Research Article Detergent-Enzymatic Decellularization of ...Detergent-Enzymatic Decellularization of Swine Blood Vessels: Insight on Mechanical Properties for ... BioMed Research

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 918753 8 pageshttpdxdoiorg1011552013918753

Research ArticleDetergent-Enzymatic Decellularization ofSwine Blood Vessels Insight on Mechanical Properties forVascular Tissue Engineering

Alessandro F Pellegata12 M Adelaide Asnaghi1 Ilaria Stefani12

Anna Maestroni3 Silvia Maestroni3 Tommaso Dominioni4 Sandro Zonta4

Gianpaolo Zerbini3 and Sara Mantero1

1 Department of Chemistry Materials and Chemical Engineering ldquoGiulio Nattardquo Politecnico di Milano Milan Italy2 PhD program in Bioengineering Politecnico di Milano Milan Italy3 Complication of Diabetes Unit Division of Metabolic and Cardiovascular Sciences San Raffaele Scientific InstituteMilan Italy

4General Surgery I Fondazione IRCCS Policlinico San Matteo Pavia Italy

Correspondence should be addressed to Alessandro F Pellegata alessandropellegatamailpolimiit

Received 8 January 2013 Revised 29 April 2013 Accepted 23 May 2013

Academic Editor George E Plopper

Copyright copy 2013 Alessandro F Pellegata et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Small caliber vessels substitutes still remain an unmet clinical need few autologous substitutes are available while synthetic graftsshow insufficient patency in the long term Decellularization is the complete removal of all cellular and nuclear matters from atissue while leaving a preserved extracellular matrix representing a promising tool for the generation of acellular scaffolds fortissue engineering already used for various tissues with positive outcomes The aim of this work is to investigate the effect of adetergent-enzymatic decellularization protocol on swine arteries in terms of cell removal extracellular matrix preservation andmechanical properties Furthermore the effect of storage at minus80∘C on the mechanical properties of the tissue is evaluated Swinearteries were harvested frozen and decellularized histological analysis revealed complete cell removal and preserved extracellularmatrix Furthermore the residual DNA content in decellularized tissues was far low compared to native one Mechanical testingswere performed on native defrozen and decellularized tissues no statistically significant differences were reported for Youngrsquosmodulus ultimate stress compliance burst pressure and suture retention strength while ultimate strain and stress relaxation ofdecellularized vessels were significantly different from the native ones Considering the overall results the process was confirmedto be suitable for the generation of acellular scaffolds for vascular tissue engineering

1 Introduction

Cardiovascular diseases still represent one of the leadingcauses of death in the world [1] Current clinical approachesshowed a poor efficacy especially in small diameter arterysubstitutions (lt6mm) Autologous transplantation usingsaphenous vein or mammary artery is currently the bestsolution however this approach is not always feasible sincemost vessels are often affected by diffuse atheroscleroticabnormalities and only few vessels remain indeed suitablefor this purpose Another option is represented by the use of

synthetic grafts which allow positive outcomes when usedin large diameter vessels substitution but often lead to thefailure of the implant due to thrombotic phenomena orintimal hyperplasia when applied in small caliber ones [2]Themechanical behavior of the implant was proven to be oneof themain causes of lumen occlusions because of compliancemismatch between the native vessel and the synthetic graft [34] in order to compensate for the mechanical difference anin vivo remodeling occurs leading to progressive vessel wallthickening until complete occlusion Furthermore mechan-ical mismatching could generate nonphysiological blood

2 BioMed Research International

flows encouraging the formation of thrombi or aneurysmsBased on these observations the ideal vascular graft shouldresist the physiological arterial pressure and should avoidany mechanical mismatch Tissue engineering can overcomethe limitations of the currently available vessel substitutesthrough the generation of biologically based functional ves-sels which could more closely replicate the physiologicaltissue Better tuned biochemical and mechanical propertiesof tissue-engineered blood vessels (TEBV) can lead to long-term patency even in the case of small caliber conduitsDifferent approaches have already been investigated in thiscontext and have shown promising results in the last years[5ndash8]

One technique that has shown good preclinical andclinical results in several tissue engineering applications [9ndash12] including blood vessels [7] is the use of decellularizedscaffolds Decellularization is the complete removal of allcellular and nuclearmatters from a tissue preserving its nativeextracellular matrix (ECM) and could be performed throughphysical chemical and enzymatic agents [13] The obtainedacellular scaffolds represent a great substrate able to promotecell adhesion differentiation and proliferation thanks tothe specific ECM components and structure Moreoverdecellularized scaffolds sport mechanical properties similarto those of native tissues a key feature for blood vessels tissueengineering as discussed earlier

