSS-PCL FTIR spectra were not significantly affected by the othersterilisation techniques FTIR spectra of POSS-PCU sampleswere unaffected by different sterilisation techniques

315 Gel Permeation Chromatography (GPC) Gel perme-ation chromatography (GPC) results are summarised inTable 2The untreated POSS-PCUwas found to have a weightaverage molecular weight (119872w) of 91200 gmol and a numberaverage molecular weight (119872n) of 47700 gmol whereasPOSS-PCL had an 119872w of 361100 and 119872n of 141000 gmolAfter ethanol bleachUV radiation and antibiotic treatments

there was a negligible impact on119872w for any of the samplesHowever solely amongst POSS-PCL samples ethanol causeda decrease of 21 in119872

119899 whilst UV increased119872

119899by 10 in

comparison to unsterilised POSS-PCL No major changes inmolecular weight distributions were detected in autoclavedsamples of POSS-PCUHowever autoclaved POSS-PCL sam-ples showed a 52 decrease in119872w and 38 decrease in119872nExposure to gamma irradiation had a significant impact onall of the samples 119872n of POSS-PCU decreased significantlyby 16 whereas 119872

119908increased by 48 Meanwhile gamma

irradiation decreased both 119872n and 119872w of POSS-PCL by68 and 58 respectively Ethylene oxide increased 119872w ofPOSS-PCU by 23 and decreased119872n of POSS-PCU by 28Concurrently it decreased119872w and119872n of POSS-PCL by 23and 31 respectively Plasma had no significant impact on


Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200


Control Bleach (SDIC)

AntibioticAntimycotic

UVEthanol

EthyleneOxide

GammaPlasma

Control Bleach(SDIC)

UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m


Figure 3 Scanning electron microscopy (SEM) images of POSS-PCL (left) and POSS-PCU (right) surfaces after different sterilisationtechniques

the molecular weight distribution of POSS-PCU or POSS-PCL polymers

316 Scanning Electron Microscopy (SEM) SEM imagesof POSS-PCL and POSS-PCU after different sterilisationtechniques are shown in Figure 3 Unsterilised POSS-PCLsample surface exhibits tufts and pits on the surface Suchtufts were lost after using ethanol antibioticantimycoticand gamma sterilisation with polymer melting and irregularreformation into larger and flatter ridges SDIC treatmentwas associated with a marked increase in pits whereas UVirradiation was associated with a pronounced increase in thenumber of tufts Ethylene oxide was associated with largertufts whereas plasma treatment caused larger pits and tuftson the POSS-PCL surface (Figure 3) POSS-PCU samplesgenerally showed little surface alterations after sterilisation

Ethanol SDIC UV EO and gamma were associated withincreased tufts The antibioticantibiotic autoclaving andplasma sterilisation caused minimal changes on the POSS-PCU surface

32 Cell Biocompatibility

321 AlamarBlue The results of the alamarBlue viabilityassay after 1 3 7 10 and 14 days of incubation are presentedin Figure 4 ADSC cultured on POSS-PCU and POSS-PCLsamples showed similar metabolic activity At Days 7 and 10plasma treated POSS-PCL was associated with the highestADSC metabolic activity compared to any other sterilisationtechnique (p lt 005) Ethanol and bleach (SDIC) ster-ilised POSS-PCL had a statistically significant higher ADSCmetabolic activity compared to ADSC on gamma ethylene


0

20

40

60

80

100

120

Time points (days)

POSS-PCLAlamarBlue Viability Assay

1 141073

Abs

orba

nce

570

nm

Bleach (SDIC)EthanolGammaEthylene Oxide

UVAntibioticAntimycoticPlasma

lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120

POSS-PCU AlamarBlue Viability Assay

Abs

orba

nce

570

nm


UV

Autoclaving

AntibioticAntimycoticPlasma

3 7 10 141Time points (days)

lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)

Figure 4 AlamarBlue viability assay of adipose derived stem cells (ADSCs) after 1 3 7 10 and 14 days of incubation on (a) POSS-PCLand (b) POSS-PCU samples Statistical significance was shown using two-way ANOVA and Turkeyrsquos multiple comparisons test lowast indicatesstatistically significant differences (lowastp lt 005)

oxide UV radiation antibioticantimycotic and autoclavingsterilised POSS-PCL At Day 14 the same observations weremade In addition UV radiation sterilised POSS-PCL hada statistically significant higher ADSC metabolic activitycompared to ADSC on antibioticantibiotic treated andgamma irradiated POSS-PCL (p lt 005) Bleach (SDIC)sterilised POSS-PCL had a statistically significantly higherADSC metabolic activity compared to ADSC on ethanolsterilised samples (p lt 005) Amongst POSS-PCU samplessimilar observations were made At Days 7 and 10 plasmatreated POSS-PCU was associated with the highest ADSCmetabolic activity compared to any other sterilisation tech-nique (p lt 005) Ethanol and bleach sterilised POSS-PCUhad a statistically significant higher ADSC metabolic activitycompared toADSC on gamma ethylene oxide UV radiationantibioticantimycotic and autoclaving sterilised POSS-PCU(p lt 005) In addition gamma ethylene oxide and UVradiation treated samples had a statistically significant higherADSCmetabolic activity compared to antibioticantimycoticand autoclaving (p lt 005) At Day 14 the same observationswere made In addition gamma irradiation was associatedwith significantly higher ADSC metabolic activity comparedto UV and ethylene oxide (p lt 005)

322 Immunofluorescence Rhodamine Phalloidin and DAPIConfocal laser scanning microscopy images indicated thatADSC developed different morphologies when grown ondifferently sterilised surfaces (Figure 5) Cells grown onbleach sterilised POSS-PCL exhibited a more spread-out

phenotype compared to cells grown on surfaces exposed toother sterilisation techniques which demonstrated a moreround character In general ADSC on POSS-PCL had amore round morphology compared to ADSC on POSS-PCUsurfaces Circularity measurements with Image J softwareshowed that ADSCgrown on bleach sterilised POSS-PCLhada significantly less circular morphology compared to ADSCon ethanol ethylene oxide gamma antibioticantibiotic andplasma (p lt 005) but not on UV sterilised POSS-PCL(Figure 5(a)) There was no statistically significant differencebetween the morphology of the ADSCs on the POSS-PCUsamples (Figure 5(b))

