• Journal of Chest Surgery
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• 제26권2호
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• pp.80-85
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• 1993

Synthetic and biosynthetic vascular grafts of small diameter have long been considered to be prone to thrombosis, ultimately leading to the complete graft occlusion. Endothelial cell seeding onto synthetic blood-contacting surfaces has been suggested to be an ideal means to solve this problem. This study described a culture method of bovine endothelial cells and evaluated blood-compatibility and seeding efficiency of cultured endothelial cells. Bovine pulmonary artery endothelial cells were harvested enzymatically and grown to confluence on polystyrene culture flask surfaces using established techniques. The identification of endothelial cells was made through the demonstration of expression of factor VIII R:Ag by immunofluorescent technique. To quantitate the effect of improvement in blood-compatibility of viable endothelial cells, endothelial monolayers were exposed to blood containing $\^$111/In-oxine labeled platelets. Viable endothelial monolayers retained less labeled platelets than control surfaces. The Indium-labeled endothelial cells were seeded onto three different blood-contacting surfaces of Dacron vascular graft immobilized in specially equipped wells and incubated for specific time intervals (t=15, 30, 60, 120 minutes). Longer incubation times showed improved cell adherence in collagen-coated and fibrin-coated Dacron vascular graft groups. However in untreated Dacron grafts, no direct relationship was observed between incubation time and endothelial cell seeding efficiency. This may be due to leakage of endothelial cells through porosity of Dacron grafts in this in-vitro experimental condition.

• 대한의용생체공학회:의공학회지
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• 제16권1호
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• pp.1-8
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• 1995

A variety of experiments of endothelial cell seeding onto artificial vessels have been performed. To improve endothelialization, one or two components of the extracellular matrix (ECM) have been used as an underlying matrix. In this study, the whole ECM excreted from fibroblasts was used as an underlying matrix. Fetal human fibroblasts were cultured on a polyurethane (PU) sheet. After a conflu; ence was attained, the cytoskeleton and the nuclei of the fibroblast were destroyed using Triton-X. Mitomycin, or irradiation. Omental microvascular endothelial cells from adult human were seeded onto various substrates. After 12 days in culture, the cells were counted. It was observed that the ECM treated by irradiation had the highest cell number. In addition, the cells on this substrate exhibited the most typical endothelial cell morphology. For preliminary animal experiments the PU vessels (inner diameter, 1.5mm) coated with ECM were implanted in the infrarena] abdominal aorta of rat. After the vessels had been implanted for 5 weeks, it was found that the surface of the PU vessels was completely covered with endothelia] cells. In conclusion, we can state that the fibroblast-derived whole ECM makes a better underlying substrate for the endothelialization of small diameter artificial vessels.

• 대한의용생체공학회:의공학회지
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• 제11권1호
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• pp.131-140
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• 1990

To improve antithrombogenicity of polymer that used in vascular graft and artificial organs, seeding of human endothelial cells on the polyurethane was studied. Human endothelial cells were ismlated from human umbilical veins, using type I collagenase, and identified with goat anti vWF antibodies. Human endothilial cell seeding was tried upon the polyurethane which has good mechanical property and resists stresses. The hydrophobic polyurethane surface was changed hydrophilic by corona discharf:e treatment. Surface hydrophilicity was measured with Wilhemly plate method and the goniometer. To evaluate matrix protein adsorption, fibronectin adsorption test was done. To eveluate cell adhesion, human endothelial cell attachment forces were measured rising a perfusion chamber of , ism diamter. Less cells were detached from the hydrophilically treated polyurethane. This showed that corona discharge on the polyurethane could improve matrix adsorption and endothelial cell attachment.

