2016 - Zhou et al

Decellularization and Recellularization of Rat Livers WithHepatocytes and Endothelial Progenitor Cells

*†Pengcheng Zhou, *Yan Huang, ‡Yibing Guo, *Lei Wang, *Changchun Ling,*Qingsong Guo, *Yao Wang, *Shajun Zhu, *Xiangjun Fan, *Mingyan Zhu, §Hua Huang,

*‡Yuhua Lu, and *Zhiwei Wang

*Department of General Surgery; †Department of Emergency Surgery; ‡Surgical Comprehensive Laboratory; and§Department of Pathology, Affiliated Hospital of Nantong University, Nantong, China

Abstract: Whole-organ decellularization has been identi-fied as a promising choice for tissue engineering. The aimof the present study was to engineer intact whole rat liverscaffolds and repopulate them with hepatocytes andendothelial progenitor cells (EPCs) in a bioreactor.Decellularized liver scaffolds were obtained by perfusingTriton X-100 with ammonium hydroxide. The architectureand composition of the original extracellular matrix werepreserved, as confirmed by morphologic, histological, andimmunolabeling methods. To determine biocompatibility,the scaffold was embedded in the subcutaneous adiposelayer of the back of a heterologous animal to observe the

infiltration of inflammatory cells. Hepatocytes werereseeded using a parenchymal injection method and cul-tured by continuous perfusion. EPCs were reseeded usinga portal vein infusion method. Morphologic and functionalexamination showed that the hepatocytes and EPCs grewwell in the scaffold. The present study describes an effec-tive method of decellularization and recellularization ofrat livers, providing the foundation for liver engineeringand the development of bioartificial livers. KeyWords: Decellularization—Extracellular matrix—Liver—Tissue Engineering—Endothelial progenitor cells—Recellularization.

Liver transplantation is currently regarded as theonly definitive and curative therapy for end-stage liverdisease. However, the increasing demand for trans-plantable livers far exceeds the availability of donorlivers (1). In addition, surgical complications, chronicrejection, and high expenses limit the wide applicationof liver transplantation. Hepatocyte transplantationoffers an alternative method to treat patients withliver diseases (2,3). Although clinical studies haveshown the efficacy of hepatocyte transplantation(3–6), it is associated with problems predominantlyrelated to the limited cell supply and low engraftmentefficiency (7–9). Bioartificial livers (BALs) are a tem-porary alternative to liver transplantation; they can beused to sustain the patient until a suitable donor organ

becomes available (10). However, BAL cannot sub-stitute liver transplantation permanently. Using theconcept of tissue engineering, artificial three-dimensional scaffolds have been generated and shownto successfully enhance the attachment and survival ofhepatocytes (11–14). However, the liver is a complexorgan that requires a constant delivery of nutrientsand oxygen, and the removal of metabolic products.In addition, the artificial scaffolds are not tissue-specific because of the lack of specific cell bindingfactors for cell functions. In recent years, with thedevelopment of regenerative medicine, a promisingapproach for organ replacement has emerged.Bioscaffolds derived from decellularized organs havebeen used to create materials for tissue engineeringapplications. Using this technology, organs such as theheart (15–19), lung (20–28), liver (29–37), and kidney(38–45) have been decellularized and recellularizedsuccessfully. The decellularized scaffolds, consistingof extracellular matrix (ECM), show good biocompat-ibility, provide tissue microarchitecture and intactvascular systems, and maintain biological factors thatpromote cell attachment, migration, and proliferation(46). With these advantages, decellularized scaffolds

doi:10.1111/aor.12645

Received February 2015; revised August 2015.Address correspondence and reprint requests to Dr. Zhiwei

Wang, Department of General Surgery, Affiliated Hospital ofNantong University, No. 20, XISI Road, Nantong, Jiangsu Prov-ince 226001, China. E-mail: [email protected] or Dr. Yuhua Lu,Departments of General Surgery, Affiliated Hospital of NantongUniversity, No. 20, XISI Road, Nantong, Jiangsu Province 226001,China. E-mail: [email protected]

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Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Artificial Organs 2015, ••(••):••–••

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are regarded as a promising choice for liver tissueengineering.

The ultimate goal of the decellularization processis to remove cellular material while minimizing thedamage to the ECM. Commonly used methods ofdecellularization include a combination of physical,chemical, and enzymatic approaches (47). Because ofthe thickness and complex intrinsic structures of theliver, perfusion decellularization is the best choice forthe construction of liver scaffolds. In the presentstudy, we used Triton X-100 (Amresco, Solon, OH,USA), which is a non-ionic detergent, as the maindetergent to remove cellular components. After thedecellularization process, we examined thedecellularization efficiency, ultrastructure and ECMcomponents, and the biocompatibility of the scaffold.

Revascularization remains the major challenge forcreating an artificial organ. All organs require a vascularnetwork to supply oxygen and nutrients, and the intactvascular structure of the artificial organ can be directlyconnected to the circulation of the recipient. The advan-tage of decellularized scaffolds over polymer scaffolds isthe presence of an intact vascular network. Researchershave used microvascular endothelial cells and humanumbilical vein endothelial cells for endothelialization ofdecellularized liver scaffolds (29,34,48,49). Endothelialprogenitor cells (EPCs) are a cell population that isreleased mainly from the bone marrow into the periph-eral blood circulation and have been confirmed to par-ticipate in vasculogenesis (50–52). EPCs have been usedin the endothelialization of blood vessels (53,54) andheart valves (55,56); however, they have not been usedin the endothelialization of liver decellularized scaf-folds. In the present study, we cultured EPCs from ratbone marrow and repopulated them into decellularizedliver scaffolds.

The methods used for reseeding of the whole organinclude direct parenchymal injection and infusion. Inour study, we compared these two methods forhepatocyte reseeding. After the reseeding process,the scaffold was transferred to the chamber and cul-tured under continuous perfusion. During the cultureprocess, the specific function of the cells in the scaffoldwas monitored. At the end of the culture, the scaffoldwas harvested and histological examination was per-formed to observe the repopulation status.

MATERIALS AND METHODS

AnimalsC57BL/6 mice weighing 20–25 g, Sprague Dawley

(SD) rats weighing 300–350 g, and 4-week-old SDrats were purchased from the Experimental AnimalCenter of Nantong University, Nantong, China. All

the animals were kept under constant environmentalconditions with a 12-h light/dark cycle and free accessto water and food. All animal procedures wereperformed according to institutional and nationalguidelines and approved by the Animal Care EthicsCommittee of Nantong University.