33 Sterility Testing

331 Visual Inspection The polymeric materials were incu-bated in TSB and THY for 7 days to test the efficiency of ster-ilisation with the resultant level of bacterial growth reportedin Table 3 THY is a viscous growth medium with reducedoxygen levels which tests the growth of anaerobic bacteriaand other organisms capable of growing in reduced oxygentension No evidence of bacterial growth was observed on anyof thematerials tested after incubation in THY TSB howeveris a general growth media for aerobic microorganisms andis designed for the growth of aerobic bacteria and yeastsandmoulds Amidst POSS-PCU sterilised samples there wasno sign of infection Only the unsterilised control samplesshowed signs of infection Amongst POSS-PCL samples all


POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV

Bleach (SDIC)Bleach(SDIC)

UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m

(a) Confocal microscope images of rhodamine phalloidin and DAPI stained adipose derived stem cells(ADSCs) cultured onPOSS-PCL and POSS-PCU surfaces for 24 hours after different sterilisation techniques

POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving


(b) The circularity quantification of the cultured adipose derived stemcells (ADSCs) seeded on POSS-PCL and POSS-PCU surfaces for 24hours after different sterilisation techniques

Figure 5


Table 3 Summary of POSS-PCL and POSS-PCU sterilisation efficacy for bleach (SDIC) ethanol ethylene oxide gamma plasmaantibioticantimycotic UV and autoclaving sterilisation techniques All control samples (3) of POSS-PCU and POSS-PCL and 1 of 9POSS-PCL samples sterilised using UV and antibioticantimycotic were infected

Sterilisation Method Control Bleach (SDIC) Ethanol Ethylene Oxide Gamma Plasma AntibioticAntimycotic UV Autoclaving

POSS-PCL TSB 99 09 09 09 09 09 19 19 NATHY 99 09 09 09 09 09 19 19 NA

POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09

unsterilised control samples and one of nine samples ster-ilised using UV radiation and antibioticantimycotic showedsigns of infection Although there was minimal evidenceof bacterial growth in the sterility studies of scaffolds withUV and antibioticantimycotic treatment no evidence wasseen in the viability testing or the morphology assessment toinvalidate the assays

4 Discussion

Polymer degradation after sterilisation techniques can beassessed using (i) macroscopic characterisation methodsproviding information on ldquobulkrdquo properties such as mechan-ical performance (ii) microscopic characterisation lookingat molecular weight and its dispersity (iii) characterisationof the molecular structure and composition such as FTIRanalysis and (iv) surface characterisation ie scanningelectron microscopy and surface wettability [16] Accordingto these criteria in this study we performed a thoroughinvestigation of the bulk surface andmolecular properties ofPOSS-PCL and POSS-PCU scaffolds after several sterilisationtechniques including plasma gamma irradiation ethyleneoxide UV radiation antibioticantimycotic 70 ethanoland bleach treatments As the biodegradable counterpart ofPOSS-PCU POSS-PCL was considerably more susceptive tochanges in the molecular weight distribution mechanicalproperties and surface morphology and chemistry afterseveral sterilisation techniques

The three leading sterilisation methodologies with FDAapproved for medical devices are gamma irradiation auto-claving and ethylene oxide Autoclaving has been the ear-liest method for the sterilisation of biomaterials Sterilisa-tion with moist heat in an autoclave is usually performedat temperatures equal to or higher than 121∘C dry heatsterilisation requires considerably higher temperatures toeffectively inactivate bacterial sporesThe suitability of steamsterilisation has been questioned for polyurethanes as thehigh temperature may soften the polymer and deform thematerial [17] In this study POSS-PCU materialrsquos propertieswere unaffected as shown by mechanical properties surfacechemistry as shown by contact angle and FTIR and surfacetopography as shown by SEM Polymer degradation wasdetermined by measuring changes in molecular weight andmass immediately after the sterilisation process using GPCanalysis Polymeric biomaterials of low molecular weightpresent less chemical and mechanical resistance in relationto the same material of higher molecular weight [18] Auto-claving was an optimal sterilisation technique for POSS-PCU

with no evidence of changes inmolecular number andweightHowever POSS-PCL was unsuited to autoclaving as shownby visual changes after sterilisation and changes in molecularweight and number demonstrating the polymer underwenta degree of hydrolysis This finding is in accordance with theliterature where biodegradable polymers break down due tothe high temperatures of autoclaving [19]

Gamma sterilisation involves ionising radiation fromeither cobalt 60 isotope or accelerated electrons Gammairradiation can generate free radicals in the polymer caus-ing surface oxidation and subsequent degradation due tochain scission and cross-linking with increasing dosagesof radiation [20] However gamma irradiation has definiteadvantages in that it is penetrating and free of residuesAlso material temperatures are only moderately elevatedduring sterilisation which is an advantageous feature for thesterilisation of bioresorbable implants where temperatureand dose conditions need close consideration Microbiologi-cal validation experiments according to ISO 11137 [21] haveshown gamma irradiation in dry ice at doses of 16 kGy ormore effectively inactivates microorganisms In this studygamma irradiation was associated with effective sterilisationof POSS-PCL and POSS-PCU scaffolds Polyurethanes havealso shown some degradation after gamma radiation POSS-PCL was found to have a significant decrease in the molec-ular weight and number indicating degradation may haveoccurred Several biodegradable polymers have shown to besusceptible to gamma irradiation [19] Holy et al found thatpolylactide-co-glycolide scaffolds had a 50 loss in theirmolecular weight after gamma irradiation [22]

Gamma irradiation has been shown to cause the releaseof free radicals in polymers causing surface oxidation andsubsequent degradation of the polymer Surface oxidationof the polyurethanes is indicated by a strong yellowing ofsamples which was also found in this study with some POSS-PCL scaffolds Despite the degradation and potential surfaceoxidation of gamma on PCL scaffolds the water contactof POSS-PCL did not change after gamma sterilisationcompared to control There are reports in the literaturethat show no change in water contact angle of biomaterialsfollowing gamma sterilisation [23 24] This could be due tothe penetrating action of the gamma sterilisation affectingbulk properties rather than surface properties [23 24]

