• Journal of Animal Reproduction and Biotechnology
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• v.36 no.4
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• pp.253-260
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• 2021

Organ transplantation is currently the most fundamental treatment for organ failure, but there is a shortage of organ supply compared to those in need. Regenerative medicine has recently developed a decellularization technique that overcomes the limitations of conventional organ transplantation and attempts to reconstruct damaged tissues or organs to their normal state. Several decellularization methods have been suggested. In this experiment, the decellularization methods were used to find effective decellularization methods for humanlike porcine placenta. The optimal conditions for decellular support are low DNA content and high glycos amino glycans (GAGs) and collagen content. In order to satisfy this condition, SDS and Triton X-100 and SDS + Triton X-100 were used as the detergent used for decellularization in this experiment. The contents were compared according to the decellularization time (0, 12, 24, 48 and 72 hours), and the concentrations of SDS (0.2, 0.5, 0.7 and 1.0%) were mixed in 1.0% Triton X-100 to analyze the contents. When decellularized using SDS and Triton X-100, respectively, it was confirmed that the contents of DNA and GAGs were opposite to each other. And decellularization treatment for 24 hours at 0.5% SDS was able to obtain an effective decellular support. If decellularization studies of various detergents can be obtained an effective decellular support, and furthermore, cell culture experiments can confirm the effect on the cells.

• Journal of Chest Surgery
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• v.45 no.6
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• pp.368-379
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• 2012

Background: Bioprostheses for cardiovascular surgery have limitations in their use following as calicification. ${\alpha}$-galactosidase epitope is known as a stimulant of immune response and then shows a progressing calcification. The objective of this study was to evaluate histologic characteristics and mechanical properties of decellularization and treated with ${\alpha}$-galactosidase. Materials and Methods: Bovine pericardial tissues were allocated into three groups: fixation only with glutaraldehyde, decellularization with sodium dodesyl sulfate and decellularization plus treatment with ${\alpha}$-galactosidase. We confirmed immunohistological characteristics and mechanical properties as fatigue test, permeability test, compliance test, tensile strength (strain) test and thermal stability test. Results: Decellularization and elimination of ${\alpha}$-gal were confirmed through immunohistologic findings. Decellularization had decreased mechanical properties compared to fixation only group in permeability (before fatigue test p=0.02, after fatigue test p=0.034), compliance (after fatigue test p=0.041), and tensile strength test (p=0.00). The group of decellularization plus treatment with ${\alpha}$-galactosidase had less desirable mechanical properties than the group of decellularization in concerns of permeability (before fatigue test p=0.043) and strain test (p=0.001). Conclusion: Favorable decellularization and elimination of ${\alpha}$-gal were obtained in this study through immunohistologic findings. However, those treatment including decellularization and elimination of ${\alpha}$-gal implied the decreased mechanical properties in specific ways. We need more study to complete appropriate bioprosthesis with decellularization and elimination of ${\alpha}$-gal including favorable mechanical properties too.

• Journal of Chest Surgery
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• v.43 no.6
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• pp.565-575
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• 2010

Background: Various decellularization methods have been studied in order to develop tissue graft which is less immunogenic and more durable. This study was performed to investigate the physico-dynamic and histological effect of trypsin pretreatment on decellularization protocols. Material and Method: Two groups of bovine pericardium specimen each underwent decellularization process based on SDS and Triton X-100 or N-lauroylsarcosinate and Triton X-100. Two more groups additionally underwent pretreatment with 0.1% Trypsin/0.1% EDTA. After decellularization process, mechanical tensile strength was tested, then biomechanical test of permeability and compliance was tested before and after fatigue test. Light microscopy and electron microscopy was performed to observe histological findings. Result: There was no difference in mechanical tensile strength between groups, but permeability and compliance was decreased in trypsin pretreated groups. Light microscopic and electron microscopic findings revealed damage of the extracellular matrix in trypsin pretreated groups and in groups which underwent the fatigue test also. Conclusion: Trypsin pretreatment in decellularizing process of bovine pericardium damages extracellular matrix and increases permeability and compliance of the bovine pericardium, but did not decrease tensile strength. Further studies are needed to use enzymatic treatments in decellularization protocols.