Following the promising results collected in a clinicalscenario in the context of trachea engineering [14 15] we areexploring the feasibility of transposing that methodology toblood vessels in vitro regeneration To this aim the presentwork focuses on the decellularization of porcine arteriesinvestigating cell removal extracellular matrix preservationand mechanical properties of the treated vessels compared tonative ones

In addition to this analysis and considerations are maderegarding the generation of an ldquooff the shelf rdquo productdesirable for an effective translation of tissue-engineeredtissues to the clinic Since explants from cadavers are themain source for the production of decellularized scaffolds asmuch tissue as possible would need to be harvested wheneveravailable and the storage of the starting material acquiresa key relevance For this reason we also analyzed how theminus80∘C storage affects the mechanical properties of explantedvessels prior to decellularization

2 Materials and Methods

21 Tissue Harvesting Arterial segments (inner diameterfrom 2 to 11mm) were obtained from abdominal aorta andcarotid from 10 pigs (Large White Piglets) under generalanesthesia and orotracheal ventilationThe study was carriedout under Ethical Committee for Animal Experimentationsof University of Pavia (permit 12012) approval and followingItalian laws for animal care Fine dissection was performedusing scalpel and scissor providing complete removal ofloose connective tissue around the vessels Each segmentwas rinsed three times in Dulbeccorsquos phosphate bufferedsaline (PBS) containing 1 penicillin 1 streptomycin and

1 amphotericin B (AA solution all reagents Sigma-AldrichSt Louis MO USA) Vessels were either directly processedfor characterization of the native tissue or stored dry at minus80∘Cuntil decellularization

22 Decellularization Vessels were decellularized accordingto a previously tested protocol using an in-house developeddevice for the automatic decellularization of biological tissues[16] Briefly the treatment consists of 4 cycles as follows72 h washing in deionized water with 1 AA solution at 4∘Cfollowed by 4 h in sodium deoxycholate 4 solution at roomtemperature (RT) and 3 h in deoxyribonuclease I 2000 kU insodiumchloride 1Mat RTThewhole processwas done underagitation and between each step the vessel was rinsed 3 timesin PBS (all reagents Sigma-Aldrich St Louis MO USA)

23 Histology At retrieval and after decellularization spec-imens of the vessels were isolated and processed inparaformaldehyde 4 inPBS then embedded in paraffin and4 120583m slices were stained with hematoxylin-eosin and 410158406-diamidino-2-phenylindole (DAPI) as nuclear marker

24 DNA Quantification At native condition and afterdecellularization the total amount of DNA was quantifiedusing a DNA isolation kit for tissues (Qiagen Venlo TheNetherlands) Briefly 40mgwet weight samples of native anddecellularized arteries were digested using a cell lysis bufferand Proteinase K followed by a protein precipitation solutionand centrifugation to remove the protein fraction In order toisolate the DNA the supernatant was added with isopropanoland ethanol and then centrifuged and rehydratedwith aDNArehydrating solution Total DNA was finally quantified usinga spectrophotometer at 260 nm

25 Scanning Electron Microscopy Samples of native defro-zen and decellularized arteries were fixed with 25 glu-taraldehyde for 1 h and stored in sodium cacodylate 01MDehydrationwas achieved using an ethanol scale and sampleswere gold coated before being analyzed with a Zeiss EVO 50environmental scanning electron microscope (ESEM)

26 Mechanical Testing Native defrozen and decellularizedvessels were cut into ring-shaped specimens for mechanicalanalysis Native tissue samples were tested within 4 hoursof cold ischemia after retrieval Mechanical testing wasperformed according to a previously developed protocol [17]with some modifications as follows 5mm wide specimenswere mounted on custom-made holders on a computer-controlled uniaxial testing machine (Synergie 200H MTSMinneapolis MN USA) and data were acquired usingthe Test Work software (MTS Minneapolis MN USA) Apreloading of 0015N was imposed and the correspond-ing length was measured Each sample was preconditionedwith loading-unloading cycles until a reproducible responsewas achieved with a loading speed of 10mmmin anda load end point of 008Nmm The sample was left torecover for 120 seconds repeatedly rehydrated with PBS anda relaxation test was then performed with a ramp speed of

BioMed Research International 3

(a) (b)

(c) (d)