Harvesting of livers from SD ratsSD rats were anesthetized by intraperitoneal injec-

tion of chloral hydrate (5%, 0.5 mL/100 g). Underdeep anesthesia, rats were treated with topical skindisinfectant, and a laparotomy extending from thepubis to the xyphoid was performed in the abdomen.The distal end of the portal vein (PV) was ligated, andthen the vein was cannulated with a 22-G cannula andfixed with 3-0 silk sutures. A total of 2 mL heparinsodium (100 U/mL) was injected through the vein foranticoagulation. Then, the infrahepatic inferior venacava was transected to allow the outflow of theperfusate. A total of 50 mL phosphate-buffered saline(PBS) was perfused slowly through the PV clear bloodfrom the liver. Then, the suprahepatic inferior venacava, the hepatic artery, and the common bile ductwere freed. Finally, the whole liver was isolated andtransferred to a cell culture dish.

Decellularization of the liverThe cannula in the PV was connected to the peri-

staltic pump (Masterflex Technical Hoses Ltd.,Oldham, UK), and the perfusion rate for each stepwas set at 4 mL/min. The decellularization processwas initiated by PV perfusion with PBS for 1 h, fol-lowed by distilled water for 30 min. Then, the liverwas perfused with 0.02% ethylenediaminetetraaceticacid (EDTA) (Sinopharm Chemical Reagent Co.Ltd., Shanghai, China) in PBS for 30 min. Then, asthe most important step of the decellularization, 1%(w/v) Triton X-100/0.1% ammonium hydroxide(Xilong Chemical Reagent Co. Ltd.) in distilled waterwas perfused for 20 h. Subsequently, distilled waterwas used to rinse cellular lysis and circuit debris for2 h. Finally, the liver was perfused with PBS for 4 h tomaintain isotonicity. The decellularized liver scaffoldwas preserved in PBS at 4°C.

Analysis of ECM componentsTo examine the ECM components of the scaffolds,

tissues were randomly cut from fresh livers (n = 3) anddecellularized liver scaffolds (n = 3) and fixed with4% formaldehyde, dehydrated, and embedded in par-affin. Tissue sections were deparaffinized and stainedwith hematoxylin and eosin (H&E), Masson’strichrome, and Sirius red stain. Slides were visualizedand recorded under an Olympus microscope

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Artif Organs, Vol. ••, No. ••, 2015

(Olympus Microscope Corp., Tokyo, Japan) for H&Eand Masson’s trichrome stain and a Nikon E200(Nikon, Nanjing, China) for detection of the Sirius redstain. To determine whether collagen I, collagen IV,laminin, and fibronectin were retained in thedecellularized scaffolds, the slides were blockedagainst nonspecific binding with a solution consistingof 1% bovine serum albumin (BSA) (Biosharp, Hefei,China) and 0.1% Triton X-100 in PBS for 30 min at37°C. Primary antibodies were diluted with blockingbuffer, anti-collagen I (Bioss Ltd., Beijing, China),anti-collagen IV (Bioss), anti-laminin (Santa CruzBiotechnology [Shanghai] Co. Ltd., Shanghai, China),and anti-fibronectin (Abcam, Cambridge, UK), andincubated with the slides overnight at 4°C. After that,slides were incubated with secondary biotinylatedgoat anti-rabbit antibodies (Zsbio, Beijing, China).Slides were visualized and recorded under anOlympus microscope for H&E, Masson’s trichromestain, and immunohistochemical analysis, and a NikonE200 for detection of the Sirius red stain.

Scanning electron microscopy (SEM) observationSamples were randomly cut from the decellularized

liver scaffolds (n = 3) and sectioned into small blocks.First, samples were fixed in 2.5% glutaraldehyde over-night followed by three washes in PBS for 10 mineach. Then, samples were fixed in 1% osmic acid in thedark. This was followed by another three PBS washingsteps of 10 min each. After that, samples were dehy-drated in a gradient series of alcohol for 10 min each(30, 50, 70, 90, 95, and 100%). Next, isoamyl acetatewas added to exchange the alcohol twice for 10 mineach. Subsequently, samples were critical point driedand sputter-coated with gold. Finally, images wereobserved and recorded under a scanning electronmicroscope (HITACHI, Tokyo, Japan).

DNA content assayFresh livers and the decellularized liver scaffolds

(n = 3) were lyophilized. Three small samples fromeach scaffold weighing 25 mg were cut and crushedinto small pieces, and then digested with proteinaseK for 24 h at 60°C. DNA was isolated using theDneasy Tissue kit (Tiangen, Beijing, China) accord-ing to the manufacturer’s instructions. The concen-tration of DNA (ng/mL) was measured using anultraviolet spectrophotometer. Finally, the DNAcontent in the tissues was determined.

GAG content assayGlycosaminoglycan (GAG) content was measured

using the GAG assay kit (Hermes Criterion Biotech-nology, Vancouver, Canada). Fresh livers and the

decellularized liver scaffolds (n = 3) were used.Three small samples from each scaffold weighing10 mg were incubated with papain extraction reagent(Sigma-Aldrich Corp., St. Louis, MO, USA) for 3 hat 65°C. Assays were then performed according tothe manufacturer’s instructions. Absorbance wasrecorded on a microplate spectrophotometer. GAGcontent was calculated based on the standard curveobtained from the standard GAG specimen.

Biocompatibility of the decellularized liver scaffoldsin heterologous animals

Tissues cut from the decellularized liver scaffolds(n = 3) were sectioned into 5 mm in diameter and 3 mmin thickness and immersed in 0.1% peracetic acid for3 h on the shaker. Then, the blocks were washed twicewith PBS on the shaker for 30 min each. The operationwas performed under sterile conditions. A 5-mm inci-sion was made on the back of C57BL/6 mice (n = 20),and the tissue blocks were embedded in the subcutane-ous adipose layer on the back. The incision was closedwith 5-0 sutures and disinfected with iodine after theoperation for 4 consecutive days. In addition, 20 000units of penicillin were injected intraperitoneally for 3consecutive days after the operation. The tissues wereharvested on postoperative days 5, 7, 10, 14, and 21 andstained with H&E.

