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Biomaterials 34 (2013) 6422e6428

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Long-term results of cell-free biodegradable scaffolds for in situ tissueengineering of pulmonary artery in a canine model

Goki Matsumura a,*, Noriko Isayama a, Shojiro Matsuda b, Kensuke Taki b, Yuki Sakamoto b,Yoshito Ikada c, Kenji Yamazaki a

aCardiovascular Surgery, The Heart Institute of Japan, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, JapanbGunze Ltd., Research and Development Department, 1 Ishiburo, Inokurashinmachi, Ayabe, Kyoto 623-8512, JapancDepartment of Indoor Environmental Medicine, Nara Medical University, 840 Shijyo-cho, Kashihara, Nara 634-8521, Japan

a r t i c l e i n f o

Article history:Received 2 May 2013Accepted 21 May 2013Available online 5 June 2013

Keywords:SurgeryVasculatureTissue engineeringBiodegradable polymersVascular grafts

* Corresponding author. Tel.: þ81 3 3353 8111; faxE-mail addresses:n4mb4a@gmail.com, smatumur@h

0142-9612/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2013.05.037

a b s t r a c t

We previously developed a cell-free, biodegradable scaffold for in-situ tissue-engineering vasculature(iTEV) in a canine inferior vena cava (IVC) model. In this study, we investigated application of this scaffoldfor iTEV of the pulmonary artery (iTEV-PA) in a canine model. In vivo experiments were conducted todetermine scaffold characteristics and long-term efficacy. Biodegradable scaffolds comprised poly-glycolide knitted fibers and an L-lactide and ε-caprolactone copolymer sponge, with an outer glycolideand ε-caprolactone copolymer monofilament reinforcement. Tubular scaffolds (8 mm diameter) wereimplanted into the left pulmonary artery of experimental animals (n ¼ 7) and evaluated up to 12 monthspostoperatively. Angiography of iTEV-PA after 12 months showed a well-formed vasculature withoutmarked stenosis, aneurysmal change or thrombosis of iTEV-PA. Histological analysis revealed a vessel-like vasculature without calcification. However, vascular smooth muscle cells were not well-developed12 months post-implantation. Biochemical analyses showed no significant difference in hydroxypro-line and elastin content compared with native PA. Our long-term results of cell-free tissue-engineering ofPAs have revealed the acceptable qualities and characteristics of iTEV-PAs. The strategy of using this cell-free biodegradable scaffold to create relatively small PAs could be applicable in pediatric cardiovascularsurgery requiring materials.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Complex heart defects often require foreign substitutes toaccomplish vascular continuity and achieve normal circulation. Interms of repairing an anomalous pulmonary artery (PA), auto-pericardium, homograft and artificial grafts are currently used.However, during long-term follow-up some patients require furthersurgical intervention because of material-related events [1e5].Because operative survival has improved for infants and childrenwith complex congenital heart defects, the number of adults withcongenital heart disease (CHD) is drastically increasing [6]. Further-more, the number of secondary operations to replace outgrown ordegenerative prosthetic conduits has also increased. One reason forthis is that commonly used for the substitutes are foreign materials.

: þ81 3 3356 0441.ij.twmu.ac.jp (G.Matsumura).

All rights reserved.

We have therefore focused on development of optimal materials,possessing suitable repair characteristics to address current materialconcerns for CHD treatment.

We previously reported long-term efficacy results of in-situtissue-engineering vasculature (iTEV) in a canine model [7]. Theobjective of this study was to determine the feasibility of using anycell source and to identify the ideal biodegradable material toachieve a versatile cell-free protocol. iTEV using our biomaterialresulted in native tissue-like histological regeneration, withacceptable biomechanical characters [7]. To overcome graft steno-sis of the tissue engineered vasculature, we explored the use of thisbiodegradable scaffold in achieving acceptable long-term results ina venous position.