Figure 1 Hematoxylin and eosin staining of swine arteries Hematoxylin and eosin staining of native ((a)-(b)) and decellularized ((c)-(d))porcine arteries the images show a complete absence of nuclei or cellular residues and a preserved ECM structure at the end of the process

(a) (b)

(c) (d)

Figure 2 DAPI staining of swine arteries DAPI fluorescent staining of native ((a)-(b)) and decellularized ((c)-(d)) porcine arteries theresults confirm the absence of nuclei in the decellularized tissue


Native Decellularized0

1000

2000

3000

4000DNA content (ngmg)

Figure 3 Residual DNA content DNA content for native anddecellularized swine arterial vessels data are reported as mean plusmnstandard deviations

01mms The ramp end point was set at 008Nmm Thetest was stopped after 500 seconds and the sample left torecover for additional 120 seconds repeatedly rehydratedwith PBS A failure tensile test was finally carried out with anactuator speed of 10mmmin until complete sample ruptureMeasurements of the vessels wall thickness were made usinga digital camera Canon EOS 350D (Canon Tokyo Japan)equipped with a macro lens to avoid optical distortionImages were processed using ImageJ software (ImageJ NIHUSA)measuring themean inner diameter (ID) and themeanwall thickness (WT)Thirty samples representatives of the 2ndash11mm ID range were analyzed and an ID-WT relation wascalculated by interpolation of the experimental dataset Thedata from the mechanical testing were processed using OpenOffice Calc (Apache Software Foundation Forest Hill USA)From the loading ramp of the relaxation test the Stiffness(119870mdash[Nmm]) was evaluated as

119870 =

119879

008minus 119879

004

120576

008minus 120576

004

(1)

where 119879 is the tensile stress (Nmm) and 120576 the circumfer-ential strain (mmmm) The vessel wall thickness allowed tocalculate the circumferential Young modulus (119864circmdash[MPa])derived as

119864circ =119870

2WT

(2)

whereWT is the wall thickness derived from the ID-WT rela-tion (see previous paragraph) Compliance was derived froma mathematical model previously described [18] introducingan equivalence criteria between 119879 and the internal pressure(119875) and between 120576 and the diameter (119863) Moreover fromthe relaxation test we evaluated the residual stress (119866) as the

ratio between 119879 after 500 s and 119879 at the end of the loadingramp

119866 =

119879

500 s119879

0

(3)

From the rupture test we estimated the ultimate ten-sile stress (119879maxmdashNmm) and the ultimate strain (120576maxmdashmmmm) as themaximum119879 and 120576 values before failure of thesample like Youngrsquos modulus the circumferential ultimatestress (120590maxmdashMPa) was calculated using the ID-WT relationas

120590max =119879max2WT

(4)

The burst pressure (BPmdashmmHg) was calculated from therupture test using the same equivalence model used forthe compliance the pressure was derived from the ultimatetensile stress and the diameter was derived from the ultimatestrain

The suture retention test was performed according to thestandard ISO 7198 vessel segments of 20mm in length wereclamped to the fixed holding of the testing machine and onthe other end of the vessel a single bite of 5ndash0 Prolene wasplaced 2mm under the edge and pulled at a constant rate of80mmmin until failure For each vessel the test was repeatedthree times with each suture bite at 120∘ with respect to theotherThe force needed to pull away the suture was measuredand reported as suture retention strength (SRmdashg)

27 Statistical Analysis Statistical analysis was performedusingGraphPad Prism (GraphPad Software Inc La Jolla CAUSA) and data were compared using a Kruskal-Wallis testwith Dunn post-test with significance limit set for 119875 lt 005

3 Results

31 Histological Results Native and decellularized vesselsamples were stained with hematoxylin-eosin and DAPITheoverall histological results as shown in Figures 1 and 2revealed the absence of cells or nuclear matter Hematoxylinand eosin staining (Figure 1) showed a preserved ECMstructure at the end of the decellularization process withno residual cells confirmed by DAPI fluorescent staining(Figure 2)

32 DNA Quantification Results The spectrophotometricanalysis revealed that the decellularization process removedthe majority of the DNA content in the treated tissuecompared to the native one (286 plusmn 83 ngmg versus 2586 plusmn542 ngmg) (Figure 3)

33 Scanning Electron Microscopy Results Native defrozenand decellularized vessels samples were freeze dried goldcoated and observed with ESEM The reported images referto the luminal surface of the vessels (Figure 4) no evidenceof damage can be observed after neither the freezing step(Figure 4(b)) nor the decellularization process (Figure 4(c))


