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KMID : 0829220100340040191
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Korean Journal of Oral and Maxillofacial Pathology
2010 Volume.34 No. 4 p.191 ~ p.200
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Histologic and Histomorphometric Analysis of rhBMP-2 and Heparin Mixture’s Osteoinductive Effect
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Jang Won-Hyuk
Chung Chin-Hyung
Hong Ki-Seok
Park Gyeong-Ju
Lim Sung-Bin
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Abstract
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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 μ g/㎖ 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 μ g/㎖ 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 μ g/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 μ g/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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KEYWORD
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Adipose stem cell, Heparin, RhBMP-2, Osteoblast, Differentiation
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