조직공학을 이용한 손상된 척추 신경의 기능적 복원 = Regeneration of Injured Spinal Cord by Tissue Engineered Methods

다국어 초록 (Multilingual Abstract)

Regeneration of spinal cord using bone marrow stromal stem cell
Bone marrow stromal stem cells (BMSCs) are widely known as a source of stem-like and progenitor cells. When BMSCs was transplanted into the normal animal central nervous systems, the cells are able to differentiate into neuronal cells. We tested experiment based on our assumption that transplantation of BMSCs into the spinal cord after a severe cord injury.
Fabrication of nerve guidance channeled poly (L-lactide - co- glycolide) (75:25 by mole ratio of lactide to glycolide, PLGA) scaffold performed by ice-particle leaching method. It investigated as potential guidance channel. BMSCs were harvested from the femurs and tibias of adult female Fischer rats. BMSCs were suspended at 2 x 10^(6) cells/scaffold in PLGA scaffold with 200 ngBDNF. Fischer rats with the hiatus (T8-T9 : 5 mm, 3 mm ,1 mm and Cutting) created through the spinal cord resection were implanted by the PLGA scaffold consisting of BMSCs and BDNF. For histological evaluation, the implants were removed after 4 and 8 weeks. Thin sections were cut from paraffin embedded tissue and histological section were stained by hematoxylin and eosin (H&E) staining. Tissue slides also stained by immunohistochemical staining for neurofilament (NF), neuro-specific enolase (NSE). We measured the movement of regeneration of spinal cord using Basso-Beattie-Bresnehan Score and Motor Evoked Potentional (MEP) performed weekly up to 8 weeks post-injury. The BBB locomotor rating score measured in the four groups up to 8 weeks after a transaction. MEP designed a high-voltage transcranial electrical stimulator that exited motor cortex using electrode which were placed over the scalp. Histological examination confirmed the appearance of neural tissue in a group, which consists of PLGA scaffold with BMSCs and BDNF. Although the laminar organization observed in the native was lost, we were observed in the regenerated tissue.
Preparation and Release profile of BDNF-loaded PLGA Scaffold
Recently, brain-derived neurotrophicfactor (BDNF) is known for a protein that can induce the neuron. BDNF is a kind of neurotrophin family of proteins which have roles in regulating the existence, growth and differentiation of neurons within the developing nervous system. The goal of this study was to investigate release tendency of BDNF’s quality.
A biodegradable and porous poly (L-lactide-co-glycolide) (75:25 by mole ratio of lactide to glycolide, PLGA) scaffold has been suggested for the application to cytokine carrier and tissue engineering. The brain-derived neurotrophic factor (BDNF) loaded PLGA scaffolds. The scaffold made by ice-particle leaching method and purpose of using this method was controlled release within porous polymer scaffold. The release amount of BDNF from BDNF loaded PLGA scaffold were observed for 4 weeks period in vitroat phosphate buffered saline (PBS), pH 7.4 at 37 ℃ incubator and the release amount of BDNF loaded in PLGA analyzed enzyme-linked immunosorbent assay (ELISA).
It can be observed the open cell pore structure of porous scaffold and can be easily controlled the pore structure by the controlling of formulation factors resulting in the controlling of the release rate and the release period. The stability of BDNF during the preparation of PLGA scaffold was evaluated by comparing the released amount of total BDNF, assayed BDNF by ELISA.
These results suggest that the released BDNF from BDNF loaded PLGA scaffold such as conduit type can be very useful for the nerve regeneration in the neural tissue engineering area. Prepared PLGA scaffold had definite porosity and amount of release increased with high content of BDNF. Also we observed that BDNF loaded in PLGA scaffolds had faster initial burst. In our study, these results propose that the released BDNF from BDNF loaded in PLGA scaffolds can regulate and controlled release for tissue engineering.
Characterization of PLGA scaffold with SIS
Tissue engineering holds great promise as away to repair and regenerate defectiveof damaged tissues and organs. Tissue engineering consists of three components: cells, scaffolds, and growth factors. From among these, the biomaterials that serve as scaffold substrates play a central role. The goal of this study was to investigate characterization of PLGA scaffold impregnated SIS for apply to tissue engineered spinal cord regeneration.
PLGA is known for biodegradable, biocompatible polymer. SIS also has widely used as biocompatible material with minimum immune response and contains lots of variety of cytokines such as collagen and glycosaminoglycan. It was explored that the most suitable content of SIS to include in PLGA scaffold and the property of fabricated scaffolds by analyzing degradation behavior and cell proliferation in vitro.
