## 3 Results
### Gene Expression of Osteogenic Markers in HDFs Supplemented with VPA  
HDFs were cultured in osteogenic medium supplemented with VPA for 16 or 24 days, and analyzed for the expression of osteogenic markers (Figure 3). As the concentration of VPA increased to 2 mM, there was an increase in the mRNA level of alkaline phosphatase (ALP), an early osteogenic marker, at Day 16 (Figure 3a). Similarly, when the concentration of VPA was increased to 2 mM, an increase in the mRNA level of osteopontin (OPN) and bone sialoprotein (BSP), both of which are late osteogenic markers, was observed at Day 24 (Figure 3b,c). The results demonstrate that VPA induces all early and late osteogenic differentiation-related gene (ALP, OPN, BSP) expression patterns during direct conversion of HDFs cells in vitro. Co-Treatment of VPA and Specific Inhibitor for Activin-Like Kinase 5 (ALK5i II) Enhances Osteoblast-Like Phenotypes of HDFs  
To further enhance the efficiency of the direct conversion, ALK5i II, a TGF-\(\beta\) R inhibitor was co-supplemented with VPA. HDFs were cultured in osteogenic medium co-supplemented with VPA and ALK5i II for 24 days, and stained with ARS to observe the calcium deposition efficiency (Figure 4a). VPA alone at 2 mM concentration induced relatively large amounts of calcium deposition, whereas VPA alone at 1 mM induced little calcium deposition, similar to the osteogenic medium alone. However, when ALK5i II and VPA were used for co-treatment, synergistic effects were shown on the direct conversion into osteoblasts. Treatments with 1 mM VPA and 100 nM ALK5i II induced relatively large amounts of calcium deposition that were similar to the 2 mM VPA alone. Figure 1: Valproic acid (VPA) promotes osteogenesis via inhibition of histone deacetylase (HDAC). (**a–c**) Human dermal fibroblasts (HDFs) were cultured in the osteogenic medium supplemented with VPA as indicated. (**a**) After culturing for 16 days, cells were stained with alkaline phosphatase (ALP). Scale bar, 200 μm. (**b**) After culturing for 24 days, calcium deposition was assessed by Alizarin Red S (ARS) staining. Scale bar, 100 μm. (**c**) Quantified ARS fold change after the treatment of VPA. Error bars indicate the standard deviation (\(n\) = 3 per group) and statistical significance was determined using Welch’s ANOVA with a Games-Howell post hoc test in comparison to the non-treated control, * \(p\) < 0.05, *** \(p\) < 0.005. Figure 3: Gene expression of osteogenic markers in HDFs supplemented with VPA. (**a**–**c**) Quantitative real time-PCR was used to determine mRNA levels of (**a**) alkaline phosphatase (ALP) on Day 16, (**b**) osteeopontin (OPN), (**c**) bone sialoprotein (BSP) on Day 24 after HDFs were treated with the osteogenic medium supplemented with VPA as indicated. Error bars indicate the standard deviation (\(n=3\) per group) and statistical significance was determined using one-way ANOVA with [PERSON]’s post hoc test in comparison to the non-treated control, * \(p<0.05\), ** \(p<0.01\), *** \(p<0.005\). Figure 2: Cytotoxicity of VPA on HDFs. (**a,b**) HDFs were cultured in the osteogenic medium supplemented with VPA as indicated. (**a**) Morphologies were observed in cells treated with VPA for 72 h. The concentration of VPA is indicated in the upper left of each image. Scale bar, 200 \(\upmu\)m. (**b**) Cell viability was measured by the MTT assay after VPA treatment for 24, 48 h, or 72 h, respectively. Error bars indicate the standard deviation (\(n=3\) per group) and statistical significance was determined using one-way ANOVA with [PERSON]’s post hoc test in comparison to the non-treated control, * \(p<0.05\), ** \(p<0.01\), n.s. = not significant. (**c**) Cell viability was measured by the MTT assay after VPA treatment in complete media or osteogenic media for 16 days, respectively. Error bars indicate the standard deviation (\(n=3\) per group) and statistical significance was determined using one-way ANOVA with [PERSON]’s post hoc test in comparison to the non-treated control, *** \(p<0.005\).