Hepatocyte cultureThe rat normal liver cell line BRL was obtained

from the cell bank of the Chinese Academy ofScience, Shanghai. Cells were cultured in Dulbecco’smodified Eagle’s medium (DMEM) (Hylcone,Shanghai, China) supplemented with 10% fetalbovine serum (FBS) (Gibco, Grand Island, NY,USA), penicillin (1 × 105 U/L), and streptomycin(100 mg/L) (Gibco) on tissue culture flasks. The cellswere cultured in an incubator at 37°C and 5% CO2.The medium was changed every other day.

Toxicity assay of the decellularized liver scaffoldsTissues were immersed in 0.1% peracetic acid for

3 h and then washed three times with PBS on theshaker for 30 min each. DMEM was added to thescaffolds at a concentration of 0.1 mL/g and incu-bated at 37°C for 24 h to extract liquid. The extractedliquid was filtered through 0.22-μm filters before use.Two types of medium were prepared: a, DMEM with10% FBS; and b, the extracted liquid with 10% FBS.BRL cells were seeded in DMEM with 10% FBS at adensity of 2000 cells/well on four 96-well plates anddivided into two groups, A and B. The medium wasremoved overnight. A total of 100 μL of a and bmedia was added to groups A and B, respectively.

RECELLULARIZATION OF HEPATOCYTES AND EPCs 3


Then, 10 μL of the CCK-8 (Dojindo, Laboratories,Kumamoto, Japan) solution was added into each wellof one plate at 0, 12, 24, and 48 h and incubated at37°C for 2 h. Absorbance was measured at 450 nm ona microplate reader.

Repopulation of BRL cells into the decellularizedliver scaffolds in vitro

A circulation perfusion device (EQUL Ltd.,Houston, TX, USA) consisting of a peristaltic pump,oxygenator, and chamber was set up (Fig. 7b,c). Thedevice was filled with 50 mL of DMEM supplementedwith 10% FBS, 10 ng/mL epidermal growth factor(Peprotech, Rocky Hill, NJ, USA), penicillin(1 × 105 U/L), and streptomycin (100 mg/L) andplaced in the incubator filled with 5% CO2 at 37°C. Torecellularize the scaffold in vitro, a total of 2 × 107 cellswere prepared. We compared the two methods, themultiposition parenchymal injection method andinfusion method. For the multiposition parenchymalinjection method, the prepared cells were dissolved in2 mL of medium and injected at 10 sites (four sites inthe left lateral lobe, two in the left median lobe, two inthe right median lobe, and two in the right laterallobe) with a 29-G needle (BD, Suzhou, China)(Fig. 7a). For the infusion method, the prepared cellswere dissolved in 2 mL of medium, and injectedthrough the PV in four steps, at 15 min intervals. Eachmethod was performed a total of four times. Themedium was collected and the engraftment rate wascalculated from the total number of cells minus thenumber of cells floating in the medium. After 40 minof stationary culture, the scaffold was transferred tothe chamber under aseptic conditions. The cannula inthe PV was connected with the circulation as the inlet,and the other vessels worked as the outlet.

Dynamic three-dimensional culture of therecellularized liver scaffold and the functionof hepatocytes

The medium was continuously perfused throughthe PV at 1 mL/min for 2 days and changed to 2 mL/min thereafter. The medium was changed daily anddetected using an automatic biochemical analyzer(Siemens Healthcare Diagnostics Ltd., Tarrytown,NY, USA) for assessment of liver function accordingto albumin and total bile acid secretion. 2-D culture indishes was set as a control. After 7 days of culture invitro, the recellularized liver scaffold was fixed andevaluated by H&E staining, SEM examination, andalbumin immunofluorescence. For the assessment ofalbumin immunofluorescence, tissue sections wereblocked against nonspecific binding with 1% BSA for30 min and then incubated in 0.1% Triton X-100 for

5 min at 37°C. Then, the sections were incubated withrabbit anti-rat ALB (Santa, Cruz Biotechnology,1:50) overnight at 4°C and washed with PBS. Next, thesections were incubated with Alexa Fluor 488-conjugated donkey anti rabbit (Proteintech, Wuhan,China, 1:200) at room temperature for 1 h. In addi-tion, the slides were stained with Hoechst (Sigma-Aldrich Corp., St. Louis, MO, USA) to label thenucleus for 10 min. Slides were visualized andrecorded under an Olympus fluorescence microscope.

EPC isolation and culture from the ratbone marrow

Bone marrow mononuclear cells (BMMNCs) wereisolated from rat bones as described previously by ourgroup (57). The femur and tibia of hind legswere removed from 4-week-old SD rats afterheparinization and euthanasia. The bones were repeat-edly washed with endothelial basal medium (EBM2,Lonza, Allendale, NJ, USA) until the bone marrowcavity became white. The washing fluid was filteredthrough a 40 μm mesh (Franklin Lakes, NJ) to obtain asingle-cell suspension. Single-cell suspensions werelysed with ACK lysing buffer (Gibco) and then washedwith EBM2 twice. The BMMNCs were then suspendedin endothelial growth medium (EGM2-MV, Lonza)containing VEGF, hFGF, IGF, hEGF, ascorbic acid,hydrocortisone, and 5% FBS and plated in 10 μg/mLhuman fibronectin (Sigma) coated T25 plates (Corning,Shanghai, China). The cells were cultured in an incuba-tor under 5% CO2 at 37°C. After 24 h, the nonadherentcells were transferred to a new plate coated withfibronectin to remove rapidly adherent cells. Afteranother 3 days, the nonadherent cells were aspiratedand the EGM2-MV medium was changed every dayuntil day 7 and on alternate days thereafter. When thecells reached 80–90% confluency, they were digestedwith 0.25% trypsin/EDTA (Gibco) and passaged at theratio of 1:2. EPCs were seeded on fibronectin-coatedslides after digestion and cultured for 24 h in EGM2-MV. The slides were blocked with 1% BSA for 30 minto prevent nonspecific antibody-antigen binding. Then,the slides were incubated with the following primaryantibodies: rabbit anti-rat CD133 (Abcam, 1:100) andgoat anti-rat CD31 (Santa, Cruz Biotechnology, 1:50)overnight at 4°C and washed with PBS. Next, the slideswere incubated with the following secondary antibodies(Proteintech, 1:200): Alexa Fluor 488-conjugateddonkey anti-rabbit for CD133 and TRITC-conjugateddonkey anti-goat for CD31 at room temperature for 1 h.In addition, the slides were stained with Hoechst(Sigma) to label the nucleus for 10 min. Slides werevisualized and recorded under an Olympus fluorescencemicroscope.