Molecular weight of used PLGA was 90,000 g/mol and SIS was isolated from pig’s jejunum and was pulverized by freezer mill. SIS powder was impregnated in PLGA scaffold to 0, 10 and 20% of weight of PLGA. The scaffold was fabricated by ice particle leaching method using 180 ~ 250㎛ size of ice particle. The porosity of these scaffolds wasexamined by mercury porosimetry. We observed morphology of scaffold by SEM. The degradation behavior of PLGA/SIS scaffold examined in 37℃ shaking incubator for 7 weeks under phosphate buffered saline (PBS, pH 7.4). Variation of pH and weight with degradation were analyzed by pH meter. The changed molecular weight of scaffold was measured by GPC. Morphology of cells on the scaffolds was analyzed by fluorescent microscope. BMSCs were harvested from the femurs and tibias of adult female Fischer rats and cultured in Dulbeco's modified Eagle's mediumsupplemented with 10% fetal bovine serum and penicilline-streptomycine. We seeded 3 × 10⁴ cells of BMSCs in each scaffold. MTT assay carried out for the cell viability in PLGA/SIS.
We confirmed that fabricated PLGA/SIS scaffold consisted of pores up to 95% and size of pores was uniformed. The morphology of the PLGA/SIS scaffold showed formation of interconnected and uniformed pores by SEM. We observed decrease of scaffold weight following to decrease of pH. The result of GPC showed that molecular weightdiminished with time. Cytotoxicity in PLGA scaffold with 10% SIS powder was better than PLGA scaffold contained 20% SIS and only PLGA scaffold. In these results indicate that the PLGA/SIS scaffold can be veryuseful for application in the tissue engineered spinal cord regeneration.
This experiment progressedPLGA/SIS for regeneration of spinal cord injury. Through result of this study, physical property and cell viability was high in scaffold contained 10% SIS. Regeneration of spinal cord should follow axons regeneration. Current result could expect that the scaffold by ice particle leaching method contained 10% SIS suitable to nerve guidance channel. In addition, it might be secreting substance from SIS helps BMSC get into neuron cell and axons regrowth. It is progressing that experiment on neurogenesis of BMSC in PLGA/SIS in vitro, and implantation of PLGA/SIS seeded BMSCs in vivo.
AdehesionBehaviorofHumanBoneMarrow StromalCells
Adehesion Behavior of Human Bone Marrow Stromal Cells on a Wettable Polyethylene Surface
Tissue engineering has the potential to revolutionize reconstructive surgery through the provision of engineeredtissues and organs. Tissue engineering consists of three components: cells, scaffolds, andgrowth factors. From among these, tissue-engineered construct that serve as scaffolds such as the adhesion and proliferation of cell to implanted surface depend on the surface characteristics. In this study, we investigated how human bone marrow stromal cells (hBMSCs) respond to surface property of scaffolds. We prepared modified low density polyethylene (LDPE) surfaces by treatment of radio frequency corona discharge, which gradually oxidizes the LDPE surface. As the surface wettability increased, that is moderate hydrophobic positions. The modified PE surfaces were characterized by measuring the static water contact angle. The viability of cells on PE films was characterized by MTT assay. And Cells adhered, oriented and grown on the microgrooved surfaces were observed by scanning electron microscope (SEM) and Optical microscope. In conclusion, these results indicate that the biological signals induced by cell adhesion depend on the wettability of the surface to which the cells attach.