To investigate the use of our biomaterial for PA regeneration,in vivo experimentation was conducted to evaluate the applicability,basic characteristics and long-term efficacy of our biodegradablescaffold for cell-free in-situ tissue-engineering vasculature of thepulmonary artery (iTEV-PA), in a canine model.

G. Matsumura et al. / Biomaterials 34 (2013) 6422e6428 6423

2. Materials and methods

2.1. In vivo experimentation

All animals received care according to the Principles of Laboratory Animal Care(NIH, 1985). The ethical committee of Tokyo Women’s Medical University reviewedand approved the study protocol (Permit Number: 11-75). Seven healthy adult fe-male beagles (NARC, Tomisato, Japan) with a mean weight of 9.6 kg (8.6e10.2 kg)were used in this study. Two animal was euthanized at 6months and 5 animals wereeuthanized at 12 months for angiographies, echocardiograms, and for histologicaland biochemical analyses. We assessed 5 dogs after 12 months because the histo-logical results in 2 dogs after 6 months were acceptable. For surgeries, animals wereanesthetized with pentobarbital (1 mg/kg body weight) and heparin (700 U/kg bodyweight) was administered intravenously for anticoagulation during anastomoses.

Under general anesthesia, the fifth intercostal was opened in a right lateralposition. After heparinization, native left PA (LPA) was resected between the bifur-cation of LPA and the superior lobar artery using a simple clamp technique. Thedistal end of the LPA was then anastomosed with the scaffold. The main PA (mPA)was then partially C-clamped and longitudinally incised followed by anastomosis ofthe scaffold. Finally, each remaining end on the scaffold was sutured together. Thecomposite tubular scaffold used in this study was 8 mm in diameter and 5e6 cmlong. The lengths of the scaffolds were trimmed according to the design of theanastomoses. The scaffold completely loses its strength within 6 months owing tonon-enzymatic hydrolysis as previously described [7]. The scaffold was cut andtrimmed to obtain natural curvature and anastomosis. All animals were assessed atthe time of implantation and 3, 6 (n ¼ 2), and 12 (n ¼ 4) months post-implantationusing a Terason t3000 ultrasound system (Terason, Burlington, MA) to evaluateblood flow in the iTEV-PA. Angiographies of iTEV-PA were carried out 6 and 12months post-implantation. Subject animals were anesthetized using the protocoldescribed above and placed in sterilized conditions for femoral vein dissection. An8-Fr long catheter sheath (Terumo, Tokyo, Japan) was placed through the femoralvein adjacent to the inferior vena cava, close to the diaphragm. An X-ray fluoroscopewas used for digital angiography. Approximately 10 ml of angiographic agent wasinjected to evaluate iTEV-PA features during the regeneration process. Animals weresubsequently maintained without anticoagulant until euthanasia was performed aspreviously described [7e9]. After dissection, iTEV-PA length was measured directlybetween the two suture lines and the iTEV-PA and was then longitudinally dissectedto perform histological and biochemical analyses. Samples of LPA and iTEV-PA weredissected, rinsed with phosphate-buffered saline (PBS) and stored at �20 �C untilrequired for analysis.

2.2. Macroscopic and histological examination

After dissecting the iTEV-PA, macroscopic sample overviews were taken with adigital camera. Longitudinally incised iTEV-PA and control LPA samples were placedon hard cardboard and fixed at both ends using metal surgical clips to maintain theoriginal length. The samples were then fixed in 4% paraformaldehyde in pH 7.0 PBS,embedded in paraffin and sectioned at 4e5 mm. Some sections were subjected tohematoxylin and eosin (H&E), Masson’s trichrome, Victoria blueevan Gieson, ormodified von Kossa staining. Immunostaining of remaining sections was performedusing antibodies against factor VIII (1:1000; Abcam, Tokyo, Japan), alpha smooth

