## 4 Discussion  
The human skeletal system relies on osteoblasts for bone formation, maintenance, and repair. Osteoblasts collaborate with osteoclasts and osteocytes to regulate bone remodeling [2]. Thus, osteoblasts can be used in the field of tissue engineering and regenerative medicine to promote bone repair and regeneration. Fetal bovine serum (FBS) is traditionally used for in vitro cell cultures, but its ethical concerns and risk of contamination have led to the use of other serum alternatives such as HPL. HPL, derived from donated human blood, offers ethical, efficient, and animal pathogen-safe cell culture. Fibrinogen in HPL may cause clotting; thus, anticoagulants like heparin are used. Fibrinogen-depleted HPL (Fd-HPL) eliminates this need. In our previous study, a calcium chloride-based protocol  
Figure 5: Osteogenic marker expression in He-HPL and Fd-HPL groups. The He-HPL group showed significantly higher expression of Runx2 and ALP compared to the Fd-HPL group. Data represent mean \(\pm\) SEM (N = 3 per group). Statistical significance was determined with *+ representing \(p\) \(<\) 0.01 and *+ representing \(p\)\(<\) 0.05. Figure 6: Concentrations of growth factors and cytokines in He-HPL and Fd-HPL. (**A**) BDNF, (**B**) VEGF, (**C**) IL-6, and (**D**) TNF-\(\alpha\). There were no notable differences in the concentrations of BDNF, VEGF, and IL-6 between He-HPL and Fd-HPL. However, Fd-HPL exhibited a decreased concentration of TNF-\(\alpha\) compared to He-HPL. Values expressed as mean \(\pm\) SEM (N = 6 per group). *+ represents \(p\)\(<\) 0.05.  
was developed to prepare Fd-HPL [14]. However, the effects of HPL with heparin (He-HPL) and Fd-HPL on human osteoblasts have yet to be elucidated. Thus, this study investigates the effects of HPL with heparin and Fd-HPL without heparin on human fetal osteoblast (hFOB) growth and phenotype. Previous studies have shown that HPL is widely utilized in stem cell cultures due to its rich growth factor content, but its high fibrinogen levels can lead to unwanted fibrin gel formation, disrupting cell expansion [11; 14]. To counteract this, heparin, an animal-derived anticoagulant, is often added to prevent clot formation by inhibiting thrombin. While heparin is commonly used to prevent fibrin gel formation in HPL due to its anticoagulant properties, it can negatively impact osteogenic differentiation. Research has shown that high doses of heparin can interfere with the mineralization process crucial for osteoblast differentiation, reducing ALP activity and calcium deposition, which are markers of osteogenesis [15; 16]. Data further indicate that heparin disrupts the CXCR4/SDF-1 signaling axis and may interfere with the migration and homing capacity of bone marrow-derived mononuclear cells [17]. Therefore, it has been suggested that heparin should be supplied at lowest possible concentrations in media containing HPL to prevent gel formation [18]. Additionally, fibrinogen, a pro-inflammatory component, can increase immune cell adhesion and negatively impact MSCs' immune-modulating abilities. Thus, alternatives like Fd-HPL, which support heparin-free culture conditions, are being explored to avoid these negative impacts while still enabling effective cell expansion. This heparin-free approach also addresses safety concerns related to animal-derived components, making it a more viable option for clinical applications [14]. The findings from the cell culture experiments on cell viability, yield, and PDT revealed no statistically significant differences between the two groups. Both He-HPL and Fd-HPL supported high cell viability, exceeding 90%, while the final cell yields and population doubling times remained comparable. These findings suggest that both He-HPL and Fd-HPL are equally effective in supporting the proliferation and maintenance of human osteoblasts. These results align with previous studies demonstrating the robustness of HPL in supporting cellular growth across various preparation methods.