Cartilage Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells in Three-Dimensional Silica Nonwoven Fabrics

Featured Application: Silica nonwoven fabrics are materials with mechanical strength and cellular functions for potential cartilage regeneration applications. Abstract: In cartilage tissue engineering, three-dimensional (3D) scaffolds provide native extracellular matrix (ECM) environments that induce tissue ingrowth and ECM deposition for in vitro and in vivo tissue regeneration. In this report, we investigated 3D silica nonwoven fabrics (Cellbed ® ) as a scaffold for mesenchymal stem cells (MSCs) in cartilage tissue engineering applications. The unique, highly porous microstructure of 3D silica fabrics allows for immediate cell inﬁltration for tissue repair and orientation of cell–cell interaction. It is expected that the morphological similarity of silica ﬁbers to that of ﬁbrillar ECM contributes to the functionalization of cells. Human bone marrow-derived MSCs were cultured in 3D silica fabrics, and chondrogenic differentiation was induced by culture in chondrogenic differentiation medium. The characteristics of chondrogenic differentiation including cellular growth, ECM deposition of glycosaminoglycan and collagen, and gene expression were evaluated. Because of the highly interconnected network structure, stiffness, and permeability of the 3D silica fabrics, the level of chondrogenesis observed in MSCs seeded within was comparable to that observed in MSCs maintained on atelocollagen gels, which are widely used to study the chondrogenesis of MSCs in vitro and in vivo. These results indicated that 3D silica nonwoven fabrics are a promising scaffold for the regeneration of articular cartilage defects using MSCs, showing the particular importance of high elasticity. 3D silica fabrics, and atelocollagen gels, and the COL1A1 and COL10A1 mRNA expression increased in the order of 3D silica fabrics, atelocollagen gels, and spheroids. These results indicate that MSCs cultured in spheroids promoted the formation of fibrocartilage 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 atelocollagen 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 chondrodysplasia. These results support the superiority or equivalence of 3D silica fabric culture under chondrogenic differentiation condition to atelocollagen gels and spheroids, which are commodity scaffolds used as controls.


Introduction
Articular cartilage is hyaline cartilage covering the articular surface of bones. Because of its avascular characteristic, once injured, articular cartilage has poor self-repair abilities. The management microstructures were developed by electrospinning through the sol-gel process. The random orientation of the silica fibers produces many interconnected pores that may promote cellular migration and tissue ingrowth and prevent shrinkage because of the sufficient mechanical strength of the fabrics [31,32]. As a result of these characteristics, fibroblasts migrated into and penetrated the 3D silica fabrics and showed remarkable growth rates compared with those in traditional 2D culture [33][34][35]. Moreover, the functions of hepatocytes co-cultured with fibroblasts in 3D silica fabrics were significantly enhanced compared with those of 2D fibroblasts because of the abundance of fibroblast-secreted soluble factors, which are important for maintaining hepatocytes [35]. We have recently demonstrated that osteogenic differentiation was significantly promoted in 3D silica fabrics.
Silica-based hybrid materials have been widely used as implantable materials for cartilage and bone tissue regeneration. For example, Kascholke et al. developed biodegradable and adjustable sol-gel glass-based hybrid scaffolds for cartilage and bone tissue regeneration [36]. This scaffold was biodegradable, and MSCs embedded in the scaffold proliferated and displayed promoted cell function. However, this scaffold lacks molding properties, and its porosity and shrinking property are limitations. Thus, 3D silica nonwoven fabrics are useful as scaffolds for cartilage tissue regeneration because of their easy molding, ECM-like network structure, high mechanical strength, and porosity.

Preparation of Silica Nonwoven Fabrics
Tetraethoxysilane as a metal compound, ethanol as a solvent, water for hydrolysis, and 1 N hydrochloric acid as a catalyst were mixed at a molar ratio of 1:5:2:0.003 and refluxed at 78 • C for 10 h. The solvent was then removed, and the mixture was heated to form a sol solution. By using the resulting sol solution as a spinning solution, gel silica fiber webs were prepared according to a plate spinning technique, which is an electrospinning method.