Fig. 1. (A) Macroscopic images of scaffold implantation for iTEV-PA (MPA, main pulmonaryimplantation. Black arrowheads represent the iTEV-PA when dissected. (C) Macroscopic viewresected iTEV-PA looking through the mPA and inferior lobar artery. Bars, 1 cm.

muscle actin (ASMA, clone 1A4, 1:1000; Dako Japan, Tokyo, Japan), myosin heavychain (MHC, 1:1000; Sigma), smooth muscle myosin heavy chain 1 (SM1, 1:1000;Yamasa, Tokyo, Japan and smooth muscle myosin heavy chain 2 (SM2, 1:1000,Abcam), as previously described [7e9]. LPA and iTEV-PA wall thicknesses weremeasured at 10 different sites in these sections.

To confirm endothelial cell distribution in iTEV-PAs at 6 months, specimenswere co-incubated with Texas Red� Lycopersicon Esculentum (TOMATO) Lectin (1/15 mg/ml; Vector Laboratories, Burlingame, CA) for 1 h at 37 �C. To confirm ASMAdistribution stereology, whole staining was performed on specimens as follows;iTEV-PA specimens were fixed in methanol/DMSO (4:1) at 4 �C overnight, washedwith 100% methanol (3 � 10 min) at room temperature (RT) and stored in 100%methanol at �20 �C for 4 days. Dehydrated specimens were then bleached inmethanol/DMSO/30% H2O2 (4:1:1) at RT overnight. Bleached specimens were thenwashed (1 � 10 min) in 75% methanol/0.1% Tween20/PBS (PBS-Tw) at RT, followedby washing in 50% methanol/PBS-Tw, 25% methanol/PBS-Tw (1 � 10 min) and PBS-Tw (2 � 10 min). Blocking was carried out by incubating specimens in 2% skimmedmilk/PBS/1% Triton X-100 (PBSMT, 2 � 1 h) at RT, and then overnight at 4 �C. Afterblocking, specimens were incubated with Fluorescein isothiocyanate (FITC)-conju-gated anti-ASMA (clone 1A4; Sigma, St. Louis, MO, USA) diluted 1:1000 in PBSMT for3 nights at 4 �C. Specimens were then washed with PBSMT (5 � 1 h) at 4 �C with afurther overnight PBSMT wash at 4 �C. iTEV-PA specimens were post-fixed for 1 h at4 �C in 4% paraformaldehyde. Fixed specimens were washed in PBS (2 � 15 min) atRT. Finally, specimens were washed in PBS-Tw (2 � 15 min), prior to washes in eachof 30% 50% and 80% Glycerol (1 � 15 min, diluted in 5% BADCO/PBS). After the an-alyses, the remaining sample was then embedded in paraffin and sectioned asdescribed above to evaluate the sample in cross-section. The sectioned sampleswere labeled with rabbit anti-Von Willebrand Factor (1:1000; Abcam) and bio-tinylated anti-rabbit IgG antibodies (Vector), followed by Alexa Fluor 594 conjugatedstreptavidin (1:400; Molecular Probes) to detect the endothelial cells. The sampleswere also labeled with fluorescein isothiocyanate (FITC) conjugated anti-ASMAantibody (1:500; Sigma) to detect ASMA.

All histological examinations and measurements were performed using lightmicroscopy (Biozero BZ-8000; Keyence, Osaka, Japan and MVX10; Olympus, Tokyo,Japan) and accompanying analytical software. Specimens were labeled with DAPI(Sigma) to identify cell nuclei when necessary.

2.3. Biochemical analyses of protein, elastin, hydroxyproline and calcium content

LPA and iTEV-PA samples were prepared and biochemical analyses were per-formed as previously described [7]. For western blotting, ASMA (1:1000, clone 1A4;Dako) for vascular smooth muscle cell (VSMC) detection and CD146 (1:1000; Epi-tomics, Burlingame, CA, USA) for endothelial cell detection were used. An anti-GAPDH antibody (1:1000, clone I-19; Santa Cruz Biotechnology, Santa Cruz, CA,USA) was used as an internal control.

