연골 조직 공학을 위한 생적합성 지지체의 개발과 Chondrocytes-PDPCs co-culture

Title
연골 조직 공학을 위한 생적합성 지지체의 개발과 Chondrocytes-PDPCs co-culture
Authors
공미은
Keywords
연골조직공학을위한생적합성지지체의개발과chondrocytespdpcscoculture
Issue Date
2011
Publisher
인하대학교
Abstract
Tissue engineering and regenerative medicine are new research areas investigating how to repair and regenerate impaired organs and tissues using the natural signaling pathway and components of the organism. In the case of joint cartilage, there are no blood vessels and the damaged tissues are not usually regenerated by themselves. Autologous chondrocyte implantation (ACI) is currently performed as a surgical treatment of arthritis. For the treatment of damaged cartilage or initial symptom of osteoarthritis, transplant surgery should be carried out with scaffold beads to support the cells transplanted. Hyaluronic acid (HA) is known to be a good scaffold to make artificial cartilage. In this study, we investigated the possibility of a manufactured HA-atelo scaffold bead as a support material for cartilage cell therapy. Also, we tried to confirm a positive influence on cell proliferation by using co-culture of chondrocytes with Periosteum-derived progenitor cells ( PDPDs). Within 24 hours after surgery, chondrocytes and PDPCs were isolated from the cartilage. HA-atelo scaffold bead was manufactured through a five-step process including HA dilution, collagen mixture, bead formation, washing, and bead collection by using umbilical cord of an embryo. In the first experiment, cell proliferation of cultured chondrocytes on Cellgen, Cytodex3, HA-atelo scaffold bead confirmed and ration of secreted glycosaminoglycan (GAG) analyzed quantitatively by using Blyscan GAG assay kit. Therefore the HA-atelo scaffold bead which we manufactured in autologous chondrocyte implantation confirmed that we were available. In the second experiment, we co-cultured on chondrocytes and PDPCs in two ways. Chondrocytes were co-cultured with PDPCs of various ratios. In other ways, chondrocytes were co-cultured with PDPCs without contacts between cells by using Inserts Membrane(CC Inserts-PC Membrane, Nunc, USA). Cell counting was measured by using hemocytometer and trypan blue dye exclusion meth
Description
1. 서론 1 1.1 조직공학 및 재생의학 1 1.2 국내외 세포치료제 시장 1 1.2.1 국내동향 1 1.2.2 국외동향 2 1.3 연골 5 1.3.1 연골조직의 특성 5 1.4 퇴행성질환 8 1.4.1 관절염 8 1.4.2 외과적 수술 12 1.4.3 문제점 14 1.5 증식에 있어서의 문제점을 위한 해결 방안 제시 14 1.5.1 생체재료의 응용 14 1.5.2 Co-culture 15 2. 연구 목적 16 3. 재료 및 방법 17 3.1 연골세포 분리 및 배양 17 3.2 골막 유래 세포의 분리 및 배양 17 3.2.1 골막 유래 세포의 초대배양 17 3.2.2 골막 유래 전구세포의 분리 및 배양 17 3.3 3가지 micro-bead와 연골 세포의 부착 실험 18 3.4 3가지 micro-bead와 연골 세포의 정적-동적 배양 19 3.5 Co-culture 19 3.5.1 직접적인 세포 접촉이 있는 co-culture 19 3.5.2 직접적인 세포 접촉이 없는 co-culture (Membrane 이용) 19 3.5.3 연골세포의 conditioned media 이용한 co-culture 19 3.7 분석 21 3.7.1 세포 수 측정 21 3.7.1.1 Trypan blue dye exclusion method 21 3.7.1.2 MTT assay 21 4. 결과 및 고찰 22 4.1 연골세포의 분리 22 4.2 골막 유래 전구세포의 분리 24 4.3 HA-atelo scaffold bead의 제조와 독성시험 26 4.3.1 HA-atelo scaffold bead의 제조 26 4.3.2 독성시험 26 4.4 3가지 micro-bead와 연골 세포의 부착 및 증식확인 28 4.4.1 부착 확인 및 제조한 HA-atelo scaffold bead의 접종 농도 확인 28 4.4.2 각 beads에서 연골세포의 증식 비교 31 4.5 3가지 micro-bead와 연골 세포의 정적-동적 배양 31 4.5.1 정적-동적 배양의 증식 비교 31 4.6 Co-culture 36 4.6.1 세포 접촉이 있는 co-culture에서 mixture ratio별 증식 확인 36 4.6.2 세포 접촉이 없는(Membrane을 이용) co-culture에서 증식 확인 36 4.6.3 Conditioned media와 새 배지의 혼합비율에 따른 증식확인 41 5. 결론 43 6. 참고문헌 44
URI
http://dspace.inha.ac.kr/handle/10505/22826
Appears in Collections:
College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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