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.

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.

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

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.