Induction of Chondrogenic Differentiation of MSCs
MSCs (3 × 10 5 ) dispersed in 500 mL of D-MEM(+) were seeded on 3D silica fabrics in 12-well culture inserts (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and precultured for 1 week. Then, the cell culture medium was replaced with chondrogenic differentiation medium (PromoCell, GmbH, Heidelberg, Germany). As a control experiment, 0.5% atelocollagen gels were prepared by diluting the original 3% atelocollagen gel with normal medium. Hydrogelation was performed in a 1.5 mL sampling tube, and 50 µL of hydrogel containing cells at a density of 1 × 10 7 cells/mL was incubated at 37 • C for 3 h and then cultured with normal medium. The encapsulated cells were precultured for three days, and the cell culture medium was changed to chondrogenic differentiation medium. In addition, spheroid culture was performed in a 96-well U-bottom cell culture plate. MSCs (1.0 × 10 5 ) dispersed in 100 µL of normal medium were seeded in each well of a 96-well U-bottom plate and precultured for 3 days. The cell culture medium was then replaced with chondrogenic differentiation medium. Media were changed every 3 days and cells were further cultured for 4 weeks.

Confocal-Laser Scanning Microscopic (CLSM) Observation
Cells (3 × 10 5 ) dispersed in 500 µL of D-MEM(+) were seeded on 3D silica fabrics in 12-well culture inserts and cultured at 37 • C and precultured for 1 week. Then, the cell culture medium was replaced with chondrogenic differentiation medium, and the medium was changed every 3 days. After 2 weeks and 4 weeks of culture, CLSM (LSM-710, Carl Zeiss Co, Ltd., Oberkochen, Germany) images were obtained. Cells were treated with 5 mg/mL Hoechst 33342 (Dojindo laboratories, Kumamoto, Japan) in D-MEM(+) for 1 h in the dark. Then, the cells were washed three times with phosphate-buffered saline (PBS) and fixed with 4 wt% paraformaldehyde solution in PBS for 20 min followed by permeation with 0.5% Triton X-100 solution for 2 min. Cells were treated with 1% Alexa Fluor 594 phalloidin (Life Technologies, Corp., Carlsbad, CA, USA) for 2 h. After washing with PBS three times, cells were observed by CLSM.

Cell Number Analysis
After 4 weeks of culture, cells in 3D silica fabrics were evaluated using a hemocytometer. Briefly, the 3D silica fabrics were exposed in 0.5% trypsin-EDTA for 5 min, pipetted for some time, and washed with PBS. The typsinized solution was collected, and cell number was counted using a hemocytometer. Data are shown as mean ± standard error of the mean of triplicates.

Quantification of Glycosaminoglycan and Collagen Content
The production of sulfate glycosaminoglycan (GAG) was determined by the modified dimethyl-methylene blue (DMMB) method [38]. The cultured sample was washed three times with PBS and digested individually in 2 N NaOH at 60 • C for 24 h. The digested sample was stored at −20 • C until further analysis. To determine GAG content, the digested sample was assayed by DMMB dye, and the absorbance was monitored at 570 nm with a microplate reader. The GAG content was extrapolated from a standard curve using shark chondroitin sulfate (Sigma-Aldrich Japan Co., Tokyo, Japan). Three parallels were averaged for each specimen.
Total collagen content was estimated by measuring the hydroxyproline content according to a previous report [39]. The collected sample was hydrolyzed in 5 N HCl at 110 • C for 24 h. The hydroxyproline content of hydrolysate was determined using chloramine-T/Ehrlich's reagent assay, and the color change was quantified spectrophotometrically at 560 nm. The standard curve was generated with l-hydroxyproline, and a conversion factor of 10 was used to convert from hydroxyproline to total collagen content [40].

