## 4 Discussion  
The present study investigated whether the osteogenic activity of MC3T3-E1 cells could be enhanced using inclusion nanocarier of ICA and \(\beta\)-CD-ALG (ICA/\(\beta\)-CD-ALG). The particle sizes of \(\beta\)-CD-ALG with or without ICA measured by TEM and DLS ranged from approximately 430 to 480 nm in diameter. [PERSON] et al. [23] demonstrated that simvastatin (SIM)-incorporated hyaluronic acid (HA)-conjugated \(\beta\)-CD (HA-\(\beta\)-CD) led to sustained release of SIM for up to 63 days. More recently, [PERSON] et al. [24] showed that berberine chloride (BE)-loaded sulfonate-\(\beta\)-CD-chitosan self-assembly nanoparticle led to steady, controlled drug release. Consistent with these studies, we found that ICA release profiles from \(\beta\)-CD-ALG exhibited a controlled release pattern for 7 days. Previous studies reported that ICA treatments not only promoted Runx-2 (runt-related transcription factor 2) gene levels in pre-osteoblastic MC3T3-E1 cells, but also induced expression of the Id-1 (inhibitor of DNA-binding 1) gene in mouse primary osteoblasts [25]. Previous studies demonstrated that, in combination with various materials such as porous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds, calcium phosphate cement tablets, and porous \(\beta\)-tricalcium phosphate ceramic disks, ICA significantly improved cell proliferation in vitro and induced new bone formation and blood vessels in vivo [26; 27; 28]. Based on the previous studies, we evaluated the osteogenic effects of ICA/\(\beta\)-CD-ALG on MC3T3-E1 cells via ALP activity, calcium content, and gene expression. ALP, a cell membrane-related enzyme, previously showed early expression consistent with osteoblast differentiation. Alkaline phosphatase has been widely proposed as an early osteoblast differentiation marker [29; 30; 31]. Furthermore, ALP activity has been shown to be associated with matrix development in osteoblasts before mineralization [31]. The ALP levels of MC3T3-E1 cells treated with \(\beta\)-CD-ALG, ICA (1 \(\upmu\)M)/\(\beta\)-CD-ALG, ICA (5 \(\upmu\)M)/\(\beta\)-CD-ALG, and ICA (10 \(\upmu\)M)/\(\beta\)-CD-ALG were measured after days 3 and 9 of incubation. The ALP activity of MC3T3-E1 cells incubated with \(\beta\)-CD-ALG (regardless of ICA) increased in a time- and dose-dependent manner. After three days, ALP activity was not significantly different in  
Figure 8: The gene levels of (**a**) OCN and (**b**) OPN in cells treated with \(\beta\)-CD-ALG, ICA (1 \(\upmu\)M)/\(\beta\)-CD-ALG, ICA (5 \(\upmu\)M)/\(\beta\)-CD-ALG, and ICA (10 \(\upmu\)M)/\(\beta\)-CD-ALG at 7 and 21 days (n = 4). **\(p<0.01\) and * \(p<0.05\). MC3T3-E1 cells treated with bare \(\beta\)-CD-ALG or \(\beta\)-CD-ALG with ICA. However, a noticeable difference was observed in ALP levels between the MC3T3-E1 cells handled with ICA (10 \(\upmu\)M)/\(\beta\)-CD-ALG and those handled with ICA (1 \(\upmu\)M)/\(\beta\)-CD-ALG after nine days of incubation. Calcium deposition has generally been used as a late osteogenic differentiation marker [32; 33]. Thus, the amount of calcium deposited by MC3T3-E1 cells treated with \(\beta\)-CD-ALG with or without ICA was investigated after 7 or 21 days of cultivation. Calcium deposition of MC3T3-E1 cells cultivated with \(\beta\)-CD-ALG with ICA was higher than that in MC3T3-E1 cells cultivated with \(\beta\)-CD-ALG at 7 days. The deposited calcium contents were significantly different between MC3T3-E1 cells treated with ICA (10 \(\upmu\)M)/\(\beta\)-CD-ALG and ICA (1 \(\upmu\)M)/\(\beta\)-CD-ALG at 21 days. [PERSON] et al. [34] described increased ALP activity and mineralization of MC3T3-E1 cells stimulated by ICA through up-regulation of bone morphogenetic protein-2. More recently, [PERSON] et al. [35] showed that ICA promoted ALP activity and mineral content in MC3T3-E1 cells through microRNA-153 that targets the Runx2 pathway. These prior results were consistent with our results and imply that ICA promotes ALP activity and calcium deposition.