## 4 Discussion
In articular cartilage, _SOX9_ is crucial for the maintenance of the ECM as chondrocyte-specific postnatal deletion of _SOX9_ results in reduced matrix proteoglycan content probably as a result of increased _ADAMTS5_ expression [52]. Thus, each scaffold in the study is useful for cartilage tissue formation. Moreover, as mentioned above, _ACAN_ and _COL1A1_ were used as markers of chondrogenesis with _COL1A1_ being a marker of fibro cartilage formation. The gene expression of chondogenic markers _COL2A1_, _ACAN_, and _SOX9_ indicates that each scaffold significantly enhanced chondrogenic differentiation of MSCs under differentiation condition. _COL2A1_ mRNA expression increased in the order of spheroids,3D silica fabrics, and atelocolagen gels, and the _COL1A1_ and _COL10A1_ mRNA expression increased in the order of 3D silica fabrics, atelocolagen gels, and spheroids. These results indicate that MSCs cultured in spheroids promoted the formation of fibro cartilage or hypertrophic cartilage and, as a result, the accumulation of COL in spheroids was greater than that in 3D silica fabrics. Furthermore, _COL10A1_ mRNA in atelocolagen gels was overexpressed compared with that in 3D silica fabrics. _COL10A1_ is the only known hypertrophic chondrocyte-specific molecular marker. Mutations in _COL10A1_ in humans have been associated with Schmid metaphyseal chondodysplasia. These results support the superiority or equivalence of 3D silica fabric culture under chondrogenic differentiation condition to atelocolagen gels and spheroids, which are commodity scaffolds used as controls. We have already demonstrated that 3D silica fabrics promoted the osteogenic differentiation of MSCs. In bone-cartilage regeneration, hardness and absorbability of the scaffold are particularly important factors [53; 54]. Previously, we concluded that osteogenic differentiation was strongly promoted in 3D silica fabrics because of the high rigidness, porosity, and permeability of the scaffold compared with those of 2D polystyrene dish. Thus, in this report, we supposed that chondrogenic differentiation in 3D silica fabrics was promoted because of these factors. Commonly, chondrogenic differentiation of MSCs has been investigated by utilizing 2D or 3D scaffolds, such as nano and microfibrous or porous scaffolds including poly (\(\epsilon\)-caprolactone) and poly (l-lactide), which have comparable stiffnesses to that of native cartilage [54; 55]. Aside from soluble factors, factors affecting chondrogenic differentiation of MSCs are classified into two categories. The first is cell-cell and the second, cell-ECM interactions. The 3D nanofibrous structures of the silica fabrics share morphological similarities to collagen fibrils and enable favorable biological responses to be promoted for chondrogenic differentiation. Furthermore, the high interconnectivity of 3D silica fabrics allows cells to interact with surrounding cells. The latter factor is the elasticity of substrates. Chondrogenic differentiation of MSCs was strongly affected by elastic substrates and rigid matrices [56; 57]. The 3D silica fabrics are highly interconnected network structures and rigid substrates compared with atelocolagen gels. Therefore, these features contributed to the promotion of chondrogenic differentiation in MSCs. Furthermore, atelocolagen gels are animal-derived scaffold and have its shrinking property, so the clinical application is limited. Thus, the application of 3D silica fabrics that replace atelocolagen gels is expected. On the other hand, the primary goal of cartilage tissue engineering is to regenerate neo-tissues that have similar biomechanical functions as those of native tissue. Chitosan, hyaluronic acid, and collagen provide suitable environments for chondrocyte and matrix deposition. However, the mechanical properties of the engineered tissue are not strong enough to endure physiological loading upon clinical implantation. Ideally, cartilage constructs should show compressive stiffness of 0.5-6.0 MPa [58].