인간의 유전자 재조합 골형성 단백질-2과 헤파린 혼합물의 골유도 효과에 관한 조직학적 및 조직형태학적 분석
Bone morphogenetic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underwayevaluating 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 differentiationinduced by BMPs in vitro, and another study demonstrated that heparin enhanced the bone formation induced by BMP‐2in vivo. This study was performed to examine adipose stem cell responses to rhBMP‐2 alone and rhBMP‐2 with heparin at0.25, and 25 μg/㎖ concentrations, respectively, in culture media. Adipose stem cells were cultured for 2, 4, and 8 daystoward 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 acontinuous sequential culture until 5th generation. The second method was using monoclonal antibodies for STRO‐1 andCD 90. Naphthol AS phosphate‐fast blue BB staining for alkaline phosphatase was used for verifying osteoblastic differentiationbecause Alkaline phosphatase activity had been used as an osteoblastic differentiation marker and degree ofosteoblastic activity. Alizarin red staining was also used as an osteoblastic differentiation marker because it quantifiesthe calcium levels in cells or tissues. During the 5th generation culture, cultured cells actively proliferated, and thesecultured cells showed a positive reaction to STRO‐1 and CD90 cell surface molecules. Naphthol AS phosphate‐fast blue BBstaining and Alizarin red staining were positive in most samples of each group at 2, and 4 days and positive reactionwas proportioned to degree of morphological differentiation. In the concentration of 25 μg/ml of heparin, the ALP activitywas highest at the 2nd day in the culture, and then the activities of ALP were decreased significantly at 4, and 8days. The ALP activity was greatest at the 4th day of the culture, and then decreased significantly at the 8th day in 0 μg/ml and 0.25 μg/ml of heparin concentrations, Adipose stem cells could be differentiated in rhBMP‐2 in culture media, andthe addition of heparin to BMP‐2 promoted differentiation of osteoblasts. Moreover, morphological differentiation was associatedwith the activity of osteoblasts. This study was shown that, when heparin concentration increases, the early differentiationof the cells was brought about, but the early differentiated cells were rapidly progressed to degenerative changes.