(a) (b) (c)

Figure 4 Environmental Scanning ElectronMicroscopy ESEM analysis of the inner lumen of native (a) defrozen (b) and decellularized (c)porcine arteries Bars 10 120583m

04

06

08

1

12

14

16

18

2

2 4 6 8 10 12

Wal

l thi

ckne

ss (m

m)

Diameter (mm)

Figure 5 Wall thickness to inner diameter relation Wall thicknessto inner diameter relation derived from optical acquisition of pigarterial samples sections

34 Vessel Inner Diameter-Wall Thickness Relation In orderto calculate circumferential stress and Youngrsquos modulus thearea of the vessel wall has to be estimated [17] While thelength of the ring specimen is known (5mm) the wallthickness was measured by using an optical setup and relatedto the diameter of the vessel The results are shown inFigure 5

The interpolating function is

WT = 06151198900088ID (5)

where WT is the wall thickness and ID the inner diameter119877

2-value of the curve is 06894

35 Mechanical Testing Results Themechanical testing anal-ysis (Figure 6) resulted in no statistically significant differ-ences for Youngrsquos modulus compliance ultimate circumfer-ential stress burst pressure and suture retention strength onthe other hand there was a significant loss in ultimate strainbetween native and decellularized vessels moreover residualstress after relaxation was increased for decellularized sam-ples compared to native ones

4 Discussion

Decellularized scaffolds have been used to substitute varioustissues with promising results reaching clinical applicationThe extracellular matrix represents a good substrate in termsof biochemical properties able to drive cell adhesion prolif-eration and differentiation [19] moreover acellular scaffoldsusually show good biomechanical properties The presentwork is focused on the decellularization of arterial tissuetranslating the process we have previously used in the contextof engineered trachea transplantation with positive results interms of both biochemical and mechanical properties of thetreated matrix [14] Cell removal and mechanical propertiesof decellularized swine blood vessels are here investigated andcompared to the native tissue

The process showed good results no residual cells wereobserved in the hematoxylin-eosin (HE) staining and thiscondition was confirmed by DAPI analysis where no lastingnuclei were marked Furthermore the HE staining revealeda well-maintained structure of the vessel with preserved andorganized laminae

The residual DNA content after decellularization was farlow from the quantity found in the native tissue by now itis unclear whether this few residual DNA could elicit or notan adverse response from the host [20] and indeed manycommercial products with positive clinical outcomes containremnant DNA and it seems unlikely that residual fragmentscould play a role in any host response [21]

SEM analysis reported a preserved basal layer on theinner lumen of the vessel without evidence of any damagedarea Having a well-preserved inner layer is very importantfor tissue remodeling in the vascular field since this avoidsthe promotion of early thrombi and the molecular signalingof the ECM can promote the re-endothelialization Thislast aspect takes even more importance when consideringthe possibility of naked implants (decellularized scaffoldimplantedwithout cell seeding) as suggested byDahl et al [7]investigating whether a prior-to-grafting endothelial seedingis necessary or not becomes intriguing in the presenceof a signal-rich surface like the acellular one A propercharacterization of the decellularized lumen considering themost important proteins related to endothelium (or thrombi)formation will help in the interpretation of the phenomena


Suture retention strength (g)

N DF DC0

500

1000

1500

Ultimate stress (MPa)

0

1

2

3

N DF DC

Youngrsquos modulus (MPa)

0

01

02

03

N DF DC

Compliance (1mmHg)

0

10

20

30

40

N DF DC

0

Ultimate strain (mmmm)

05

1

15

2

25

N DF DC

lowast

Burst pressure (mmHg)

0

1000

2000

3000

4000

N DF DC

Stress relaxation ()

04

05

06

07

08

09

1

N DF DC

lowast

Figure 6 Mechanical analysis Mechanical testing results for native (N) defrozen (DF) and decellularized (DC) swine arterial vessels Dataare reported as median and 5ndash95 percentiles lowast

