## 4 Discussion  
In this report, we evaluated the effects of IGF-2 on stem cells that were cultured on deproteinized bovine bone at the concentration of 10 and 100 ng/mL. Applying IGF-2 increased alkaline phosphatase  
Figure 7: Quantification by real-time polymerase chain reaction. * Statistically significant increase was seen when compared with 0 ng/mL at Day 7 (\(p<\) 0.05). activity and mineralization of the bone marrow derived stem cells by enhancing the expression of BGLAP. IGFs are protein hormones that share structural similarity to insulin, having affinity and specificity to IGF receptors and IGF-binding proteins [18]. IGF-1 is reported to be one of the most abundant growth factors in the bone matrix and it is involved in the maintenance of bone mass in adulthood [19]. In a previous report, IGF-1 promoted the osteogenic differentiation of stem cells and it was suggested that IGF-1 treated stem cells may serve as a candidate for bone regeneration [19]. Alternately, IGF-2 is reported to be involved in the development and function of various organs, including liver and brain [20,21]. IGF-2 is reported to be involved in reversing or preventing the aging-related memory loss in animal model [20]. IGF-2 also acted as an important mitogen regarding liver repopulation in animal model [21]. IGF-2 was reported to be at higher level in the osteoblast-conditioned medium when compared with the control medium conditioned with the stem cells [22]. The expression of IGF-2 in developing osteophyte was strongly associated with a degree of osteogenesis, with the highest expression in active osteoblasts [23]. IGF-2 was shown to produce the increase in the total protein synthesis and collagenous protein synthesis in osteoblast like cells isolated from human bone [24]. Moreover, IGF-2 is reported to be an essential component of stem cell niche and to be involved in the sproliferation and differentiation of stem cells [16]. In this study, the use of IGF-2 on stem cell loaded graft produced higher cellular viability and higher mineralization. The various concentrations of IGF-2 were tested to evaluate/suggest the optimal concentration [16,25]. IGF-2 was used at different concentrations and concentrations ranging from 50 to 200 ng/mL promoted the proliferation of stem cells [16]. The application of IGF-2 at 200 ng/mL concentration enhanced the cellular proliferation of human marrow stromal cell line, which was shown by increased [3H]thymidine incorporation and increase in the cell number [25]. In this study, the significant increase of osteogenesis and mRNA expression was seen both at 10 and 100 ng/mL, with the highest at 10 ng/mL concentration. It was shown that IGF-2 had no effects on cell proliferation in the presence of 10% fetal bovine serum, but it promoted the cellular proliferation in the presence 1% fetal bovine serum [16]. It was shown that IGF-2 promoted the adipogenic differentiation of stem cells at 100 ng/mL concentration [26]. Likewise, the net effects or optimal dosage may differ between the studies due to culturing conditions, type, and passages of cells [27]. Moreover, IGF-2 may have additional functions. It was shown that IGF-2 promoted the adipogenic differentiation of stem cells at 100 ng/mL concentration [26]. IGF have been shown to be involved in the chondrogenesis and the addition of IGF-2 increased the induction of chondrogenic differentiation while using the three-dimensional culture system [28,29]. The expressions of various genes were tested to evaluate the osteogenic differentiation including Runx 2 and BGLAP [17,30]. Runx2 and BGLAP are widely used biomarkers for osteogenic differentiation [30]. BGLAP was reported to play an important role in the modulation of osteogenic differentiation of mesenchymal stem cells and the maturation of mineral species [31]. In a previous report, attempts were made to achieve the increase in osteogenic potential of stem cells by applying hypoxic conditions [32].