Gamma irradiation showed no changes in material prop-erties including mechanical surface chemistry or molecularnumber for POSS-PCU scaffolds However SEM did showsome surface changes after gamma sterilisation on POSS-PCU Although suitable for POSS-PCU POSS-PCL was


unsuitable for gamma sterilisation with significant changes inmolecular weight and number

EtO is the final sterilisation technique which has beenapproved for medical implants EtO is a liquid below 11∘so in contrast to steam sterilisation it is a low temperaturemethod [17] In addition to being toxic and explosive causinga significant occupational health and safety hazard thereare concerns on removing all traces of the EtO from theimplant after sterilisation [15] In this study SEM showedsome surface material changes after EtO without changingthe bulk properties including tensile strength or molecularweight for POSS-PCU For POSS-PCL EtO caused significantloss in molecular weight and number and changes to thesurface morphology by SEM EtO has also been shown inthe literature to be an inappropriate sterilisation techniquefor biodegradable scaffolds due to changes in the structuraland biochemical properties [17 22] For example Hooperet al showed that EtO of polycarbonate materials causeda reduction in yield strength and faster degradation ratescompared to nonsterilised controls [25]

Although not considered sterilisation techniques formedical devices we also evaluated the effect of different disin-fectantmethods Techniques such as bleach using antibioticsandUV radiation are used in the laboratory setting to sterilisescaffolds for in vitro and in vivo examination The use of SDICprevented contamination of the samples and was not asso-ciated with any significant changes in mechanical propertiesor any visual deformation of both nanocomposite polymersAlthough longstanding polymer exposure to bleach has beenassociated with increased hydrophobicity [26] we did notobserve any significant changes in surface wettability ofPOSS-PCL scaffolds Bleach has been shown to increaseroughness and porosity of polymeric scaffolds [26] In thisstudy we observed similar submicron changes to POSS-PCLscaffold structure mainly an increase in the number of pitsSDIC was the sole sterilisation method which showed slightchanges in the surface chemistry of the scaffolds on the FTIRspectra likely due to hydrolysis

Ethanol and UV are all useful sterilisation techniquesfor laboratory analysis and provided sterility of the POSS-PCU and POSS-PCL nanocomposite scaffolds For POSS-PCU scaffolds UV and ethanol did cause some changesto the surface as shown by SEM although bulk mechanicalproperties were not affected UV decreased the contact angleof POSS-PCU which is consistent with other reports of UVcreating hydrophilic surfaces [27ndash29] UV sterilisation mayhave oxidized the surface and caused a drop in the watercontact angle [27ndash29]

POSS-PCL scaffolds also maintained their bulk mechani-cal properties after ethanol and UV modification Howeverreports in the literature show that whilst Gram-positiveGram-negative acid-fast bacteria and lipophilic virusesshow high susceptibility to concentrations of ethanol in waterranging from 60 to 80 hydrophilic viruses and bacterialspores are resistant to the microbial effects of ethanol [30]Antibiotic solution had minimal effect on the surface or bulkproperties of POSS-PCUor POSS-PCL Furthermore reportshave shown that different microorganisms have varying sen-sitivity to UV [19] For example UV easily destroys vegetative

bacteria but bacterial spores and prions are more resistantSome viruses are considered inactivated whilst others areresistant [19] Antibiotic solutions inactivate bacteria by inter-fering with DNA replication [19] However such solutionsare only effective against vegetative bacteria and spores whilstfungi moulds and viruses are not affected [19] Thereforeethanol UV and antibiotics are considered to be chemicaldisinfectants instead of sterilising techniques and not used inthe sterilisation of biomedical devices [19]

Plasma technology is defined as neutral ionised gasand includes photons electrons positive and negative ionsatoms free radicals and nonexcited molecules [31] Severaltypes of plasma techniques exist but in this study radiofre-quency plasma was utilised Argon plasma demonstrated nochanges in the material characteristics of both POSS-PCUincludingmechanical molecular number and weight surfacechemistry by FTIR or surface topography by SEM Howeverargon plasma did cause some surface topographical changesof POSS-PCL due to potential etching effect of plasmatreatment The surface wettability significantly decreasedafter argon plasma for both POSS-PCU and POSS-PCL Thesterilisation process itself may affect the surface and bulkproperties to negatively influence its cytocompatibility andbiocompatibility The in vitro compatibility of the sterilisedscaffolds was performed to evaluate the release of cytotoxicmolecular weight products from the sterilisation Viabilitydata demonstrated that significantly more cells were ableto adhere to POSS-PCU and POSS-PCL after argon plasmasurface modification compared to other sterilisation tech-niques by Day 7 (Figure 4) Several studies have found thatdecreasing the water contact angle of scaffolds below 90∘ dueto plasma treatment induces a hydrophilic surface causinga greater number of cells adhere to the biomaterial surface[32 33] Both POSS-PCU and POSS-PCL became morehydrophilic after argon plasma surface modification withoutcausing bulk mechanical properties (mechanical and molec-ular numberweight) With optimal sterilisation efficacymaintenance of mechanical properties and improvementin cell biocompatibility plasma demonstrated the optimalsterilisation process in this study

Menashi et al first reported the use of argon for sterilisa-tion in 1968 after sterilising the surface of vials contaminatedby bacterial spores [34] Since then the gas type and thepower of discharge have shown to influence the efficacy ofthe treatment Analysis of RF plasmas has suggested thatthe chemical species created in the discharge are responsiblefor the destruction of the microorganisms and the UV orthermal effects are secondary effects [35 36] Argon plasmahas shown to sterilise titanium implant surfaces [37] Othertypes of plasma have been investigated for the sterilisationof materials The comparison of the ethanol dry ovenautoclave UV radiation and hydrogen peroxide plasma assterilisation techniques for electrospun PLA scaffolds [38]showed that UV irradiation and hydrogen peroxide werethe most effective without affecting the chemical and mor-phological features The authors demonstrated the hydrogenperoxide produced destructive hydroxyl free radicals whichcan attack membrane lipids DNA and other essential cellcomponents to deactivate the microorganisms [38]


