바이러스 안전성이 보증된 무세포 소 양막 생물창상피복재 제조 공정 개발

배정은,김찬경,김성포,양은경,김인섭 한국미생물·생명공학회 2010 한국미생물·생명공학회지 Vol.38 No.4

A process for manufacturing virally-safe bovine amniotic membrane(BAM) has been developed for biological dressing. BAM was harvested from a healthy bovine placenta, and then the epithelium was removed. The remaining stromal layer was consecutively disinfected with 70% ethanol and 0.05% sodium hypochlorite. The stromal layer was incubated in a decellularization solution containing 0.25%(w/v) trypsin to remove the cellular components. The resulting acelluar BAM was lyophilized to preserve its biochemical and structural integrity. The BAM was packed and exposed to 25 kGy of gamma irradiation for sterilization purpose. Histological, electron microscopical, and biochemical observations showed that the acellualr BAM had intact structural integrity of three dimensional collagen fibers and contained several growth factors, accelerating wound healing, such as EGF (Epidermal growth factor), KGF (Keratinocyte growth factor), and FGF (Fibroblast growth factor). Bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), and bovine parvovirus (BPV) were chosen as the biological indicators for validation of viral safety of the acellular BAM. Samples from relevant stages of the production process were spiked with each virus and subjected to viral inactivation processes. Viruses were recovered from the samples and then titrated immediately. All the viruses tested were completely inactivated to undetectable levels within 1 h of 70% ethanol treatment. Enveloped viruses such as BHV, BVDV, and BPIV-3 were more effectively inactivated than BPV by 0.05% sodium hypochlorite treatment. BHV, BVDV, and BPIV-3 were completely inactivated to undetectable levels by 25kGy of gamma irradiation. Also BPV was effectively inactivated by 25 kGy of gamma irradiation. The cumulative log reduction factors of BHV, BVDV, BPIV-3, and BPV were ≥13.30, ≥14.32, ≥15.22, and ≥7.57,respectively. These results indicate that the production process for acelluar BAM has a sufficient virus-reducing capacity to achieve a high margin of the virus safety. 바이러스 안전성이 보증된 무세포 소 양막 생물창상피복재 제조공정을 확립하고자 하였다. 기질세포를 제거하기 위해 효소(트립신)를 처리하는 공정과 바이러스를 불활화하기위해 70% 에탄올, 0.05% sodium hypochlorite, 25 kGy 감마선 처리 공정을 포함하는 무세포 소 양막 제조공정을 확립하였다. 무세포 소 양막의 조직학적 분석과 전자현미경 분석 결과 면역거부반응을 일으킬 수 있는 상피층과 기질세포들이 잘 제거되었으며, 소 양막 콜라겐 섬유의 3차원적 구조가 잘 유지되어 있음을 확인하였다. 또한 상처치유효과가있는 EGF, KGF, FGF와 같은 성장인자를 포함하고 있었다. 바이러스 불활화 효과를 검증하기 위해 국제적 가이드에 따라 4종의 바이러스(BHV, BVDV, BPIV-3, BPV)를 생물학적지표로 사용하여 소 양막에 각 생물학적 지표를 첨가한 후각 바이러스 불활화 공정을 실시한 다음 각 바이러스를 회수하여 정량한 후 불활화 정도를 비교하였다. 24시간 70%에탄올 처리 공정에서 BHV, BVDV, BPIV-3, BPV 모두 처리 시간 1시간 안에 검출한계 이하로 완벽하게 불활화되었다. 30분 0.05% sodium hypochlorite 처리 공정에서 BHV,BVDV, BPIV-3 같은 외피 바이러스는 BPV 같은 비-외피 바이러스에 비해 효과적으로 불활화되었다. 25 kGy 감마선 조사에 의해 BHV, BVDV, BPIV-3는 검출한계 이하로 완벽하게 불활화되었고, BPV도 효과적으로 불활화되었다. 3가지바이러스 불활화 공정에서 BHV, BVDV, BPIV-3, BPV에대한 log 바이러스 감소인수 합은 각각 ≥13.30, ≥14.32,≥15.22, ≥7.57이었다. 이와 같은 결과 본 연구를 통해 확립된 무세포 소 양막 제조공정은 바이러스 안전성을 보증할 수있는 충분한 바이러스 불활화 능력을 갖고 있는 것으로 판단된다.