• 대한의용생체공학회:의공학회지
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• 제12권3호
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• pp.165-170
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• 1991

Many experiments about endothelial cell seeding on artificial vessels were studied and conducted For this one or a combination of the extramatrix was used for the underlying matrix. But we used the whole ECM(extracellular matrix) that made excreated from flbroblasl. In thls study, we obtained human adult omental microvascular endothelium by collagenase digestion and used polyurthane sheets in order to make a new artificial vessel material. We cultured fibroblast on the polyurethane and gelatin - coated polyurethane. After confluent ingrowth we treated the polyure thane with triton in order to destroy the cytoskeleton and nucleus. We observed the preformed extra cellular matrix on the ployurethane and cultured the isolated microvascular endothelium. We also ok served the growth of microvascular endothelium on the polyurethane and gelatin. We conclude that the use of the whole ECM is promising fair as a new underying substrate for endothelial cell seeding on artificial vessels.

• 대한의용생체공학회:의공학회지
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• 제17권3호
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• pp.355-364
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• 1996

Construction of the stable monolayer of endothelial cells onto physicochemically modified polymeric surFace is one of the appropriate method to develop the small caliber vascular graft with the long-term patency. In this study, we constructed the monolayer of endothelial cells on the fibronectin rind the extracellular matrix-coated polyurethane surface derived from human fibroblast cells. To elucidate the adhesion strength of endothelial cells on the extracellular matrix-coated polyurethane, a laminar flow chamber apparatus was developed to exposure the shear stress on the apical membrane of ondothelial cells. Endothelial cells show the strongest adhesion after two days of seeding onto the fibronectin-coated polyurethane surface, whereas endothelial cells on the extracellular matrix derived from the human flbroblast cells show the minimal doubling time of cellular growth.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권4호
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• pp.246-251
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• 2003

The blood-brain barrier (BBB) is composed of the brain capillaries, which are lined by endothelial cells displaying extremely tight intercellular junctions. Several attempts at creating an in vitro model of the BBB have been met with moderate success as brain capillary endothelial cells lose their barrier properties when isolated in cell culture. This may be due to a lack of recreation of the in vivo endothelial cellular environment in these models, including nearly constant contact with astrocyte foot processes. This work is motivated by the hypothesis that growing endothelial cells on one side of an ultra-thin, highly porous membrane and differentiating astrocyte or astrogliomal cells on the opposite side will lead to a higher degree of interaction between the two cell types and therefore to an improved model. Here we describe our initial efforts towards testing this hypothesis including a procedure for membrane fabrication and methods for culturing endothelial cells on these membranes. We have fabricated a 1 $\mu\textrm{m}$ thick, 2.0 $\mu\textrm{m}$ pore size, and 55% porous membrane with a very narrow pore size distribution from low-stress silicon nitride (SiN) utilizing techniques from the microelectronics industry. We have developed a base, acid, autoclave routine that prepares the membranes for cell culture both by cleaning residual fabrication chemicals from the surface and by increasing the hydrophilicity of the membranes (confirmed by contact angle measurements). Gelatin, fibronectin, and a 50/50 mixture of the two proteins were evaluated as potential basement membrane protein treatments prior to membrane cell seeding. All three treatments support adequate attachment and growth on the membranes compared to the control.

• Journal of Chest Surgery
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• 제37권4호
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• pp.297-306
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• 2004

