조직공학은 장기이식의 발달로 인하여 그 필요성이 제기되었고, 다양한 생체 재료를 만들어 이식함으로써 조직의 재생을 유도하는 것을 목적으로 하고 있다. 본 연구에서는 조직공학적 생체재료로서 돼지 해면골을 이용하여 골유도능과 생체적합성을 평가하고자 하였다. 이상적인 골 재생을 위해서는 충분한 골형성 세포와 적절한 골 대체물이 결합 할 수 있는 생체 환경을 제공하여야 한다.
이 연구에서 세포는 New Zealand White rabbit으로부터 분리한 bone marrow stromal cells (BMSCs)을 사용하였고, 골 대체물로서 열처리하여 얻어진 무기물화된 돼지 해면골(bone block)을 이용하였다.
Bone block의 특성을 분석하기 위하여 SEM (scanning electron micorscopy), XRD (X-ray diffracbon), EDX (energy-dispersive X-ray), compressive test를 통하여 mechanical strength를 측정하였다.
Bone block에 세포를 배양한 후, in vitro experiment는 collagen coating에 따른 실험 과, 정적 배양과 동적 배양 실험에 따른 비교 실험을 하였으며, in vivo study는 beagle (canine)의 경골에 bone block을 이식하는 동물 실험을 수행하였다.
열처리한 bone block은 다공성 구조를 가졌으며, 수산화인회석(hydroxyapatite)을 기초로한 생체재료이다. 갈슘과 인산염의 비율은 1,65-1.66로써, 사람의 해면골과 유사하 였다. 10개의 시편으로 얻어진 탄성계수(Young's moduli) 값은 346.33 MPa로, 사람의 대퇴골과 경골에서 얻어진 값과 비슷했다.
열처리한 bone block은 4군으로 나누어서 실험하였다: 정적 배양에서 collagen coating을 하지 않은 군과 collagen coating한 군을 라각 1군과 11군으로, 동적 배양에서 collagen coating을 하지 않은 군과 collagen coating한 군을 각각 m군과 IV군으로 나누었다,
Irt ultro experiment로서 cell count, MTT assay, alkaline phosphatase (ALP) activity, total Drotein content를 측정하였는데, 시간이 지남에 따라 bone block에서 BMSCs의 성장과 분화는 유의한 차이를 나타내며 증가하였다(p<0.05). Cell count는 정 적 배양과 동적 배양을 비교했을 때, collagen coating block (p=0.033)과 collagen non-coating block ()9-o,003) 둘 다 유의한 차이를 나타냈다. Collagen coating을 한 bone block의 정적 배양이 collagen coating을 하지 않은 bone block의 동적 배양에 비 해 높은 유의한 차이를 나타냈다(p=0.000). MTT assay에서는 14일에 가장 높게 나타났으며, collagen coating을 하지 않은 bone block의 정적 배양과 collagen coating을 한 bone block의 동적 배양이 유의한 차이를 나타냈다(p=0.0l5). ALP acbvity는 큰 유의한 수준을 나타내지 않았다, MTT assay는 배양 기간 초반에 유의한 차이를 나타냈고, ALP activity는 배양 기간 후반에 유의한 차이를 나타냈다(p<0.05). 이러한 생화학적 분석을 통해 10-14일 정도에 세포가 가장 많이 증식되면서 분화되기 시작하는 것을 알 수 있다, Total protein content는 10일까지 정적 배양과 동적 배양사이에서 유의한 차이를 나타냈다(p<0,05), SEM (scanning electron microscopy)으로 세포가 부착된 형태를 관찰하였고, H&E 염색과 TEM (transmission electron microscopy) 결과, 4주째에 골세포 주위로 골기질(osteoid)이 보였고, 7, 8주째에 무층골과 층판골이 나타나기 시작 했다, 미세구조적으로 collagen 섬유 형성이 관찰되었다.
In vivo study는 동물 실험을 3군으로 나누어 수행하였다: 대조군(A군)과 illiac crest 에서 복은 bone marrow를 묻히지 않은 bone block을 사용한 군(B군)과 bone marrow 를 묻힌 bone block을 사용한 군(C군)으로 나누었다, B군과 C군의 Implantation 결과, radiological examination에서는 bone block과 beag1e의 정상골이 잘 유합된 부분을 관찰할 수 있었고, HKE 염색에서는 새로운 골형성과 섬유 조직들이 관찰되었다. 그러나, A군(대조군)에서는 골유합과 골형성이 일어나지 않았다.
대체적으로 동적 배양은 정적 배양에 비해 골아세포(osteob1astic cell)의 증식과 분화 에 더 효과적이라는 것을 알 수 있었다. 그리고, collagen coabng의 유무에 따른 변화는 초기 세포 부착에만 영향을 주었으나, 배양시간이 지남에 따라 세포 증식과 분화에는 큰 차이를 보이지 않았다,
결론적으로, 돼지 해면골에서 얻어진 열처리한 bone block은 이종이식을 통한 골재생을 위해 골대체물로서의 가능성을 제시하였다.

