## 4 Discussion
Accordingly, we selected ALP as the early stage marker and provided the staining analysis in Figure 1a and gene expression analysis in Figure 3a. Based on the previous finding, it was expected that the Runx2 level would also increase along with ALP level. Taken together, it was hypothesized that that small molecule VPA promotes the induction of osteoblasts and mineralization of calcium matrices via formation of hyper-acetylated histones through inhibition of HDAC activity (Figure 5). Meanwhile, in previous studies, ALK5i II was used as a TGF-\(\beta\) R inhibitor in direct conversion, whereby ALK5i II induced fibroblasts to exhibit osteoblast phenotypes with remarkable abilities [62]. Among various ALK5 inhibitors, ALK5i II exhibited the highest inhibition of smad2/3 signaling and induced osteoblastic conversion at the highest efficiency. Previously, it was shown that a TGF-\(\beta\) R inhibitor substituted transcription factors, such as Oct4 or Sox2 in the induction of reprogramming and maintenance of pluripotency [63; 64]. Treatment of a TGF-\(\beta\) R inhibitor resulted in an 8-fold increase in the efficiency of the direct conversion of fibroblast into cardiomyocytes induced by cardiomyocyte-specific reprogramming factors [65; 66]. A TGF-\(\beta\) R inhibitor was also used as an essential component when tested as a small molecule in the induction of direct conversion of fibroblasts into neural cells [67; 68] and cardiomyocytesocytes [69]. It is understood that osteoblasts and osteocytes produce TGF-\(\beta\), but its roles in osteogenesis is more complicated. It was demonstrated that the TGF-\(\beta\) signal enhances osteoblast differentiation at early stages, whereas at later stages of differentiation, it inhibits proliferation and mineralization of osteoblasts [70]. Other reports have revealed that TGF-\(\beta\) may either stimulate or inhibit the differentiation of osteoblasts as a function of the composition, concentration, density, and differentiation stage of the cells [71; 72]. Herein, we employed small molecule VPA, a HDAC inhibitor, in combination with a TGF-\(\beta\) R inhibitor, ALK5i II, for cell fate conversion to efficiently reprogram fibroblasts into osteoblasts. Simultaneous treatment demonstrated a synergistic effect on the direct conversion of somatic cells to osteogenic lineage. It is anticipated that the addition of other small molecules, transcription factors, or supplements may further elevate the expression levels of osteoblast-specific genes and promote the direct conversion efficiency. The generation of functional osteoblasts from human somatic cells could provide an alternative means of bone regenerative therapy for bone disorders such as osteoporosis. The process is attractive because of its advantages, that is, it is a highly efficient conversion step for the production of bone matrix and promoting bone tissue regeneration, and additionally, it is a transgene-free method. Further investigation of the VPA mechanism associated with histone acetylation and the promotion of direct reprogramming efficiency could support the findings of our study. It is anticipated that these results may be beneficial in providing a safer approach and higher reprogramming efficiency for practical use in human therapies. Figure 5: Schematic illustration of increased direct conversion efficiency of somatic cells into osteoblasts and mineralization by supplementation of VPA in osteogenic medium (O.M.). VPA promotes chromosomal remodeling and maintains acetylated histones as an “open form” of the chromatin structure by acting as an inhibitor of HDAC. **Author Contributions:** [PERSON] and [PERSON] collected data and evidence; [PERSON] and [PERSON] conceived and designed the study; [PERSON] and [PERSON] wrote the manuscript; [PERSON] and [PERSON] drew the figures; [PERSON] and [PERSON] directed and validated the data analysis. All authors have read and agreed to the published version of the manuscript.