Proliferation and Morphology of Chondrogenic MSCs Cultured in 3D Silica Fabrics
The proliferation and morphology of chondrogenic-differentiated MSCs cultured in 3D silica fabrics were observed using CLSM ( Figure 1). CLSM images of MSCs in silica fabrics cultured with normal medium (Diff (−)) and chondrogenic differentiation medium (Diff (+)) after 2 weeks and 4 weeks in culture are shown in Figure 1. After 2 weeks of culture, MSCs were detected at a depth of approximately 48 μm under both culture conditions, and no difference was observed in terms of MSC morphology. After 4 weeks of culture, differentiated MSCs but not normal MSCs were detected at a depth of approximately 73 μm. Moreover, normal MSCs were crowded on the 3D silica fabric surface. These results suggested that differentiated cells cultured in Diff (+) preferentially migrated vertically in 3D silica fabrics without proliferation. These proliferation results are related to cell number.

Quantification of GAG and Collagen Content
To evaluate the biochemical functionality of these scaffolds, chondrogenic differentiation behavior was investigated in terms of the accumulation of glycosaminoglycan (GAG) and collagen (COL) in the scaffold. Atelocollagen gels and spheroids cultured in 96-well plates, which are commodity scaffolds, were used as controls. Figure 3 shows the accumulation of GAG after 4 weeks of culture in atelocollagen gels, spheroids, and 3D silica fabrics under normal and differentiation conditions. GAG is one of the chondrogenic factors in cartilages. In spite of cultivation in both atelocollagen gel and spheroid culture under normal and differentiation conditions, no difference in accumulation was observed in either condition. Although accumulation in the 3D silica fabrics was inferior to those in the other scaffolds, accumulation under differentiation condition was significantly superior to that under normal condition.

Quantification of GAG and Collagen Content
To evaluate the biochemical functionality of these scaffolds, chondrogenic differentiation behavior was investigated in terms of the accumulation of glycosaminoglycan (GAG) and collagen (COL) in the scaffold. Atelocollagen gels and spheroids cultured in 96-well plates, which are commodity scaffolds, were used as controls. Figure 3 shows the accumulation of GAG after 4 weeks of culture in atelocollagen gels, spheroids, and 3D silica fabrics under normal and differentiation conditions. GAG is one of the chondrogenic factors in cartilages. In spite of cultivation in both atelocollagen gel and spheroid culture under normal and differentiation conditions, no difference in accumulation was observed in either condition. Although accumulation in the 3D silica fabrics was inferior to those in the other scaffolds, accumulation under differentiation condition was significantly superior to that under normal condition.

Quantification of GAG and Collagen Content
To evaluate the biochemical functionality of these scaffolds, chondrogenic differentiation behavior was investigated in terms of the accumulation of glycosaminoglycan (GAG) and collagen (COL) in the scaffold. Atelocollagen gels and spheroids cultured in 96-well plates, which are commodity scaffolds, were used as controls. Figure 3 shows the accumulation of GAG after 4 weeks of culture in atelocollagen gels, spheroids, and 3D silica fabrics under normal and differentiation conditions. GAG is one of the chondrogenic factors in cartilages. In spite of cultivation in both atelocollagen gel and spheroid culture under normal and differentiation conditions, no difference in accumulation was observed in either condition. Although accumulation in the 3D silica fabrics was inferior to those in the other scaffolds, accumulation under differentiation condition was significantly superior to that under normal condition.  Figure 4 shows the accumulation of COL after 4 weeks of culture in 3D silica fabrics, atelocollagen gels, and spheroids under normal and differentiation conditions. Here, because atelocollagen itself was detected by the hydroxyproline assay, COL accumulation could not be accurately quantified. The accumulation of COL under differentiation condition was superior to that under normal condition in both spheroid and 3D silica fabric culture. However, COL accumulation in 3D silica fabrics was inferior to that in spheroid culture under differentiation condition. Figure 3. Accumulation of glycosaminoglycan (GAG) in normal medium (Diff (−)) and chondrogenic differentiation medium (Diff (+)) on 3D silica fibers, atelocollagen gels, and spheroids for 4 weeks. Figure 4 shows the accumulation of COL after 4 weeks of culture in 3D silica fabrics, atelocollagen gels, and spheroids under normal and differentiation conditions. Here, because atelocollagen itself was detected by the hydroxyproline assay, COL accumulation could not be accurately quantified. The accumulation of COL under differentiation condition was superior to that under normal condition in both spheroid and 3D silica fabric culture. However, COL accumulation in 3D silica fabrics was inferior to that in spheroid culture under differentiation condition.