The purpose of this study is to investigate the potential of tissue engineered porcine trabecular bones as a bone substitute. Generally, there are three major requirements to have a successful bony tissue regeneration: (1) osteoprogenitor cells, (2) osteoconductive matrix, and (3) osteoinductive environments.
In this study, bone marrow stromal cells (BMSCs) harvested from the femurs of New Zealand White rabbits followed by the successive treatments were used. For osteoconductive matrix, mineralized porcine trabecular bones with various chemical and heat treatments were chosen. Before seeding the cells onto the matrix, mechanical strength was evaluated through the compressive test. The effects of seeding and culturing conditions on the tissue generation were also examined. For these, the results of static and dynamic culturing were investigated through various biochemical analyses techniques. In addition to in vivo experiments, animal study was also carried out up to the week 12 after operation.
The morphological observation said that the heat-treated bone block was highly porous. Also, it was found to be hydroxyapatite (HA)-based material. The ratio of calcium to phosphate was ranged from 1.65 to 1.66 which is very similar to that of human trabecular bones. The average value of Young's moduli obtained from 10 specimens was 346.33 MPa, which is close to that of trabecular bone in proximal tibia or femur of human.
The heat-treated bone blocks were divided into 4 groups: intact heat-treated blocks with static culture (Group I), collagen coated blocks with static culture (Group II), intact heat-treated blocks with dynamic culture (Group III), and collagen coated blocks with dynamic culture (Group IV).
The dynamic seeding provided more preferable environment in cell attachment. When the blocks were coated with collagen, synergy effect was observed. The results of MTT assay showed that the proliferation was most active at the week 2 after culture in all groups. The alkaline phosphatase (ALP) activities were still increased even after the week 2 of culture. Therefore, differentiation activity took over the proliferation around the week 2 after the culture. Total protein contents were also investigated and maximum value was obtained around the day 10.
Observation by scanning electron microscopy (SEM), H&E staining and transmission electron microscopy (TEM) revealed bony tissue regeneration such as the cell adhesion, the formation of osteoids, and generation of lamellar bone and woven bone.
For animal study, three groups were set: control (Group A), group for implantation without bone marrow (Group B) and group for implantation with bone marrow (Group C). There were no apparent differences between group B and C. No resorption was observed in group B and C.
Histological examination for animal study also confirmed the osteoblast lining, osteoids at the week 12 after operation. Fibrotic tissues were also observed in all groups. Even the control group showed most fibrotic tissues, the remaining two groups also showed considerable amount of fibrotic tissues.
The result said that the dynamic culture is superior to static culture. Even there was no significant effect of the collagen coating on the cell activities, the attachment was improved in collagen coated bone block.
Based on all the results obtained from this study, we could come to the conclusion that this heat-treated porcine trabecular bone has the potential of the xenograft for bone regeneration.

• 목차
• ABSTRACT = i
• 국문 초록 = iv
• I. Introduction = 1
• A. Backgrounds = 2
• 1. Bone graft = 2
• 2. Natural biomaterial = 3
• 3. Bone marrow stromal cells (BMSCs) = 4
• B. Tissue engineered backgrounds = 6
• 1. Tissue engineering = 6
• 2. Collagen coated bone block = 7
• 3. Threedimensional culturing system = 8
• 4. Dynamic seeding and dynamic culture = 8
• 5. Animal study = 9
• II. Objectives = 10
• III. Materials and Methods = 11
• A. Natural bone block materials = 11
• 1. Preparation of bone block = 11
• 2. Collagen coating on the bone block = 11
• B. Chemical and mechanical characteristics of bone block = 12
• 1. Scanning electron microscopy (SEM) = 12
• 2. Xray diffraction (XRD) = 12
• 3. Energydispersive Xray (EDX) = 13
• 4. Measurement of mechanical strength = 13
• C. Isolation of bone marrow stromal cells (BMSCs) = 15
• 1. Dynamic seeding = 15
• 2. Static and dynamic cultures = 16
• 3. Experimental design = 16
• D. In vitro experimentation = 18
• 1. Cell count = 18
• 2. MTT assay = 18
• 3. Alkaline phosphatase (ALP) activity = 18
• 4. Total protein content = 19
• 5. Scanning electron microscopy (SEM) = 19
• 6. Histological examination . = 20
• 7. Transmission electron microscopy (TEM) = 20
• E. Statistical analysis = 21
• F. Animal study = 22
• IV. Results = 24
• A. Characteristics of bone block = 24
• 1. Scanning electron microscopy (SEM) = 24
• 2. Xray diffraction (XRD) = 26
• 3. Energydispersive Xray (EDX) = 27
• 4. Mechanical strength = 29
• B. In vitro experimentation = 30
• 1. Cell count = 30
• 2. MTT assay = 32
• 3. Alkaline phosphatase (ALP) activity = 34
• 4. Total protein content = 36
• 5. Scanning electron microscopy (SEM) = 38
• 6. Histological examination = 40
• 7. Transmission electron microscopy (TEM) = 42
• C. In vivo implantation = 44
• 1. Radiological examination = 44
• 2. Histological examination = 48
• V. Discussion = 51
• VI. Conclusions = 53
• References = 54