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Endothelialization of the decellularizedscaffold with EPCs

Decellularized liver scaffolds were perfused withEGM2 through the PV before endothelialization. Atotal of 5 × 106 EPCs were suspended in 1 mL ofEGM2 and seeded through the PV very slowly. Afterstationary culture for 4 h, the scaffold was transferredto the chamber of the circulation perfusion device.The circulation culture was performed at the flowrate of 1 mL/min for another 3 days. Finally, thescaffold was harvested for H&E staining andimmunofluorescence for CD31.

Statistical analysisStatistical analysis was performed using SPSS 22

(SPSS Inc., Chicago, IL, USA). Statistical differenceswere evaluated by the Kruskal–Wallis test. A P value<0.05 was considered significant. The mean values ofDNA, GAG, albumin synthesis, and total bile acidsecretion were expressed as the mean ± standarddeviation (SD).

RESULTS

Perfusion decellularization of rat liversWhole liver decellularization was achieved by

portal perfusion with Triton X-100 and otherreagents. The PV was successfully cannulated with a22-G cannula and fixed with 3-0 silk sutures (Fig. 1a).The liver color turned soft red after the blood waswashed out (Fig. 1b,c). After rinsing with distilledwater, the liver turned yellowish brown and showedmild swelling (Fig. 1d). During the perfusion of 1%(w/v) Triton X-100/0.1% ammonium hydroxide, livercells were washed out in great numbers, and the livercolor quickly became semi-transparent. At the end ofthe process, the liver was transparent and theacellular scaffold retained the gross shape of the liver(Fig. 1e). The vascular system was visualized clearlythrough the capsule of the acellular scaffold (Fig. 1f).

Histological and immunohistochemicalexaminations of the scaffold

H&E staining of the decellularized liver scaffoldsshowed the absence of cells compared with the nativeliver (Fig. 2a,d). Masson’s trichrome staining con-firmed these result. Most of the collagen fiber com-ponents (stained blue) were retained and maintaineda tubular structure (Fig. 2b,e). Sirius red stainingshowed that the collagen structure of the vascularwalls was retained after the decellularization process(Fig. 2c,f). Immunohistochemical analysis of thenative liver and scaffold showed that collagen I(Fig. 2h,m), collagen IV (Fig. 2i,n), fibronectin(Fig. 2j,o), and laminin (Fig. 2k,p) were preservedand distributed around the vascular structures andparenchymal areas.

SEM examinationSEM showed the three-dimensional ECM micro-

structure of the decellularized liver scaffolds and con-firmed the absence of cell components. The spacesurrounded by the collagen fibers in the ECM wasthought to be the original position of hepatocytes(Fig. 3a,b).

DNA and GAG content quantificationQuantitative DNA assay showed that the DNA

content of the decellularized liver scaffolds was48.6 ± 17.1 ng/mg dry weight, and the total DNAcontent of the normal liver was 5263.1 ± 336.9 ng/mg dryweight (Fig. 4a). The GAG content in the decellularizedliver scaffolds was 28.2 ± 0.79 ng/mg wet weight. Normalliver GAG content was 34.2 ± 0.61 ng/mg wet weight.These results show that about 80% of GAG was pre-served in the decellularized live scaffold during thedecellularization process (Fig. 4b).

Biocompatibility assayAfter the operation, the mice behaved normally

regarding eating and drinking. On days 5, 7, 10, 14,and 21 postsurgery, the scaffolds were removed. The

FIG. 1. Sequential whole-organ decellularization progress. (a) The PV was cannulated with a 22-G cannula and (b) after 50 mL PBS wasperfused through the PV to clear blood. (c) The liver was harvested. (d) After rinsing with distilled water, the liver turned yellowish brownand swelled a little. (e) After perfusion of EDTA and 1% (w/v) Triton X-100/0.1% ammonium hydroxide, the liver became transparent, andthe acellular scaffold retained the gross shape of the liver. (f) The vascular system was intact.



FIG. 2. H&E staining of the normal liver (a) and decellularized liver scaffold (d). Masson’s trichrome staining of the normal liver (b) anddecellularized liver scaffold (e). Sirius red staining of the normal liver (c) and decellularized liver scaffold (f). Immunohistochemical analysisof the normal liver and decellularized liver scaffold: negative control (g, l), collagen I (h, m), collagen IV (i, n), fibronection (j, o), and laminin(k, p).

FIG. 3. ECM microstructure of the decellularized liver scaffolds (a, b). Recellularized scaffold showed that BRL cells engrafted into theparenchymal area of the scaffold (c, d).

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scaffolds retained their appearance and texture. Theresults of H&E staining are shown in Fig. 5. Inflam-matory cells began to infiltrate into the scaffold insmall amounts on day 5. On days 7 and 10, thenumber of inflammatory cells began to increase,reaching a peak value before starting to decrease,showing obviously reduced numbers on day 14. Onday 21, few inflammatory cells were present in thescaffold. In addition, the scaffold retained its originalstructure. No macrophagocytes or other pathologicalsigns of graft rejection were observed, suggesting thatthe decellularized liver scaffolds were biocompatible.

BRL cell growthBRL cells were cultured in two types of medium

for 48 h, and cell proliferation was assessed in eachmedium (Fig. 6). The proliferation curves showedthat the extract liquid of the decellularized liver scaf-folds did not inhibit the growth of BRL cells, andinstead showed mild growth promoting activity.

In vitro recellularization of decellularizedlive scaffolds

Two recellularization methods were used, namelymultipositional parenchymal injection and the infu-sion method. The infusion method resulted in a lowengraftment rate (83.4 ± 5.2%) and poor distribution

of the seeding cells. The infused cells tended to blockthe vessel lumen or were washed out of the scaffolds(Fig. 7d). The multipositional parenchymal injectionmethod resulted in a better engraftment rate(93.2 ± 4.0%), and cells were dispersed in the paren-chymal space after 24 h (Fig. 7e). Therefore, thesecond method was selected in our study. After astationary culture of 40 min, the scaffold was trans-ferred to the chamber for dynamic perfusion culture

FIG. 4. (a) DNA content of the native liver versus the decellularized liver scaffolds (P < 0.001). (b) GAG content of the native liver versusthe decellularized liver scaffold (P < 0.001).