119875

lt 005

Considering mechanical properties Youngrsquos moduluscompliance circumferential ultimate stress burst pressureand suture retention strength showed no significant differ-ences between nontreated and decellularized samples whileacellular specimens had a lower ultimate strain and a higherresidual stress compared to the native tissue Among theseparameters the compliance is identified as the most crucialfactor in eliciting intimal hyperplasia in case of mismatch atthe anastomosis sites [4] and in the vessel response to bloodpressure affecting tissue remodeling too With no statisticallysignificant differences in terms of compliance compared tonative tissue samples the decellularized vessels hold promisesof in vivo performance closely resembling the physiologicalones Considering the overall outcome the decellularizationprocess leads to limited modifications of the treated tissuescompared to native ones The histological analysis reportedno modifications in the ultrastructure of the matrix with nounfolded laminae and a well-preserved compact structure

however the loss of the cellular component could introducesome alterations in the lamina-to-lamina interactions whichcould be responsible for the slight modifications we haveobserved in the mechanical behavior (adhesiveness betweentwo adjacent layers) Regarding this various studies reportedchanges in the mechanical properties of decellularized tis-sues However discrepancies among the reported resultsexist and the data are hardly comparable because of thedifferent testing methods applied and the diverse parametersinvestigated Only few works have evaluated time-dependentmechanical parameters modifications on decellularized scaf-folds Zou and Zhang [22] reported the same trend wehave observed for stress relaxation and hypothesized thecause being the absence of smooth muscle cells (SMCs)Williams et al [23] claimed no differences between native anddecellularized arteries however the reference native vesselsin that study were stored at minus20∘C prior to characteriza-tion a condition that can alter SMCs functionality These


findings corroborate our results where no statistically sig-nificant differences were found in both nativedefrozen anddefrozendecellularized comparisons while some parameterswere significantly different between native and decellularizedsamples

The translation of tissue engineering techniques to theclinic setting is currently in the eye of the storm A crucialfactor in the case of decellularized scaffolds is the availabilityof the starting material at the time of need since the sourcesfor these acellular scaffolds are cadaveric donors the tissueavailability often does not match with patient needs Expect-ing a prior-to-process storage as the most frequent situationwe analyzed the effect of minus80∘C freezing on fresh arterialtissue Our results showed that this storage technique doesnot bring any significant mechanical alteration confirmingand integrating (stress-relaxation tests) previous results fromStemper et al [24] In order to properly address the wholedecellularized scaffold production for future clinical usefurther work will be aimed at investigating suitable storagemethods for the decellularized matrices

5 Conclusions

In the last years decellularized scaffolds emerged inside thetissue engineering scenario as a promising solution forvarious tissue the complete removal of cellular and nuclearmatter provides a promising substrate in terms of biochem-ical and mechanical properties This method reaches a keyimportance in the small caliber blood vessels field where fewclinical substitutes are available

The aim of this work was to assess the outcome on swinearteries of a detergent-enzymatic decellularization protocolin summary the proposed method led to complete cellremoval preserved ECM and limited alteration of the mech-anical properties of the tissues moreover freezing of thestarting material was also taken into account and evaluatedas a possible storage step in a future clinical scenario

In conclusion the present work confirmed decellular-ization as a promising solution for the production of bloodvessel acellularmatrices for vascular tissue engineering appli-cations

Conflict of Interests

The authors declare no conflict of interests

Acknowledgment

This work was supported by ldquoProgetti di Ricerca Indipen-dente (Sanita)rdquo Biovart-Decreto 13848 del 11122009 (Reg-ione Lombardia)

References

[1] M Peck D Gebhart N Dusserre T N McAllister and NLrsquoHeureux ldquoThe evolution of vascular tissue engineering andcurrent state of the artrdquo Cells Tissues Organs vol 195 no 1-2pp 144ndash158 2011

[2] O E Teebken and A Haverich ldquoTissue engineering of smalldiameter vascular graftsrdquo European Journal of Vascular andEndovascular Surgery vol 23 no 6 pp 475ndash485 2002

[3] R J van Det B H R Vriens J van der Palen and R HGeelkerken ldquoDacron or ePTFE for femoro-popliteal above-knee bypass grafting short- and long-term results of a mul-ticentre randomised trialrdquo European Journal of Vascular andEndovascular Surgery vol 37 no 4 pp 457ndash463 2009

[4] H L Prichard R JManson L DiBernardo L E Niklason J HLawson and S L M Dahl ldquoAn early study on the mechanismsthat allow tissue-engineered vascular grafts to resist intimalhyperplasiardquo Journal of Cardiovascular Translational Researchvol 4 no 5 pp 674ndash682 2011

[5] N Hibino E McGillicuddy G Matsumura et al ldquoLate-termresults of tissue-engineered vascular grafts in humansrdquo Journalof Thoracic and Cardiovascular Surgery vol 139 no 2 pp 431ndash436 2010