목차 (Table of Contents)

• 제 1 장 서 론 = 1
• 1.1 조직공학 = 1
• 1.1-1. 조직공학의 개념 = 1
• 1.1-2. 조직공학의 구성 요소 = 3
• 1.1-2-1. 세포 = 3
• 1.1-2-2. 생체재료 = 4
• 1.1-2-2-1. 생분해성 고분자 = 5
• 1.1-2-2-2. 생분해성 고분자의 생체적합성 = 6
• 1.1-2-2-3. 생체조직공학용 재료로서의 생분해성 고분자 = 6
• 1.1-2-2-4. 천연유래 지지체용 생체재료 = 7
• 1.1-2-3. 생리활성 물질 = 8
• 1.1-3. 조직세포의 배양 = 9
• 1.1-4. 이식 = 9
• 1.2 참고문헌 = 11
• 제 2 장 골수간엽줄기세포 및 다공성 지지체를 이용한 조직공학적 척추신경재생 = 24
• 2.1 이론적 배경 = 24
• 2.2 재료 및 방법 = 26
• 2.2-1. 골수간엽 줄기세포의 분리 및 배양 = 26
• 2.2-2. 이식재료의 제조 = 27
• 2.2-3. 척수절제 수술 = 27
• 2.2-4. 수술 후 관리 = 28
• 2.2-5. 운동력 평가 = 28
• 2.2-6. 조직학적 평가 = 29
• 2.3 결과 및 고찰 = 30
• 2.3-1. 이식재료의 제조 = 30
• 2.3-2. 운동력 평가 = 31
• 2.3-3. 육안적 소견 = 32
• 2.3-4. 조직학적 평가 = 32
• 2.4 결 론 = 33
• 2.5 참고문헌 = 36
• 제 3 장 BDNF를 함유한 PLGA/SIS 지지체의 제조 및 특성 평가 = 53
• 3.1 이론적 배경 = 53
• 3.2 재료 및 방법 = 55
• 3.2-1. 시약 및 재료 = 55
• 3.2-2. SIS 파우더의 제조 = 56
• 3.2-3. BDNF를 함유한 다공성 PLGA/SIS 지지체의 제조 = 56
• 3.2-4. SEM에 의한 다공도 분석 = 57
• 3.2-5. BDNF를 함유한 지지체의 방출 실험 = 57
• 3.3 결과 및 고찰 = 58
• 3.3-1. BDNF를 함유한 다공성 PLGA/SIS 지지체 = 58
• 3.3-2. BDNF를 함유한 PLGA 지지체에서의 방출 거동 = 59
• 3.4 결 론 = 60
• 3.5 참고문헌 = 62
• 제 4 장 친수성을 첨가한 다양한 PLGA 지지체 내에서의 BDNF의 방출 경향 = 67
• 4.1 이론적 배경 = 67
• 4.2 재료 및 방법 = 69
• 4.2-1. 시약 및 재료 = 69
• 4.2-2. MPEG-PLGA 공중합체의 합성 = 69
• 4.2-3. BDNF를 함유한 다공성 MPEG-PLGA 지지체의 제조 = 70
• 4.2-4. BDNF를 함유한 지지체의 방출 실험 = 71
• 4.3 결과 및 고찰 = 71
• 4.3-1. MPEG-PLGA 공중합체의 합성 = 72
• 4.3-2. BDNF를 함유한 다공성 MPEG-PLGA 지지체 = 72
• 4.3-3. BDNF를 함유한 MPEG-PLGA 지지체에서의 방출 거동 = 73
• 4.4 결 론 = 74
• 4.5 참고문헌 = 76
• 제 5 장 표면 적심성에 따른 NIH/3T3 섬유아세포의 생물학적 평가 = 86
• 5.1 이론적 배경 = 86
• 5.2 재료 및 방법 = 89
• 5.2-1. LDPE 필름 시료의 준비 = 89
• 5.2-2. 적심성이 다른 PE 표면의 준비 = 89
• 5.2-3. 표면개질 된 PE 표면의 특성 분석 = 90
• 5.2-4. hBMSC 세포의 배양 = 90
• 5.2-5. 적심성을 달리한 PE 표면에서의 hBMSC의 생존율 = 90
• 5.2-6. 세포의 모폴로지 = 91
• 5.3 결과 및 고찰 = 92
• 5.3-1. 접촉각 측정 = 92
• 5.3-2. 세포의 생존율 분석 = 92
• 5.3-3. 세포의 모폴로지 확인 = 93
• 5.4 결 론 = 93
• 5.5 참고문헌 = 96
• 제 6 장 SIS를 함유한 PLGA 지지체의 특성 평가 = 109
• 6.1 이론적 배경 = 109
• 6.2 재료 및 방법 = 109
• 6.2-1. 시약 및 재료 = 109
• 6.2-2. SIS 파우더의 제조 = 110
• 6.2-3. 지지체의 제조 = 110
• 6.2-4. 물 흡수성 평가 = 111
• 6.2-5. 지지체의 다공 크기와 다공도의 측정 = 111
• 6.2-6. in vitro 생분해성 실험 = 112
• 6.2-7. 골수간엽 줄기세포의 분리 및 배양 = 112
• 6.2-8. 세포 생존율 평가 실험 = 113
• 6.2-9. 세포의 부착 확인 실험 = 113
• 6.2-10. in vivo 생체적합성 평가 = 114
• 6.2-11. 조직학적 분석 = 114
• 6.2-12. 통계학적 분석 = 115
• 6.3 결과 및 고찰 = 115
• 6.3-1. PLGA/SIS 지지체의 제조 = 115
• 6.3-2. PLGA/SIS 지지체의 물 흡수성 = 116
• 6.3-3. in vitro 생분해성 평가 = 117
• 6.3-4. 세포 생존율 평가 = 117
• 6.3-5. in vivo 생체 적합성 평가 = 118
• 6.3-6. Host tissue response = 118
• 6.4 결론 = 119
• 6.5 참고문헌 = 121