FIG. 5. Histologic change at 5th, 7th, 10th, 14th, 21st day postsurgery. Inflammatory cells began to infiltrate into the scaffold in smallamount at day 5 and reached the peak at day 7, 10. Inflammatory cells began to fade away at day 14, and only a few inflammatory cellscan be seen in the view at day 21. The scaffold retained almost the original structure as before.

FIG. 6. The proliferation curve of BRL cells cultured in two kindsof medium.



FIG. 7. (a) The multiposition parenchymal injection method. (b, c) The circulation perfusion device which consisted of a peristaltic pump,oxygenator, and chamber. (d) H&E staining after 24 h by infusion method. (e) H&E staining after 24 h by multiposition parenchymalinjection method. (f, g) H&E staining of the normal liver and the recellularized liver scaffold. (h, i) Immunofluorescence of ALB of the normalliver and the recellularized liver scaffold.

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with daily culture media changes. To assess the meta-bolic function of the engrafted BRL cells, we detectedalbumin production and total bile acid secretion daily.The results showed that albumin production in thescaffold was lower than that in the 2-D dish culturefor the first 3 days, whereas it was higher from day4 to day 7. The 7-day cumulative albumin productionin the scaffold was significantly higher than thatin the 2-D dish culture (48.14 ± 3.97 μg/106 cells vs.42.76425 ± 0.448 μg/106 cells, P < 0.05) (Fig. 8a). Totalbile acid secretion in the scaffold was higher than thatin the 2-D dish culture for the first 4 days, whereas itwas lower from day 5 to day 7. The difference in thecumulative total bile acid secretion was not statisti-cally significant (Fig. 8b). The production of albuminand total bile acid increased gradually. These resultsindicated that the decellularized live scaffold is suit-able for BRL cell attachment and growth. After 7 daysof perfusion culture, the scaffold was harvested. H&Estaining, immunofluorescence staining for albumin,and SEM were performed. H&E staining and albuminimmunofluorescence revealed that the BRL cellswere distributed in the matrix in a pattern resemblingthe structure of the normal liver (Fig. 7f–i). SEMshowed that BRL cells engrafted into the parenchy-mal area of the scaffold (Fig. 3c,d).

Morphology and characteristics of EPCsNonadherent rat BMMNCs cultured for 24 h were

harvested and cultured in EGM2. On day 4,nonadherent cells were aspirated and adherent cellsformed scattered colonies of spindle-shaped cells(Fig. 9a). After 6–8 days, the colonies expandedquickly and formed clusters (Fig. 9b,c). With increas-ing passages, the shape of cells gradually changedfrom spindle-shaped to round. After passage 2, theendothelial cell-like “cobblestone” morphologybegan to be observed (Fig. 9d). EPCs were positive

for the endothelial cell markers CD133 and CD31(Fig. 9e,f).

Endothelialization of the decellularized scaffoldThe vascular tree structure of the scaffold was

observed by phase contrast microscopy. Forendothelialization of the scaffold, EPCs from passage2 were seeded via the PV and cultured for 3 days.H&E (Fig. 10a,b) and immunofluorescence stainingfor CD31 (Fig. 10b–d) showed that EPCs coveredthe internal surface of the tubular structures in thescaffold.

DISCUSSION

Scaffold materials composed of ECM derived fromdecellularized organs are increasingly being used forregenerative medicine and tissue engineering. Theultimate goal of organ decellularization is maximizingthe removal of cellular material while minimizingalterations in the composition, biologic activity, ormechanical integrity of the ECM. The efficacy ofdecellularization methods depend on certain factorsas follows: (i) the density of the organ, (ii) the cellulardensity of the organ, (iii) the lipid content of theorgan, and (iv) the thickness of the organ. Unlikediffusion techniques used for the decellularization ofsimple and thin tissues, solid organs require antegradeor retrograde perfusion techniques to efficientlyremove cellular components (58–61). Commonlyused methods of decellularization include the combi-nation perfusion of acids and bases, hypotonic andhypertonic solutions, detergents, alcohols, enzymes,and nonenzymatic agents. In the present study, con-sidering that the liver is an organ with a complicatedstructure that is formed of an abundance of cells andcontains many cellular enzymes, we selected continu-ous perfusion at low speed with detergent containingEDTA, a hypotonic solution, Triton X-100, and

FIG. 8. Function of the BRL cells in the recellularized liver matrix. (a) Albumin secretion over time during the 7 days. (b) Total bile Acid(TBA) secretion over time during the 7 days.



ammonium hydroxide for decellularization. EDTAaids in cell dissociation from ECM proteins bychelating metal ions (62). Hypotonic solutions areused to lyse the cells within the organs. Triton X-100 isthe most widely used nonionic detergent to disruptlipid–lipid and lipid–protein interactions withoutaffecting protein–protein interactions (63). Sodiumdodecyl sulfate (SDS), an ionic detergent, is moreeffective than Triton X-100. However, studies haveshown that SDS tends to damage the component ofthe ECM (20,31,64). We therefore chose the milddetergent Triton X-100 instead of SDS. The hypo-tonic ammonium hydroxide solution was used to lysecell membranes.

After the decellularization process, it is necessaryto evaluate the efficiency of the decellularizationmethod. The removal of cellular material can be

confirmed by the lack of nuclear material in scaffoldsections stained with H&E and DNA contentassays. Visualization of the gross appearance allowsassessment of the integrity of the vascular structure.The retention of ECM components is visualized byhistological staining, such as Masson’s trichromeand Sirius red stains for collagens. In addition, SEMis often performed to examine the ultrastructurewithin the decellularized scaffolds and to verify theabsence of cellular material. The protection of col-lagen types I and IV, fibronectin, and laminin isconfirmed by immunohistochemical examinations.Collagen type I is the most abundant form of colla-gen in the body, whereas collagen type IV is foundin the basement membrane and provides bioactivesignals to endothelial cells in the vasculature (59).Fibronectin is an ECM glycoprotein that plays a

FIG. 9. (a–c) Morphologic characteristics of the EPCs after separation from the bone marrow. (d) Morphology of cobblestone-shapedEPCs. (e, f) Cobblestone-shaped EPCs were positive for CD133 and CD31.

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role in cell adhesion, growth, migration, and differ-entiation (65). Laminin is another protein that existson the basement membrane and the vasculature ofmost organs. GAG is important for the protectionof free growth factors from enzymatic degradation,as free growth factors are anchored to GAG(66).

