## 4 Discussion  
In the present study, chondrogenic differentiation of human bone marrow-derived MSCs in 3D silica fabrics was investigated. For the chondrogenic differentiation of MSCs, we utilized 3D silica fabrics as previously reported, as it is a reasonable scaffold for tissue engineering [34; 35; 36; 38]. Briefly, 3D silica fabrics were developed via the sol-gel process to create highly interconnected porous microstructures of electrospun fabrics for cell growth. 3D silica fabrics have a high porosity of approximately 95% and an average pore size of 7.6 m (n = 5). The diameter of the fibers constituting the 3D silica fabrics was 704 \(\pm\) 280 nm (n = 50). The random orientation of the silica fibers produced many interconnected pores that may promote cellular infiltration and tissue ingrowth while preventing shrinkage and contraction during cell culture due to the inherent mechanical strength. The thickness of the MSC layers in the 3D silica fabrics under normal and differentiation conditions was dependent on the culture, and MSCs under normal culture were laminated compared with those in differentiation condition. These results indicate that DNA synthesis under normal condition was promoted in 3D silica fabrics. We have previously demonstrated that fibroblasts cultured in 3D silica fabrics showed remarkable growth rates compared with those in traditional 2D culture [34]. The results in this study suggested that MSCs migrated vertically and proliferated effectively in 3D environments without receiving signals of contact inhibition. Moreover, the MSC layers in silica fabrics under differentiation condition were thicker, which indicated that the cell attachment region in 3D silica fabrics was limited by the accumulation of ECM components, such as aggrecan and collagen. This result also supported the vertical migration of MSCs under chondrogenic condition and formation of 3D cellular layers in 3D silica fabrics. The number of cells in 3D silica fabrics under differentiation condition stimulated by medium containing chondrogenic factors, TGF-\(\beta\), ascorbic acid, and dexamethasone was controlled compared with that in normal condition. TGF-\(\beta\) activates TGF-\(\beta\) receptor II on the cell surface and then binds with TGF-\(\beta\) receptor I, inducing the combination of TGF-\(\beta\) receptor I and II into a heteromeric complex and activating the downstream Smad pathway [46]. In addition, TGF-\(\beta\) is known to control the increase in cell number, such as TGF-\(\beta\), feedback and aggravate the production of ECM proteins, such as fibronectin, aggrecan, and collagen [47; 48; 49]. Thus, the number of cells in 3D silica fabrics under differentiation condition is inferior to that under normal condition, which strongly indicates that TGF-\(\beta\) works in 3D silica fabrics without deactivation. These results are highly correlated to the CLSM images. In chondrogenic differentiation, the accumulation of GAG and COL in 3D silica fabrics was increased compared with that in normal condition. GAG production is one of the key early indicators of a chondrocyte-like phenotype. Native cartilage comprises primarily sulfated GAG chains and type II collagen [50]. Thus, effective accumulation in 3D silica fabrics is a promising result for cartilage tissue engineering. Finally, we investigated chondrogenic behavior in 3D silica fabrics in detail. _SOX9_ is an early-stage marker and a major regulator of chondrogenesis, and it was expressed significantly at the mRNA level in MSCs under differentiation condition in each scaffold. _SOX5_ is required for _SOX5_ and _SOX6_ expression and, together, _SOX5_, _SOX6_, and _SOX9_ (the SOX trio) coordinately regulate the expression of several cartilage matrix genes including _ACAN_, _COL2A1_, and _COL1A1_[50]. During endochondral bone formation, _SOX9_ expression is downregulated in growth plate chondrocytes as they undergo maturation and begin to express markers of hypertrophy, such as _RINX2_ and _COL10A1_[51].