• Journal of Biomedical Engineering Research
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• v.38 no.2
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• pp.62-73
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• 2017

To identify a solution for the restricted availability of healthy lungs and the high risk of immune rejections following organ transplantation, tissue engineering techniques for culturing lungs have been studied by many research groups. The most promising method for culturing lungs is the utilization of a bio-scaffold that was prepared using harvested organs from human donors or other animals by removing their original cells. In this study, a pulsatile perfusion pump was used to alleviate the cell removal effect with the high fluid-dynamic power of the perfusion stream during the decellularization process, while other conventional studies focused on chemical methods to identify efficient detergents. The purpose of this study was to analyze the developed device by using energy equivalent pressure (EEP), which is an indicator of pulsatility, to understand the characteristics of pulsatile energy transmitted according to the load size by using the artificial model and compare it with the measured EEP. The pulsatility of the device can be estimated with the concept of fluid-dynamic energy during a particular constant time period or fluid-dynamic power represented as EEP and EEP increment. Because the measured EEP of perfusion flow during decellularization can be changed by the amount of fluid leakage and the degree of clogging in the capillary vessels, EEP should be measured to determine whether the decellularization is progressing without problems. The decrement of EEP caused by the high perfusion resistance was observed from some experimental results that were obtained with artificial models. EEP can be used to monitor the decellularization process after analyzing the varying EEP according to the amount of load. It was confirmed that the EEP was maintained at a high level in the experiment using the harvested lungs from 12-13-week-old rats. In addition, it was confirmed that the cell removal time was faster than when continuous perfusion was performed. In this study, pulsatile power delivered to the lungs was measured to monitor the process of cell removal, and it serve as the evidence for efficient decellularization.

• Journal of Chest Surgery
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• v.41 no.6
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• pp.679-686
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• 2008

Background: Cellular remnants in the bioprosthetic heart valve are known to be related to a host's immunologic response and they can form the nidus for calcification. The extracellular matrix of the decellularized valve tissue can also be used as a biological scaffold for cell attachment, endothelialization and tissue reconstitution. Thus, decellularization is the most important part in making a bioprosthetic valve and biological caffold. Many protocols and agents have been suggested for decellularization, yet there ave been few reports about the effect of a treatment with hypotonic solution prior to chemical or enzymatic treatment. This study investigated the effect of a treatment with hypotonic solution and the appropriate environments such as temperature, the treatment duration and the concentration of sodium dodecylsulfate (SDS) for achieving proper decellularization. Material and Method: Porcine aortic valves were decellularized with odium dodecylsulfate at various concentrations (0.25%, 0.5%), time durations (6, 12, 24 hours) and temperatures ($4^{\circ}C$, $20^{\circ}C$)(Group B). Same the number of porcine aortic valves (group A) was treated with hypotonic solution prior to SDS treatment at the same conditions. The duration of exposure to the hypotonic solution was 4, 7 and 14 hours and he temperature was $4^{\circ}C$ and $20^{\circ}C$, respectively. The degree of decellularization was analyzed by performing hematoxylin and eosin staining. Result: There were no differences in the degree of decellularization between the two concentrations (0.25% 0.5%) of SDS. Twenty four hours treatment with SDS revealed the best decellularization effect for both roups A and B at the temperature of $4^{\circ}C$, but there was no differences between the roups at $20^{\circ}C$. Treatment with hypotonic solution (group A) showed a better ecellularization effect at all the matched conditions. Fourteen hours treatment at $4^{\circ}C$ ith ypotonic solution prior to 80S treatment revealed the best decellularization effect. The treatment with hypotonic solution at $20^{\circ}C$ revealed a good decellularization effect, but his showed significant extracellular matrix destruction. Conclusion: The exposure of porcine heart valves to hypotonic solution prior to SDS treatment is highly effective for achieving decellularization. Osmotic treatment with hypotonic solution should be considered or achieving decellularization of porcine aortic valves. Further study should be carried out to see whether the treatment with hypotonic solution could reduce the exposure duration and concentration of chemical detergents, and also to evaluate how the structure of the extracellular matrix of the porcine valve is affected by the exposure to hypotonic solution.