[6] T N McAllister M Maruszewski S A Garrido et al ldquoEffec-tiveness of haemodialysis access with an autologous tissue-engineered vascular graft a multicentre cohort studyrdquo TheLancet vol 373 no 9673 pp 1440ndash1446 2009

[7] S L M Dahl A P Kypson J H Lawson et al ldquoReadily avail-able tissue-engineered vascular graftsrdquo Science TranslationalMedicine vol 3 no 68 Article ID 68ra9 2011

[8] L E Niklason J Gao W M Abbott et al ldquoFunctional arteriesgrown in vitrordquo Science vol 284 no 5413 pp 489ndash493 1999

[9] Y Eitan U Sarig N Dahan andMMacHluf ldquoAcellular cardiacextracellular matrix as a scaffold for tissue engineering invitro cell support remodeling and biocompatibilityrdquo TissueEngineering C vol 16 no 4 pp 671ndash683 2010

[10] T H Petersen E A Calle L Zhao et al ldquoTissue-engineeredlungs for in vivo implantationrdquo Science vol 329 no 5991 pp538ndash541 2010

[11] A Soto-Gutierrez L Zhang CMedberry et al ldquoAwhole-organregenerative medicine approach for liver replacementrdquo TissueEngineering C vol 17 no 6 pp 677ndash686 2011

[12] A Asnaghi PMacchiarini and SMantero ldquoTissue engineeringtoward organ replacement a promising approach in airwaytransplantrdquo International Journal of Artificial Organs vol 32 no11 pp 763ndash768 2009

[13] S F Badylak D O Freytes and T W Gilbert ldquoExtracellularmatrix as a biological scaffold material structure and functionrdquoActa Biomaterialia vol 5 no 1 pp 1ndash13 2009

[14] P Macchiarini P Jungebluth T Go et al ldquoClinical transplan-tation of a tissue-engineered airwayrdquo The Lancet vol 372 no9655 pp 2023ndash2030 2008

[15] M A Asnaghi P Jungebluth M T Raimondi et al ldquoAdouble-chamber rotating bioreactor for the development oftissue-engineered hollow organs from concept to clinical trialrdquoBiomaterials vol 30 no 29 pp 5260ndash5269 2009

[16] A F Pellegata M A Asnaghi S Zonta G Zerbini and SMantero ldquoA novel device for the automatic decellularization ofbiological tissuesrdquo International Journal of Artificial Organs vol35 pp 191ndash198 2012

[17] A Remuzzi S Mantero M Colombo et al ldquoVascular smoothmuscle cells on hyaluronic acid culture and mechanical char-acterization of an engineered vascular constructrdquo Tissue Engi-neering vol 10 no 5-6 pp 699ndash710 2004

[18] V Quaglini T Villa FMigliavacca et al ldquoAn in vitromethodol-ogy for evaluating the mechanical properties of aortic vascularprosthesesrdquo Artificial Organs vol 26 no 6 pp 555ndash564 2002


[19] C A Barnes J Brison R Michel et al ldquoThe surface molecularfunctionality of decellularized extracellular matricesrdquo Biomate-rials vol 32 no 1 pp 137ndash143 2011

[20] T J Keane R Londono N J Turner and S F Badylak ldquoCon-sequences of ineffective decellularization of biologic scaffoldson the host responserdquo Biomaterials vol 33 no 6 pp 1771ndash17812012

[21] TW Gilbert J M Freund and S F Badylak ldquoQuantification ofDNA in biologic scaffoldmaterialsrdquo Journal of Surgical Researchvol 152 no 1 pp 135ndash139 2009

[22] Y Zou and Y Zhang ldquoMechanical evaluation of decellularizedporcine thoracic aortardquo Journal of Surgical Research vol 175 pp359ndash368 2012

[23] C Williams J Liao E M Joyce et al ldquoAltered structural andmechanical properties in decellularized rabbit carotid arteriesrdquoActa Biomaterialia vol 5 no 4 pp 993ndash1005 2009

[24] BD StemperN YoganandanMR Stineman TAGennarelliJ L Baisden and F A Pintar ldquoMechanics of fresh refrigeratedand frozen arterial tissuerdquo Journal of Surgical Research vol 139no 2 pp 236ndash242 2007

