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Acta Biomaterialia 10 (2014) 875–882

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Acta Biomaterialia

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Mechanostructure and composition of highly reproducibledecellularized liver matrices

1742-7061/$ - see front matter � 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.10.023

⇑ Corresponding author at: Research Centre ‘‘E. Piaggio’’, University of Pisa, LargoLucio Lazzarino 1, 56126 Pisa, Italy. Tel.: +39 050 2217050; fax: +39 050 2217051.

E-mail address: [email protected] (G. Mattei).

G. Mattei a,b,⇑, V. Di Patria b, A. Tirella c, A. Alaimo b, G. Elia b, A. Corti d, A. Paolicchi d, A. Ahluwalia b

a Department of Civil and Industrial Engineering, University of Pisa, Largo Lucio Lazzarino 1, 56122 Pisa, Italyb Research Centre ‘‘E. Piaggio’’, University of Pisa, Largo Lucio Lazzarino 1, 56122 Pisa, Italyc National Research Council, IFC, Via Moruzzi 1, 56124 Pisa, Italyd Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Via Roma 55, 56126 Pisa, Italy

a r t i c l e i n f o

Article history:Received 21 June 2013Received in revised form 17 September2013Accepted 22 October 2013Available online 30 October 2013

Keywords:Extracellular matrixDecellularizationBiologic three-dimensional scaffoldBiomaterialTissue engineering

a b s t r a c t

Despite the increasing number of papers on decellularized scaffolds, there is little consensus on theoptimum method of decellularizing biological tissue such that the micro-architecture and protein con-tent of the matrix are conserved as far as possible. Focusing on the liver, the aim of this study wastherefore to develop a method for the production of well-characterized and reproducible matrices thatbest preserves the structure and composition of the native extra cellular matrix (ECM). Given theimportance of matrix stiffness in regulating cell response, the mechanical properties of the decellular-ized tissue were also considered. The testing and analysis framework is based on the characterization ofdecellularized and untreated samples in the same reproducible initial state (i.e., the equilibrium swol-len state). Decellularized ECM (dECM) were characterized using biochemical, histological, mechanicaland structural analyses to identify the best procedure to ensure complete cell removal while preservingmost of the native ECM structure and composition. Using this method, sterile decellularized porcineECM with highly conserved intra-lobular micro-structure and protein content were obtained in a con-sistent and reproducible manner using the equilibrium swollen state of tissue or matrix as a reference.A significant reduction in the compressive elastic modulus was observed for liver dECM with respect tonative tissue, suggesting a re-examination of design parameters for ECM-mimicking scaffolds for engi-neering tissues in vitro.

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

1. Introduction

Maintaining hepatic functional characteristics in vitro has longbeen the holy grail of many researchers involved in the study ofin vitro liver models. A number of scientists have suggested thatthe target has been elusive because hepatocytes in vitro aredeprived of the multi-parametric, multi-stimuli in vivo milieu[1]. Reproducing the three-dimensional (3-D) features of the nativeliver is crucial, since the in vivo environment affects cell behaviourand function through a variety of soluble and insoluble signallingfactors, including physical, chemical and mechanical cues(e.g., oxygen concentration, substrate stiffness) [2,3]. No singlescaffold or scaffold material has been identified as optimal forhepatocytes, leaving this an open terrain for exploitation anddevelopment. The consensus is that a 3-D scaffold should providean adhesive substrate that mimics extracellular matrix (ECM)ligands and matches both the stiffness and the porosity of the

physiological matrix. Ideally, it should also be well characterized,reproducible and easy to fabricate, sterilize and store [4].