There were limitations to this work During the plasmatreatment the scaffolds were exposed to the unsterile envi-ronment whilst moving them from the plasma chamber Tofurther optimise the plasma treatment the apparatus will beplaced in a sterile laminar flow cabinet in the future

In summary argon plasma maintained the propertiesof the nanocomposite scaffolds in addition to improvingcell adhesion (Figure 4) Plasma sterilisation is easily trans-ferrable to clinical practice for sterilisation of materials as itis simple to operate and the lack of toxic residuals makes itsafe for the operator

5 Conclusion

In conclusion the FDA approved sterilisation technique ofautoclaving maintained POSS-PCU characteristics but wasunsuitable for biodegradable POSS-PCL scaffolds Taking allresults into account we argue that plasma surface modifi-cation using argon gas may effectively sterilise polyurethanebiomaterials as well as improve cytocompatibility of tissueengineering scaffolds by modification in surface wettabilityand topography Argon plasma should be explored andoptimised for the sterilisation of further biomaterials

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Disclosure

Michelle Griffin and NaghmehNaderi are equal first authors

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

We would like to thank the funding from Medical ResearchCouncil and Action Medical Research which providedMichelle Griffin a clinical fellowship to conduct this workGN 2339

References

[1] M T Lam and J C Wu ldquoBiomaterial applications in car-diovascular tissue repair and regenerationrdquo Expert Review ofCardiovascularTherapy vol 10 no 8 pp 1039ndash1049 2012

[2] J P Guggenbichler ldquoIncidence and clinical implication ofnosocomial infections associated with implantable biomateri-als - catheters ventilator-associated pneumonia urinary tractinfectionsrdquo GMS Krankenhhyg Interdiszip vol 6 2011

[3] L Montanari M Costantini E C Signoretti et al ldquoGammairradiation effects on poly(dl-lactictide-co-glycolide) micro-spheresrdquo Journal of Controlled Release vol 56 no 1ndash3 pp 219ndash229 1998

[4] FDAstandards httpwwwfdagovMedicalDevicesDeviceReg-ulationandGuidanceStandardsdefaulthtm

[5] N Hirata K-I Matsumoto T Inishita Y Takenaka Y Sumaand H Shintani ldquoGamma-ray irradiation autoclave and ethy-lene oxide sterilization to thermosetting polyurethane Steril-ization to polyurethanerdquo Radiation Physics and Chemistry vol46 no 3 pp 377ndash381 1995

[6] A Simmons J Hyvarinen and L Poole-Warren ldquoThe effect ofsterilisation on a poly(dimethylsiloxane)poly(hexamethyleneoxide) mixed macrodiol-based polyurethane elastomerrdquo Bio-materials vol 27 no 25 pp 4484ndash4497 2006

[7] M Ahmed G Punshon A Darbyshire and A M SeifalianldquoEffects of sterilization treatments on bulk and surface prop-erties of nanocomposite biomaterialsrdquo Journal of BiomedicalMaterials Research Part B Applied Biomaterials vol 101 no 7pp 1182ndash1190 2013

[8] N Naderi M Griffin E Malins et al ldquoSlow chlorine releasingcompounds A viable sterilisation method for bioabsorbablenanocomposite biomaterialsrdquo Journal of Biomaterials Applica-tions vol 30 no 7 pp 1114ndash1124 2016

[9] L Yildirimer and A M Seifalian ldquoSterilization-induced chan-ges in surface topography of biodegradable POSS-PCLU andthe cellular response of human dermal fibroblastsrdquo TissueEngineering - Part C Methods vol 21 no 6 pp 614ndash630 2015

[10] T Jacobs H Declercq N De Geyter et al ldquoEnhanced cell-material interactions onmedium-pressure plasma-treated poly-hydroxybutyratepolyhydroxyvaleraterdquo Journal of BiomedicalMaterials Research Part A vol 101 no 6 pp 1778ndash1786 2013

[11] Y Wan X Qu J Lu et al ldquoCharacterization of surface propertyof poly(lactide-co-glycolide) after oxygen plasma treatmentrdquoBiomaterials vol 25 no 19 pp 4777ndash4783 2004

[12] S G Wise A Waterhouse A Kondyurin M M Bilek andA S Weiss ldquoPlasma-based biofunctionalization of vascularimplantsrdquo Nanomedicine vol 7 no 12 pp 1907ndash1916 2012

[13] M F Griffin R G Palgrave A M Seifalian P E Butler andDM Kalaskar ldquoEnhancing tissue integration and angiogenesisof a novel nanocomposite polymer using plasma surface poly-merisation an in vitro and in vivo studyrdquo Biomaterials Sciencevol 4 no 1 pp 145ndash158 2016

[14] P A Zuk M Zhu H Mizuno et al ldquoMultilineage cells fromhuman adipose tissue implications for cell-based therapiesrdquoTissue Engineering Part A vol 7 no 2 pp 211ndash228 2001

[15] M F Griffin A Ibrahim A M Seifalian P E M ButlerD M Kalaskar and P Ferretti ldquoChemical group-dependentplasma polymerisation preferentially directs adipose stem celldifferentiation towards osteogenic or chondrogenic lineagesrdquoActa Biomaterialia vol 50 pp 450ndash461 2017

[16] P Vermette P S Levesque and H J Griesser ldquoBiomedicaldegradation of polyurethanesrdquo in Biomedical Applications ofPolyurethanes P Vermette Ed pp 97ndash159 Landes Bioscience2001

[17] MK LambaKAWoodhouse andS LCooperPolyurethanesin Biomedical Applications CRC Press 1997

[18] E F Lucas B G Soares and E E C Monteiro Caracterizacaode polımeros determinacao de peso molecular e analise termicaE E-papers Ed Rio de Janeiro Brazil 1st edition 2001

[19] Z Dai J Ronholm Y Tian B Sethi and X Cao ldquoSterilizationtechniques for biodegradable scaffolds in tissue engineeringapplicationsrdquo Journal of Tissue Engineering vol 7 2016