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flows encouraging the formation of thrombi or aneurysmsBased on these observations the ideal vascular graft shouldresist the physiological arterial pressure and should avoidany mechanical mismatch Tissue engineering can overcomethe limitations of the currently available vessel substitutesthrough the generation of biologically based functional ves-sels which could more closely replicate the physiologicaltissue Better tuned biochemical and mechanical propertiesof tissue-engineered blood vessels (TEBV) can lead to long-term patency even in the case of small caliber conduitsDifferent approaches have already been investigated in thiscontext and have shown promising results in the last years[5ndash8]

One technique that has shown good preclinical andclinical results in several tissue engineering applications [9ndash12] including blood vessels [7] is the use of decellularizedscaffolds Decellularization is the complete removal of allcellular and nuclearmatters from a tissue preserving its nativeextracellular matrix (ECM) and could be performed throughphysical chemical and enzymatic agents [13] The obtainedacellular scaffolds represent a great substrate able to promotecell adhesion differentiation and proliferation thanks tothe specific ECM components and structure Moreoverdecellularized scaffolds sport mechanical properties similarto those of native tissues a key feature for blood vessels tissueengineering as discussed earlier

Following the promising results collected in a clinicalscenario in the context of trachea engineering [14 15] we areexploring the feasibility of transposing that methodology toblood vessels in vitro regeneration To this aim the presentwork focuses on the decellularization of porcine arteriesinvestigating cell removal extracellular matrix preservationand mechanical properties of the treated vessels compared tonative ones

In addition to this analysis and considerations are maderegarding the generation of an ldquooff the shelf rdquo productdesirable for an effective translation of tissue-engineeredtissues to the clinic Since explants from cadavers are themain source for the production of decellularized scaffolds asmuch tissue as possible would need to be harvested wheneveravailable and the storage of the starting material acquiresa key relevance For this reason we also analyzed how theminus80∘C storage affects the mechanical properties of explantedvessels prior to decellularization

2 Materials and Methods

21 Tissue Harvesting Arterial segments (inner diameterfrom 2 to 11mm) were obtained from abdominal aorta andcarotid from 10 pigs (Large White Piglets) under generalanesthesia and orotracheal ventilationThe study was carriedout under Ethical Committee for Animal Experimentationsof University of Pavia (permit 12012) approval and followingItalian laws for animal care Fine dissection was performedusing scalpel and scissor providing complete removal ofloose connective tissue around the vessels Each segmentwas rinsed three times in Dulbeccorsquos phosphate bufferedsaline (PBS) containing 1 penicillin 1 streptomycin and

1 amphotericin B (AA solution all reagents Sigma-AldrichSt Louis MO USA) Vessels were either directly processedfor characterization of the native tissue or stored dry at minus80∘Cuntil decellularization

22 Decellularization Vessels were decellularized accordingto a previously tested protocol using an in-house developeddevice for the automatic decellularization of biological tissues[16] Briefly the treatment consists of 4 cycles as follows72 h washing in deionized water with 1 AA solution at 4∘Cfollowed by 4 h in sodium deoxycholate 4 solution at roomtemperature (RT) and 3 h in deoxyribonuclease I 2000 kU insodiumchloride 1Mat RTThewhole processwas done underagitation and between each step the vessel was rinsed 3 timesin PBS (all reagents Sigma-Aldrich St Louis MO USA)

23 Histology At retrieval and after decellularization spec-imens of the vessels were isolated and processed inparaformaldehyde 4 inPBS then embedded in paraffin and4 120583m slices were stained with hematoxylin-eosin and 410158406-diamidino-2-phenylindole (DAPI) as nuclear marker

24 DNA Quantification At native condition and afterdecellularization the total amount of DNA was quantifiedusing a DNA isolation kit for tissues (Qiagen Venlo TheNetherlands) Briefly 40mgwet weight samples of native anddecellularized arteries were digested using a cell lysis bufferand Proteinase K followed by a protein precipitation solutionand centrifugation to remove the protein fraction In order toisolate the DNA the supernatant was added with isopropanoland ethanol and then centrifuged and rehydratedwith aDNArehydrating solution Total DNA was finally quantified usinga spectrophotometer at 260 nm

25 Scanning Electron Microscopy Samples of native defro-zen and decellularized arteries were fixed with 25 glu-taraldehyde for 1 h and stored in sodium cacodylate 01MDehydrationwas achieved using an ethanol scale and sampleswere gold coated before being analyzed with a Zeiss EVO 50environmental scanning electron microscope (ESEM)

