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Kafe 바로가기주관연구기관 | 국립농업과학원 National Institute of Agricultural Sciences |
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보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2015-02 |
과제시작연도 | 2014 |
주관부처 | 농촌진흥청 Rural Development Administration(RDA) |
등록번호 | TRKO201500010536 |
과제고유번호 | 1395035252 |
사업명 | 차세대바이오그린21 |
DB 구축일자 | 2015-07-11 |
DOI | https://doi.org/10.23000/TRKO201500010536 |
Ⅳ. 연구개발결과
○ 1세부과제명 : 실크단백질을 이용한 뼈 헝성 소재 개발
- 유용 물질 담지용 실크 지지체, 유기복합 실크단백질 비드, 인공시멘트용 저분자 실크 소재를 제조하여 그 구조와 물성을 분석한 후 협동연구과제에 재료를 공급하였음
○ 2세부과제명 : 실크단백질을 이용한 생체막 소재 개발
- 투명도와 유연성을 갖는 복합 실크 생체막 소재, 혈액 흡수도와 구조적 안정성이 우수한 혼합 실크 스펀지 개발하고 협동연구과제에 재료를 공급하였음
○ 1협동과제명 : 신경외과용 생체막 및 인공시멘트 소재 전임상
Ⅳ. 연구개발결과
○ 1세부과제명 : 실크단백질을 이용한 뼈 헝성 소재 개발
- 유용 물질 담지용 실크 지지체, 유기복합 실크단백질 비드, 인공시멘트용 저분자 실크 소재를 제조하여 그 구조와 물성을 분석한 후 협동연구과제에 재료를 공급하였음
○ 2세부과제명 : 실크단백질을 이용한 생체막 소재 개발
- 투명도와 유연성을 갖는 복합 실크 생체막 소재, 혈액 흡수도와 구조적 안정성이 우수한 혼합 실크 스펀지 개발하고 협동연구과제에 재료를 공급하였음
○ 1협동과제명 : 신경외과용 생체막 및 인공시멘트 소재 전임상 연구
- 실크를 이용한 투명 인공뇌경막, 척추뼈 압박골절용 실크 인공시멘트/실크
-Ca-p-Hydroyapatite 인공시멘트 개발하여 전임상 동물실험으로 효과를 구명하고, 실크의 뼈 생성 분화 기전을 밝힘
○ 2협동과제명 : 조직공학용 실크지지체의 골형성 효과 연구
- 경골 골수강정 모델 개선을 통한 성장인자 탑재 실크지지체의 골형성능 분석하고 경골천공모델을 이용한 실크지지체 및 실크마이크로비드의 골형성능 연구함
○ 3협동과제명 : 실크단백질을 이용한 치과용 인공뼈 연구
- 4-HR을 이용한 실크 소재의 생분해성 및 면역 조절 기작 구명하고 실크 코팅 임플란트의 골 형성 연구 및 혈관 패치 개발함
○ 3세부과제명 : 진피용 실크 소재 연구
- 진피용 실크 소재의 세포 독성 연구하고 동물모델을 이용한 진피용 실크 소재의 생체안전성 및 생체적합성 연구함
Silkworm cocoon for biomaterials has been studied. Silk for bone regeneration and biological membrane were prepared and examined in the fields of neurosurgical, bone tissue engineering, otorhinolaryngological, and dental applications.
Silk scaffold for bone tissue engineering was prepared with po
Silkworm cocoon for biomaterials has been studied. Silk for bone regeneration and biological membrane were prepared and examined in the fields of neurosurgical, bone tissue engineering, otorhinolaryngological, and dental applications.
Silk scaffold for bone tissue engineering was prepared with porogen and measured porosity, micromorphology, crystalline struccture. Bioactive materials were immobilized in silk materials and examined its structural characteristics and release behavior. Complex silk materials with synthetic biomaterials were also prepared and investigated. Silk beads prepared from silk itself, silk and natural/synthetic biomaterials, alginic acid/polyethyleneglycol were prepared and examined its morphology using scanning electron microscope, thermal properties using differential scanning calorimeter, structural characteristics using infrared spectromer and X-ray diffractomer. And then Swelling and releasing behaviour of silk beads were examined. Also silk powder using Korean silkworm cocoon was prepared and confirmed its molecular weight and supplied for the medical applications.