Chemiluminescent images were acquired using a cooled CCD camera (LAS-3000Mini; Fujifilm, Tokyo, Japan) and band intensities were analyzed using bundledsoftware. Data were quantified as the ratio between the luminescence of the spec-ified protein and the internal control. A commercially available elastin assay kit(Fastin Elastin Assay Kit; Biocolor, Belfast, Northern Ireland) was used to quantifyelastin content and high-performance liquid chromatography (HPLC) was used tomeasure hydroxyproline content of the samples. The calcium concentrations of the

artery; LAA, left atrium appendage). (B) Macroscopic view of iTEV-PA 6 months post-of resected iTEV-PA (LPA, left pulmonary artery). (D) Proximal site and (E) distal site of

G. Matsumura et al. / Biomaterials 34 (2013) 6422e64286424

tissue samples were determined using a Zeeman polarized atomic absorptionspectrophotometer (ModelZ-6100; Hitachi Co., Ltd., Tokyo, Japan) as previouslydescribed [7].

2.4. Statistical analysis

The Mann-Whitney U-test was used to compare results from the controls andiTEV-PA samples. KruskaleWallis test was used to evaluate statistical significancebetween samples at each time point. All data are expressed as mean � standarderror of the mean (SEM) and a probability value of p < 0.05 was considered to bestatistically significant. IBM SPSS Statistics 19 software (IBM Japan, Tokyo, Japan) wasused for statistical analysis.

Fig. 2. (A) Macroscopic view of iTEV-PA 6 months post-implantation. The black arrow indicwas labeled with TOMATO-Lectin to identify endothelial cells (bar, 2 mm). (C) Higher magniWhole staining of iTEV-PA labeled with an anti-alpha smooth muscle actin (ASMA) antibodusing FITC-labeled ASMA and DAPI staining (bars, 50 mm). (F) Cross-section examinationendothelialization, visualized with anti-ASMA antibodies (bars, 100 mm). (GeI) Negative contfluorescence). (JeL) Scanning electron microscopy demonstrates the internal surface of iTE

3. Results

3.1. Macroscopic overviews of iTEV-PA

All animals remained alive and healthy without any seriouscomplications until euthanasia. All 7 iTEV-PAs were dissected andexamined. Scaffolds, which were sewn between themPA just abovethe commissure of the PA valve and adjacent to the left superiorlobar artery, remodeled into iTEV-PAs in an extra-anatomicalposition (Fig. 1A, B). Inner surfaces of the iTEV-PAs 12 months

ates where the following specimens were observed. (B) Fresh iTEV-PA (inset, bar, 1 cm)fication of the mid-portion of iTEV-PA labeled with TOMATO-Lectin (bars, 500 mm). (D)y (bar, 500 mm). (E) The steric structures of actin filaments in iTEV-PA were observedof iTEV-PA. The sample was treated with Von Willbrand factor (vWf) to confirm therols of the samples are shown to rule out auto-fluorescence ((H) for green and (I) for redV-PA and endothelialization (bars: I, 2 mm; J, 1 mm; K, 200 mm).

G. Matsumura et al. / Biomaterials 34 (2013) 6422e6428 6425

post-implantation were endothelialized in the absence of throm-bosis or aneurysmal change (Figs. 1C, D, E and 2A). Length mea-surements of implanted scaffolds compared with excised iTEV-PAsfrom individual animals indicated that all iTEV-PAs changed inlength over the 12 month period, ranging from 0.1 to 1.2 cm(n ¼ 5, mean 0.48 � 0.20 cm). Mean diameters of proximal anddistal iTEV-PAs were 6.38 � 0.55 and 4.41 � 0.23 mm, respectively,at 12 months. All iTEV-PA diameters changed during the 12-monthperiod compared with the initial implanted 8 mm tubular-shapedscaffold. The mean pressure gradient between the mPA and distaliTEV-PA was 1.45 � 0.30 mmHg.