Graft rejection, which occurs as a response to theantigenic components of xenogeneic tissues, is themain barrier to the use of xenogeneic scaffolds intranslational applications (67). Xenogeneic antigensare usually recognized as foreign by the host andcause a destructive inflammatory response (68). Totest the biocompatibility of the decellularized scaf-fold, transplantation was performed in heterologousanimals. Decellularized liver scaffolds derived fromSD rats were embedded in the subcutaneousadipose layer of the back of C57BL/6 mice. Inflam-matory cellular infiltration and the original structurewere monitored at different time points. Twenty-onedays after transplantation, the inflammatory cellswere not obvious, and the structure was preserved.However, the mechanisms of constructive remodel-ing and degradation of the ECM are only partiallyunderstood. The ECM can promote a switch fromTh1 effector cells to Th2 effector cells and evoke anM2 phenotypic macrophage immune response (69).Both Th2 and M2 are responsible for anti-inflammatory, wound-healing, and constructiveremodeling responses. Ineffective decellularizedscaffolds containing both DNA and cellularepitopes, such as the Gal epitope, will promote a

more M1-type response and induce rejectionresponses after implantation (69). These results indi-cate that the decellularized scaffold did not induceapparent xenograft rejection and shows good bio-compatibility.

In the present study, we used the BRL cell line forrecellularization, which has been rarely reported.Continuous perfusion of medium through the scaf-fold imitated the blood perfused through the liver invivo. The results showed that BRL cells attached andfunctioned well in the scaffold. After 7 days ofculture, the BRL cells were laid out in a streakpattern that was comparable to the hepatocytearrangement in the native liver.

Revascularization remains the major challenge forcreating an artificial organ. The intact vascular struc-ture of the artificial organ can be directly connectedto the circulation of the recipient. Decellularizedwhole-organ scaffolds retain the vasculature;however, the use of this vascular system to directblood in vivo without thrombus formation requiresproper endothelialization. EPCs can be induced todifferentiate into mature endothelial cells in vitroand in vivo and have been used to coat the vasculargrafts to produce an intact surface (69). We usedEPCs to revascularize the decellularized liver scaf-fold. However, there is controversy regarding theidentification and method of culture of this celltype. Amiel et al. (70) first reported that CD34+

mononuclear cells from the peripheral blood couldbe differentiated into EPCs with endothelial struc-ture and function. Asahara et al. (71) showed that

FIG. 10. (a, b) H&E staining showed that EPCs covered the internal surface of the tubular structures in the scaffold. (c–e)Immunofluorescence of CD31 staining of the EPCs in the vessels of the scaffold.



CD34− /CD133+ subpopulations can also be differ-entiated into EPCs. As reliable cell surface markersfor the detection of EPCs have not been identified,the separation of EPCs by magnetic-activated cellsorting is not accurate. Currently, the isolation ofEPCs is achieved by a differential attachmentmethod. Recent studies (72–74) showed that slowadherent BMMNCs exhibit the genetic phenotypeof both immature and endothelial lineage cells and ahigh potential for forming tube-like structures,whereas fast adherent BMMNCs are less likely toshow the genetic phenotype of endothelial cells andlow tube-like structure forming activity, which areconsidered to be monocyte/macrophage orinflammation-related cell-rich BMMNC popula-tions. As a result, we discarded the 24-h adherentcell population and harvested the nonadherent cellpopulation for further culture. Cells were positivefor CD133 and CD31. The EPCs were repopulatedinto the vascular lumen through the PV by slowinfusion, and medium was perfused through the vas-cular system by pressure gradient. The large vesselshad a higher pressure than the microvessels, result-ing in more medium perfused than that inmicrovessels. As a result, large vessels were betterendothelized than microvessels. To obtain betterresults, we may try to infuse fibronectin beforerepopulating EPCs, which is beneficial for the settle-ment and proliferation of EPCs.

CONCLUSIONS

In summary, the present study showed that wholerat decellularized liver scaffolds can be successfullyobtained by continuous perfusion of EDTA, a hypo-tonic solution, and Triton X-100/ammonium hydrox-ide. Analyses verified the preservation of theultrastructure and biomechanical properties of theextracellular matrix. Heterologous transplantationshowed good biocompatibility of the scaffold, whichlaid the foundation for xenografts. BRL cellsattached and functioned well in the three-dimensional scaffold. Endothelial progenitor cellswere successfully used for re-endothelialization ofthe vessels of the scaffolds. Although the presentstudy was limited to rat livers, we have also success-fully applied the decellularization method to porcinelivers, which could be scaled up to human liver size(data not shown). Additional experiments onco-reseeding the nonparenchymal liver cells withhepatocytes are necessary, and the efficiency ofendothelialization needs to be optimized before thebioengineered liver can be transplanted and functionin large animals. Ultimately, future clinical studies

could develop such an artificial liver by combiningxenogeneic liver scaffolds with autologous stem cell-derived hepatocytes. Taken together, our resultsindicated that the decellularized scaffold is a promis-ing choice for liver tissue engineering and regenera-tive medicine.

Acknowledgments: This work was supported bythe National Natural Science Foundation of China(no.81471801), Jiangsu province’s key project ofhealth department (K201101), and the Science andTechnology Innovation Project of Jiangsu Provinceand Nantong University for postgraduates. Weacknowledge the technical assistance from SurgicalComprehensive Laboratory (Affiliated Hospital ofNantong University), Pathology Department (Affili-ated Hospital of Nantong University), and KeyLaboratory of Neuroregeneration (NantongUniversity).

Conflict of Interest: None.

REFERENCES

1. Klein AS, Messersmith EE, Ratner LE, Kochik R, Baliga PK,Ojo AO. Organ donation and utilization in the United States,1999–2008. Am J Transplant 2010;10:973–86.

2. Fox IJ, Roy-Chowdhury J. Hepatocyte transplantation. JHepatol 2004;40:878–86.

3. Fisher RA, Strom SC. Human hepatocyte transplantation:worldwide results. Transplantation 2006;82:441–9.

4. Horslen SP, McCowan TC, Goertzen TC, et al. Isolatedhepatocyte transplantation in an infant with a severe ureacycle disorder. Pediatrics 2003;111:1262–7.

5. Dhawan A, Puppi J, Hughes RD, Mitry RR. Humanhepatocyte transplantation: current experience and futurechallenges. Nat Rev Gastroenterol Hepatol 2010;7:288–98.

6. Horslen SP, Fox IJ. Hepatocyte transplantation. Transplanta-tion 2004;77:1481–6.

7. Nagata H, Nishitai R, Shirota C, et al. Prolonged survival ofporcine hepatocytes in cynomolgus monkeys. Gastroenterol-ogy 2007;132:321–9.