[20] D Mohr M Wolff and T Kissel ldquoGamma irradiation forterminal sterilization of 17120573-estradiol loaded poly-(DL-lactide-co-glycolide) microparticlesrdquo Journal of Controlled Release vol61 no 1-2 pp 203ndash217 1999


[21] ISO 11137 ldquoSterilisation of Health Care Products Requirementsfor Validation and Routine Control Radiation Sterilisationrdquo2011

[22] C E Holy C Cheng J E Davies and M S ShoichetldquoOptimizing the sterilization of PLGA scaffolds for use in tissueengineeringrdquo Biomaterials vol 22 no 1 pp 25ndash31 2001

[23] T J Kinnari J Esteban N Zamora et al ldquoEffect of surfaceroughness and sterilization on bacterial adherence to ultra-high molecular weight polyethylenerdquo Clinical Microbiology andInfection vol 16 no 7 pp 1036ndash1041 2010

[24] R Colaco E Pires T Costa and A P Serro ldquoInfluence ofsterilization with y-irradiation in the degradation of plasmasprayed hydroxyapatite coatingsrdquoMaterials Science Forum vol514ndash516 no 2 pp 1054ndash1058 2006

[25] K A Hooper J D Cox and J Kohn ldquoComparison of the effectof ethylene oxide and 120574-irradiation on selected tyrosine-derivedpolycarbonates and poly(L-lactic acid)rdquo Journal of AppliedPolymer Science vol 63 no 11 pp 1499ndash1510 1997

[26] B Madsen ldquoEffect of sterilisation techniques on the physic-ochemical properties of polysulfone hollow fibersrdquo Journal ofApplied Polymer Science pp 3429ndash3436 2011

[27] T TunaMWeinM Swain J Fischer andWAtt ldquoInfluence ofultraviolet photofunctionalization on the surface characteristicsof zirconia-based dental implant materialsrdquo Dental Materialsvol 31 no 2 pp e14ndashe24 2015

[28] T Zubkoy D Stahl T L Thompson D Panayotov O Diwaldand J T Yates Jr ldquoUltraviolet light-induced hydrophilicity effecton TiO2(110) (1times1) Dominant role of the photooxidation ofadsorbed hydrocarbons causing wetting by water dropletsrdquoTheJournal of Physical Chemistry B vol 109 no 32 pp 15454ndash154622005

[29] H Zhang S Komasa C Mashimo T Sekino and J OkazakildquoEffect of ultraviolet treatment on bacterial attachment andosteogenic activity to alkali-treated titanium with nanonetworkstructuresrdquo International Journal of Nanomedicine vol 12 pp4633ndash4646 2017

[30] J F Gardner and M M Peel Introduction to SterilisationDisinfection and Infection Control Churchill Livingstone NewYork NY USA 1991


[32] T Hoshino I Saito R Kometani et al ldquoImprovement ofneuronal cell adhesiveness on parylene with oxygen plasmatreatmentrdquo Journal of Bioscience and Bioengineering vol 113 no3 pp 395ndash398 2012

[33] L Canullo T Genova H-L Wang S Carossa and F MussanoldquoPlasma of argon increases cell attachment and bacterial decon-tamination on different implant surfacesrdquo The InternationalJournal of Oral ampMaxillofacial Implants vol 32 no 6 pp 1315ndash1323 2017

[34] S S Block Disinfection Sterilization and Preservation Lippin-cott Williams andWilkins 2001

[35] M Laroussi and F Leipold ldquoEvaluation of the roles of reactivespecies heat and UV radiation in the inactivation of bacterialcells by air plasmas at atmospheric pressurerdquo InternationalJournal of Mass Spectrometry vol 233 no 1ndash3 pp 81ndash86 2004

[36] M Moreau N Orange and M G J Feuilloley ldquoNon-thermalplasma technologies New tools for bio-decontaminationrdquoBiotechnology Advances vol 26 no 6 pp 610ndash617 2008

[37] M Annunziata L Canullo G Donnarumma P Caputo LNastri and L Guida ldquoBacterial inactivationsterilization byargon plasma treatment on contaminated titanium implantsurfaces In vitro studyrdquoMedicina Oral Patologıa Oral y CirugıaBucal vol 21 no 1 pp e118ndashe121 2016

[38] A Rainer M Centola C Spadaccio et al ldquoComparative studyof different techniques for the sterilization of poly-L-lactideelectrospunmicrofibers Effectiveness versusmaterial degrada-tionrdquo The International Journal of Artificial Organs vol 33 no2 pp 76ndash85 2010

CorrosionInternational Journal of

Hindawiwwwhindawicom Volume 2018

Advances in

Materials Science and EngineeringHindawiwwwhindawicom Volume 2018


Journal of

Chemistry

Analytical ChemistryInternational Journal of


ScienticaHindawiwwwhindawicom Volume 2018

Polymer ScienceInternational Journal of



Advances in Condensed Matter Physics


International Journal of

BiomaterialsHindawiwwwhindawicom

Journal ofEngineeringVolume 2018

Applied ChemistryJournal of


NanotechnologyHindawiwwwhindawicom Volume 2018

Journal of


High Energy PhysicsAdvances in

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

TribologyAdvances in



ChemistryAdvances in


Advances inPhysical Chemistry


BioMed Research InternationalMaterials

Journal of


Na

nom

ate

ria

ls


Journal ofNanomaterials

Submit your manuscripts atwwwhindawicom


[2 3] For economical and practical reasons the latter strategyhas been employed with polymeric scaffolds intended forin vivo use and is considered a more realistic approach toachieve sterile implantable scaffolds [1 2] Nevertheless thechallenge remains to determine an efficient and nondestruc-tive sterilisation procedure for polymer scaffolds that pre-serves their structure and surface properties [3] Sterilisationtechniques may influence a materialrsquos structural chemicaland biological properties thus it is important to ensure themodality implemented does not affect biocompatibility [3]Sterilisation of biomaterials accepted by the FDA for medicaldevices includes ethylene oxide autoclaving and gammasterilisation [4] The success of an implant for sterilisationis dependent not only on the implant remaining sterilebut also on achieving sterility without adversely affectingthe materialrsquos properties Different sterilisation agents haveshown that they can attack polymers causing hydrolysismelting or depolymerisation [5 6]