3.2. Distribution of endothelial cells and ASMA-positive cells iniTEV-PA at 6 months

An iTEV-PAmacroscopic section at 6 months is shown in Fig. 2A.Following reaction with TOMATO-Lectin, Fig. 2B and C shows thedistribution of endothelial cells in iTEV-PA. Using the same spec-imen, whole staining of iTEV-PA for anti-ASMA antibody is shown(Fig. 2D and E). ASMA-positive cells proliferated 3-dimensionally iniTEV-PA. To confirm the endothelialization and ASMA distributionin iTEV-PA, the samples were also examined in cross-section usinganti-Von Willebrand factor and anti-ASMA antibodies (Fig. 2F).Finally, the sample was fixed in 2.5% glutaraldehyde and examinedwith scanning electron microscopy to observe the features of theinternal surface of iTEV-PA (Fig. 2J, K, L).

3.3. iTEV-PA histological analysis

H&E, Masson’s trichrome and Victoria blue staining showedcomponents of the four basic vascular layers, endothelial cells,VSMCs, elastic fibers and collagen fibers in iTEV-PA sections at 12months (Fig. 3AeC). Immunohistological analysis revealed endo-thelialization (Fig. 3D) and VSMCs proliferation (Fig. 3EeH) iniTEV-PA sections. Calcified lesions were not observed in any sam-ples (Fig. 3I). A comparison of wall thicknesses between LPA and

Fig. 3. Representative iTEV-PA histological sections: (A) hematoxylin and eosin (H&E) staininVIII, (E) ASMA, (F) myosin heavy chain (MHC), (G) SM1 and (H) SM2 immunostaining demModified von Kossa staining showing no calcified lesions; bars, 100 mm. (J) Box-whisker plothistological samples (lines, lower, median, and upper quartile values; whiskers, extent of remsignificant.

iTEV-PA showed walls were 0.59 � 0.07 and 0.61 � 0.03 mm thick,respectively, with no significance difference (p ¼ 0.372, Fig. 3J).

3.4. Echocardiography and angiography

Echocardiograms were used to follow the patency of iTEV-PAsand demonstrated that iTEV-PA branched off from the mPA bodyin an extra-anatomical position (Fig. 4A). Doppler echocardiogramsof iTEV-PA recorded a mean velocity of 1.37 � 0.15, 1.38 � 0.06,1.14 � 0.34 and 1.45 � 0.12 m/s at 0, 3, 6 and 12 months, respec-tively (Fig. 4B). There was no significant difference in the meanvelocity for iTEV-PA by the KruskaleWallis test (p ¼ 0.121). Sevenanimals were evaluated by angiography whilst in a right lateralposition to confirm scaffold features and patency. During tissueremodeling, although the scaffold diameter changed, the scaffoldmaintained its patency with concurrent material degradation.

3.5. Biochemical findings

Immunoreactive band densitometry for ASMA (40 kDa), CD146(113 kDa) and GAPDH (37 kDa) in LPA and iTEV-PA is shown(Fig. 5A). After normalization to GAPDH, there was no significantdifference in CD146 protein content between LPAs and iTEV-PAs at12 months, with densitometry ratios measuring 2.45 � 0.18 and2.78 � 0.57, respectively (p ¼ 0.841). However, the densitometryratio of ASMA protein was significantly different between LPAs andiTEV-PAs, measuring 2.33 � 0.39 and 1.16 � 0.07, respectively(p ¼ 0.007, Fig. 5B).

