Re-endothelialization of biological tissue-engineered conduits and scaffold for its potential use in bioprosthesis

Abstract Introduction In vitro and in vivo approaches are currently being used for guided cell repopulation of both biological and synthetic scaffolds. Till now, researchers have used different strategies to decellularize biological samples such as the use of detergents like SDS, 1× Triton-X and EDTA solutions. However, their widespread clinical acceptance is very limited. In the present study, different tissueengineered biological conduits and scaffolds made from bovine carotid artery (BCA) have been developed using cultured endothelial cells from sheep external jugular vein endothelial cells (SEJVECs). Materials and methods Conduit preparation was achieved as follows: (a) 0.2% glutaraldehyde fixation and (b) scaffold preparation by decellularization method using 75% alcohol–0.25% trypsin treatment at two different time intervals. The percentage of decellularization was calculated for all the prepared conduits (n = 3), and it was observed that longer exposure to alcohol and trypsin leads to better decellularization. Endothelial cells were seeded on the conduits prepared by the above methods. Results About 0.2% glutaraldehyde-fixed conduits showed better attachment of endothelial cells, as compared with ethanol-trypsin–treated decellularized conduits. These results demonstrate the use of BCA as a source of biological conduits in the creation of a new small-diameter tissue-engineered vascular graft. Conclusion This study shows that it is possible to prepare conduit and scaffold (glutaraldehyde-treated and decellularized) for seeding endothelial cells on their luminal surface, which can be used for vascular grafting.


Introduction
Atherosclerosis is the leading cause of death in the developed world and is on the rise in developing countries 1,2 .The vascular endothelium is a complex modulator of a variety of biological systems and may be the key to definitive success in the treatment of cardiovascular disorders.The lack of endothelial cells (ECs) on the luminal surface contributes to thrombogenicity and promotes proliferation of pseudointima.The EC matrix avoids exposure of circulating white blood cells to the basement membrane followed by the tunica media.The interaction of blood with medical grade biomaterials commonly results in thrombus formation 1 .We have focused on lining the luminal surface of conduits from animal sources with ECs.The EC lining of the blood vessels reduces the thrombogenicity.The adverse immune response from the host can be avoided in future by transplanting autologous ECs.One of the significant aspects in developing a biological conduit is avoidance of graft rejection.
In the present work, we have treated bovine carotid arteries (BCAs) in two different ways: (a) exposure to 0.2% glutaraldehyde and (b) decellularization.Glutaraldehyde treatment provides structural stability and is an excellent sterilizing agent 17 .Glutaradehyde-treated tissue correlates well with the concept of stabilized tissue of low antigenicity and reactivity.The mechanism by which glutaraldehyde is believed to reduce connective tissue antigenicity is through the conversion of tissue proteins into insoluble crosslinked structures, with greatly enhanced resistance to proteolytic degradation and masking through antigenic sites and also strongly increases hydrophilia of the surface, thus enhancing cellular attachment due to strong bonding of 0.2% glutaraldehyde to tissue collagen that provides a robust and rubbery nature to the conduit 3 .Decellularized tissue scaffolds have evidence of similar compliance and burst strength to the native arteries.One important advantage is the absence of cellular components in the different layers of the decellularized conduit, leaving behind the only scaffold structure that could reduce antigenicity and rejection after implantation.
We further hypothesize that a tissue-engineered biological conduit composed of biological materials and expanded sheep external jugular vein endothelial cells (SEJVECs) would have advantages.A viable graft will show responsive and self-renewing tissue.Biological components will be remoulded at in vivo conditions.Absence of synthetic material will preclude foreign body reaction to allow complete graft integration.These coverslips were thoroughly washed with distilled water for 5 min and stained with haematoxylin (1%) stain for 15 min, followed by 1% acid alcohol treatment to decolourize excess stain.These coverslips were counterstained with eosin (1%).After a final wash with absolute alcohol, stained ECs were mounted in DPX and observed under phase-contrast microscope (Carl Zeiss Meditec AG) 7 .

Conduit/scaffold preparation
BCAs and BJVs were used for conduit/scaffold preparation.We have used two different methods for conduit preparation: (a) glutaraldehyde treatment and (b) enzymatic decellularization of vessels.

