Asian Cardiovasc Thorac Ann 2007;15:91-96
© 2007 Asia Publishing EXchange Ltd
Histological/Biological Characterization of Decellularized Bovine Jugular Vein
Wu Li, MD,
Wei-Yong Liu,
Ding-Hua Yi, MD,
Shi-Qiang Yu, MD,
Zhen-Xiao Jin, MD
Department of Cardiovascular Surgery Xijing Hospital, The Fourth Military Medical University Xian, China
For reprint information contact: Wei-Yong Liu, MD Tel: 86 29 8477 5565 Fax: 86 29 8321 0092 Email: weiyongl001{at}163.com, 15 Changlexi Street, Xian 710032, Shaanxi Province, China.
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ABSTRACT
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Several deficiencies in currently available right ventricular valved conduits make them problematic for use in infants and children. A solution would be to develop a tissue-engineered valved conduit containing autologous cells. A method was devised to produce a decellularized bovine matrix scaffold for developing a tissue-engineered right ventricular valved conduit. Fresh bovine jugular veins were treated with sodium deoxycholate and Triton X-100. The major structural proteins of the fresh and decellularized jugular venous valves and vessel walls were detected by histological methods. Thickness, water absorption rate, water maintenance rate, disruption strength, and extensibility were determined. Circumferential and radial specimens of valves and vessel walls were subjected to tensile testing. Histological analysis showed that no cell fragments were retained within the decellularized matrix scaffold and the major structural proteins had been retained intact. There were no significant differences in thickness, rates of absorption and maintenance of water, disruption strength, and extensibility between the decellularized and fresh veins. It was concluded that this treatment can successfully remove cellular components while maintaining the major structural components and the histological and biological properties of bovine jugular veins.
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INTRODUCTION
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Current right ventricular outflow tract replacements have problems during long-term implantation.1 Tissue valved conduit substitutes, including xenografts and homografts, have a much lower risk of thromboembolism but a life expectancy of only 10–15 years.2 These conduits are difficult to size-match for infants and children.3,4 One solution would be tissue engineering. A tissue-engineered right ventricular valved conduit containing living autologous cells would be able to maintain its histological and biological properties yet grow in infants and children. Bovine jugular vein contains natural valves with extremely thin and mobile leaflets.5 The venous vessel wall is soft and ideal for suturing, and usually does not necessitate interposition of other materials to enable it to be anastomosed to the right ventricle and the pulmonary artery. Moreover, its availability in various diameters from 12 to 22 mm makes its use possible in infants and children.6 It is important that the decellularization technique would leave no remaining cells or cell fragments in the matrix as there is evidence that cellular components are associated with inflammation and calcification, which may ultimately lead to calcified tissue deterioration and limited conduit longevity.7,8 However, a decellularization process that damages the integrity of matrix structural proteins would result in conduit tearing or valve prolapse after implantation. It is vital that the extraction procedure preserves the extracellular matrix proteins of the venous conduit, particularly those associated with biological function. We assessed the matrix of bovine jugular vein histologically and biologically after sodium deoxycholate and Triton X-100 were used for decellularization.
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MATERIALS AND METHODS
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Whole bovine jugular veins were obtained aseptically from a local abattoir within 2 hr of slaughter, and transported to the laboratory in cold sterile 10 mM phosphate-buffered saline (PBS) solution at pH 7.2 containing 1.0 x 104 kIU·L–1 aprotinin, 1.0 x 105 U·L–1 penicillin, 1.0 x 105 U·L–1 streptomycin, and 1.0 x 105 U·L–1 nystatin. The veins were stored in fresh PBS containing protease inhibitors (EDTA 0.1% w/v, and aprotinin 1.0 x 104 kIU·L–1) prior to the next step in the treatment. Detergents and chemicals were purchased from Sigma (USA). The jugular veins were randomly assigned to 2 groups of 24 veins each: one group comprised fresh veins; the other comprised decellularized veins. For decellularization, the specimens were individually immersed in the solutions described below, and shaken continuously. Specimens were first shaken in Tris-HCl solution (0.01 mol·L–1, pH 7.2) at 37°C for 8 hr, then in Triton X-100 in PBS (5% v/v) at 37°C for 12 hr, in Tris-HCl (0.01 mol·L–1, pH 7.2) at 37°C for 3 hr, in sodium deoxycholate in PBS (4% w/v) for 6 hr, and finally in PBS for 12 hr. The volume of solution required for specimen treatment was calculated according to the length of the specimen, based on 5 mL per centimeter. Triton X-100 is a nonionic detergent that can eliminate cellular components by destroying the cellular biomembrane when it combines with the plasma membrane protein and phosphatide to give a soluble complex. All specimens were stored in PBS at 4°C after treatment.
