## 3 Results
### Cytokine Profiling of hUC-MSCs Treated with Various Concentrations of HA  
A multitude of cytokines and chemokines secreted by hUC-MSCs are known to have critical roles in biological processes such as immunomododulation, vascular stabilization, angiogenesis and tissue repairs. We profiled the cytokine expression of the supernatant from hUC-MSCs treated with or without 0.1% HA for 48 h. Based on Figure 2A,B, the cytokine secretion patterns of hUC-MSCs and hUC-MSCs treated with 0.1% HA were similar. Twenty-two out of 105 cytokines or chemokines were detected in both groups. Among these factors, eight are known for their roles in cell growth and proliferation, such as hepatocyte growth factor (HGF) and growth differentiation factor-15 (GDF-15); twelve for immunomododulation, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and Serpin Family E Member 1 (Serpin E1) and two for anti-apoptosis, such as urokinase-type plasminogen activator receptor (uPAR) (Figure 2C). Figure 1: Cell viability of hUC-MSCs (human umbilical cord mesenchymal stem cells) in treatments by different concentrations of HA (hyaluronan acid). hUC-MSCs were exposed to varying concentrations of HA for 48 h, and the cell viability was measured by trypan blue staining. The data showed the mean \(\pm\) SD (standard deviation) of three independent experiments. Figure 2: _Cont._  
However, it was noted that, in the 0.1% HA-treated group, the expression of Chitinase-3-like protein 1 (CHI3L1), DKK-1, HGF, interleukin-8 (IL-8), interleukin-17A (IL-17A), Osteopontin (OPN) and uPAR were elevated higher than 5% (Figure 2C). Among the upregulated protein, DKK-1 and HGF were known to ameliorate osteoarthritis (OA) pathogenesis. DKK-1 exerts a protective effect on its capacity to inhibit the Wnt-mediated expression of catabolic factors, such as matrix metalloproteinase 13 (MMP-13), and inhibit OA cartilage destruction [18; 19]. It has been noted that HGF stimulates the proliferation, motility and proteoglycan synthesis of chondrocytes and is involved in the repair mechanism of matrix damage [12; 20; 21]. Taken together, DKK-1 and HGF might serve as therapeutic targets in osteoarthritis. The results from the single cytokine array experiment showed that the expression of DKK-1 and HGF were elevated in hUC-MSC treatments with 0.1% HA for 48 h (Figure 2C). To investigate whether the cytokine secretion of the hUC-MSCs was dependent on the concentration of HA, we tested six different supernatants from the culture of hUC-MSCs with 0, 0.025, 0.05, 0.1, 0.2 and 0.5% of HA. Next, the amount of DKK-1 and HGF were quantified by a specific ELISA to validate our single cytokine array result. Our data demonstrated that HA treatment significantly induced the expression of DKK-1 secreted by hUC-MSCs. DKK-1 expression was increased in the 0.1%, 0.2% and 0.5% HA groups but not in the 0.05% and 0.025% HA groups. Interestingly, the secretion of DKK-1 was elevated in a dose-dependent manner in the 0.1%, 0.2% and 0.5% HA groups (Figure 2D). Subsequently, we sought to examine the expression of HGF under HA treatment. It could be noted that the expression of HGF was only increased in the 0.1% HA group (Figure 2E). These results were similar to the cytokine array results, in which hUC-MSCs cultured the 0.1% HA condition increased the expression of DKK-1 and HGF. Notably, both cytokines have been shown to regulate the  
Figure 2: Effects of HA on the hUC-MSC cytokine profile. Representative images of the cytokine array analysis performed with supernatants of hUC-MSCs previously incubated with MEM\(\alpha\) (control) (**A**) or MEM\(\alpha\) with 0.1% HA (**B**) for 48 h. (**C**) Densistometric analysis of the cytokine patterns on the membrane (**A,B**). Positive controls were framed in black boxes. Data were normalized after background subtraction.