Dehydrothermal Treatment로 제작한 흡수성 콜라겐 골유도재생술 차단막

방강미,정한울,김성포,양은경,김기호,김성민,김명진,장정원,이종호,Pang, Kang-Mi,Choung, Han-Wool,Kim, Sung-Po,Yang, Eun-Kyung,Kim, Ki-Ho,Kim, Soung-Min,Kim, Myung-Jin,Jahng, Jeong-Won,Lee, Jong-Ho 대한악안면성형재건외과학회 2011 Maxillofacial Plastic Reconstructive Surgery Vol.33 No.2

Purpose: Collagen membranes are used extensively as bioabsorbable barriers in guided bone regeneration. However, collagen has different effects on tissue restoration depending on the type, structure, degree of cross-linking and chemical treatment. The purpose of this study was to evaluate the inflammatory reaction, bone formation, and degradation of dehydrothermal treated porcine type I atelocollagen (CollaGuide$^{(R)}$) compared to of the non-crosslinked porcine type I, III collagen (BioGide$^{(R)}$) and the glutaldehyde cross-linked bovine type I collagen (BioMend$^{(R)}$) in surgically created bone defects in rat mandible. Methods: Bone defect model was based upon 3 mm sized full-thickness transcortical bone defects in the mandibular ramus of Sprague-Dawley rats. The defects were covered bucolingually with CollaGuide$^{(R)}$, BioMend$^{(R)}$, or BioGide$^{(R)}$ (n=12). For control, the defects were not covered by any membrane. Lymphocyte, multinucleated giant cell infiltration, bone formation over the defect area and membrane absorption were evaluated at 4 weeks postimplantation. For comparison of the membrane effect over the bone augmentation, rats received a bone graft plus different covering of membrane. A $3{\times}4$ mm sized block graft was harvested from the mandibular angle and was laid and stabilized with a microscrew on the naturally existing curvature of mandibular inferior border. After 10 weeks postimplantation, same histologic analysis were done. Results: In the defect model at 4 weeks post-implantation, the amount of new bone formed in defects was similar for all types of membrane. Bio-Gide$^{(R)}$ membranes induced significantly greater inflammatory response and membrane resorption than other two membranes; characterized by lymphocytes and multinucleated giant cells. At 10 weeks postoperatively, all membranes were completely resorbed. Conclusion: Dehydrotheramal treated cross-linked collagen was safe and effective in guiding bone regeneration in alveolar ridge defects and bone augmentation in rats, similar to BioGide$^{(R)}$ and BioMend$^{(R)}$, thus, could be clinically useful.

Virus Inactivation during the Manufacture of a Collagen Type I from Bovine Hides

배정은,김찬경,김성포,양은경,김인섭,Bae, Jung Eun,Kim, Chan Kyung,Kim, Sungpo,Yang, Eun Kyung,Kim, In Seop The Microbiological Society of Korea 2012 미생물학회지 Vol.48 No.4