Our group have developed and patented two familiesof nanocomposite polymers for the development of organsand tissues [7ndash9] The nonbiodegradable polymer incorpo-rates POSS nanoparticles into polycarbonate-based urea-urethane (POSS-PCU UCL-Nano) Its biodegradable coun-terpart modifies poly(caprolactone urea-urethane) POSS-PCL Understanding an appropriate sterilisation techniquefor POSS-PCU and POSS-PCL is crucial for translation toclinical practice A brief previous study compared threetechniques of sterilising POSS-PCU and POSS-PCL scaffoldsincluding autoclaving gamma irradiation and ethanol [7]Autoclaving was found to be effective in maintaining steril-isation of the scaffolds without degrading the material Thefirst authors of this paper have also demonstrated that bleachmay be useful for sterilising POSS-PCL scaffolds comparedto ethanol and autoclaving sterilisation [8] Adipose derivedstem cells (ADSCs) were shown to adhere to the POSS-PCL scaffolds following ethanol and bleach sterilisation [8]Lastly a study comparing the effects of autoclave microwaveantibiotics and 70 ethanol sterilisation on POSS-PCLscaffolds found ethanol to be a suitable sterilisation techniquewith maintained fibroblast attachment [9] The aim of thisstudy was to compare all available sterilisation techniquesfor POSS-PCU and POSS-PCL in a single study includinga new method using argon plasma sterilisation buildingon previous studies to understand the optimal sterilisationtechnique for nanocomposite scaffolds

An increasingly popular method of modifying the sur-face functionality to enhance cell behaviour on scaffolds isplasma modification [10 11] Plasma consists of electronsions energy rich neutrals molecules fragments atomsand photons It can be under low atmospheric or highpressureThe distinct behaviour of gases led to the suggestionthat plasma is the ldquofourth state of matterrdquo [12] Plasmamodification (PM) is an easy reliable clean way of creatingreactive functional groups on the surfaces of biomaterials andfor creating anchoring sites for further chemical reactions[12]

This study compared ethylene oxide argon plasmableach antibioticantimycotic ethanol ultraviolet radiationautoclaving and gamma irradiation sterilisation procedures

for morphological alteration chemical damage effects onpolymer degradation and biocompatibility The viability ofADSCs was assessed following the sterilisation of POSS-PCUand POSS-PCL scaffolds to assess biocompatibility of thesterilisation techniques

2 Materials and Methods

21 Polymer Synthesis

211 POSS-PCU The nanocomposite scaffolds were man-ufactured as previously described [7 13] In brief polycar-bonate polyol 2000mwt and trans-cyclohexanechloroydrin-isobutyl-silsesquioxane (Hybrid Plastics Inc) was heated to135∘C and then cooled to 70∘C Flakes of 441015840-methylenebis(phenyl isocyanate) (MDI) were then added to the mixtureat 75ndash85∘C for 90minutes to form the prepolymerThenNN-dimethylacetamide (DMAc) was to form a solution Furtherchain extension was completed by the drop-wise additionof ethylenediamine and diethylamine in DMAc This thencreated the POSS-modified polycarbonate urea-urethane inDMAc All chemicals and reagents were purchased fromAldrich Limited Gillingham UK

212 POSS-PCL POSS-PCL nanocomposites solution wasmanufactured as described previously [7] In brief polyca-prolactone diol (2000 gmol) and trans-cyclohexanechloro-hydrinisobutyl-polyhedral oligomeric silsesquioxane (POSS)were mixed and heated to 135∘C Then 94 g of 441015840-methylenebis(cyclohexylisocyanate) was added to form aprepolymer Following this 100 g of DMAC was added tothe prepolymer Chain extension was performed by drop-wise addition of 1 g of ethylenediamine in 80 g of dry DMACFollowing this 2 g of 1-butanol in 5 g of DMAC was addedto form the nanocomposite All chemicals and reagents werepurchased from Aldrich Limited Gillingham UK

213 Sample Preparation Polymers were fabricated as 3Dscaffolds using the phase separationparticulate-leachingtechnique as described previously [13] Firstly NaCL (200-250120583m) was dissolved in POSS-PCL and POSS-PCU inDMAc containing Tween-20 surfactant (11 weight ratio)Thesolution was dispersed and degassed in a Thinky AER 250mixer (Intertonics Kidlington UK) The polymer mixturewas spread onto steel moulds The moulds were then washedin deionised water to dissolve the solvent and DMAC for 7days Following washing polymer sheets with 700-800120583mthicknessweremanufactured For cell culture analysis 16 mmpolymer disks were cut from the sheets using a steel manualshape cutter

22 Sterilisation

221 Gamma Irradiation Scaffolds were irradiated with adose of 25 kGy at room temperature using a 60Co gamma-ray source (Synergy Health Swindon UK) Scaffolds wereexposed on a continuous path for 10 hours as describedpreviously [7]
















24 Cytotoxicity










3 Results







Table1Mecha

nicalP

rope

rtieso

fPOSS

-PCUan

dPO

SS-PCLaft

ersterilisatio

nprocessesYo

ungrsquos

mod

ulusm

axim

umstr

esselon

gatio

natbreakandthickn

esso

fcon

trolP

OSS-PCL

andPO

SS-PCU

andsamples

subjectedto

ethano

lbleach


oxideUVradiation

antib

iotic

antim

ycotictre


n

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Gam

ma

Ethy

lene

Oxide

UV

Antibiotic

Antim

ycotic

Plasma

Autoclaving

Youn

grsquosMod

ulus

(MPa)PO

SS-PCL

018plusmn00

029plusmn002

020plusmn002

032plusmn004

040plusmn011

035plusmn002

025plusmn003

030plusmn005

NA

POSS-PCU

055plusmn004

057plusmn002

056plusmn002

056plusmn004

055plusmn002

055plusmn002

053plusmn001

055plusmn003

054plusmn002

Maxim

umStress(M

Pa)