기존의 인공 심장 판막은 혈전 형성, 내구성, 세균 감염에 대한 저항력, 성장성 등에서 문제점을 가지고 있다 최근 이상적인 심장 판막을 개발하기 위한 노력으로 조직공학 기법을 이용하고 있다. 본 연구는 이종 판막을 지지체로 하여 이종 세포 제거 후 자가 세포를 파종한 조직공학 심장 판막을 개발하기 위하여 이종 세포 제거와 자가 세포 파종 방법을 비교하였고, 얻어진 심장 판막첨의 생체 적합성을 살펴보았다. 대상 및 방법: 이종 지지체로 두 마리의 돼지에서 각각 3개의 폐동맥 판막첨을 채취하여, Triton-X, freeze-thawing, NaCl-SDS 등 세 방법 중 하나로, 각각 두 개의 판막첨을 처리하고, 세포 제거 정도를 비교하였다. 염소의 경정맥에서 분리한 내피 세포를 배양하여, 상기한 방법 중 하나로 이종 세포가 제거된 돼지 판막첨에 파종한 후, 내피 세포의 보존 상해를 비교하였다. 생체 실험을 위해, 6마리 돼지에서 각각 2개의 폐동맥 판막첨을 채취해 이종 세포를 제거하였고, 6개의 판막첨은 염소의 내피 세포를 파종하고(이종-자가 이식편),다른 6개의 판막첨은 내피 세포를 파종않고(이종 이식편) 6마리 염소의 폐동맥 판막첨 두 개와 치환하였다. 치환되지 않은 하나의 판막첨을 대조군으로 비교하였다. 염소는 수술 6시간, 24시간, 1주일, 1개월, 3개월, 그리고 6개월 후 희생시키고 폐동맥 판막첨을 채취하여 분석하였다 걸과: Triton-X와 freeze-thawing 처리한 판막첨에서는 이종세포가 남아 있었으나, NaCl-SDS 처리한 판막첨에서는 세포가 완전히 제거되었고, 이종 세포 제거 후 파종한 자가 내피 세포도 NaCl-SDS 처리한 판막첨에서 다른 두 방법으로 처리한 판막첨에 비해 잘 보존되어 있었다. 6마리 염소는 혈색전증의 소견 없이 예정된 기간 동안 모두 생존하였으며, 희생 후 적출한 이종 이식편과 이종-자가 이식편 모두 대조군 보다 두꺼워져 있었다. 두 이식편 모두 24시간 후부터 섬유아세포의 증식이 관찰되었고, 1개월 후 세포의 개형(remodeling)과정을 볼 수 있었고, 3개월 후 세포 기능을 수행하였고, 6개월 후 대조군보다 세포수가 더 많이 증가하였다. 파종한 내피 세포는 잘 보존되어 있었고, 이종 이식편도 6개월 후에는 내피 세포가 피복 되어 있었다. 걸론: 이종 세포의 제거와 자가 세포의 파종에 가장 적합한 방법은 NaCl-SDS 처리였다 동물 실험에서 이종 이식편과 이종-자가 이식편 모두 6개월 간 지지체의 역할을 수행하면서, 자가 세포의 증식을 가능하게 하였고, 자가 조직으로 발전할 가능성을 보여 주었다.

• Archives of Plastic Surgery
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• 제36권4호
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• pp.380-384
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• 2009

Purpose: Autologous vessels remain the gold standard for vascular grafts in microanastomoses. However, they are sometimes unavailable and have a limited long - term patency. Synthetic vessels have high success rates in large - diameter reconstructions but failed when used as small - diameter grafts due to graft occlusion. It has been proved that endothelial cell seeding improves prosthesis performance and long - term patency. Among polyurethane, PET and ePTFE, polyurethane has the best affinity to endothelial cells and mechanical properties closest to human vessels. We examined the ability of endothelial cells to attach to a polyurethane graft manufactured by the electrospinning method. Methods: Endothelial cells, which were cultured from porcine internal jugular veins, were attached to polyurethane grafts with an internal diameter of 3 mm. The same cells were attached to allogeneic decellularized porcine internal carotid artery grafts as controls. Both of the 10 mm - long grafts were exposed to endothelial cells in a well for 1 hour. Each well contained $2{\times}10^5$ endothelial cells. The graft materials were rotated through 90 degrees every 15 minutes in order to minimize the effect of gravity. The extent of cell attachment was examined with the MTT assay. Results: The MTT assay showed good incorporation of endothelial cells into both grafts. For the evaluation of affinity, the number of attached cells was counted at 10 fields of microscopic examination with ${\times}40$ magnification. Endothelial cells adhered more to polyurethane grafts (mean, $127.4{\pm}6.2cells$) compared to porcine artery grafts (mean $45.8{\pm}5.1cells$)(p<0.05,Mann - Whitney test). Conclusion: In this study, we attached porcine endothelial cells to polyurethane grafts, manufactured by electrospinning. The grafts exhibited a better affinity to endothelial cells than allogeneic decellularized porcine internal carotid artery grafts. It is suggested that the time required for endothelial cells to attach to decellulized artery grafts may be longer than that which is required for attachment to polyurethane grafts.