Biological scaffolds from decellularized tissues and organs havebeen used successfully for myriad applications in reconstructivesurgery and regenerative medicine [5,6]. Recently, the success ofwhole organ perfusion, which involves sending detergents throughthe vasculature, as well as the deeper understanding of the role ofmatrix signals in guiding cell function, has reawakened an interestin matrix-derived materials and scaffolds [7]. Besides whole organperfusion [8–10], common decellularization techniques includepressure gradients [11–13], and immersion and agitation [14–16]. Most methods involve washing and saponification phases toremove blood and debris, respectively, and to hydrolyse cell mem-branes. Following an immersion and agitation approach, Lang et al.described a study to identify the most suitable combination ofwashing and saponification cocktail to decellularize thin slices ofpig liver [14]. The authors optimized different decellularization/oxidation procedures based on Triton X-100, ammonium hydrox-ide, phosphate buffered saline (PBS) and sodium chloride, coupledwith a peracetic acid (PAA) treatment. They report 93% removal ofcellular components from porcine liver tissue and preservation of

876 G. Mattei et al. / Acta Biomaterialia 10 (2014) 875–882

the key molecular components in the ECM. Others have used SDSand Triton X-100 perfused through the portal vein to remove cel-lular material from whole organs. Despite the recent explosion ofinterest, there is little consensus on the optimum method of decell-ularizing hepatic tissue such that the micro-architecture and pro-tein content of the matrix are conserved as far as possible.Indeed, there is some concern that aggressive physical and chem-ical treatments may alter the protein structure, content and porousarchitecture of the matrices. In this context, the aim of this studywas to generate well-characterized and reproducible off-the-shelfscaffolds of liver-derived ECM for studying hepatic tissue regener-ation and for drug-related applications. Focusing on the techniqueof agitation and immersion, the present authors investigated theeffects of different decellularization procedures on matrix compo-sition and mechanostructural parameters, paying particular atten-tion to the experimental design and initial testing conditions ofsamples in order to enable meaningful comparisons between dif-ferent decellularization methods and controls.

2. Materials and methods

2.1. Hepatic tissue harvesting

Four porcine livers were collected from 1-year-old healthyswine as a slaughter by-product. Pig liver consists of five lobes(right lateral, right medial, left medial, left lateral and caudatelobe) wrapped in a tough fibrous capsule, i.e., Glisson’s capsule[17]. Individual lobes, except for the small caudate one, were sec-tioned from collected livers. After some fresh samples had been ta-ken for mechanical tests (see Section 2.7), liver lobes were frozenat �20 �C until use. Frozen livers were thawed at 4 �C overnight,then punched with a tool to obtain 14-mm-diameter cylinders,which were subsequently cut in 3-mm-thick liver discs, avoidingmacroscopic vasculature and Glisson’s capsule. Liver discs werefrozen at �20 �C until use.

2.2. Liver decellularization methods

Several decellularization methods (Table 1), based on immer-sion and agitation as described in Supplementary material S1, wereinvestigated, varying chemical detergent and treatment duration.The decellularization methods tested can be grouped into three

Table 1Decellularization protocols used to obtain liver dECM; percentages refer to the weight/volufinal washing day for DF protocols, since the contribution of 0.1% w/v Triton X-100 to cell r0.1% w/v SDS used in the other protocols.

Family Protocol Day 1 Day 2 Day 3

Ionic I3 PBS1�

SDS 0.1% ½ d Triton X-100 0.1% + ½1�

I4 PBS1�

SDS 0.1% SDS 0.1%

I5 PBS1�

SDS 0.1% SDS 0.1%

Non-ionic NI3 PBS1�

Triton X-1001%

½ d Triton X-100 0.1% + ½1�

NI4 PBS1�

Triton X-1001%

Triton X-100 1%

NI5 PBS1�

Triton X-1001%

Triton X-100 1%

Detergent-free DF3 PBS1�

PBS 1� PBS 1�

DF4 PBS1�

PBS 1� PBS 1�

DF5 PBS1�

PBS 1� PBS 1�

Not treated(control)