POSS-PCL

056plusmn008

061plusmn010

031plusmn011

117plusmn015

171plusmn

019

145plusmn004

110plusmn014

080plusmn026

NA

POSS-PCU

083plusmn003

082plusmn006

085plusmn003

083plusmn001

084plusmn001

083plusmn001

081plusmn012

083plusmn002

082plusmn004

Elon

gatio

natbreak(

)PO

SS-PCL

5896plusmn262

5497plusmn68

2930plusmn61

4622plusmn16

4716plusmn39

3875plusmn22

4695plusmn11

5001plusmn

30NA

POSS-PCU

2833plusmn95

72736plusmn1001

2792plusmn174

2880plusmn305

2886plusmn14

62693plusmn855

2738plusmn90

12811plusmn412

2805plusmn78

8

Thickn

ess(mm)

POSS-PCL

20plusmn0

143plusmn015

1525plusmn013

16plusmn022

12plusmn025

138plusmn005

155plusmn017

2433plusmn

012

NA

POSS-PCU

075plusmn004

077plusmn002

084plusmn002

077plusmn005

076plusmn002

077plusmn002

075plusmn003

076plusmn002

074plusmn006


POSS-PCL POSS-PCU

lowastlowastlowast

0

50

100

150

Con

tact

Ang

le (

∘)

lowast



PlasmaAutoclaving

lowastlowastlowast

lowast



Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Abs

Wavenumber (cm -1)

04

03

02

01

0


Autoclaving


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm -1)

07

06

04

02

0

minus01

Abs







119899by 10 in





Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200




UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls



















24 Cytotoxicity










3 Results







Table1Mecha

nicalP

rope

rtieso

fPOSS

-PCUan

dPO

SS-PCLaft

ersterilisatio

nprocessesYo

ungrsquos

mod

ulusm

axim

umstr

esselon

gatio

natbreakandthickn

esso

fcon

trolP

OSS-PCL

andPO

SS-PCU

andsamples

subjectedto

ethano

lbleach


oxideUVradiation

antib

iotic

antim

ycotictre


n

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Gam

ma

Ethy

lene

Oxide

UV

Antibiotic

Antim

ycotic

Plasma

Autoclaving

Youn

grsquosMod

ulus

(MPa)PO

SS-PCL

018plusmn00

029plusmn002

020plusmn002

032plusmn004

040plusmn011

035plusmn002

025plusmn003

030plusmn005

NA

POSS-PCU

055plusmn004

057plusmn002

056plusmn002

056plusmn004

055plusmn002

055plusmn002

053plusmn001

055plusmn003

054plusmn002

Maxim

umStress(M

Pa)

POSS-PCL

056plusmn008

061plusmn010

031plusmn011

117plusmn015

171plusmn

019

145plusmn004

110plusmn014

080plusmn026

NA

POSS-PCU

083plusmn003

082plusmn006

085plusmn003

083plusmn001

084plusmn001

083plusmn001

081plusmn012

083plusmn002

082plusmn004

Elon

gatio

natbreak(

)PO

SS-PCL

5896plusmn262

5497plusmn68

2930plusmn61

4622plusmn16

4716plusmn39

3875plusmn22

4695plusmn11

5001plusmn

30NA

POSS-PCU

2833plusmn95

72736plusmn1001

2792plusmn174

2880plusmn305

2886plusmn14

62693plusmn855

2738plusmn90

12811plusmn412

2805plusmn78

8

Thickn

ess(mm)

POSS-PCL

20plusmn0

143plusmn015

1525plusmn013

16plusmn022

12plusmn025

138plusmn005

155plusmn017

2433plusmn

012

NA

POSS-PCU

075plusmn004

077plusmn002

084plusmn002

077plusmn005

076plusmn002

077plusmn002

075plusmn003

076plusmn002

074plusmn006


POSS-PCL POSS-PCU

lowastlowastlowast

0

50

100

150

Con

tact

Ang

le (

∘)

lowast



PlasmaAutoclaving

lowastlowastlowast

lowast



Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Abs

Wavenumber (cm -1)

04

03

02

01

0


Autoclaving


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm -1)

07

06

04

02

0

minus01

Abs







119899by 10 in





Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200




UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls












3 Results







Table1Mecha

nicalP

rope

rtieso

fPOSS

-PCUan

dPO

SS-PCLaft

ersterilisatio

nprocessesYo

ungrsquos

mod

ulusm

axim

umstr

esselon

gatio

natbreakandthickn

esso

fcon

trolP

OSS-PCL

andPO

SS-PCU

andsamples

subjectedto

ethano

lbleach


oxideUVradiation

antib

iotic

antim

ycotictre


n

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Gam

ma

Ethy

lene

Oxide

UV

Antibiotic

Antim

ycotic

Plasma

Autoclaving

Youn

grsquosMod

ulus

(MPa)PO

SS-PCL

018plusmn00

029plusmn002

020plusmn002

032plusmn004

040plusmn011

035plusmn002

025plusmn003

030plusmn005

NA

POSS-PCU

055plusmn004

057plusmn002

056plusmn002

056plusmn004

055plusmn002

055plusmn002

053plusmn001

055plusmn003

054plusmn002

Maxim

umStress(M

Pa)

POSS-PCL

056plusmn008

061plusmn010

031plusmn011

117plusmn015

171plusmn

019

145plusmn004

110plusmn014

080plusmn026

NA

POSS-PCU

083plusmn003

082plusmn006

085plusmn003

083plusmn001

084plusmn001

083plusmn001

081plusmn012

083plusmn002

082plusmn004

Elon

gatio

natbreak(

)PO

SS-PCL

5896plusmn262

5497plusmn68

2930plusmn61

4622plusmn16

4716plusmn39

3875plusmn22

4695plusmn11

5001plusmn

30NA

POSS-PCU

2833plusmn95

72736plusmn1001

2792plusmn174

2880plusmn305

2886plusmn14

62693plusmn855

2738plusmn90

12811plusmn412

2805plusmn78

8

Thickn

ess(mm)