8. Basma H, Soto-Gutierrez A, Yannam GR, et al. Differentia-tion and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 2009;136:990–9.

9. Smets F, Najimi M, Sokal EM. Cell transplantation in thetreatment of liver diseases. Pediatr Transplant 2008;12:6–13.

10. Demetriou AA, Brown RS Jr, Busuttil RW, et al. Prospective,randomized, multicenter, controlled trial of a bioartificial liverin treating acute liver failure. Ann Surg 2004;239:660–7, dis-cussion 667–70.

11. Du Y, Chia SM, Han R, Chang S, Tang H, Yu H. 3Dhepatocyte monolayer on hybrid RGD/galactose substratum.Biomaterials 2006;27:5669–80.

12. Lan SF, Safiejko-Mroczka B, Starly B. Long-term cultivationof HepG2 liver cells encapsulated in alginate hydrogels: astudy of cell viability, morphology and drug metabolism.Toxicol in Vitro 2010;24:1314–23.

13. Huang H, Hanada S, Kojima N, Sakai Y. Enhanced functionalmaturation of fetal porcine hepatocytes in three-dimensionalpoly-L-lactic acid scaffolds: a culture condition suitable forengineered liver tissues in large-scale animal studies. CellTransplant 2006;15:799–809.

P. ZHOU ET AL.12


14. Mei J, Sgroi A, Mai G, et al. Improved survival of fulminantliver failure by transplantation of microencapsulatedcryopreserved porcine hepatocytes in mice. Cell Transplant2009;18:101–10.

15. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: using nature’s platform to engineer abioartificial heart. Nat Med 2008;14:213–21.

16. Wainwright JM, Czajka CA, Patel UB, et al. Preparation ofcardiac extracellular matrix from an intact porcine heart.Tissue Eng Part C Methods 2010;16:525–32.

17. Remlinger NT, Wearden PD, Gilbert TW. Procedure fordecellularization of porcine heart by retrograde coronaryperfusion. J Vis Exp 2012;e50059.

18. Ng SL, Narayanan K, Gao S, Wan AC. Lineage restrictedprogenitors for the repopulation of decellularized heart.Biomaterials 2011;32:7571–80.

19. Lu TY, Lin B, Kim J, et al. Repopulation of decellularizedmouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells. Nat Commun2013;4:2307.

20. Ott HC, Clippinger B, Conrad C, et al. Regeneration andorthotopic transplantation of a bioartificial lung. Nat Med2010;16:927–33.

21. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungsfor in vivo implantation. Science 2010;329:538–41.

22. Jensen T, Roszell B, Zang F, et al. A rapid lungde-cellularization protocol supports embryonic stem cell dif-ferentiation in vitro and following implantation. Tissue EngPart C Methods 2012;18:632–46.

23. Petersen TH, Calle EA, Colehour MB, Niklason LE. Matrixcomposition and mechanics of decellularized lung scaffolds.Cells Tissues Organs 2012;195:222–31.

24. Mahdavi Shahri N, Baharara J, Takbiri M, Khajeh Ahmadi S.In vitro decellularization of rabbit lung tissue. Cell J2013;15:83–8.

25. Bonvillain RW, Danchuk S, Sullivan DE, et al. A nonhumanprimate model of lung regeneration: detergent-mediateddecellularization and initial in vitro recellularization withmesenchymal stem cells. Tissue Eng Part A 2012;18:2437–52.

26. Nichols JE, Niles J, Riddle M, et al. Production and assess-ment of decellularized pig and human lung scaffolds. TissueEng Part A 2013;19:2045–62.

27. O’Neill JD, Anfang R, Anandappa A, et al. Decellularizationof human and porcine lung tissues for pulmonary tissue engi-neering. Ann Thorac Surg 2013;96:1046–55, discussion 1055–6.

28. Gilpin SE, Guyette JP, Gonzalez G, et al. Perfusiondecellularization of human and porcine lungs: bringing thematrix to clinical scale. J Heart Lung Transplant 2014;33:298–308.

29. Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organreengineering through development of a transplantablerecellularized liver graft using decellularized liver matrix. NatMed 2010;16:814–20.

30. Shupe T, Williams M, Brown A, Willenberg B, Petersen BE.Method for the decellularization of intact rat liver. Organo-genesis 2010;6:134–6.

31. Ren H, Shi X, Tao L, et al. Evaluation of two decellularizationmethods in the development of a whole-organ decellularizedrat liver scaffold. Liver Int 2013;33:448–58.

32. Kajbafzadeh AM, Javan-Farazmand N, Monajemzadeh M,Baghayee A. Determining the optimal decellularization andsterilization protocol for preparing a tissue scaffold of ahuman-sized liver tissue. Tissue Eng Part C Methods2013;19:642–51.

33. Yagi H, Fukumitsu K, Fukuda K, et al. Human-scale whole-organ bioengineering for liver transplantation: a regenerativemedicine approach. Cell Transplant 2013;22:231–42.

34. Baptista PM, Siddiqui MM, Lozier G, Rodriguez SR, Atala A,Soker S. The use of whole organ decellularization for thegeneration of a vascularized liver organoid. Hepatology2011;53:604–17.

35. Barakat O, Abbasi S, Rodriguez G, et al. Use of decellularizedporcine liver for engineering humanized liver organ. J SurgRes 2012;173:e11–25.

36. Lang R, Stern MM, Smith L, et al. Three-dimensional cultureof hepatocytes on porcine liver tissue-derived extracellularmatrix. Biomaterials 2011;32:7042–52.

37. Ji R, Zhang N, You N, et al. The differentiation of MSCs intofunctional hepatocyte-like cells in a liver biomatrix scaffoldand their transplantation into liver-fibrotic mice. Biomaterials2012;33:8995–9008.

38. Ross EA, Williams MJ, Hamazaki T, et al. Embryonic stemcells proliferate and differentiate when seeded into kidneyscaffolds. J Am Soc Nephrol 2009;20:2338–47.

39. Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, OttHC. Regeneration and experimental orthotopic transplanta-tion of a bioengineered kidney. Nat Med 2013;19:646–51.

40. Ross EA, Abrahamson DR, St. John P, et al. Mouse stem cellsseeded into decellularized rat kidney scaffolds endothelializeand remodel basement membranes. Organogenesis 2014;8:49–55.