세포치료제 또는 조직공학제제에 사용되는 동물 유래 콜라겐은 원료물질 유래 바이러스가 오염될 가능성이 있기 때문에 생산과정 중 바이러스가 오염되지 않도록 하여야 한다. 이를 위해 콜라겐 생산공정은 오염될 가능성이 있는 바이러스들을 불활화 하거나 제거하는 과정을 포함하여야 하며, 바이러스 불활화/제거 능력은 제품의 안전성을 보증하는 중요한 지표로 사용된다. 본 연구의 목적은 소 가죽을 원료로 하여 type I 콜라겐을 생산하는 공정에서 소 유래 바이러스들의 불활화/제거 효능을 평가하는 데 있다. 이를 위해 70% 에탄올 처리 공정과 펩신 처리 공정(pH 2)에서 바이러스 불활화 효과를 평가하였다. 바이러스 불활화 효과 평가를 위해 bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza 3 virus (BPIV-3), bovine parvovirus (BPV)를 모델 바이러스로 선정하였다. 바이러스 불활화를 위해 24시간 동안 70% 에탄올을 처리하는 공정에서 BHV, BVDV, BPIV-3, BPV 모두 처리 1시간 안에 검출 한계 이하로 불활화되었으며, 바이러스 로그 감소 값은 각각 ${\geq}5.58$, ${\geq}5.32$, ${\geq}5.11$, ${\geq}3.42$이었다. 또한 소 조직으로부터 콜라겐을 추출하기 위한 14일간의 펩신 처리 공정에서 BHV, BVDV, BPIV-3, BPV 모두 처리 5일 안에 검출한계 이하로 불활화되었으며, 바이러스 로그 감소 값은 각각 ${\geq}7.08$, ${\geq}6.60$, ${\geq}5.60$, ${\geq}3.59$이었다. 두 공정에서 BHV, BVDV, BPIV-3, BPV의 누적 바이러스 로그 감소 값은 각각 ${\geq}12.66$, ${\geq}11.92$, ${\geq}10.71$, ${\geq}7.01$이었다. 이상의 결과에 의하면, 소 가죽 유래 type I 콜라겐제조공정은 바이러스 안전성 보증을 위한 충분한 바이러스 불활화 능력을 가지고 있는 것으로 판단된다. Most types of collagen used for biomedical applications, such as cell therapy and tissue engineering, are derived from animal tissues. Therefore, special precautions must be taken during the production of these proteins in order to assure against the possibility of the products transmitting infectious diseases to the recipients. The ability to remove and/or inactivate known and potential viral contaminants during the manufacturing process is an ever-increasingly important parameter in assessing the safety of biomedical products. The purpose of this study was to evaluate the efficacies of the 70% ethanol treatment and pepsin treatment at pH 2.0 for the inactivation of bovine viruses during the manufacture of collagen type I from bovine hides. A variety of experimental model viruses for bovine viruses including bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), bovine parainfluenza 3 virus (BPIV-3), and bovine parvovirus (BPV), were chosen for the evaluation of viral inactivation efficacy. BHV, BVDV, BPIV-3, and BPV were effectively inactivated to undetectable levels within 1 h of 70% ethanol treatment for 24 h, with log reduction factors of ${\geq}5.58$, ${\geq}5.32$, ${\geq}5.11$, and ${\geq}3.42$, respectively. BHV, BVDV, BPIV-3, and BPV were also effectively inactivated to undetectable levels within 5 days of pepsin treatment for 14 days, with the log reduction factors of ${\geq}7.08$, ${\geq}6.60$, ${\geq}5.60$, and ${\geq}3.59$, respectively. The cumulative virus reduction factors of BHV, BVDV, BPIV-3, and BPV were ${\geq}12.66$, ${\geq}11.92$, ${\geq}10.71$, and ${\geq}7.01$. These results indicate that the production process for collagen type I from bovine hides has a sufficient virus-reducing capacity to achieve a high margin of virus safety.

양막 콘텍트렌즈의 임상적 효용성

정현철,이재훈,최수영,김성포,박완기,박우찬 대한안과학회 2022 대한안과학회지 Vol.63 No.7

목적: MS-Amnion 양막 콘택트렌즈의 임상적 효용성에 대해 알아보고자 하였다. 대상과 방법: 비수술적 치료에 반응하지 않는 심한 점상각막염 또는 각막상피결손 환자를 대상으로 MS-Amnion 양막 콘텍트렌즈를1주일 동안 착용하였다. 3개월 이상 경과 관찰한 18안을 대상으로 시술 전과 시술 후 시력, 눈물막파괴시간, 각막지각과 각막혼탁정도를 비교하였으며, 경과 관찰 기간 중 치료 성공률 및 병변 재발률과 기타 합병증에 대해 분석하였다. 결과: 18안 중 15안(83.3%)은 2주 이내에 병변이 호전되어 추가 양막 치료 없이 회복하였고, 3안(16.7%)은 치료 이후에도 병변이 지속되어 추가적인 양막이식술을 하였다. 회복되었던 15안 중 3안(20%)은 기존 병변이 재발하였다. 양막 콘택트렌즈 착용 기간 동안 5안(27.8%)은 양막이 저절로 탈락했고, 2안(11.1%)은 반으로 접혀 있었다. 최대교정시력은 시술 전 평균 0.89 logarithm of the minimal angle of resolution (logMAR)에서 3개월 후 0.27 logMAR로 호전되었고, 각막혼탁 정도는 시술 전 평균 0.38에서 3개월 후 0.11로감소하였으며 두 수치는 통계적으로 유의한 차이를 보였다. 눈물막파괴시간과 각막지각은 호전되는 양상을 보였으나 통계적으로 유의한 차이는 없었고, 특별한 합병증은 발견되지 않았다. 결론: MS-Amnion 양막 콘택트렌즈는 봉합사를 이용한 일시적 양막이식술과 달리 외래에서 간편하게 적용할 수 있는 장점이 있고, 난치성 안구 표면 질환을 치료하는 데 있어 일시적 양막이식술을 대체할 수 있는 효과적인 방법이 될 수 있을 것이라 기대된다.

