Interact CardioVasc Thorac Surg 2009;8:301-305. doi:10.1510/icvts.2008.194076
© 2009 European Association of Cardio-Thoracic Surgery
Work in progress report - Congenital |
Decellularized and photooxidatively crosslinked bovine jugular veins as potential tissue engineering scaffolds
Wei-Dong Lüa,b,
Ming Zhanga,
Zhong-Shi Wua,* and
Tie-Hui Hua
a Department of Thoracic and Cardiovascular Surgery, Second Xiangya Hospital of Central South University, Changsha, Hunan Province, 410011, People's Republic of China
b Department of Thoracic Surgery, Tumor Hospital of Shaanxi Province, Xi'an, Shaanxi Province, 710061, People's Republic of China
Received 14 September 2008;
received in revised form 23 November 2008;
accepted 25 November 2008
 This work was supported by Special Project of Hunan Science and Technology Plan of People's Republic of China (04SK1005).
*Corresponding author. Tel.: +86-731-5295108; fax: +86-731-5292133.
E-mail address: owenzswu{at}yahoo.cn (Z.-S. Wu).
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Abstract
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Decellularization means and altering crosslinking approaches were two promising alternatives for glutaraldehyde fixation to biological tissues. Bovine jugular veins (BJVs) were decellularized by a multi-step detergent–enzymatic extraction method, then photooxidatively crosslinked. Gross and histological integrity of which was retained. Ultrastructures showed integrity of collagen fibrils and elastic fibers, and a basement membrane free luminal surface. Mechanical strength test and tissue protein extraction assay demonstrated their tissue stability. After being pre-coated with gelatin, collagen IV and fibronectin, cultured human umbilical vein endothelial cells were planted in the luminal surface of decellularized plus photooxidized BJV patches for seven days. Endothelial cells were denser in pre-coated patches than in uncoated controls. A rat subcutaneous implantation model revealed more resistance against in vivo degradation for further crosslinked BJV patches than decellularized patches at 12-week retrieval. Host cells were all layer repopulated for both. Histological examination and content assay demonstrated collagen and glycosaminoglycan components synthesis for decellularized plus photooxidized BJV patches. Decellularized and photooxidatively crosslinked BJV patches possess tissue integrity, excellent in vitro and in vivo tissue stability and repopulation patterns. Thus, they have potentials as tissue engineering scaffolds in future cardiovascular surgery.
Key Words: Tissue engineering; Bovine jugular vein; Decellularization; Photooxidation; Extracellular matrix
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1. Introduction
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The glutaraldehyde-fixed bovine jugular vein (BJV) conduit [Contegra (Medtronic Inc, Minneapolis, MN, USA)] constitutes a promising alternative to homograft. However, several authors expressed their concern about an unpredictable incidence of fibrous supravalvular stenosis, thrombosis and aneurysmal dilatation at mid-term follow-up [1–3]. Immune response was found to play a significant role in the process [4]. Presence of cellular remnants and residual glutaraldehyde released from the implant were thought to be partly responsible for immunogenicity of the conduits [5]. Decellularization means and altering crosslinking approaches were two promising alternatives to glutaraldehyde fixation. Liang et al. [6] demonstrated that decellularized natural materials that underwent proper cross-linking have potentials as tissue engineering scaffolds. The dye-mediated photooxidation technique has little cytotoxicity, and possesses chemical, enzymatic, and in vivo stability [7]. We suppose that decellularization plus photooxidation (DP) treatment to BJVs could produce scaffolds that have both tissue stability and regeneration patterns. This research was focused on the histological change, tissue stability, in vitro cell planting, and in vivo performance of DP-treated BJVs.
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2. Material and methods
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2.1. Decellularization procedure
BJVs of 300–500 kg bovines were obtained fresh from a local slaughterhouse. The multi-step detergent–enzymatic decellularization procedure was developed by Teebken et al. [8] with several modifications. Briefly, BJVs were decellularized by detergent Triton X-100 (0.5%, Amresco Inc, Solon, OH, USA) for 48 h, Trypsin (0.025%)/EDTA (0.02%) (both Sigma Ltd, Poole, Dorset, UK) for 30 min, and DNase I (30 U/ml)/RNase A (0.3 mg/ml) (both Invitrogen Corp, Carlsbad, CA, USA) with 50 mmol/l MgCl2 included for 24 h. Then the conduits were washed several times with PBS. All steps were conducted under continuous shaking condition at 37 °C.