41. Sullivan DC, Mirmalek-Sani SH, Deegan DB, et al.Decellularization methods of porcine kidneys for whole organengineering using a high-throughput system. Biomaterials2012;33:7756–64.

42. Orlando G, Farney AC, Iskandar SS, et al. Production andimplantation of renal extracellular matrix scaffolds fromporcine kidneys as a platform for renal bioengineering inves-tigations. Ann Surg 2012;256:363–70.

43. Nakayama KH, Batchelder CA, Lee CI, Tarantal AF.Decellularized rhesus monkey kidney as a three-dimensionalscaffold for renal tissue engineering. Tissue Eng Part A2010;16:2207–16.

44. Orlando G, Booth C, Wang Z, et al. Discarded human kidneysas a source of ECM scaffold for kidney regeneration technolo-gies. Biomaterials 2013;34:5915–25.

45. Katari R, Peloso A, Zambon JP, et al. Renal bioengineeringwith scaffolds generated from human kidneys. Nephron ExpNephrol 2014;126:119.

46. Wang Y, Cui CB, Yamauchi M, et al. Lineage restriction ofhuman hepatic stem cells to mature fates is made efficient bytissue-specific biomatrix scaffolds. Hepatology 2011;53:293–305.

47. Gilbert TW, Sellaro TL, Badylak SF. Decellularization oftissues and organs. Biomaterials 2006;27:3675–83.

48. Shirakigawa N, Ijima H, Takei T. Decellularized liver as apractical scaffold with a vascular network template for livertissue engineering. J Biosci Bioeng 2012;114:546–51.

49. Shirakigawa N, Takei T, Ijima H. Base structure consisting ofan endothelialized vascular-tree network and hepatocytes forwhole liver engineering. J Biosci Bioeng 2013;116:740–5.

50. Kirton JP, Xu Q. Endothelial precursors in vascular repair.Microvasc Res 2010;79:193–9.

51. Murasawa S, Asahara T. Endothelial progenitor cells forvasculogenesis. Physiology (Bethesda) 2005;20:36–42.

52. Li DW, Liu ZQ, Wei J, Liu Y, Hu LS. Contribution ofendothelial progenitor cells to neovascularization (Review).Int J Mol Med 2012;30:1000–6.

53. Conklin BS, Wu H, Lin PH, Lumsden AB, Chen C. Basicfibroblast growth factor coating and endothelial cell seeding ofa decellularized heparin-coated vascular graft. Artif Organs2004;28:668–75.

54. Quint C, Kondo Y, Manson RJ, Lawson JH, Dardik A,Niklason LE. Decellularized tissue-engineered blood vessel asan arterial conduit. Proc Natl Acad Sci U S A 2011;108:9214–9.

55. Fang NT, Xie SZ, Wang SM, Gao HY, Wu CG, Pan LF.Construction of tissue-engineered heart valves by usingdecellularized scaffolds and endothelial progenitor cells. ChinMed J 2007;120:696–702.

56. Lee DJ, Steen J, Jordan JE, et al. Endothelialization of heartvalve matrix using a computer-assisted pulsatile bioreactor.Tissue Eng Part A 2009;15:807–14.



57. Ling CC, Ng KT, Shao Y, et al. Post-transplant endothelialprogenitor cell mobilization via CXCL10/CXCR3 signalingpromotes liver tumor growth. J Hepatol 2014;60:103–9.

58. Crapo PM, Gilbert TW, Badylak SF. An overview of tissueand whole organ decellularization processes. Biomaterials2011;32:3233–43.

59. Arenas-Herrera JE, Ko IK, Atala A, Yoo JJ.Decellularization for whole organ bioengineering. BiomedMater 2013;8:014106.

60. Song JJ, Ott HC. Organ engineering based on decellularizedmatrix scaffolds. Trends Mol Med 2011;17:424–32.

61. Badylak SF, Taylor D, Uygun K. Whole-organ tissueengineering: decellularization and recellularization of three-dimensional matrix scaffolds. Annu Rev Biomed Eng2011;13:27–53.

62. Klebe RJ. Isolation of a collagen-dependent cell attachmentfactor. Nature 1974;250:248–51.

63. Seddon AM, Curnow P, Booth PJ. Membrane proteins, lipidsand detergents: not just a soap opera. Biochim Biophys Acta2004;1666:105–17.

64. Kasimir MT, Rieder E, Seebacher G, et al. Comparison ofdifferent decellularization procedures of porcine heart valves.Int J Artif Organs 2003;26:421–7.

65. Miyamoto S, Katz BZ, Lafrenie RM, Yamada KM.Fibronectin and integrins in cell adhesion, signaling, and mor-phogenesis. Ann N Y Acad Sci 1998;857:119–29.

66. Saksela O, Moscatelli D, Sommer A, Rifkin DB. Endothelialcell-derived heparan sulfate binds basic fibroblast growth

factor and protects it from proteolytic degradation. J Cell Biol1988;107:743–51.

67. Wong ML, Griffiths LG. Immunogenicity in xenogeneic scaf-fold generation: antigen removal vs. decellularization. ActaBiomater 2014;10:1806–16.

68. Erdag G, Morgan JR. Allogeneic versus xenogeneic immunereaction to bioengineered skin grafts. Cell Transplant2004;13:701–12.

69. Keane TJ, Badylak SF. The host response to allogeneic andxenogeneic biological scaffold materials. J Tissue Eng RegenMed 2015;9:504–11.

70. Amiel GE, Komura M, Shapira O, et al. Engineering of bloodvessels from acellular collagen matrices coated with humanendothelial cells. Tissue Eng 2006;12:2355–65.

71. Asahara T, Murohara T, Sullivan A, et al. Isolation of putativeprogenitor endothelial cells for angiogenesis. Science 1997;275:964–7.

72. Friedrich EB, Walenta K, Scharlau J, Nickenig G, Werner N.CD34-/CD133+/VEGFR-2+ endothelial progenitor cell sub-population with potent vasoregenerative capacities. Circ Res2006;98:e20–5.

73. Brandl A, Yuan Q, Boos AM, et al. A novel early precursorcell population from rat bone marrow promotes angiogenesisin vitro. BMC Cell Biol 2014;15:12.

74. Kahler CM, Wechselberger J, Hilbe W, et al. Peripheralinfusion of rat bone marrow derived endothelial progenitorcells leads to homing in acute lung injury. Respir Res 2007;8:50.

P. ZHOU ET AL.14


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