FF Fresh liver frozen at �20 �C, then thawed at 4 �C ovFF-NS Same as FF, but not equilibrium swollen prior to tes

major families (i.e., detergent-free (DF), ionic (I) and non-ionic(NI)). First, a DF control family was provided to decouple thedecellularizing effect due to mechanical agitation from that dueto chemical agents (i.e., detergents). Triton X-100 and sodiumdodecyl sulfate (SDS) were chosen as chemical decellularizationdetergents on the basis of their different modes of action and thereported effects on ECM [18]. Triton X-100 is a NI detergent thatdisrupts DNA–protein interactions as well as lipid–lipid, lipid–pro-tein and, to a lesser degree, protein–protein interactions. SDS is an Idetergent (i.e., anionic) that solubilizes cytoplasmic and nuclearcellular membranes, better at removing cell nuclei from dense tis-sues and organs than Triton X-100 is, but tends to denature pro-teins, disrupting the ultrastructure [19] and eliminating keygrowth factors [20].

Each protocol began with a rinsing day, to remove blood resi-dues, and ended with a final washing day, to remove detergent res-idues. Furthermore, the capability of NI Triton X-100 to formmicelles with anionic SDS [21] was used in this work to enhanceresidual SDS removal from the I protocols.

2.3. Decellularization assessment

Cell removal from liver discs obtained with each of the nineprotocols was assessed by hematoxylin and eosin (H&E) stainingand DNA quantification. Untreated frozen and thawed liver sam-ples, henceforth termed fresh-frozen (FF), were used as controls.Decellularized liver ECM (dECM) discs were fixed and paraffinembedded. The dECM samples were then cut into 5-lm sections,stained with H&E and examined using an Olympus IX 81 micro-scope (Olympus Italia, Milan, IT).

2.4. Swelling tests

Liver dECM samples obtained with all I and NI protocols weretested in triplicate (6 protocols � 3 samples). Decellularized liverdiscs were freeze-dried at �50 �C, 0.45 mbar for 48 h to determinetheir dry weight Wd. Then they were swollen in PBS 1X at roomtemperature and weighed every 12 h until a stable weight was ob-tained, i.e., the equilibrium swollen weight Weq. The equilibriummass swelling ratio was calculated as Q eq ¼Weq=Wd. All measure-ments refer to the samples’ equilibrium swollen state, unless sta-ted otherwise.

me ratio (w/v) of detergent solutions in deionized H2O; only PBS 1� was used in theemoval is very poor and not significant compared with that of 1% w/v Triton X-100 or

Day 4 Day 5

d PBS

½ d Triton X-100 0.1% + ½ d PBS1�SDS 0.1% ½ d Triton X-100 0.1% + ½ d PBS

1�d PBS

½ d Triton X-100 0.1% + ½ d PBS1�Triton X-100 1% ½ d Triton X-100 0.1% + ½ d PBS

1�

PBS 1�

PBS 1� PBS 1�

ernightting

G. Mattei et al. / Acta Biomaterialia 10 (2014) 875–882 877

2.5. DNA content

Total DNA content (micrograms per milligram equilibriumswollen sample) was determined for FF and all decellularized liversamples. DNA was extracted using the procedure outlined in Sup-plementary material S2.

2.6. Biochemical characterization

Total protein content (TPC) was determined using the Bio-RadProtein Assay (Bio-Rad Laboratories Inc, Hercules, CA). FF and alldecellularized liver samples were first equilibrium swollen in PBS1� to determine Weq, then solubilized in 1 M NaOH and subse-quently diluted. To obtain an absolute and repeatable measure-ment of protein concentration, care was taken to obtain areproducible initial matrix and a meaningful standard curve sub-ject to the same solubilization process as the samples (see Supple-mentary material S3).

Key liver ECM proteins (i.e., laminin, fibronectin and collagenIV) were selectively analysed using Western blot (see Supplemen-tary material S4 for experimental details). According to decellular-ization outcomes and TPC results (see Sections 3.1 and 3.4),Western blot analysis was performed in triplicate for protocols I3and NI3 as representatives of I and NI decellularization families,respectively. The results were then compared with those obtainedfor protocol DF3 (from the DF family) and FF liver. Once again, toobtain meaningful comparisons between protocols, the same totalprotein quantity was loaded for each analysis.