Specimens for light microscopy were fixed in 10% (v/v) neutral buffered formalin, dehydrated, and embedded in paraffin. Tissue sections were assessed by routine hematoxylin and eosin staining, trinitrophenol-Sirius red staining, and elastic fiber staining to determine the degree of decellularization, integrity of collagen I and III, and elastic fibers, respectively. After trinitrophenol-Sirius red staining and elastic fiber staining, photographs were taken with a Nikon microscope at a magnification of x100. Three 10–4 x 10–4 mm2 fields in each photomicrograph were randomly selected to determine the areas of red, yellow, green, and brown colors with Spot software. The red and yellow areas indicate the content of collagen I, green indicates collagen III, and brown indicates elastin. Specimens for scanning electron microscopy were fixed in 25% (v/v) glutaraldehyde in PBS, processed as usual for electron-microscopy specimens, examined and photographed with a Hitachi S-520 scanning electron microscope.
Genome DNA was extracted from specimens with an EZNA kit (Aldevron, Fargo, ND, USA), according to the manufacturer’s instructions, and determined by electrophoresis in agarose 0.7% (w/v) with ethidium bromide (5.0 x 104 g·L–1) using lambda Hind III DNA markers as references. Electropherograms were analyzed with a Kodak digital scanning analyzer.
Fresh and decellularized specimens were freeze-dried and immersed in distilled water for 24 hr. The weights (W) of the dry and wet specimens were measured to calculate the water absorption rate: [(Wwet - Wdry)/Wdry] x 100%. The specimens were placed in centrifuge tubes pre-filled with filter paper, centrifuged at 1,500 rpm for 3 min, and weighed again (W’wet) to calculate the water maintenance rate: [(W’wet - Wdry)/Wdry] x 100%. Samples of up to 10 x 10 mm of valve and vein wall of the fresh and treated bovine jugular veins were cut and mounted on a purpose-built clamp and holder. During clamping, care was taken to mount the specimens under zero strain. Specifically, the wet specimens were floated onto the smooth surface of the clamp with minimum handling, and secured in a completely relaxed state. The clamp and holder were mounted in a Howden tensile testing machine (Instron Ltd., High Wycombe, Buckinghamshire, UK). This device can be used to increase stress gradually, the maximum loading capability is 20 Newtons. After mounting, the holder was removed. The specimens were preconditioned under cyclic loading over 50 cycles, at a rate of 500 mm·min–1 and strains of 35% for circumferential and 70% for radial specimens. After preconditioning, the specimens were loaded to failure at a rate of 10 mm·min–1. The experiment was conducted in saline at room temperature (20°C). During testing, force and distance data between the cross-head of the testing machine were recorded at a rate of 20 Hz. Specimen stress was calculated according to the formula: 
= F/A, where is stress in mPa, F is the recorded force in Newtons, A is the original cross-sectional area of the specimen at zero strain, expressed in mm2 and calculated as: width x thickness; the thickness was measured with a HD-10 thickness gauge.
All statistical analyses were completed using the Statistical Package for Social Sciences version 10.0 (SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation. The two-tailed paired Student’s t test was used to compare intergroup means. A p value < 0.05 was considered significant.
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RESULTS
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Tissue sections stained with hematoxylin and eosin indicated that the decellularization treatment had completely removed the cellular components from the bovine jugular vein tissue, with no apparent disruption of the tissue and fibril structure. There were no significant differences ( p > 0.05) in the contents of collagen I (red/yellow), collagen III (green), and elastin (brown) between the decellularized and fresh veins (Figures 1
–5). Scanning electron microscopy demonstrated that there were no endothelial cells on the surface of the decellularized bovine jugular vein wall and valve (Figure 6). Most of the genome DNA was removed by the decellularization process. Compared with fresh specimens, the DNA content of decellularized bovine jugular vein wall and valve was only 2.75% and 2.42% respectively (Figure 7). The thickness, water absorption rate and water maintenance rate of decellularized bovine jugular vein tissues were slightly increased compared with fresh specimens, but the differences did not reach statistical significance (Table 1). The disruption strength and tissue extensibility of fresh and decellularized bovine jugular vein were not significantly different (Table 2).