Glutaraldehyde treatment 8
BCAs (n = 3) were cleaned by flushing its luminal surface with sterile 1× PBS (Hi-Media) till all blood clots were removed.These samples were fixed in 0.2% glutaraldehyde (SD Fine Chemicals, Mumbai, India) for 7 days and stored at 4°C.On the eighth day, these samples were treated with 10% citric acid for 5 min to neutralize glutaraldehyde.Finally, treated BCAs were washed with sterile distilled water till neutral pH was achieved.
Decellularization of BCA and BJV 9 Cleaned BCAs (n = 3) and BJVs (n = 3) of 10-12 cm length were immersed in 75% ethanol solution for 48 h with solvent to tissue mass ratio of 20:1.Decellularization of BCA and BJV were achieved by incubating them in trypsin-EDTA (Invitrogen) (1×) for 48 h at 37°C.These decellularized BCAs and BJVs were crosslinked with methylene green for photo-oxidation.The decellularized vessels were processed for histological analysis by 10% formaldehyde treatment followed by paraffin-embedded block preparation.These blocks were trimmed and sectioned (5 µm) on fully automated rotary microtome (Lecia) at their midpoints and stained by H&E.fibronectin-coated culture flasks.After 12 h of incubation, ECs were fed with complete ECGM containing 20% FBS, 2 mM L-glutamine, 10 µg/ml heparin, penicillin 5 U/ml and streptomycin 5 mg/ml and 6 mg/ml ciprofloxacin and incubated at 37°C at 5% CO 2 .The split ratio for the subculture was kept at 1:2, and cells were reseeded in fresh tissue culture flasks.Subculturing of ECs was done at 70% confluency 4,5 .
Harvested and cultured SEJVECs were maintained for up to five passages.The proliferation rate was calculated by plating fixed population densities of ECs and their recovery after a number of days.

Characterization of ECs
The cells from early passages (two to three) were used for the characterization studies.For immunocytochemistry, study cells were grown in monolayers on coverslips and were fixed using 4% paraformaldehyde 6 .The cells were then permeabilized using 50% methanol for 5 min followed by treatment with 5% bovine serum albumin (BSA) in PBS for 1 h to block nonspecific binding sites.The cells were then exposed to antivon-Willebrand factor (vWF), a primary non-labelled antibody IgG fraction of anti-serum developed in the rabbit (1:200 dilution) (Chemicon, Temecula, CA), for 12 h at 4°C, followed by respective FITC-tagged anti-rabbit secondary antibody (Invitrogen) for 1 h at 37°C.The coverslips were mounted on a mounting medium containing anti-fade (Vectashield, Vector Laboratory, Burlingame, CA) and 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).The slides were then viewed using a confocal laserscanning microscope-LSM 510 Zeiss workstation (Carl Zeiss Meditec AG, Jena, Germany).DAPI was used for nuclei visualization.

Haematoxylin and eosin (H&E) staining
SEJVECs grown on coverslips were fixed in absolute alcohol for 10 min.

Reduced graft infection may result in increased overall patency.
The present study demonstrates two different tissue-engineered biological conduits based exclusively on the use of cultured SEJVECs seeded on the luminal aspect.

Materials and methods
All experimental protocols were approved by the KEM Hospital and GS Medical College Animal Ethics Committee.Animal care was in accordance with the institution guidelines.
Sample collection: BCAs, bovine jugular vein (BJV) and SEJVs were collected from Deonar Abattoir, Govandi, Mumbai.An external jugular vein segment was isolated by a vertical incision over the left side of the neck parallel to the trachea.Isolated external jugular vein segment was collected in a phosphate buffer saline (PBS) (Hi-Media, Mumbai, India) containing antibiotics (penicillin 25 U/ml and streptomycin 25 mg/ ml-Gibco-BRL, Burlington, OHand ciprofloxacin 30 mg/ml-Cipla, Mumbai, India) and immediately transported to the tissue culture laboratory at the KEM Hospital.