2.7. Unconfined compression experiments

Samples were characterized mechanically in compression,using a twin column Zwick/Roell ProLine Z005 testing machine(Zwick/Roell, Ulm, Germany) equipped with a 10 N load cell. Thetesting setup and the method used to determine the elastic modu-lus are described in Supplementary material S5.

The first analysis was performed to assess whether the bulkcompressive modulus of fresh liver depends on the sample har-vesting site (i.e., intra-swine dependence) and whether it changessignificantly between animals (i.e., inter-swine dependence). Then,variations in the liver compressive modulus after a freeze–thawcycle were investigated, collecting samples from FF liver lobesstored at �20 �C and thawed at 4 �C overnight. Finally, dECM fromprotocols I3 and NI3 were mechanically tested in compression asrepresentative samples of I and NI decellularization families,respectively. The dECM samples obtained by decellularizing eachof the four swine livers with the two aforementioned protocolswere tested in triplicate (4 animals � 2 decellularization proto-cols � 3 samples). The resultant moduli were compared with thatof untreated liver to assess whether and how different decellular-ization procedures affected liver bulk compressive properties.

As the present results showed that protocol NI3 yielded the bestliver dECM in terms of preserving ECM structure/ultrastructureand its constituents, micro-computed tomography (l-CT), histol-ogy and cytotoxicity tests were subsequently carried out on liverdisc samples obtained using this NI protocol only.

2.8. l-CT and further histology

l-CT imaging was performed on freeze-dried dECM from proto-col NI3 using a SkyScan 1174 system (Skyscan, Aartselaar, Bel-gium) with a resolution of 6.5 lm pixel�1, 180� rotation.Histological staining was also performed on protocol NI3 dECMto identify connective components using silver, Mallory’s tri-chrome and Alcian blue/PAS stains (10 lm slices were used, asthinner sections were lacking in contrast).

2.9. Sterilization of liver dECM and cytotoxicity tests

Several sterilization procedures were investigated to obtaindecellularized matrices usable as scaffolds for 3-D cell cultures orfurther processing to produce tissue derivatives. Protocol NI3matrices were freeze dried and then treated by (i) exposing to chlo-roform vapour, (ii) H2O2 gas plasma, (iii) a combination of (i) and(ii), or (iv) PAA, before being placed in contact with hepatocytesin culture for cytotoxicity tests. Cell viability was evaluated usingCellTiter Blue (Promega, Madison, WI). Details are given in Supple-mentary material S6.

2.10. Statistical analysis

All experiments were carried out at least in triplicate. TPC, his-tology, mechanical tests and swelling measurements were per-formed on samples from at least two different pig livers.Comparisons between n groups of data (e.g., TPC, equilibrium massswelling ratio) referring to one factor only (e.g., different decellu-larization protocols) were performed using one-way ANOVA fol-lowed by Tukey’s Multiple Comparison Test. Two-way ANOVAfollowed by Tukey’s Multiple Comparison Test was used to analysethe mechanical compressive results of fresh liver samples har-vested from different lobes (1st factor of variability) of differentpig livers (2nd factor). The same type of analysis was applied tothe mechanical properties of liver dECM obtained by decellulariz-ing samples from different pigs (1st factor) with two different pro-tocols (2nd factor), and to cell viability assays after sterilization.Differences were considered statistically significant at P < 0.05.

3. Results

3.1. Liver decellularized matrices

Fig. 1 reports the histological analysis from the panel of proto-cols outlined in Table 1. The present authors did not detect anycells in either I or NI dECM at high magnification, but several cellswere observed in all the DF protocols (Supplementary material S7).Notably, there is a gradual collapse in lobule structure with time,independent of the nature of the detergent. The use of SDS clearlypromotes gradual removal of the intra-lobular matrix, and the re-moval occurs from the centre of the lobule outwards.

