## 4 Discussion  
This research examined the effects of CTGF on cellular viability, osteogenic differentiation, and mRNA expression of stem cell spheroids. The effects of CTGF on various models have been reported previously [8; 21; 22]. The present study showed no significant difference in cellular viability between the groups. However, in one study, the effects of recombinant CTCF on osteoblast proliferation were evaluated using MTT and BrdU assays (a colorimetric assay used to quantify cell proliferation) on Day 3 after adding different concentrations (1, 10, 50, and 100 ng/mL) of CTCF to rat osteoblasts [8]. The results showed dose-dependent increases in cell number and proliferation. ALP activity is considered to be a relative early marker of osteoblast differentiation [23; 24]. In a previous report, the maximal ALP activity of mesenchymal stem cells was achieved at day 7 [25]. This study showed that the application of CTGF led to significant increase in the 200 ng/mL group on Day 1. The previous report evaluated the effects of recombinant CTGF on ALP activity in animal osteoblasts after applying 50 ng/mL for 48 h, resulting in significantly increased activity [8]. Similarly, this study showed a statistically significant increase in Alizarin Red S staining values at 25, 100, and 200 ng/mL. In a previous report, cultures treated with 100 ng/mL recombinant CTCF exhibited a significant increase in calcium deposits on Day 11 [8]. GMSCs showed positive marker expression greater than 90% for CD29, CD44, CD73, CD90, and CD105 at passage 13 and expression greater than 85% at passage 16 [26]. Moreover, GMSCs have the ability to undergo multilineage differentiation when specific culture conditions are used in the third to eighth passages. The differences in cellular viability may be due to the type of culture, type of cells, or culture period [21; 22]. Passage number may also influence the results [27]. Various attempts have been made to evaluate or suggest the optimal concentration of CTCF [28; 29]. One study found CTGF in 10% of basal tears, with a maximum level of 17 ng/mL [30]. CTGF was detected in the subretinal fluid of all research cases at a mean concentration of 10.1 \(\pm\) 3.68 ng/mL [28]. Another study evaluated the effects of CTGF on type I collagen transcription in quiescent hepatic stellate cells at concentrations of 10, 50, and 100 ng/mL for 16 and 48 h [29]. Incubation for 16 h did not produce any notable difference, but significant increases in collagen transcription were seen in the 50 and 100 ng/mL CTCF groups after 48 h. In the present study, the highest values were seen at 200 ng/mL for alkaline phosphatase activity and Alizarin Red S staining. qPCR results showed the highest expression of RUNX2, BSP, OCN, and COL1A1 at 100 ng/mL group. Our study shows some similarity pattern of previous report, which evaluated osteogenic capacity of elderly osteoblasts in three-dimensional cultivation. It showed that expression of OCN was in high level on Day 0, which went down and rose again [31]. Expression levels of various genes were tested to evaluate osteogenic capacity, including those of RUNX2, BSP, OCN, and COL1A1 [1; 18]. RUNX2 is a widely used biomarker for osteogenic differentiation and is considered an important transcription factor for osteoblasts [32]. BSP is highly expressed in bone and is reported to play a functional role in bone formation [33]. OCN is reported to be a bone-specific marker and is associated with the maturation of osteogenesis [34]. The expression of OCN is indicator of maturation of extracellular matrix [35]. COL1A1 is considered an early marker of osteoblasts, and increased COL1A1 expression was seen with the transition of osteoprogenitor cells to pre-osteoblasts [36]. Moreover, early expression of the COL1A1 is indicates an early stage of maturation [31]. In a previous report, CTCF treatment was found to lead to significant enhancement in the synthesis of collagen I [37].