사림 (詞林) : 통도사계단락성회후 (通度寺戒壇落成會後)

허남사,조야운,김간전,서석재,김구하,김성포,최구고,윤우산,권퇴경,약생국영 조선불교월보사 1911 朝鮮佛敎月報 Vol.6 No.-

배양된 인공피부와 인공각막 등 인체조직을 이용한 화장품원료의 안전성 및 효능평가 기술

양은경 ( Eun Kyung Yang ),고강일 ( Kang Ii Ko ),김성포 ( Sung Po Kim ),이종원 ( Jong Won Lee ),박정극 ( Jung Keug Park ),김기호 ( Ki Ho Kim ) 대한화장품학회 2001 대한화장품학회지 Vol.27 No.2

가토모델에서 배양 구강상피를 이용한 근-점막 피판의 형성에 관한 연구

신영민,정헌종,안강민,박희정,성미애,김성민,황순정,김명진,장정원,김성포,양은경,송계영,이종호,Shin, Young-Min,Chung, Hun-Jong,Ahn, Kang-Min,Park, Hee-Jung,Sung, Mi-Ae,Kim, Soung-Min,Hwang, Soon-Jung,Kim, Myung-Jin,Jahng, Jeong-Won,Kim, Sung-P 대한악안면성형재건외과학회 2005 Maxillofacial Plastic Reconstructive Surgery Vol.27 No.3

Purpose : Extensive defect of oral and maxillofacial area is usually reconstructed with composite flap including skin paddle. However, if the defects are lined with only skin components, the mucosa's role in mastication and texture are not restored. Furthermore, stiffness and hair-growing prevent denture rehabilitation and good oral hygiene. This study was performed to overcome the disadvantages of composite soft tissue flaps including the skin and to make a model for myo-mucosal flaps. Materials and methods : Buccal mucosa sized $0.5\times1.0\;cm^2$ from New Zealand rabbit (around 1.5kg) was harvested and cultivated by the modification of Rheinwald and Green's keratinocyte culture method. Cultured mucosa was grafted on the fascia of latismus dorsi as form of mucosal sheet. After 7, 10, 14 days, the myomucosal flap was excised and evaluated under light microscope with H & E and immunohistochemical staining. As control group, harvested buccal mucosa from rabbit was transplanted to gracilis muscle(n=6). Results : From 7 days after prelamination, the basal layer of the grafted mucosa resembled that of normal mucosa. As control group, transplanted mucosa had original shape but there's slight inflammatory reaction. Prelaminated mucosa has 19.8$\pm$4.59 cell layers and some samples have more than 20 layers. The expression rate of PCNA was relatively strong (42.9%$\pm$14.1) at the basal layer of grafted mucosa and the laminin was found at the basal layer. On the contrary, prelaminated mucosa at 10 days showed moderate expression rate of PCNA(32.4%$\pm$4.62). We found the mucosal layer was somehow disappeared and there is strong inflammatory reaction. After 14 days prelamination, the grafted oral keratinocytes were almost disappeared and expression of PCNA was not observed. Conclusion : We can make 75 fold large mucosal($3850mm^2$) sheet from small samples of mucosa $(50mm^2)$. Epithelial sheet that grafted on the fascia of muscle underwent differentiation and proliferation. But after 10, 14 days, there was strong inflammatory reaction and the grafted mucosa was destroyed from surface layer. In rabbit model, transfer of fascio-mucosal flap should be done from 7 to 10 days after prelamination.