2.2. Photooxidation technique
Decellularized BJV conduits were photooxidatively crosslinked according to the method of Moore et al. [7]. The treated conduits were stored in PBS and sterilized by  -ray irradiation (25 KGy).
2.3. Histology
Decellularized and DP-treated BJV walls and valves were fixed in 10% phosphate-buffered formalin. Paraffin sections were cut at 5-µm thickness, stained in a 0.1% Sirius-red solution (Direct Red 80, Sigma Ltd) in saturated picric acid, and analyzed by fluorescence microscopy [9].
2.4. Ultrastructures
Decellularized and DP-treated BJV walls were fixed in 2.5% glutaraldehyde in PBS (pH 7.4) and underwent transmission electron microscopy (TEM) and scanning electron microscopy (SEM) examination.
2.5. Biomechanical test
The mechanical tensile strengths of native, decellularized and DP-treated BJV walls (n=10) were determined by uniaxial measurements using a Lloyd LRX material testing machine (Lloyd Instruments Ltd, Fareham, Hampshire, UK). The specimens (5 mmx20 mm) were stress-loaded to rupture at a constant speed of 20 mm/min. The tensile strength denoted the amount of force required for rupture to occur.
2.6. Tissue protein extraction assay
Samples of each group (n=3) were placed in separate 1.5 ml microcentrifuge tubes and underwent tissue protein extraction assay as previously reported by Moore et al. [10].
2.7. Cell planting study
Cultured human umbilical vein endothelial cells (HUVECs) were planted in the luminal surface of DP-treated BJV patches (n=6). The HUVEC line (purchased from the American Type Tissue Collection, Catalog #CRL-2480, Manassas, VA, USA) was maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Invitrogen Corp), containing 10% fetal bovine serum and 1% penicillin—streptomycin in a humidified incubator (37 °C, 5% CO2). The cultured cells were trypsinized and resuspended in culture medium, seeded (approximately 2.5x105 cells/cm2) onto the scaffolds, and cultured in static DMEM for seven days in an incubator. Cell medium was carefully changed on a daily basis. Before seeding, DP-treated BJV patches were sheared to disks (diameter 1.5 cm), placed into 4-well plates (one patch per well), and pre-coated with 100 mg/ml fibronectin, 10 mg/ml gelatin and 50 mg/ml collagen IV (all Sigma Ltd) for 2 h. Uncoated controls were treated with PBS. The 7th day samples had SEM checks (n=3). Paraffin sections (n=6) were studied by hematoxylin–eosin (H&E) staining.
2.8. Animal study
All animals received humane care in compliance with the European Convention on Animal Care and the study was approved by the Animal Care and Use Committee of Central South University. The decellularized and DP-treated BJV patches (n=12, size, 10 mmx20 mm) were implanted subcutaneously in a growing rat model (3-week-old male Sprague–Dawley rats) under aseptic conditions. On retrieval at 12 weeks postoperatively, samples were grossly examined. Parallel sections were stained with H&E, elastica-van Gieson (EVG) and Scott's alcian blue method. Immunohistochemistry was performed using goat anti-procollagen-I antibody (1:200, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) to detect neo-collagen fibrils.
Retrieved tissues and their adjacent segments at pre-implantation (n=12) were lyophilized for 24 h to dry weight. Hydroxyproline was extracted according to the method of Kivirikko et al. [11] and was converted to collagen content by assuming a factor of 12.5%. Elastin was extracted with 0.25N oxalic acid (Sigma Ltd) at 95 °C for 1 h for four times and elastin content was measured using a Fastin Elastin Assay kit (Biocolor Ltd, Belfast, Northern Ireland). Glycosaminoglycans (GAGs) were assayed according to the method of Farndale et al. [12].
2.9. Statistics
All values were expressed as mean±S.E.M. Statistical analysis was performed with a one-way ANOVA followed by analysis between groups with the Student–Newman–Keuls test. A value of P<0.05 was considered significant.
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3. Results
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3.1. Gross appearance
The wall and valves kept their integrity after decellularization (Fig. 1a). After being further photooxidatively crosslinked, the wall and valves appeared blue and also retained their integrity (Fig. 1b).