3.2. Swelling behaviour

The duration of decellularization procedures (3–5 days) did notaffect the equilibrium mass swelling ratios (Qeq), which were foundto be dependent only on the decellularization family, as reported inFig. 2. The I samples were characterized by a significantly higherQeq than the NI ones (respectively, 10.3 ± 2.1 and 6.7 ± 0.5,P = 0.018), reached in a shorter time, starting from the freeze-driedstate (24 h compared with 36 h). This swelling behaviour can beexplained by considering that NI liver dECM are characterized bya cell-free but ECM-rich intra-lobular network that hinders PBSabsorption within the matrix, resulting in a lower Qeq, reachedafter a longer time interval with respect to I samples. The I matri-ces are characterized by a more open porous network composedprincipally of inter-lobular connective tissue, which allows forgreater and more rapid penetration of water. However, the Qeq ofFF and DF samples were similar (respectively, 1.9 ± 0.4 and2.3 ± 0.4, P = 0.96), but significantly lower than those of I(P = 7.19 � 10�5 for FF vs. I, and P = 1.03 � 10�4 for DF vs. I) andNI (P = 3.35 � 10�3 for FF vs. NI, and P = 5.70 � 10�3 for DF vs. NI)with equilibrium after 48 h. These values can be related to struc-tural differences, as seen from histology, DNA and TPC analysis.

Fig. 1. H&E staining micrographs for all tested protocols as a function of time of immersion and agitation. There is a progressive shrinking of lobule dimensions with time andthe intra-lobular matrix of the I family is clearly eroded. Scale bar 200 lm.

Fig. 2. Equilibrium swelling ratios (Qeq) for different sample families. The I samplesare characterized by a significantly higher Qeq than the NI ones, owing to the moreporous network, which allows for a greater and more rapid penetration of water.However, FF and DF samples have a high cell density and a tighter network, leadingto significantly lower values of Qeq with respect to fully decellularized I and NIsamples. Significant differences in equilibrium swelling ratio between protocols aredenoted by an asterisk (⁄P < 0.05).

878 G. Mattei et al. / Acta Biomaterialia 10 (2014) 875–882

In particular, FF and DF samples have a high cell density and, there-fore, fewer pores for water to penetrate with respect to the cell-free I and NI samples.

3.3. Total DNA analysis

The total DNA in all samples is reported in Fig. 3A. Both the his-tology and swelling analyses are confirmed by these data. The DFprotocols are clearly insufficient to remove cells, demonstratingthe need to use chemical reagents along with forced mixing. Differ-ences between the I and NI families were insignificant, suggestingthat either reagent can be used for complete cell removal, whichwas reached after 3 days.

3.4. Evaluation of ECM constituents

The FF and decellularized liver TPC are summarized in Fig. 3B.All TPC results were normalized to the equilibrium swollen weightof the sample, apart from FF-NS, i.e., FF not-swollen liver sample.FF-NS served to account for proteins lost during swelling. As ex-pected, swelling samples to equilibrium results in a significant de-crease in TPC for FF liver, as a result of elimination of bloodproteins and, in the present authors’ opinion, FF represents thecontrol against which dECM should be compared. TPC was foundto be 185 ± 7 mg protein g�1 wet sample for FF-NS, in agreementwith published data [22].

One-way ANOVA analysis considering all the data showed thatTPC did not change significantly within I, NI and DF families, butthere were significant differences between groups, as reported inFig. 3B. NI procedures were found to be less aggressive than I pro-cedures in terms of protein retention within liver dECM. As con-firmed by histology results, the DF family did not allow forcomplete cell removal, in fact the TPC values were found to bein-between those of the NI and FF liver. However, considering eachfamily independently, the one-way ANOVA analysis showed a sig-nificant decrease in TPC with time for both I and NI protocols, inagreement with the histological observations in Fig. 1.