가토모델에서 배양 구강상피를 이용한 점막근조직판의 형성에 관한 연구

신영민,정헌종,안강민,박희정,성미애,김성민,황순정,김명진,장정원,김성포,양은경,송계영,이종호 대한악안면성형재건외과학회 2005 Maxillofacial Plastic Reconstructive Surgery Vol.27 No.4

바이러스 안전성이 보증된 무세포 소 양막 생물창상피복재 제조 공정 개발

김인섭 ( In Seop Kim ),배정은 ( Jung Eun Bae ),양은경 ( Eun Kyung Yang ),김성포 ( Sung Po Kim ),김찬경 ( Chang Kyong Kim ) 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 2010 한국미생물·생명공학회지 Vol.38 No.4

바이러스 안전성이 보증된 무세포 소 양막 생물창상피복재 제조공정을 확립하고자 하였다. 기질세포를 제거하기 위해 효소(트립신)를 처리하는 공정과 바이러스를 불활화하기 위해 70% 에탄올, 0.05% sodium hypochlorite, 25 kGy 감마선 처리 공정을 포함하는 무세포 소 양막 제조공정을 확립하였다. 무세포 소 양막의 조직학적 분석과 전자현미경 분석 결과 면역거부반응을 일으킬 수 있는 상피층과 기질세포들이 잘 제거되었으며, 소 양막 콜라겐 섬유의 3차원적 구조가 잘 유지되어 있음을 확인하였다. 또한 상처치유효과가 있는 EGF, KGF, FGF와 같은 성장인자를 포함하고 있었다. 바이러스 불활화 효과를 검증하기 위해 국제적 가이드에 따라 4종의 바이러스(BHV, BVDV, BPIV-3, BPV)를 생물학적 지표로 사용하여 소 양막에 각 생물학적 지표를 첨가한 후각 바이러스 불활화 공정을 실시한 다음 각 바이러스를 회수하여 정량한 후 불활화 정도를 비교하였다. 24시간 70%에탄올 처리 공정에서 BHV, BVDV, BPIV-3, BPV 모두 처리 시간 1시간 안에 검출한계 이하로 완벽하게 불활화되었다. 30분 0.05% sodium hypochlorite 처리 공정에서 BHV, BVDV, BPIV-3 같은 외피 바이러스는 BPV 같은 비-외피 바이러스에 비해 효과적으로 불활화되었다. 25 kGy 감마선 조사에 의해 BHV, BVDV, BPIV-3는 검출한계 이하로 완벽하게 불활화되었고, BPV도 효과적으로 불활화되었다. 3가지 바이러스 불활화 공정에서 BHV, BVDV, BPIV-3, BPV에 대한 log 바이러스 감소인수 합은 각각 ≥13.30, ≥14.32, ≥15.22, ≥7.57이었다. 이와 같은 결과 본 연구를 통해 확립된 무세포 소 양막 제조공정은 바이러스 안전성을 보증할 수있는 충분한 바이러스 불활화 능력을 갖고 있는 것으로 판단된다. A process for manufacturing virally-safe bovine amniotic membrane(BAM) has been developed for biological dressing. BAM was harvested from a healthy bovine placenta, and then the epithelium was removed. The remaining stromal layer was consecutively disinfected with 70% ethanol and 0.05% sodium hypochlorite. The stromal layer was incubated in a decellularization solution containing 0.25%(w/v) trypsin to remove the cellular components. The resulting acelluar BAM was lyophilized to preserve its biochemical and structural integrity. The BAM was packed and exposed to 25 kGy of gamma irradiation for sterilization purpose. Histological, electron microscopical, and biochemical observations showed that the acellualr BAM had intact structural integrity of three dimensional collagen fibers and contained several growth factors, accelerating wound healing, such as EGF (Epidermal growth factor), KGF (Keratinocyte growth factor), and FGF (Fibroblast growth factor). Bovine herpes virus (BHV), bovine viral diarrhoea virus (BVDV), bovine parainfluenza virus type 3 (BPIV-3), and bovine parvovirus (BPV) were chosen as the biological indicators for validation of viral safety of the acellular BAM. Samples from relevant stages of the production process were spiked with each virus and subjected to viral inactivation processes. Viruses were recovered from the samples and then titrated immediately. All the viruses tested were completely inactivated to undetectable levels within 1 h of 70% ethanol treatment. Enveloped viruses such as BHV, BVDV, and BPIV-3 were more effectively inactivated than BPV by 0.05% sodium hypochlorite treatment. BHV, BVDV, and BPIV-3 were completely inactivated to undetectable levels by 25 kGy of gamma irradiation. Also BPV was effectively inactivated by 25 kGy of gamma irradiation. The cumulative log reduction factors of BHV, BVDV, BPIV-3, and BPV were ≥13.30, ≥14.32, ≥15.22, and ≥7.57, respectively. These results indicate that the production process for acelluar BAM has a sufficient virus-reducing capacity to achieve a high margin of the virus safety.