26 Mechanical Testing Native defrozen and decellularizedvessels were cut into ring-shaped specimens for mechanicalanalysis Native tissue samples were tested within 4 hoursof cold ischemia after retrieval Mechanical testing wasperformed according to a previously developed protocol [17]with some modifications as follows 5mm wide specimenswere mounted on custom-made holders on a computer-controlled uniaxial testing machine (Synergie 200H MTSMinneapolis MN USA) and data were acquired usingthe Test Work software (MTS Minneapolis MN USA) Apreloading of 0015N was imposed and the correspond-ing length was measured Each sample was preconditionedwith loading-unloading cycles until a reproducible responsewas achieved with a loading speed of 10mmmin anda load end point of 008Nmm The sample was left torecover for 120 seconds repeatedly rehydrated with PBS anda relaxation test was then performed with a ramp speed of


(a) (b)

(c) (d)


(a) (b)

(c) (d)




1000

2000

3000




119870 =

119879

008minus 119879

004

120576

008minus 120576

004

(1)


119864circ =119870

2WT

(2)



119866 =

119879

500 s119879

0

(3)


120590max =119879max2WT

(4)




3 Results





(a) (b) (c)


04

06

08

1

12

14

16

18

2

2 4 6 8 10 12

Wal

l thi

ckne

ss (m

m)

Diameter (mm)




WT = 06151198900088ID (5)




4 Discussion







N DF DC0

500

1000

1500


0

1

2

3

N DF DC


0

01

02

03

N DF DC

Compliance (1mmHg)

0

10

20

30

40

N DF DC

0


05

1

15

2

25

N DF DC

lowast


0

1000

2000

3000

4000

N DF DC


04

05

06

07

08

09

1

N DF DC

lowast


119875

lt 005






5 Conclusions






Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)


(a) (b)

(c) (d)




1000

2000

3000




119870 =

119879

008minus 119879

004

120576

008minus 120576

004

(1)


119864circ =119870

2WT

(2)



119866 =

119879

500 s119879

0

(3)


120590max =119879max2WT

(4)




3 Results





(a) (b) (c)


04

06

08

1

12

14

16

18

2

2 4 6 8 10 12

Wal

l thi

ckne

ss (m

m)

Diameter (mm)




WT = 06151198900088ID (5)




4 Discussion







N DF DC0

500

1000

1500


0

1

2

3

N DF DC


0

01

02

03

N DF DC

Compliance (1mmHg)

0

10

20

30

40

N DF DC

0


05

1

15

2

25

N DF DC

lowast


0

1000

2000

3000

4000

N DF DC


04

05

06

07

08

09

1

N DF DC

lowast


119875

lt 005






5 Conclusions






Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















1000

2000

3000




119870 =

119879

008minus 119879

004

120576

008minus 120576

004

(1)


119864circ =119870

2WT

(2)



119866 =

119879

500 s119879

0

(3)


120590max =119879max2WT

(4)




3 Results





(a) (b) (c)


04

06

08

1

12

14

16

18

2

2 4 6 8 10 12

Wal

l thi

ckne

ss (m

m)

Diameter (mm)




WT = 06151198900088ID (5)




4 Discussion







N DF DC0

500

1000

1500


0

1

2

3

N DF DC


0

01

02

03

N DF DC

Compliance (1mmHg)

0

10

20

30

40

N DF DC

0


05

1

15

2

25

N DF DC

lowast


0

1000

2000

3000

4000

N DF DC


04

05

06

07

08

09

1

N DF DC

lowast


119875

lt 005






5 Conclusions






Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b) (c)


04

06

08

1

12

14

16

18

2

2 4 6 8 10 12

Wal

l thi

ckne

ss (m

m)

Diameter (mm)




WT = 06151198900088ID (5)




4 Discussion







N DF DC0

500

1000

1500


0

1

2

3

N DF DC


0

01

02

03

N DF DC

Compliance (1mmHg)

0

10

20

30

40

N DF DC

0


05

1

15

2

25

N DF DC

lowast


0

1000

2000

3000

4000

N DF DC


04

05

06

07

08

09

1

N DF DC

lowast


119875

lt 005






5 Conclusions






Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















N DF DC0

500

1000

1500


0

1

2

3

N DF DC


0

01

02

03

N DF DC

Compliance (1mmHg)

0

10

20

30

40

N DF DC

0


05

1

15

2

25

N DF DC

lowast


0

1000

2000

3000

4000

N DF DC


04

05

06

07

08

09

1

N DF DC

lowast


119875

lt 005






5 Conclusions






Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusions






Acknowledgment


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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