Since both I and NI protocols removed 97% of cells from day 3onwards (Fig 3A), while clearly lobular shrinking with time(Fig. 1) was accompanied by protein erosion (Fig. 3B), only repre-sentative samples of NI, I and DF from day 3 were used for furthertests.

These three representative samples as well as FF untreated he-patic tissue were then analysed by Western blot in order to evalu-ate the presence of specific ECM proteins. Since the conservation ofcell adhesion proteins and the basal lamina is crucial for the recell-ularization of 3-D scaffolds or materials derived from these matri-ces, the study focused on fibronectin, laminin and collagen IV. Asthe TPC of control and fresh frozen livers is made up largely ofcytoplasmic proteins, the ECM proteins are almost undetectable(Fig. 3C). However, both I and NI decellularization protocols

Fig. 3. Liver biochemical characterization before and after decellularization. (A)Total DNA content of FF and decellularized liver samples; data are expressed as lgDNA per mg of sample equilibrium swollen weight. (B) TPC of FF and decellularizedliver samples; data are expressed as mg of protein per g of sample equilibriumswollen weight, except for the fresh tissue FF-NS, which was processed as it was. (C)Immunoblot analysis of ECM proteins; the same quantity of protein was loaded intoeach lane. Different letters indicate significant differences between samples(P < 0.05), whereas the same letter means non-significant differences.

G. Mattei et al. / Acta Biomaterialia 10 (2014) 875–882 879

increased the signal of all investigated ECM proteins. Although theobserved signal increase appeared to be more pronounced in sam-ple I3, more extensive banding patterns (suggesting the presence

of protein degradation products) were observed, compared withthe NI3 sample. Since all the DF protocols had traces of cells, fur-ther testing on DF samples was not carried out.

3.5. Compressive mechanical properties

No significant differences in the bulk compressive modulus (k)of fresh liver were found among liver lobes and different pigs.Moreover, k did not change significantly between fresh and FF liversamples (1.62 ± 0.13 kPa). Based on these results, liver disc sam-ples harvested from all lateral and medial lobes were pooled to-gether for all experiments. The resultant k of the decellularizedsamples was not dependent on pig, as expected. Moreover, no dif-ferences in k were observed between protocol I3 and NI3 liverdECM (respectively, 1.25 ± 0.07 kPa and 1.31 ± 0.09 kPa, P = 0.28).These values are significantly different from that of untreated(i.e., fresh or FF) liver samples (P = 2.65 � 10�5), concluding that(i) cell removal was the main cause of the sharp decrease in k be-tween untreated and decellularized liver samples, while (ii) k isindependent of the decellularization protocol, indicating that itdoes not depend on the TPC of liver dECM.

3.6. Two-dimensional and 3-D architecture

The 3-D architecture of dECM from protocol NI3 is depicted inthe l-CT view in Fig. 4A (a video and slice are available in the Sup-plementary material files). NI3 has a dense matrix with an average(dry) pore size of 500 lm. Micrographs in Fig. 4B–D show a retic-ular intra-lobular mesh rich with connective components, includ-ing mucosubstances. The fibrillar matrix in the micrograph inFig. 4B has pores of �10 lm, similar to cellular dimensions (seeSupplementary material S7). In comparison with the pink connec-tive rich intra-lobular matrix in Fig. 4D, the cells in FF liver (Fig. 4E)stain bright red with Mallory’s trichrome.

3.7. Liver dECM sterilization and cytotoxicity

Fig. 5 shows the relative cytotoxic effects of the different steril-ization methods investigated. Exposure to chloroform vapour andimmersion in PAA were identified as the best methods of sterilizingliver dECM. The worst method was gas plasma treatment (with orwithout chloroform). The germicidal action of hydrogen peroxideplasma sterilization is based on the generation of free radicals,and it is likely that the low vitality observed was due to the actionof free radicals on the matrix.