There was no significant difference in elastin and hydroxypro-line content between LPAs and iTEV-PAs (Fig. 6A and B), withelastin content measuring 54.4 � 3.67 and 59.4 � 4.08 mg/g wettissue weight, respectively (p ¼ 0.690). Based on hydroxyprolineconcentration, collagen concentration in LPAs and iTEV-PAs were74.8 � 6.72 and 81.0 � 5.48 mmol/g wet tissue weight, respectively(p ¼ 0.463). However, there was a significant difference in calciumcontent between LPAs and iTEV-PAs (Fig. 6C), with the calcium

g, (B) Masson’s trichrome staining and (C) Victoria blueevan Gieson staining. (D) Factoronstrate endothelial cells, vascular smooth muscle cells (VSMCs) and its isoforms. (I)s illustrate wall thickness of the left PA (LPA) and iTEV-PA at 12 months (n ¼ 5 each) inaining data); 10 sites for each sample (p ¼ 0.372 by the ManneWhitney U-test); NS, not

Fig. 4. (A) Representative echocardiography of iTEV-PA (top). Color Doppler echocardiography showing iTEV-PA branching off from the main PA (middle), and blood flow velocity ofiTEV-PA (bottom; AoV, aortic valve; mPA, main PA; rt PA, right PA). (B) Mean velocities of iTEV-PA immediately, 3 months, 6 months and 12 months post-implantation are shown.The data are shown as box-whisker plots (KruskaleWalis test, p ¼ 0.121; NS, not significant). (C) Representative iTEV-PA angiography 6 months post-implantation. Black arrowsindicate extra-anatomically anastomosed iTEV-PA (mPA, main pulmonary artery; lt PA, left PA; rt PA, right PA; RV, right ventricle).

G. Matsumura et al. / Biomaterials 34 (2013) 6422e64286426

content measuring 0.17 � 0.03 and 0.42 � 0.03 mg/g wet tissueweight, respectively (p ¼ 0.004).

4. Discussion

The first report of tissue-engineering PA in vivo used a lambmodel, whereby cultured venous cell mixtures were seeded onto a

Fig. 5. (A) Western blot immunoreactive protein bands (CD146, ASMA and GAPDH) weretometry normalized to GAPDH in left PA (LPA) and iTEV-PA samples at 12 months; lines, lp ¼ 0.841 and ASMA, p < 0.01 by ManneWhitney U-test); NS, not significant.

biodegradable scaffold prior to implantation [10]. However, thisprotocol proved difficult to envisage clinically because the cellpreparation process took several weeks. Accordingly, the procedurewas altered to incorporate mononuclear bone marrow cells(MN-BMCs), which can be easily prepared on the procedure day tocreate a tissue engineered autograft [8,9]. Although this protocolwas applied in human clinical trials with good results [11e13],

measured by densitometry. (B) Box-whisker plots of CD146 and ASMA protein densi-ower, median, and upper quartile values; whiskers, extent of remaining data (CD146,

Fig. 6. (A) Elastin, (B) hydroxyproline and (C) calcium content in left PA (LPA) and iTEV-PA at 12 months expressed as box-whisker plots; lines, lower, median, and upper quartilevalues; whiskers, extent of remaining data. (Elastin, p ¼ 0.690, hydroxyproline, p ¼ 0.463 and calcium p < 0.01 by ManneWhitney U-test); NS, not significant.

G. Matsumura et al. / Biomaterials 34 (2013) 6422e6428 6427

it still requires a complex cell preparation processes, which repre-sents a critical barrier for broad clinical application. Consequently,we developed a scaffold for simplified tissue engineering ofvasculature that does not require prior cell seeding [7]. This scaffoldproved to be useful in both venous and PA positions. Initial datafrom the study are presented herein.

In the clinical setting, surgical results following PA reconstruc-tion are variable but generally satisfactory. However, concernsregarding current approaches continue to exist. The sheer numberof possible surgical procedures and strategies, anatomical differ-ences, patient factors, diagnosis, thrombosis formation [2,5] andmaterial used, all represent risks for further intervention post-surgery [1]. Surgical intervention is still crucial for critical PA dis-eases, including small and occluded PA cases. Therefore, an opti-mized material for the surgical procedures is still desired.