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Fig. 1. (a,b) The luminal gross appearance of decellularized (a) and decellularization plus photooxidation (DP)-treated BJVs (b). (c–f) Walls and valves of decellularized (c and d) and DP-treated BJVs (e and f) stained by Sirius red and observed under fluorescent light (scale bar=50 µm). The collagen fibrils were red-orange color whereas elastic fibers showed strong green. (g–j) Ultrastructures of decellularized and DP-treated BJV walls. Locations of elastic fibers (black arrowheads) and collagen fibrils (white arrowheads) are indicated in TEM for decellularized (g) and DP-treated BJV walls (h) (6000x, scale bar=5 µm). SEM examination of the luminal surface of decellularized (i) and DP-treated BJV walls (j) showed a basement membrane free luminal surface for both treatments and non-orientation collagen fibrils for DP-treated walls (scale bar=10 µm).
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3.2. Histology
Images of decellularized BJVs (Fig. 1c,d) showed collagen fibrils were retained well in the wall and valve, and elastic fibers were distributed abundantly in the wall but sparsely in the base of the valve. DP-treated BJVs (Fig. 1e,f) were comparable to decellularized BJVs.
3.3. Ultrastructures
TEM investigation showed freedom of cellular components with normal appearing collagen fibrils and elastic fibers for decellularized BJV walls (Fig. 1g). DP-treated BJV walls also retained their integrity of collagen fibrils and elastic fibers (Fig. 1h).
SEM micrographs of the decellularized BJV walls revealed lack of cellular remnants in the luminal surface, with collagen fibrils exposed (Fig. 1i). Collagen fibrils lost their orientation after further photooxidation crosslinking (Fig. 1j).
3.4. Biomechanical test
Tensile strength test (Fig. 2a) demonstrated decrease of mechanical stability with decellularization treatment. Further photooxidation crosslinking recovered the mechanical stability.
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Fig. 2. (a) Tensile strength comparison of native (N), decellularized (De), and DP-treated BJV walls. Investigation of decellularized BJV conduits revealed a dramatical loss of strength, but the strength recovered for DP-treated BJV conduits. All presented results were mean±S.E.M. [*P<0.05 vs. native BJV conduits (n=10);  P<0.05 vs. decellularized BJV conduits (n=10)]. (b) Polyacrylamide gel electrophoretic analysis of extractant supernatants of native (N), decellularized (De) and DP-treated BJV walls. Note the decrease of extractable protein in decellularized BJV tissues and lack of extractable protein in DP-treated BJV tissues.
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3.5. Tissue protein extraction assay
Fig. 2b presents the protein extraction assay for tissue stability. Electrophoretic separation of decellularized tissues demonstrated removal of considerable amounts of proteins, indicating the effective reduction of potential immunogenic components. Fewer protein bands were detected in the DP-treated tissues, demonstrating the stabilizing effect of photooxidation crosslinking on decellularized BJVs, and further decrease of immunogenicity.
3.6. Cell planting
H&E staining revealed a confluent layer of cells on the luminal surface of pre-coated BJV patches (Fig. 3b). To their uncoated controls, less cells presented and cell-extracellular matrix (cell-ECM) linkage was sparse (Fig. 3a). SEM investigation showed more cells and cell–cell junctions in the pre-coated surface (Fig. 3d) than in the uncoated controls (Fig. 3c).
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Fig. 3. Endothelial cell seeding of uncoated controls and pre-coated DP-treated BJV patches. Hematoxylin–eosin (H&E) staining showed more cells in the luminal surface of pre-coated patches (b) than in uncoated patches (a) (scale bar=50 µm). SEM of the pre-coated specimens (d) showed a confluent layer of endothelial cells, whereas uncoated controls (c) revealed only a few endothelial cells (scale bar=10 µm).
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3.7. Animal study
All rats survived the prescribed period. On retrieval at 12 weeks postoperatively, decellularized BJV patches were severely degraded. In contrast, the DP-treated patches were comparable to their pre-implantation counterparts (Fig. 4a,b).
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Fig. 4. Gross appearance, histology and tissue content analysis of decellularized and DP-treated BJV patches before implantation and at 12-week rat subcutaneous implantation retrieval. For retrieved patches (b), decellularized patches were greatly degraded but DP-treated patches were comparable to their pre-implantation counterparts (a). H&E staining showed a cell-free wall before implantation (c), a thinner wall repopulated by host cells for retrieved decellularized patches (f), and a moderate wall repopulated by host cells for retrieved DP-treated BJV patches (i). Elastica-van Gieson (EVG) staining shows elastic fibers in black and collagenous materials in pink (d,g,j). Alcian blue staining shows glycosaminoglycans (GAGs) in blue (e,h,k). Procollagen-I staining shows neo-collagen fibrils in brown (l,m) (scale bar=100 µm). For tissue content analysis (n), all presented results are mean±S.E.M. [*P<0.05 vs. pre-implantation counterparts for decellularized patches (n=12); P<0.05 vs. pre-implantation counterparts for DP-treated patches (n=12)].