4. Discussion

In order to achieve complete cell removal from small samples ofhepatic tissue while preserving most of the ECM constituents andmicroscopic lobular architecture, it is necessary to establish quan-tifiable parameters that enable different methods to be compared.It is also useful to identify the most crucial factors that can modifythe outcome of the decellularization process. This study first fo-cused on establishing whether time plays a role in the decellular-ization process and which of the two most commonly useddetergents is best for complete cell removal, through an assess-ment of histological features, swelling ratio and DNA and TPC.The histology and DNA results clearly show that a reagent for dis-lodging cells from the matrix is essential, as DF decellularizationdid not allow cell removal even after 5 days. Furthermore, therewere significant differences in TPC between families, while littlechange in DNA or TPC was observed over time for a given protocolfamily (Table 1 and Fig. 3A, B), indicating that time is not a crucialfactor for cell removal. However, the decellularization processing

Fig. 4. (A–D) Three-dimensional structural and histological analysis of 3 day NI (NI3) liver dECM and (E) histology of control FF liver. (A) l-CT 3-D reconstruction of NI3showing a dense matrix; (B) PAS/Alcian blue showing the mucin component of NI3; (C) silver stain highlighting the presence of a dense reticular component in NI3; and (D)Mallory’s trichrome for NI3. The deep blue colour indicates connective tissue, while the pink stain shows elastic fibres. (E) Mallory’s trichrome of control FF liver: the deep redstains nuclei. Scale bar for histology images is 300 lm.

Fig. 5. Assessment of dECM sterilization methods through cell viability. ProtocolNI3 dECM sterilized using four different methods (chloroform, chloroform + gasplasma, gas plasma, peracetic acid) and then placed in contact with HepG2 cells inculture for up to 48 h. Different letters indicate significant differences in cellviability between sterilization methods (P < 0.05), whereas the same letter meansnon-significant differences.

880 G. Mattei et al. / Acta Biomaterialia 10 (2014) 875–882

time should be kept to a minimum to avoid structural collapse, asdemonstrated in Fig. 1. In most reports of tissue decellularization,ECM constitutents (e.g., proteins, glycosaminoglycans) are normal-ized to the dry weight of samples (e.g., Refs. [9,13,23,24]), and theevaluation of the aggressiveness of decellularization methods issomewhat arbitrary. In fact, matrix components expressed as mil-ligrams per gram of dry tissue may be higher for decellularized tis-sue than for fresh tissue, making comparisons meaningless byprivileging the more aggressive method, which removes morecomponents unselectively. However, because the equilibriumswollen state is exceptionally stable and reproducible, highlyreproducible quantitative data were obtained by normalizing all

measurements with respect to the equilibrium swollen state ofsamples, with modest standard deviations. Furthermore, the datashow that the time to reach equilibrium and the equilibrium swell-ing ratio are simple and reliable indicators of the porosity and net-work density of dECM.

After it had been established that the NI protocol is effective atcell removal and conserves more protein and matrix than the I pro-tocol, and that 3 days are sufficient to ensure elimination of 97%DNA with both detergents, immunoblotting was performed to con-firm the presence of adhesive and basal lamina proteins in selectedsamples.

As the same quantity of protein was loaded into each lane, andas (i) no differences between the FF controls and DF3 and (ii) an in-crease in the band intensity for each of the investigated proteins(fibronectin, laminin and collagen IV) were observed, the presentresults suggest that other proteins were arbitrarily removed bySDS. Moreover, the increase in band intensity was accompaniedby an increase in band smearing. The latter is probably caused bythe presence of degradation products, as suggested by Baptistaet al. [9], hence the observed smear increasing from NI to I proto-cols may reflect a higher degree of protein degradation as the treat-ment procedures become more aggressive.