Although devices and materials for surgical intervention arewell-developed, results are often unsatisfactory in patients becauseof post-operative material-related failures [1,4,5]. For example,smaller sized conduits and/or younger age are risk factors forfurther intervention in PA reconstruction using homograft [3].Right ventricle-to-pulmonary artery conduits or conduits forsystemic-to-pulmonary shunts might promote PA obstruction in ajuxtaductal position because of histopathological and/or hemody-namical reasons [2,4]. To repair stenotic lesions, autologous tissuesincluding autologous pericardium are favorable, although usingautologous tissue can still cause stenosis [1,5]. Implantation ofsmaller conduits is essential in pediatric cardiovascular surgery,therefore the use of iTEV-PA would be beneficial for biocompati-bility and its relatively small diameter. We were therefore keen toidentify a material that is suitable for pediatric cardiovascularsurgery, and is adaptable to the child’s growth.

We previously reported long-term results of in-situ tissue-engineered vasculature (iTEV) in a canine model. Following im-plantation of cell-free tubular composite scaffolds, efficacy, in termsof patency and histological, biomechanical and biochemical simi-larities to native vessels, was clearly demonstrated [7]. Because thetubular scaffold remodeled into native tissue-like features, wespeculated that this material and strategy could also be applied tothe reconstruction of PA.

According to our previous study, this scaffold has the potentialto avoid anticoagulation therapy and reduce incidence of throm-boembolic complications. This is because it permits endothelialcells to cover the inner surface of the iTEV within a few months.Furthermore, calcified lesions were avoided because the materialabsorbs within approximately 6 months. iTEV remodels into anative-like tissue with comparable size and mechanical character-istics to native tissues [7]. In the current study, iTEV-PA is an arti-ficial material when implanted, however the scaffold resorbs over

time, allowing regeneration of native-like tissue. We have shownthat iTEV-PA undergoes remodeling to meet the body’s re-quirements. This is evidenced by changes in iTEV-PA diameter,histologically reconstructed with appropriate wall thickness andlength over timewithoutmarked stenosis, dilatation or aneurysmalchange. Histologically, the internal lumen was completely coveredwith endothelial cells 6 months post-implantation, and collagenand elastin content was comparable to native tissue 12 monthspost-implantation. Furthermore, in terms of elastin content, iTEV-PA of 12 months post-implantation produced more elastin thaniTEV of 12 months which was implanted in a venous position [7].This benefit of iTEV-PA is likely a result of its exposure to the pul-satile pressure of the pulmonary artery. However, the presence ofVSMCs at 12 months was relatively low in both the previous andcurrent study [7]. Previously, we determined it would takeapproximately 1e2 years for VSMCs to proliferate in tissue-engineering vasculature. As the tissue remodeling process andduration varies among each individual difference, this time-consuming differentiation of VSMCs might be a concern for iTEV-PA as well. This phenomenon might be resolved by changing thescaffold design because scaffold features strongly influence tissuedevelopment. However, iTEV-PAs managed to maintain their orig-inal features while avoiding stenosis, aneurysmal changes andcalcification. Because the sample size in this study was limited,further research is required before moving these scaffolds into theclinical setting. However, these preliminary findings support thepossibility of applying iTEV-PAs to pediatric cardiovascular surgerywhere PA reconstruction requiring a small diameter conduit isneeded.

5. Conclusions

iTEV-PA with good long-term results can be constructedfollowing direct implantation of this biodegradable scaffold. As thescaffold degrades and regenerates into native tissue, iTEV-PAsretain their native-vessel-like characteristics. Based on the pre-sent findings, the protocol for iTEV-PA development can besimplified and made more versatile. We believe this cell-free tis-sue-engineering platform can be readily applied to treat patientswho require vascular surgical interventions with artificial grafts toprovide them with a better quality of life.

Acknowledgments

This work was supported by a Grant-in Aid for ScientificResearch (B: number 20390373) from The Ministry of Education,Culture, Sports, Science and Technology (MEXT), Japan.

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