인간의 유전자 재조합 골형성 단백질-2과 헤파린 혼합물의 골유도 효과에 관한 조직학적 및 조직형태학적 분석

서지정보

ㆍ발행기관 : 대한구강악안면병리학회 ㆍ수록지정보 : 대한구강악안면병리학회지 / 34권 / 4호
ㆍ저자명 : 장원혁, 정진형, 홍기석, 박경주, 임성빈

목차

I. Introduction
II. Materials and Methods
1. Recombinant human bone morphogeneticprotein and Heparin
2. Isolation, culture and verification of adiposestem cell
3. Osteoblastic differentiation culture
4. Determining of osteoblastic differentiation
5. Statistical analysis
III. Results
1. Verification of adipose stem cell lineage
2. Morphologic changes and Naphthol ASphosphate‐fast blue BB reaction
3. Morphologic changes and Alizarin redreaction
4. Alkaline phophatase Activity
IV. Discussion
V. Conclusion
VI. References

영어 초록

Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway evaluating the ability of BMPs to promote bone formation in grafting procedures and fracture healing. Some studies, have independently reported that sulfated polysaccharides particularly heparin, enhance the osteoblastic differentiation induced by BMPs in vitro, and another study demonstrated that heparin enhanced the bone formation induced by BMP‐2 in vivo. This study was performed to examine adipose stem cell responses to rhBMP‐2 alone and rhBMP‐2 with heparin at 0.25, and 25 μg/㎖ concentrations, respectively, in culture media. Adipose stem cells were cultured for 2, 4, and 8 days toward the osteoblastic differentiation in rhBMP‐2 alone and rhBMP‐2 with heparin at 0.25, and 25 μg/㎖ concentrations, respectively, in culture media. Verification of the stem cell lineage was performed in two ways. The first method was a continuous sequential culture until 5th generation. The second method was using monoclonal antibodies for STRO‐1 and CD 90. Naphthol AS phosphate‐fast blue BB staining for alkaline phosphatase was used for verifying osteoblastic differentiation because Alkaline phosphatase activity had been used as an osteoblastic differentiation marker and degree of osteoblastic activity. Alizarin red staining was also used as an osteoblastic differentiation marker because it quantifies the calcium levels in cells or tissues. During the 5th generation culture, cultured cells actively proliferated, and these cultured cells showed a positive reaction to STRO‐1 and CD90 cell surface molecules. Naphthol AS phosphate‐fast blue BB staining and Alizarin red staining were positive in most samples of each group at 2, and 4 days and positive reaction was proportioned to degree of morphological differentiation. In the concentration of 25 μg/ml of heparin, the ALP activity was highest at the 2nd day in the culture, and then the activities of ALP were decreased significantly at 4, and 8 days. The ALP activity was greatest at the 4th day of the culture, and then decreased significantly at the 8th day in 0 μ g/ml and 0.25 μg/ml of heparin concentrations, Adipose stem cells could be differentiated in rhBMP‐2 in culture media, and the addition of heparin to BMP‐2 promoted differentiation of osteoblasts. Moreover, morphological differentiation was associated with the activity of osteoblasts. This study was shown that, when heparin concentration increases, the early differentiation of the cells was brought about, but the early differentiated cells were rapidly progressed to degenerative changes

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