• Journal of Chest Surgery
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• v.41 no.5
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• pp.550-562
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• 2008

Background: We attempted to reproduce a previously reported method that is known to be effective for decellularization, and we sought to find the optimal condition for decellularization by introducing some modifications to this method. Material and Method: Porcine semilunar valves, arterial walls and pericardium were processed for decellularization with using a variety of combinations and concentrations of decellularizing agents under different conditions of temperature, osmolarity and incubation time. The degree of decellularization and the preservation of the extracellular matrix were evaluated by staining with hematoxylin and eosin and with alpha-Gal and DAPI in some of the decellularized tissues. Result: Decellularization was achieved in the specimens that were treated with sodium deoxycholate, sodium dodesyl sulfate, Triton X-100 and sodium dodesyl sulfate with Triton X-100 as single-step methods, and this was also achieved in the specimens that were treated with hypotonic solution ${\rightarrow}$ Triton X-100 ${\rightarrow}$ sodium dodesyl sulfate, sodium deoxycholate ${\rightarrow}$ hypotonic solution ${\rightarrow}$ sodium dodesyl sulfate, and hypotonic solution sodium dodesyl sulfate as multi-step methods. Conclusion: Considering the number and the amount of the chemicals that were used, the incubation time and the degree of damage to the extracellular matrix, a single-step method with sodium dodesyl sulfate and Triton X-100 and a multi-step method with hypotonic solution followed by sodium dodesyl sulfate were both relatively optimal methods for decellularization in this study.

• Journal of Chest Surgery
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• v.43 no.3
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• pp.235-245
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• 2010

Background: Our goal was to evaluate anti-calcification effects of decellularization and diverse fixing methods including preincubation of the bovine pericardium with ethanol. We also assessed changes in mechanical properties. Material and Method: Harvested bovine pericardium was decellularized with 0.25% sodim dodecysulfate and then treated with 5 methods of fixation: (1) 0.5% glutaraldehyde (GA) for 14 days, (2) 0.5% GA for 5 days, 2% GA for 2 days and 0.25% GA for 7 days, (3) 0.5% GA for 5 days, 2% GA for 2 days, 0.25% GA for 7 days, and then 70% ethanol for 2 days, (4) 0.5% GA for 5 days, a mixture of 2% GA and 70% ethanol for 2 days, and 0.25% GA for 7 days, (5) 0.5% GA for 5 days, a mixture of 2% GA, 65% ethanol, and 5% octanediol for 2 days and then 0.25% GA for 7 days. All treated bovine pericardia were tested for histological variables, lipid content, and mechanical properties including tensile strength and thermal stability. A total 10 kinds of differently treated bovine pericardia were implanted into rat subdermis and harvested 8 weeks later. Harvested pericardia were evaluated for calcium content. Result: No protein denaturation was observed microscopically after decellularization. There was a 32% mean decrease in tensile strength index after decellularization in the bovine pericardium group fixed. Octanediol preincubation attenuated the decrease in tensile strength and maintained thermal stability. TG and cholesterol were not affected by decellularization but were decreased by organic solvent. Calcium content was decreased after decellularization, and organic solvent preincubation decreased calcification in the non-decellularized bovine pericardium group. Conclusion: Decellularization and organic solvent preincubation have anti-calcification effects but decellularization may cause mechanical instability. A method of decellularization and fixation that does not cause damage to matrices will be needed for evaluation of the next step in using tissue-engineering for replacement of cardiac valves.

• Journal of Chest Surgery
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• v.45 no.6
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• pp.380-389
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• 2012

