KOREASCHOLAR

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

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

• 장원혁(Department of Periodontology, School of Dentistry, Dankook University) | Won Hyuk Jang
• 정진형(Department of Periodontology, School of Dentistry, Dankook University) | Chin Hyung Chung
• 홍기석(Department of Periodontology, School of Dentistry, Dankook University) | Ki Seok Hong
• 박경주(Department of Oral Histology, School of Predentistry, Dankook University) | Gyeong Ju Park
• 임성빈(Department of Periodontology, School of Dentistry, Dankook University) | Sung Bin Yim correspondence