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For decellularized BJV patches before implantation, H&E staining showed removal of all cellular components, leaving some open spaces within collagenous ECM (Fig. 4c). Black stain elastic fibers were in a parallel-oriented arrangement, including pink collagen fibrils in between, and the adventitia exhibited scanty elastic fibers (Fig. 4d). Blue stain GAGs were all layer present (Fig. 4e). Since no histological difference was found between decellularized and DP-treated BJV patches before implantation, histochemical staining for DP-treated BJV patches at pre-implantation was not shown.
The retrieved decellularized samples showed that thinner walls were repopulated by host cells, with neo-capillaries all layer present (Fig. 4f). Elastic fibers were compactly arranged (Fig. 4g). GAGs were of stronger expression in all layers than pre-implantation and procollagen-I positive staining tissues were present in the inner layer areas (Fig. 4h,l), implying synthesis of new GAGs and neo-collagen fibrils.
H&E staining showed the retrieved DP-treated patches were comparable to the pre-implantation patches. Host cells were all layer repopulated, with neo-capillaries found in the recellular areas (Fig. 4i). Elastic fibers were less in the outer and inner layer (Fig. 4j). New GAGs and neo-collagen fibrils were compactly distributed in the outer 1/3 and inner 1/3 of the walls (Fig. 4k,m).
Fig. 4n shows tissue content analysis of decellularized and DP-treated BJV patches. Collagen, elastin and GAG contents were comparable before implantation (all P>0.05, n=12). For the retrieved decellularized patches, collagen content greatly decreased, and elastin and GAG contents increased significantly compared to their pre-implantation counterparts (all P<0.05, n=12). For the retrieved DP-treated patches, elastin content decreased and GAG content increased compared to their pre-implantation counterparts (both P<0.05, n=12). Collagen content increased but did not show statistical significance (P=0.053, n=12).
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4. Discussion
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In this study, a multi-step detergent–enzymatic extraction method based on Teebken et al. [8] was developed to remove the cellular components of BJVs. The integrity of collagen fibrils and elastic fibers was retained. Decellularization to BJVs showed tissue stability was reduced, and further photooxidation was shown to effectively stabilize the decellularized BJVs by increasing mechanical tensile strength and resisting tissue protein extraction. Protein extraction assay presents the extractable protein and reflects the immunogenicity. It had also been used to test tissue stability of photooxidatively crosslinked tissues [10]. The resistance to protein extraction demonstrated photooxidation stabilized the decellularized BJVs, and lessened their immunogenicity.
Lack of basement membrane may have a negative effect on further endothelial cell seeding since intact basement membrane is significant for endothelial cell attachment for natural tissues [13]. Nevertheless, the drawbacks of basement membrane removal can be overcome by surface pre-coating. Our mixture-pre-coated BJV patches supported more endothelial cells attachment and growth than their uncoated counterparts, and a confluent layer of endothelial cells will make the luminal surface resist thrombus formation and fibrous overgrowth in future circulating implantation.
Gross and histological investigations to rat subcutaneous implantation model showed more in vivo stability of DP-treated BJV patches than their uncrosslinked counterparts. It has been shown that crosslinkers significantly reduce degradation and cellular infiltration and thereby enhance graft stability, but they do not eliminate the chronic inflammatory response toward xenogeneic biomaterials [14]. Host cells were able to infiltrate into all layers of both decellularized and DP-treated BJV patches. The infiltrated endothelial cells, fibroblasts and myofibroblasts may synthesize collagen and other ECM proteins, and produce matrix proteinases for further tissue remolding [15]. Mononuclear cells were sparsely distributed in the middle layer of DP-treated BJV patches, indicating less chronic inflammatory response and degeneration for DP treatment than sole decellularization.