양막과 콜라겐을 이용한 생체 적합 드레싱 소재 개발 및 백서 창상치유 실험

안강민(Kang-Min Ahn),이지호(Ji-Ho Lee),이의룡(Ui-Lyong Lee),이종호(Jong-Ho Lee),이종원(Jong-Won Lee),김성포(Sung-Po Kim),양은경(Eun-Kyung Yang),김기호(Ki-Ho Kim) 대한구강악안면외과학회 2006 대한구강악안면외과학회지 Vol.32 No.3

Purpose of study: Partial thickness skin graft is the golden standard regimen for full-thickness skin defect caused by burn or trauma. However, in case of extensive burns of more than 50% of total body surface area, the donor site is not sufficient to cover all defects. As a second choice, allograft, xenograft and synthetic materials have been used to treat skin defect. Among them the amniotic membrane(AM) was used as a biological dressing for centuries because of its potential for wound healing. In this study, quantification of EGF in AM and effect of AM-collagen complex on full thickness skin defects was examined. Materials & Methods: The concentration of EGF in fresh, deep frozen and freeze-dried AM was evaluated by ELISA. EGF-R immunostaining was performed in freeze-dried AM. SD rats weighing 250~300g was used for wound healing experiment. Three full thickness skin defects(28mm diameter) were made on dorsal surface of SD rat. The control group was covered by Vaselin gauze and AM-collagen complex and Terudermis was grafted in two other defects. Healing area, Cinamon’s score were evaluated before biopsy. Grafted sites were retrieved at 3 days, 1 week, 2 weeks and 4 weeks after operation. H & E and Factor VIII immunohistochemical stain was performed to evaluate the microscopic adhesion and structural integrity and microvessel formation. Results: 1. EGF concentration of fresh, deep frozen and freeze-dried AM showed similar level and EGF-R was stained in epithelial layer of freeze-dried AM. 2. At 4 weeks after grafting, the healing area of AM-collagen and Terudermis group was 99.29±0.71% and 99.19±0.77 of original size. However, that of control group was 24.88±2.90. 3. The Cinamon’s score of AM-Collagen and Terudermis group at 4 weeks was 15.6±1.26 and 14.6±3.13 and that of control group was 3.7±0.95. Significant difference was observed among control and experimental groups(p〈0.05). 4. Histologic examination revealed that AM protected leukocyte infiltration and epithelial migration was nearly completed at 4 weeks. Terudermis group showed mild neutrophil infiltration until 2 weeks and completion of epithelization at 4 weeks. Control group showed massive leukocyte infiltration until 4 weeks. 5. Microvessels were increased sharply at 1 week and control group at 1 and 4 week showed significant differences with Terudermis group of same interval(p〈0.05) but no differences were found with AM group(p〈0.05). Conclusion: EGF and EGF-R were well preserved in freeze-dried AM. AM attached to collagen acted as excellent biologic dressing which had similar effect with Terudermis. AM showed anti-inflammatory action and healing was completed at 4 weeks after full-thickness skin defect.