Although recombinant human Bone Morphogenetic Proteins-2 (rhBMP-2) is clinically useful for bone regeneration, induction of new bone formation requires a large amount of rhBMP-2 in humans. Many investigators have been concentrated efforts on searching materials which enhance the effect of rhBMP-2, and then heparin was found as a potentiating material to rhBMP-2. The purpose of this study were histomorphometrically to analyze the enhancing effect of heparin to rhBMP-2 and to study the mode of actions of heparin to rhBMP-2. Stem cells obtained from rabbit adipose tissue were divided into 4 groups according to heparin concentrations(0, 0.25, 2.5, and 25 μg/ml) with a constant rhBMP-2 concentration(150 ng/ml) and cultured for 2, 4, and 8 days. Naphtol AS phosphate-fast blue BB staining for alkaline phosphatase content and Alizarin red staining for calcium content were performed as time schedules and the morphology and osteoblastic activity were observed closely. 5 μg/ml rhBMP-2 was mixed with the following doses of heparin: 0, 0.25, 2.5, and 25 μg/ml. Each mixture was blotted into 0.5 g of multiporous anorganic bovine bone and was inserted into the critical sized calvarial defects(diameter of 0.8-mm) of rabbits. After 1, 3, and 6 weeks, the harvested tissues were processed and stained using H&E and Masson’s trichrome methods. And the areas of newly formed bone in the grafted material were measured and statistically analyzed. During culture experiment of adipose stem cells with rhBMP-2 and heparin, the degree of osteoblastic differentiation was increased with increasing heparin concentration, but the cellular degeneration was accelerated at higher concentration of heparin as time passed. Consequently the osteoblastic differentiation of progenitor cells were accelerated as the concentration of heparin increased. In addition, the progenitor cells exhibited full differentiations early showing fast degeneration. The higher the concentration of heparin, larger newly formed bone in grafted materials was obtained in initial period. However, the increased amount of the newly formed bone in grafted materials was progressively decreased at the higher concentration of heparin as time passed. In conclusion, the heparin has influence on the osteoinductive effect of rhBMP-2 in the initial stage of bone formation. The use of heparin with rhBMP-2 could offer cheaper, safer, and improve clinical results in grafting procedures.

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