Isolation and expansion of SEJVECs
In brief, the SEJV was cannulated and flushed with sterile PBS to remove blood and blood clots.One end of the vein was clamped using artery forceps, and an enzyme cocktail of 0.15% collagenase type IV (Sigma Aldrich, St. Louis, MO) and dispase II (Roche, Nutley, NJ) was injected in the vein for detachment of ECs from the luminal wall.The entire vein was incubated for 20 min at 37°C.At the end of the incubation period, the vein was flushed using M199 medium (Invitrogen, Carlsbad, CA), and the digest was centrifuged at 1,500 rpm for 10 min.Cell pellet was resuspended in the endothelial cell growth medium (ECGM) (PromoCell GmbH, Germany) containing 20% foetal bovine serum (FBS) (Invitrogen) and seeded onto adhere to the culture well within 3-4 h of seeding.
SEJVECs showed typical 'cobblestone' morphology in growing culture.H&E-stained samples of SEJVECs resemble perfect cobblestone morphology with dark-bluestained nuclei and a pink cytoplasm background (Figure 1).
The EC structure became more elongated in shape as the passage number increased.SJVECs showed contact inhibition after reaching the fifth passage.

SEJVEC characterization
Immunocytochemistry and confocal microscopy confirmed the presence of the endothelial-specific marker (vWF) in the cultured SEJVECs (Figure 2).Simultaneously, negative control showed only DAPI staining 15 .
Conduit/scaffold preparation BCA and BJV conduit was prepared by 0.2% glutaraldehyde treatment.It was observed to be very robust, rubbery and mechanically very strong.BJV conduit was treated in the same manner.

Comparative decellularization of BCA and BJV
As is clearly evident in Table 1, a 24-h treatment with ethanol-trypsin was Scanning electron microscopy (SEM) SEM of 0.2% glutaraldehyde-treated conduit and decellularized scaffold was performed.SEJVEC expansion onto the luminal surface was analysed for endothelization.Scaffold samples were fixed in 2.5% glutaraldehyde for 2 h at room temperature and thereafter washed with PBS and dehydrated with graded ethanol for 10 min each 13,14 .Samples were dried overnight and sputter coated with gold (Quorum Technologies Ltd., UK) and observed under SEM (Zeiss scanning electron microscope).

Isolation and expansion of SJVECs
Around 50-70 clusters of ECs were isolated from 10-cm long SEJVs.ECs from the SEJVs attached well within a few hours to the fibronectin-coated surface and reached 70-75% confluency within 6-8 days depending upon the initial seeding density.After subculture, the cell density at 70% confluency was found to be 6 × 10 5 cells/cm 2 .These freshly isolated cells

Comparative determination of cellularity
By using bright-field microscope interfaced with an image analysis system (Zeiss imaging system canon 32 pixel), all nuclei in the intima, media and adventitia present within a bandwidth were counted.Quadrants in two transverse sections (total of eight counts) were averaged for each fresh control (n = 3) and decellularized (n = 3) specimen.Percentage decellularization was calculated using the formula given below.BJV specimens were processed (studied) just for comparative analysis to check the degree (process) of decellularization 10 .

Evaluation of extracellular matrix
Collagen and elastin were analysed similarly, by fixing the BCA (n = 3) and BJV (n = 3) samples in 10% formalin.Fixed samples were embedded in paraffin before taking transverse section (5 µm).All the cut sections were stained by the Verhoeff-van Gieson method 11 .The collagen-and elastincovered area in the stained sections were observed under bright-field microscopy and analysed using digital image analysis.

Seeding of the graft surface with cultured ECs and its in vitro evaluation
Expanded SEJVECs from the third passage were trypsinized using 0.025% trypsin-EDTA (Sigma Aldrich) and seeded onto the luminal surface of the basement membrane, which was already coated with fibrin glue on conduit, and scaffold treated in two different ways: 0.2% glutaraldehyde-treated conduit and enzymatic decellularized BCA scaffold, which was incubated at 37°C at 5% CO 2 12 .After 12 h of incubation, arteries containing SEJVECs were fed with 20% ECGM.Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.for BCA and BJV respectively.Further treatment for 48 h resulted in better decellularization than 24-h treatment.

Extracellular matrix structure
Bright-field microscopy of fresh and 0.2% glutaraldehyde-treated BCAs (n = 3) (Figure 3) and BJVs (n = 3) (Figure 4) stained with the Verhoeff-van Gieson provided evidence of preservation of extracellular matrix and cellular elements.BCAs (Figure 5) and BJVs (Figure 6) treated with ethanol and trypsin for 48 h showed maximum decellularization with preservation of extracellular matrix structure.Collagen stained red and elastin (black) were intact in Verhoeff-Gieson sections.

