## 4 Discussion
Real-time PCR analysis revealed significantly higher expression of Runx2 and ALP in He-HPL compared to Fd-HPL and no significant differences in the mRNA expression of other osteogenic markers, including OPN, BSP, Col 1 and OCN. The increase in the mRNA expressions of ALP, an early marker of osteoblast differentiation [23], and Runx2, a master regulator of osteoblast differentiation [24] demonstrated that He-HPL might have more potent osteogenic differentiation potential compared to Fd-HPL. These discrepancies might be due to the presence of heparin, which has been reported to promote osteogenic differentiation of mesenchymal stem cells via the induction of BMP and WNT pathways [25; 26]. Additionally, Fd-HPL has a high calcium level that has been reported to suppress osteogenic gene expression [14; 27]. These findings suggest that He-HPL is more effective in maintaining the osteogenic markers of hFOB cells. There was a lack of significant differences in calcium deposition between hFOB cells cultured with He-HPL and those cultured with Fd-HPL. Despite the reduction in fibrinogen content, both He-HPL and Fd-HPL were equally capable of supporting calcium deposition, suggesting that fibrinogen depletion does not impair the ability of HPL to maintain the osteogenic potential of hFOB. This finding indicated that differences in the fibrinogen and calcium concentration in both forms of HPL did not influence the osteogenic potential of hFOB. In this study, we measured the concentration of four growth factors and cytokines, i.e., VEGF, BDNF, TNF-\(\alpha\), and IL-6. Only the concentration of TNF-\(\alpha\) dropped significantly after the depletion of fibrinogen, while the concentration of other growth factors and cytokines are maintained. When fibrinogen is depleted, TNF-\(\alpha\) may be reduced due to the loss of this stabilization effect and be removed together with fibrinogen, leading to its lower concentration in Fd-HPL [28]. Both types of HPL promoted osteoblast mineral deposition, indicating both are equally potent for the expansion of HPL in vitro without causing changes in cell phenotype. Coupled with consistency in cell growth and morphology, all these findings indicated that both types of HPL are rich in nutrients required for the in vitro expansion of osteoblasts. Despite providing valuable insights into the effects of He-HPL and Fd-HPL on osteoblast growth and osteogenic differentiation, this study has certain limitations. Firstly, the results are based on hFOB, which may not fully represent the complexities of primary osteoblasts or osteogenesis in vivo. Additionally, while the study focused on gene expression as a marker of osteogenic potential, protein-level validation through techniques like Western blot or immunofluorescence was not performed, which could have provided a more complete picture of the osteogenic process. Furthermore, the study did not explore the metabolic effects or other functional consequences of using these platelet lysates, which could offer additional insights into their potential applications. Finally, the use of He-HPL and Fd-HPL, while promising, requires further investigation, particularly in clinical or in vivo models, to better understand their long-term effects and safety profiles. These limitations suggest that future studies should include more comprehensive assays and in vivo models to validate these findings. As a key limitation, future studies will also include FBS controls to directly compare its effects alongside He-HPL and Fd-HPL, enabling a clearer understanding of how HPL compares to FBS in osteoblast proliferation, differentiation, and overall functionality. Future studies could explore additional methods to assess osteoblast proliferation and differentiation, such as incorporating proliferation assays like the BrdU incorporation assay, MTT/MTS assay, or Ki-67 staining. These assays would provide more comprehensive insights into the effects of He-HPL and Fd-HPL on osteoblast proliferation, complementing the current findings from cell number counting.