To develop a biological silk membrane, we made the silk biological membranes using silk fibroin solutions with different amount and dissolving time of silk. The characterizations of the silk biological membranes such as morphology, structure, and mechanical strength were observed. Also, we made silk sponge and porous silk tube. Although each biological membrane has the same fibroin content, there was a significant difference in the thickness and transparency. All silk products showed biocompatibility. Characteristics of the silk products, such as thickness, transparency, physical properties, biodegradability, could be controlled depending on the manufacturing method. We were established the manufacturing condition for silk fibroin biological membrane. So we expect that the conditions will help in the development of medical supplies in the future.
To develop neurosurgical artificial dura membrane, cytotoxicity of silk membrane was examined using astrocyte and silk showed no cytotoxicity and inhibit immune reaction in vitro and in vivo. Compressive strength of silk complexed with calcium phosphate and hydroxyapatite showed increased with hydroxyapatite contents.
This study aimed to examine the utility of various silk fibroin-based scaffolds in long bone reconstruction. In order to attract endogenous mesenchymal stem cells (MSCs) to implanted silk scaffold at the site of bone injury, we first excavated MSC-specific chemokine receptors by microarray-based gene expression analysis. CMKLR1 that binds chemokine RARRES2 was found to be overexpressed in MSCs. Treatment of RARRES2 could activate chemotactic migration of MSCs. Although chemotactic potential of RARRES2 was weaker than SDF-1, RARRES2 preferentially attracted MSCs over fibroblasts. Rat ibial injury model with intramedullary nail with fixing ear and rat tibial cavity model were developed to test in vivo bone forming capacity of various silk scaffolds. Bone formation in the implanted scaffold was analyzed by low dose X-ray radiographic followup, micro CT and histological examination with H & E and Masson trichrome stainings. Nanofibrous and sponge-type silk scaffolds implanted into artificial 3-mm gap injury sites of tibia by intramedullary nailing remained largely intact and bone formation into the scaffolds was negligible even after 6~10 weeks incubation. Sponge-type scaffold of silk fibroin supplemented with 10% hydroxyapatite did not enhance bone-forming capacity of the silk fibroin, either. Deposition of collagen was obvious in the crevice and its neighborhool within the silk scaffold in all of the cases. Loading RARRES2 on the nanofibrous silk scaffold and the 10% HA-silk scaffold appeared to improve deposition of collagen fiber into the scaffold in tibial intramedullary nailing model. Bone forming capacity of HA-silk scaffold loaded with growth factors including BMP-2 and VEGF with or without RARRES2 or SDF-1 was also investigated in tibial intramedullary nailing and tibial cavity models of rat. Among the reagent combinations loaded on the silk scaffold, combination of VEGF with RARRES2 or SDF-1 only manifested significantly improved collagen deposition.
The other combinations including single reagent loadings and BMP-2 plus RARRES was found not effective in collagen deposition into the scaffold. Although RARRES2 enhanced attachment and/or growth of MSCs on HA-silk scaffold, bone forming capacity of cell and cell plus RARRES2-loaded scaffold was found inconsequential. Growth and differentiation of MSCs into osteoblasts on silk microbead were also examined by measuring MTT conversion and alkaline phosphatase activity and by Alizarin staining. Attachment and/or growth of MSCs on silk microbead was significantly lower than that on a 96-well plate.
Accordingly, differentiation of MSCs on the silk microbead was not as prominent as on the culture vessel. Implantation of silk microbead with or without RARRES2 in tibial cavity model did not show significant difference in formation of bone tissue than tibial cavities left empty. Silk microbeads were pushed away from the injury sites, which suggests that novel model system should be developed to test the bone forming capacity of the microbeads. In summary, we discovered a novel MSC chemokine RARRES2 and investigated bone forming capacity of various silk scaffolds with newly developed tibial injury models. Growth factor combinations of VEGF with RARRES2 or SDF-1 only were found reasonably effective in deposition of bone materials into the silk scaffold.