Matrix stiffness has been described as one of the most impor-tant biophysical parameters regulating cell function and differen-tiation. The current consensus is that the stiffness of a substrateshould closely match that of the gross stiffness of biological tissuein order to elicit physiological cell responses. Attention was thusdedicated to the evaluation of the mechanical properties of liverdECM as a function of treatment protocol. As the mechanicalproperties of hepatic tissue are highly dependent on organ statusand the test method employed [25], the present authors preferredto set-up an independent but self-consistent and reproducibleplatform for evaluating and comparing dECM and fresh tissuestiffness, starting from the equilibrium swollen state, as describedin Supplementary material S5. To this end, a first series of testswas performed to ascertain that the compressive modulus k isinvariant for (i) different zones, (ii) different pigs and (iii) freshand FF samples. The results are in agreement with those of Mar-chesseau et al. [25] and Tamura et al. [26]. The former highly ci-ted paper lumps together data from 60 samples (5 animals � 3lobes � 4 samples) to reduce the mechanical differences betweenlobes and animals: the present data confirm their a prioriassumption. The latter performed a series of preconditioningcompression tests on porcine liver specimens to determine the

G. Mattei et al. / Acta Biomaterialia 10 (2014) 875–882 881

effects of freezing on the mechanical response of the tissue. Theyfound that the hysteresis loops obtained after freezing samplesmatched the hysteresis loops of the same samples before freezing.From these results, they concluded that freezing did not signifi-cantly change the mechanical properties of the tissue in compres-sion. Interestingly, no difference in compressive modulus wasobserved between the I and NI protocols. However, in agreementwith the trend recently reported by Evans et al. [27], there was asignificant lowering of k with respect to fresh and FF tissue, sug-gesting that the cells themselves also contribute to the stiffness oftissues. These studies relating the compressive properties of liverdECM to that of fresh tissue suggest that the design rules forECM-mimicking scaffolds for recreating organs and tissuesin vitro should be re-examined.

The NI 3-day treatment protocol (NI3) was chosen as the opti-mum, and further characterization with l-CT and histologicalstaining was performed to confirm the presence of additional ma-trix components and a well-preserved intra-lobular network.Assuming that the swelling ratio can be used to estimate the in-crease in pore size from the dry to equilibrium wet state, the drypore dimensions observed in the l-CT images (�500 lm) corre-spond to the average lobule dimensions in the H&E micrographof NI3 in Fig. 3B–D (1000 lm). The finer intra-lobular reticular net-work observed with silver staining (Figs. 3B and S7) was not re-solved with l-CT.

The sterilization of decellularized scaffolds or material derivedthereof is a prerequisite for cell culture and eventual in vivoimplantation. However, the sterilization method of choice shouldnot degrade proteins nor alter the structure of the scaffold. Meth-ods such as autoclaving, gamma irradiation, ethanol and ethyleneoxide are known to induce changes in the protein chemistry andphysical properties [28].

After assessing a variety of chemical and gas plasma-based ster-ilization techniques by measuring the viability of HepG2 cells over48 h, it was concluded that chloroform vapour and PAA are notcytotoxic, with the method of choice falling on chloroform, beingmore benign to proteins [29,30].

In conclusion, a testing and analysis framework for identifyingthe best procedure for decellularizing hepatic tissue slices wasestablished. The equilibrium swollen state and correspondingswelling ratio were shown to be useful indicators of the porosityof the scaffold and, more importantly, it was shown that usingthe equilibrium state as a reference enables reproducible quantita-tive comparisons between different decellularization protocols.Focusing on the conservation of mechanostructural parametersand protein content, the present authors singled out the NI proto-col NI3 as the optimum. Finally, a significant reduction in the stiff-ness of liver dECM was observed compared with intact hepatictissue.

Acknowledgements

The authors are grateful to Stefano Mazzoni and Andrea Pironefor the histology preparations and to the abattoir Desideri LucianoS.p.A. Via Abruzzi, 2 – Pontedera (Pisa), Italy, for kindly supplyingfresh hepatic tissue. The work leading to these results has receivedfunding from the European Union Seventh Framework Programme(FP7/2007-2013) under grant agreement 304961 (ReLiver).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.actbio.2013.10.023.

Appendix B. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1 and 4, are dif-ficult to interpret in black and white. The full colour images can befound in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013.10.023

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