Seeding of the graft surface with cultured SEJVECs and SEM
Harvested SEJVECs cultured onto both types of conduit and scaffolds (BCA), that is glutaraldehyde and decellularized, showed cell proliferation without any cell death.The expansion of cultured SEJVEC was confirmed on the basis of electron microscopy for both types of scaffold.SJVECs seeded onto glutaraldehyde-treated scaffold showed clear proliferation of SJVECs (Figure 7).Similar results were obtained for decellularized scaffold,   biological tissue.Glutaraldehyde protein interaction reactions most likely account for the unique structural stability of connective tissue treated with glutaraldehyde.The free aldehydes of monomeric or polymeric glutaraldehyde react primarily with amines of lysine, hydroxylysine or N-terminal amino acid residues present in the protein to form Schiff base, which is extremely stable at physiological temperature and pH 17 .
Glutaradehyde cross-links with collagen fibres of the artery, mainly in the adventitial region as more fibroblast cells are present in that region; it binds covalently.Although fixation of BCA for 7 days results in complete cross-links with collagen fibres, glutaraldehyde toxicity is neutralized by washing the BCA conduits with 10% citric acid.This strongly increases the hydrophilia of the surface, thus enhancing cellular attachment.Strong bonding of glutaraldehyde to BCA tissue collagen provides a rubbery nature to the conduit.
The histological analysis of both H&E and Elastin-Verhoeff-van Gieson (EVG) technique provided evidence that glutaraldehyde helps in maintaining its intact tissue structure along with nonviable cells in all three layers (intima, media and adventitia) conduit.
BCA (n = 3) and BJV (n = 3) clearly gave a comparatively similar percentage of the decellularization results after various treatment procedures followed by the development of the biological scaffold.Similar and comparable results were obtained for 48-h BCA and BJV ethanol-trypsin treatment.Histological staining by H&E and EVG technique showed that the comparative results for 24-h BCA and BJV trypsin treatment and for 48-h BCA and BJV trypsin treatment proved that the process of decellularization can even be achieved by using trypsin treatment, while decellularization with ethanol-trypsin demonstrated excellent percentage of decellularization for incubation periods was studied using H&E staining.The growth period of SEJVECs was found to be 14 days, which was studied by plating fixed densities of SEJVECs into the wells and the number of days required for reaching confluence.
Webiel-Palade bodies are unique characters of ECs, while the vWF is a molecular marker that is located in these bodies.Immunostaining of the vWF confirms the isolated cell population is ECs.
The use of glutaraldehyde has been widely used over the past few years to sterilize and stabilize the with and without expansion of ECs (Figure 8) 16 .

Discussion
The harvested and expanded SEJVECs were maintained in the culture till five passages.Contact inhibition was observed after the fifth passage, indicating similar cellular profile of SEJVEC to that of human umbilical vein endothelial cells and human saphenous vein endothelial cells, which shows contact inhibition after reaching 70% confluence.The typical cobblestone morphology of cultured SEJVECs

Conclusion
This study shows that it is possible to prepare conduit and scaffold ( glutaraldehyde-treated and decellularized) for seeding ECs on their of 48-h and 24-h BCA and BJV.From the above progressive decellularization results, it was concluded that we can prepare maximum decellularized scaffold with ethanol-trypsin treatment of 48 h each (BCA and BJV).This is likely to provide excellent patency if implanted in animal models in future, as there will be lesser chance of graft antigenicity and rejection due to lack of tissue cells in the different layers of the conduit.

Figure 1 :
SEJVECs in dark-blue-stained nuclei and pink cytoplasm background.% ( Decellularization Cell count for fresh BCA or BJV cell c = − o ount decellularized BCA or BJV Cell count for fresh B )×100 C CA or BJV Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY) For citation purposes: Almelkar SI, Kadam SS, Diwate S, Chaukar A, Bhonde RR, Patwardhan AM.Re-endothelialization of biological tissue-engineered conduits and scaffold for its potential use in bioprosthesis.OA Tissue Engineering 2013 Mar 01;1(1):1.

Figure 2 :
Figure 2: Immunocytochemistry of these isolated SJVECs showed expression of vWF, confirming their identity as endothelial cells.

Table 1 Level of significance (P value) percentage of decellularization (mean ± SD) and comparison of treatments of sample S. no. P Value Sample Comparison of Treatments Percentage of Decellularization and Standard Deviation (Mean ± SD)
more effective as compared with only trypsin treatment values (1a) and (2b)