Background: Bovine pericardium is one of the most widely used materials in bioprosthetic heart valves. Immunologic responses have been implicated as potential causes of limited durability of xenogenic valves. This study aimed to determine the effectiveness of decellularization and ${\alpha}$-galactosidase (${\alpha}$-gal) to remove major xenoreactive antigens from xenogenic tissues. Materials and Methods: Recombinant Bacteroides thetaiotaomicron (B. thetaiotaomicron) ${\alpha}$-gal or decellularization, or both were used to remove ${\alpha}$-gal from bovine pericardium. It was confirmed by ${\alpha}$-gal-bovine serum albumin-based enzyme-linked immunosorbent assay (ELISA), high-performance anion exchange chromatography, flow cytometry, 3,3'-diaminobenzidine-staining, and lectin-based ELISA. The mechanical properties of bovine pericardium after decellularization or ${\alpha}$-gal treatment were investigated by tests of tensile-strength, permeability, and compliance. Collagen fiber rearrangement was also evaluated by a 20,000${\times}$ transmission electron microscope (TEM). Results: Recombinant B. thetaiotaomicron ${\alpha}$-gal could effectively remove ${\alpha}$-gal from bovine pericardium B. thetaiotaomicron (0.1 U/mL, pH 7.2) while recombinant human ${\alpha}$-gal removed it recombinant human ${\alpha}$-gal (10 U/mL, pH 5.0). There was no difference in the mechanical properties of fresh and recombinant ${\alpha}$-gal-treated bovine pericardium. Furthermore, the TEM findings demonstrated that recombinant ${\alpha}$-gal made no difference in the arrangement of collagen fiber bundles with decellularization. Conclusion: Recombinant B. thetaiotaomicron ${\alpha}$-gal effectively removed ${\alpha}$-gal from bovine pericardium with a small amount under physiological conditions compared to human recombinant ${\alpha}$-gal, which may alleviate the harmful xenoreactive immunologic responses of ${\alpha}$-gal. Recombinant ${\alpha}$-gal treatment had no adverse effects on the mechanical properties of bovine pericardium.

• Journal of Chest Surgery
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• v.43 no.1
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• pp.1-10
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• 2010

Background: Bioprosthetic materials have been made using glutaraldehyde fixation of porcine or bovine pericardium during cardiovascular surgery. But these bioprostheses have the problems of calcification and mechanical failure. We determined changes in tensile strength and elasticity of pericardium after glutaraldehyde, solvent, decellularization and detoxification. Material and Method: Tissues were allocated to four groups: glutaraldehyde with and without solvent, decellularization, and detoxification. We studied tensile strength and strain on tissues. We measured the tensile strength of fresh pericardium stretched in six directions (with 5 mm width), and % strain, which we calculated from the breaking point when we pulled the pericardium in two directions. Result: Tensile strength was reduced when we used the usual concentrated glutaraldehyde fixation (n=83, $MPa=11.47{\pm}5.40$, p=0.006), but there was no change when we used solvent. Elasticity was increased after glutaraldehyde fixation (n=83, strain $(%)=24.55{\pm}9.81$, p=0.00), but there was no change after solvent. After decellularization of pericardium, the tensile strength was generally reduced. The decrease in tensile strength after concentrated glutaraldehyde fixation for a long time was significantly greater less than after concentrated solvent (p=0.01, p=0.00). After detoxification, the differences in strength and strain were not significant. Conclusion: After glutaraldehyde treatment of pericardium there is no loss in tensile strength (even though we did the glutaraldehyde, solvent and detoxification treatments LOGIC IS UNCLEAR). Also, these treatments had a tendency to increase elasticity. Although post-treatment decellularization led to a significant loss in strength, this effect could be attenuated using a low concentration of solvent or hypertonic solution.

• Microbiology and Biotechnology Letters
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• v.38 no.2
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• pp.168-176
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• 2010

Acellular dermal matrix (ADM), produced by decellularization from human cadaveric skin, has been used for various biomedical applications. A manufacturing process for ADM ($SureDerm^{TM}$) using tri-n-butyl phospahate (TnBP) and deoxycholic acids as the decellularization solution has been developed. The manufacturing process for $SureDerm^{TM}$ has 70% ethanol treatment and ethylene oxide gas sterilization for inactivating infectious microorganisms. The purpose of this study was to examine the efficacy of the 70% ethanol treatment, decellularization process using 0.1% TnBP and 2% deoxycholic acids, and EO gas sterilization process in the inactivation of viruses. A variety of experimental model viruses for human pathogens, including the human immunodeficiency virus type 1 (HIV-1), bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), hepatitis A virus (HAV), and porcine parvovirus (PPV) were all selected for this study. Enveloped viruses such as HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by 70% ethanol treatment. However HAV and PPV showed high resistance to 70% ethanol treatment with the log reduction factors of 1.85 and 1.15, respectively. HIV-1, BHV, and BVDV were effectively inactivated to undetectable levels by decellularization process. All the viruses tested were completely inactivated to undetectable levels by EO gas treatment. The cumulative log reduction factors of HIV-1, BHV, BVDV, HAV, and PPV were $\geq12.71$, $\geq18.08$, $\geq14.92$, $\geq6.57$, and $\geq7.18$, respectively. These results indicate that the production process for $SureDerm^{TM}$ has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.