POSS-PCL

20plusmn0

143plusmn015

1525plusmn013

16plusmn022

12plusmn025

138plusmn005

155plusmn017

2433plusmn

012

NA

POSS-PCU

075plusmn004

077plusmn002

084plusmn002

077plusmn005

076plusmn002

077plusmn002

075plusmn003

076plusmn002

074plusmn006


POSS-PCL POSS-PCU

lowastlowastlowast

0

50

100

150

Con

tact

Ang

le (

∘)

lowast



PlasmaAutoclaving

lowastlowastlowast

lowast



Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Abs

Wavenumber (cm -1)

04

03

02

01

0


Autoclaving


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm -1)

07

06

04

02

0

minus01

Abs







119899by 10 in





Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200




UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





Table1Mecha

nicalP

rope

rtieso

fPOSS

-PCUan

dPO

SS-PCLaft

ersterilisatio

nprocessesYo

ungrsquos

mod

ulusm

axim

umstr

esselon

gatio

natbreakandthickn

esso

fcon

trolP

OSS-PCL

andPO

SS-PCU

andsamples

subjectedto

ethano

lbleach


oxideUVradiation

antib

iotic

antim

ycotictre


n

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Gam

ma

Ethy

lene

Oxide

UV

Antibiotic

Antim

ycotic

Plasma

Autoclaving

Youn

grsquosMod

ulus

(MPa)PO

SS-PCL

018plusmn00

029plusmn002

020plusmn002

032plusmn004

040plusmn011

035plusmn002

025plusmn003

030plusmn005

NA

POSS-PCU

055plusmn004

057plusmn002

056plusmn002

056plusmn004

055plusmn002

055plusmn002

053plusmn001

055plusmn003

054plusmn002

Maxim

umStress(M

Pa)

POSS-PCL

056plusmn008

061plusmn010

031plusmn011

117plusmn015

171plusmn

019

145plusmn004

110plusmn014

080plusmn026

NA

POSS-PCU

083plusmn003

082plusmn006

085plusmn003

083plusmn001

084plusmn001

083plusmn001

081plusmn012

083plusmn002

082plusmn004

Elon

gatio

natbreak(

)PO

SS-PCL

5896plusmn262

5497plusmn68

2930plusmn61

4622plusmn16

4716plusmn39

3875plusmn22

4695plusmn11

5001plusmn

30NA

POSS-PCU

2833plusmn95

72736plusmn1001

2792plusmn174

2880plusmn305

2886plusmn14

62693plusmn855

2738plusmn90

12811plusmn412

2805plusmn78

8

Thickn

ess(mm)

POSS-PCL

20plusmn0

143plusmn015

1525plusmn013

16plusmn022

12plusmn025

138plusmn005

155plusmn017

2433plusmn

012

NA

POSS-PCU

075plusmn004

077plusmn002

084plusmn002

077plusmn005

076plusmn002

077plusmn002

075plusmn003

076plusmn002

074plusmn006


POSS-PCL POSS-PCU

lowastlowastlowast

0

50

100

150

Con

tact

Ang

le (

∘)

lowast



PlasmaAutoclaving

lowastlowastlowast

lowast



Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Abs

Wavenumber (cm -1)

04

03

02

01

0


Autoclaving


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm -1)

07

06

04

02

0

minus01

Abs







119899by 10 in





Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200




UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





POSS-PCL POSS-PCU

lowastlowastlowast

0

50

100

150

Con

tact

Ang

le (

∘)

lowast



PlasmaAutoclaving

lowastlowastlowast

lowast



Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Abs

Wavenumber (cm -1)

04

03

02

01

0


Autoclaving


Plasma

Control

Bleach (SDIC)

Ethanol

Gamma

Ethylene Oxide

4000

3800

3600

3400

3200

3000

2800

2600

2400

2200

2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm -1)

07

06

04

02

0

minus01

Abs







119899by 10 in





Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200




UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





Table2Su

mmaryo

fgelperm

eatio

nchromatog

raph

y(GPC

)resultsM

olecularwe

ight

(119872w)and

molecular

number(119872

n)valuesofPO

SS-PCL

andPO

SS-PCU

after

different

sterilisa

tion

techniqu

es

Sterilisatio

nMetho

dCon

trol

Bleach

(SDIC

)Etha

nol

Ethy

lene

Oxide

Gam

ma

Plasma

Antibiotic

Antim

ycotic

Autoclaving

UV

Mass-averageM

olecular

Weigh

t(Mw)(gmol)

POSS-PCL

36110

03509

00356100

276300

151200

358600

391600

1746

00365400

POSS-PCU

91200

9200

08940

0112

200

135000

90800

92700

88300

93800

Num

ber-averageM

olecular

Weigh

t(Mn)

(gm

ol)PO

SS-PCL

141000

137600

111300

97500

44700

113300

160800

88100

155300

POSS-PCU

47700

46300

47200

34200

4000

046

700

46200

45500

46200




UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls







UVEthanol

EthyleneOxide

GammaPlasma


UVEthanol

EthyleneOxide

GammaPlasma

Autoclaving

2 m









0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





0

20

40

60

80

100

120

Time points (days)


1 141073

Abs

orba

nce

570

nm



lowast lowastlowast

lowast lowast

lowast

lowast

lowastlowast

lowastlowast

(a)

0

20

40

60

80

100

120


Abs

orba

nce

570

nm


UV

Autoclaving



lowast

lowast

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(b)








POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls





POSS-PCL POSS-PCU


Ethylene Oxide

Ethanol

Gamma

Plasma

UV


UV

Ethanol

Plasma

Autoclaving

Ethylene oxide

Gamma


20 m


POSS-PCL POSS-PCU00

02

04

06

08

10

Circ

ular

ity In

dex


UV

Autoclaving



Figure 5





POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls








POSS-PCU TSB 99 09 09 09 09 09 09 09 09THY 99 09 09 09 09 09 09 09 09


4 Discussion



















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls
















5 Conclusion


Data Availability


Disclosure




Acknowledgments


References










































Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls

























Advances in



Journal of

Chemistry















Journal of





Volume 2018









Journal of


Na

nom

ate

ria

ls




Evaluation of Sterilisation Techniques for Regenerative...

Documents

Transcript of Evaluation of Sterilisation Techniques for Regenerative...