• Korean Journal of Radiology
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• 제19권6호
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• pp.1099-1109
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• 2018

Objective: In a proof of concept study, we compared no-touch radiofrequency ablation (NtRFA) in bipolar mode with conventional direct tumor puncture (DTP) in terms of local tumor control (LTC), peritoneal seeding, and tumorigenic factors, in the rabbit VX2 subcapsular hepatic tumor model. Materials and Methods: Sixty-two rabbits with VX2 subcapsular hepatic tumors were divided into three groups according to the procedure: DTP-RFA (n = 25); NtRFA (n = 25); and control (n = 12). Each of the three groups was subdivided into two sets for pathologic analysis (n = 24) or computed tomography (CT) follow-up for 6 weeks after RFA (n = 38). Ultrasonography-guided DTP-RFA and NtRFA were performed nine days after tumor implantation. LTC was defined by either achievement of complete tumor necrosis on histopathology or absence of local tumor progression on follow-up CT and autopsy. Development of peritoneal seeding was also compared among the groups. Serum hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) were measured via ELISA (Elabscience Biotechnology Co.) after RFA for tumorigenic factor evaluation. Results: Regarding LTC, there was a trend in NtRFA (80%, 20/25) toward better ablation than in DTP-RFA (56%, 14/25) (p = 0.069). Complete tumor necrosis was achieved in 54.5% of DTP-RFA (6/11) and 90.9% of NtRFA (10/11). Peritoneal seeding was significantly more common in DTP-RFA (71.4%, 10/14) than in NtRFA (21.4%, 3/14) (p = 0.021) or control (0%). Elevations of HGF, VEGF or IL-6 were not detected in any group. Conclusion: No-touch radiofrequency ablation led to lower rates of peritoneal seeding and showed a tendency toward better LTC than DTP-RFA.

• 대한의용생체공학회:의공학회지
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• 제16권4호
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• pp.463-470
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• 1995

사람의 내피세포는 동물내피세포에 비해 배양증식이 어려운 것으로 알려져 있어 이를 효율적으로 배양증식 시키기 위해서 배양액에 내피세포성장인자를 헤파린과 함께 첨가하는 방법이 많이 사용되어 오고 있다. 한편 최근에는 세포내 cyclic adenosine monophosphate(cAMP)을 증가시키는 물질들인 콜레라독소와 아이소부틸메틸산틴(isobutlmethylxanthine, IBMX)을 세포배양액에 첨가하여 내피세포 증식을 향상시킨 실험결과가 보고된바 있다. 이런 연구결과들을 토대로 할때 내피세포 배양액에 내피세포성장인자 및 헤파린과 함께 cAMP 증가물질을 같이 첨가하여 주면 내피세포의 성장증식을 보다 향상시킬수 있을 것이라는 가설이 가능할 것이다. 본 실험에서는 이와같은 가설을 검증하기 위해 사람의 대망 미세혈관(omental microvessel)으로부터 내피세포를 분리배양한뒤 내피세포성장인자 및 헤파린과 cAMP 증가물질들의 첨가가 내피세포의 증식에 미치는 영향을 분석하고, 궁극적으로는 사람 내피세포의 최적 배양증식 조건을 확립하고자 하였다. 실험 결과 사람의 대망 미세조직에서 내피세포를 분리하여 이를 효과적으로 배양증식하기 위해서는 내피세포성장인자와 헤파린을 첨가한 배지를 사용하거나, 또는 내피세포성장인자를 사용하지 않는 경우 콜레라독소와 IBMX를 병합 첨가하는 것이 좋은 것으로 관찰되었으며, 내피세포성장인자와 콜레라독소 및 IBMX를 동시에 병합 첨가하는 것은 효과가 없는 것으로 밝혀졌다.