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Figure 2. The fresh valve leaflet (A) exhibited a clear structure; bovine jugular vein treated with sodium deoxycholate and Triton X-100 (B) showed that no cells or cell fragments were retained, and there was no apparent tissue disruption. Hematoxylin and eosin stain, original magnification x 100.
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Figure 3. Fresh (A) and decellularized (B) bovine jugular vein stained with trinitrophenol-Sirius red for collagen I (red/yellow) and collagen III (green). Original magnification x 100.
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Figure 4. Fresh (A) and decellularized (B) bovine jugular vein stained for elastic fiber. Original magnification x 100.
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Figure 5. Fresh and decellularized bovine jugular vein specimens showed no significant difference in the degree of staining for collagen I (red/yellow) and collagen III (green) and elastin (brown).
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Figure 6. Scanning electron microscopy of decellularized specimen: the endothelial cells were extracted completely; (A) surface of valve, (B) endothelial lining of blood vessel wall. Original magnification x 1000.
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Figure 7. Genome DNA electrophoresis: (1) lambda Hind III DNA marker, (2) fresh valve, (3) fresh vessel wall, (4) decellularized valve, (5) decellularized vessel wall, (6) phosphate-buffered saline.
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DISCUSSION
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This process commenced with a number of criteria that needed to be addressed if the scaffold was to be suitable for later stages in the tissue engineering procedure. These included minimizing tissue immunogenicity, and maintaining the structural components and biological properties of the matrix. To address the first point, the tissue cellular components in the scaffold should be removed completely.9 With this goal in mind, a procedure using as few different reagents and stages as possible was developed. For the solubilization of lipid cell membranes, the obvious reagents for selection were detergents. The ranges of detergent concentration selected for analysis were based on their critical micelle concentrations. Detergents with lower critical micelle concentrations are easier to remove by subsequent treatment. Some of the detergents selected for analysis were already acknowledged to have cell-extracting capabilities. For example, the nonionic detergent Triton X-100, the ionic detergent sodium dodecyl sulfate, and deoxycholate have been used to decellularize bovine aortic valve.10,11
There are concerns that upon in vivo implantation, nucleic acids retained within the acellular matrices could behave as calcification nucleation factors.12 The extraction of these components was addressed in this study. The ratios of the DNA content of decellularized jugular vein wall and valve to that of fresh tissues were 2.75% and 2.42%, respectively, which showed that the procedure used in this study removed most of the residual DNA from the matrix. The thickness of decellularized jugular vein valve and vessel wall was slightly increased, indicating that decellularization removed the hydrophobic lipid, which might cause an increase of matrix hydration with a consequent increase in the water-holding ability of the highly hydrophilic glycosaminoglycans in the matrix. A hydrophilic matrix has advantages for cell sticking, cell growth, and nutritive substance transmission. The water absorption rate and water maintenance rate of decellularized jugular veins were slightly higher than those of fresh veins, with no statistically significant difference, indicating that the decellularization process did not change the pore structure or hydrophilicity of the jugular vein tissue. The disruption strength and tissue extensibility of the decellularized and fresh jugular vein tissues were not significantly different, indicating that collagen and elastic fiber triaxial structures were not substantially changed.13,14
The major goal of tissue engineering is to repopulate autologous cells on a biodegradable scaffold, and ultimately form a new functional tissue that possesses the ability to grow and remodel, with inherent anti-thrombogenicity. The goal of this study was an acellular matrix that might act as a scaffold for in vitro repopulation with autologous cells. Whether the matrix prepared by the methods herein provides a suitable microenvironment for seeded cells, whether it will be completely biocompatible, with minimal risk of infection, dilation, thromboembolic complications, and narrowing in vivo will be investigated in future preclinical animal studies. Although our early results have shown that we created an acellular scaffold for a tissue-engineered ventricular valved conduit in vitro, the results are still preliminary. Numerous issues remain to be addressed before a clinically applicable tissue-engineered right ventricular valved conduit emerges. However, the histological and biological characteristics of decellularized bovine jugular vein are stable. The absence of cellular debris as foci for calcification, the retention of collagen and elastin, as well as the unchanged matrix compliance and strength, form a promising platform for autologous cell reseeding to produce a tissue-engineered valved conduit for clinical implantation.15
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ABSTRACT
INTRODUCTION