Crevice-directd deposition of bone materials and improved efficacy in collagen deposition by VEGF implicate that structures permissible for free material exchange are preferred for scaffolds in bone tissue engineering.
Silk is produced by insect and mainly composed with fibroin and sericin. It has been used as suture materials for decades. Recently, silk fibroin has been considered as a scaffold in the tissue engineering. Silk based biomaterials have been developed for tympanic membrane, bone graft, and burn dressing. As silk is slowly biodegradable, it can be used for a membrane of guided bone regeneration. Considering its low degradability, it also can be considered as soft tissue augmentation material. However, many foreign body type giant cells are involved in silk degradation. This foreign body reaction may induce chronic inflammation.
4-Hexylresorcinol (4HR) is also bacterial origin molecular chaperone. It has been used as antiseptics, and food additives. 4HR also has anti-cancer effect and inhibits NF-kB pathway.
4HR is used as bone graft material combined with hydroxyapatite. Silk with 4HR combination graft has been shown better performance than silk without 4HR. 4HR can inhibit giant cell formation induced by silk fibroin via diacylglycerol kinase pathway. Additionally, 4HR can inhibit antibody binding capacity. Therefore, silk with 4HR combination graft may show better performance when it is used for dental applications.
In this work, silk fibroin particles (SP) had been prepared by simple ball-milling technique, and further blended with PLC to form a colloidal solution containing silk fibroin SP (PCL/SP10% and PCL/SP30%) capable of forming nanofibers by cold-plate electrospinning.
A comparative study consisting of pristine and silk modified PCL nanofibers had been assayed in the present communication. The pristine nanofibers prepared by cold plate electrospinning experienced collapse in porosity and therefore, resulted in thin membrane-like films. However, nanofibers modified with silk fibroin particles possessed intact pore architecture and therefore retained full-thickness. These nanofibers fabricated had been extensively characterized by various states of art techniques; like TEM, VP-FE-SEM, TEM, Contact angle, FT-IR and TGA. The results from these experiments revealed that silk particles can be successfully introduced in/on nanofibers. The cytotoxicity and cell infiltration studies were carried after culturing NIH 3T3 fibroblasts in presence of nanofibers. These results confirmed hybrid nanofibers exhibits better cell viability and good cell infiltration than those of pristine PCL nanofibers. Moreover, in vivo studies were conducted on back of experimental rat models to determine the usability of these nanofibers as dermal analogue than commercially available Matriderm®. The histological examination using H& E and MT staining after (5, 10, 15 and 20 days), revealed that these nanofibers can moderately be converted to artificial dermis compared than Matriderm®,However, the re sults from gross findings indicated that nanofibers results in low contraction and less scar formation than the commercially available Matriderm®.
Epistaxis is defined as active bleeding arising from the nasal mucosa. It constitutes one of the most common otolaryngology emergencies and can be severe or even fatal. Hemostasis is a process which causes bleeding to stop, meaning to keep blood within a damaged blood vessel which involves differential sequential events. This system in humans has been the most extensively researched. However, lot of research work is going on and lot more needs to be done to overcome the problem associated with this serious issue. This study was conducted to develop a new sponge type of biomaterial to be used for ideal hemostatic agent. The combination of three biomaterials; Silk fibroin (SF), Hyaluronic acid (HA), Gelatin (Gel) and Polyvinyl alcohol (PVA) as proposed hemostatic agent were tested against commercially available hemostatic pad (ChitoClot®). A comparative study of these silk based hemostasis agents designed in combination of SF/HA/PVA and SF/Gel/PVA sponges were tested on injured model of rats. Each sponge to be tested was made same size of (1×1 cm) then applied on top of injury site while applying a pressure of 5 N for 30 seconds. The In vivo experiments were carried by creating a proximal arterial injury in unilateral femoral arteries of 12 anesthetized SD rats. In conclusion, this study suggest a new developed silk fibroin-based sponges as hemostatic materials, which induced durable hemostasis and an increased blood clotting in animal studies. These initial studies explores the loopholes present in the existing ChitoClot® pad compared with that of fabricated silk based sponges.
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