The literature provided evidence for photooxidative crosslink formation in collagen proteins [7]. To the DP-treated BJV patches, thick walls can be comparable to the walls before implantation and neo-collagen fibrils also appeared in the outer layer of the walls, where elastic fibers were scanty. Tissue content analysis showed that GAG content increased, but elastin content decreased, and collagen content increased slightly despite lack of statistical significance. It was different to their decellularized counterparts. Possible mechanism is that photooxidation stabilized the collagens against in vivo proteolysis, and the undegraded collagens acted as platforms for host cells ingrowth and synthesis of neo-collagens and new GAGs. However, biosynthesis of elastin component was very limited and needs further study.
The limitation of this study is that the activity and function of endothelial cells in cell planting is not evaluated, which is important for maintenance of long-term integrity of the vascular scaffold. Moreover, for a potential clinical application, expected induction of immune responses against the surface pre-coating foreign proteins is critical.
In conclusion, DP-treated BJV patches have ideal tissue stability, support endothelial cells attachment and growth in vitro after pre-coating, and resist degradation and have regeneration patterns in vivo. Thus, they have potentials as tissue engineering scaffolds for future application in cardiovascular surgery.
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Acknowledgements
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We thank Dr Lan Song for advice during the preparation of the manuscript.
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References
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- Sekarski N, van Meir H, Rijlaarsdam ME, Schoof PH, Koolbergen DR, Hruda J, von Segesser LK, Meijboom EJ, Hazekamp MG. Right ventricular outflow tract reconstruction with the bovine jugular vein graft: 5 years' experience with 133 patients. Ann Thorac Surg 2007;84:599–605.[Abstract/Free Full Text]
- Boudjemline Y, Bonnet D, Agnoletti G, Vouhé P. Aneurysm of the right ventricular outflow following bovine valved venous conduit insertion. Eur J Cardiothorac Surg 2003;23:122–124.[Abstract/Free Full Text]
- Tiete AR, Sachweh JS, Roemer U, Kozlik-Feldmann R, Reichart B, Daebritz SH. Right ventricular outflow tract reconstruction with the Contegra bovine jugular vein conduit: a word of caution. Ann Thorac Surg 2004;77:2151–2156.[Abstract/Free Full Text]
- Boudjemline Y, Beyler C, Bonnet D, Sidi D. Surprising outcome similarities between Contegra bovine jugular vein conduit and Shelhigh No-React porcine pulmonary valve conduit: role of immunologic reaction. Eur J Cardiothorac Surg 2003;24:850–851.[Free Full Text]
- Wojtalik M, Mrowczynski W, Zeromski J, Bartkowski R. Does Contegra xenograft implantation evoke cellular immunity in children? Interact Cardiovasc Thorac Surg 2003;2:273–278.[Abstract/Free Full Text]
- Liang HC, Chang Y, Hsu CK, Lee MH, Sung HW. Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern. Biomaterials 2004;25:3541–3552.[CrossRef][Medline]
- Moore MA, Bohachevsky IK, Cheung DT, Boyan BD, Chen WM, Bickers RR, McIlroy BK. Stabilization of pericardial tissue by dye-mediated photooxidation. J Biomed Mater Res 1994;28:611–618.[CrossRef][Medline]
- Teebken OE, Bader A, Steinhoff G, Haverich A. Tissue engineering of vascular grafts: human cell seeding of decellularised porcine matrix. Eur J Vasc Endovasc Surg 2000;19:381–386.[CrossRef][Medline]
- Borges LF, Taboga SR, Gutierrez PS. Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy. Cell Tissue Res 2005;320:551–552.[CrossRef][Medline]
- Moore MA, Chen WM, Phillips RE, Bohachevsky IK, McIlroy BK. Shrinkage temperature versus protein extraction as a measure of stabilization of photooxidized tissue. J Biomed Mater Res 1996;32:209–214.[CrossRef][Medline]
- Kivirikko KI, Laitinen O, Prockop DJ. Modifications of a specific assay for hydroxyproline in urine. Anal Biochem 1967;19:249–255.[CrossRef][Medline]
- Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986;883:173–177.[Medline]
- Davis GE, Senger DR. Endothelial extracellular matrix: biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ Res 2005;97:1093–1107.[Abstract/Free Full Text]
- Courtman DW, Errett BF, Wilson GJ. The role of crosslinking in modification of the immune response elicited against xenogenic vascular acellular matrices. J Biomed Mater Res 2001;55:576–586.[CrossRef][Medline]
- Stenmark KR, Davie N, Frid M, Gerasimovskaya E, Das M. Role of the adventitia in pulmonary vascular remodeling. Physiology (Bethesda) 2006;21:134–145.[CrossRef][Medline]
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Abstract
1. Introduction