Expression of Chondrogenic Differentiation Marker Genes
To evaluate chondrogenic behavior in 3D silica fabrics in more detail, the expression of chondrogenic differentiation marker genes, COL1A1, COL2A1, COL10A1, ACAN, and SOX9, was investigated by qRT-PCR ( Figure 5). For almost all the genes, differences in expression were preferentially observed between normal and differentiation conditions. Under differentiation condition, the expression of COL2A1 in atelocollagen and 3D silica fabric culture was superior to that in spheroid culture, but the expression of ACAN was the most elevated in spheroid culture. The expression of SOX9 was the same in all scaffolds. The expression levels of COL1A1 and COL10A1 in spheroid culture were superior to those in the other scaffolds, and they were the lowest in 3D silica fabric culture. Chondrogenic differentiation of MSCs in each scaffold was also evaluated by assessing GAG and COL accumulation. These results suggested that 3D silica fabric culture was useful in the development of tissue-engineered constructs, particularly for cartilage formation, and that it is equivalent or superior to atelocollagen gel culture as a commodity scaffold.

Expression of Chondrogenic Differentiation Marker Genes
To evaluate chondrogenic behavior in 3D silica fabrics in more detail, the expression of chondrogenic differentiation marker genes, COL1A1, COL2A1, COL10A1, ACAN, and SOX9, was investigated by qRT-PCR ( Figure 5). For almost all the genes, differences in expression were preferentially observed between normal and differentiation conditions. Under differentiation condition, the expression of COL2A1 in atelocollagen and 3D silica fabric culture was superior to that in spheroid culture, but the expression of ACAN was the most elevated in spheroid culture. The expression of SOX9 was the same in all scaffolds. The expression levels of COL1A1 and COL10A1 in spheroid culture were superior to those in the other scaffolds, and they were the lowest in 3D silica fabric culture. Chondrogenic differentiation of MSCs in each scaffold was also evaluated by assessing GAG and COL accumulation. These results suggested that 3D silica fabric culture was useful in the development of tissue-engineered constructs, particularly for cartilage formation, and that it is equivalent or superior to atelocollagen gel culture as a commodity scaffold. Appl. Sci. 2018, 8, x FOR PEER REVIEW 8 of 14 Figure 5. The expression of COL1A1, COL2A1, COL10A1, ACAN, and SOX9 in mesenchymal stem cells (MSCs) cultured on 3D silica fibers in normal medium (Diff (−)) and chondrogenic differentiation medium (Diff (+)). Signal intensity was normalized using that of a control housekeeping gene (human GAPDH gene).

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, Figure 5. The expression of COL1A1, COL2A1, COL10A1, ACAN, and SOX9 in mesenchymal stem cells (MSCs) cultured on 3D silica fibers in normal medium (Diff (−)) and chondrogenic differentiation medium (Diff (+)). Signal intensity was normalized using that of a control housekeeping gene (human GAPDH gene).

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 ± 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-β, ascorbic acid, and dexamethasone was controlled compared with that in normal condition. TGF-β activates TGF-β receptor II on the cell surface and then binds with TGF-β receptor I, inducing the combination of TGF-β receptor I and II into a heteromeric complex and activating the downstream Smad pathway [46]. In addition, TGF-β is known to control the increase in cell number, such as TGF-β, 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-β 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. SOX9 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 RUNX2 and COL10A1 [51]. 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 fibrocartilage formation. The gene expression of chondrogenic 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 atelocollagen gels, and the COL1A1 and COL10A1 mRNA expression increased in the order of 3D silica fabrics, atelocollagen gels, and spheroids. These results indicate that MSCs cultured in spheroids promoted the formation of fibrocartilage 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 atelocollagen 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 chondrodysplasia. These results support the superiority or equivalence of 3D silica fabric culture under chondrogenic differentiation condition to atelocollagen 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 (ε-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 atelocollagen gels. Therefore, these features contributed to the promotion of chondrogenic differentiation in MSCs. Furthermore, atelocollagen 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 atelocollagen 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]. Several studies on engineered cartilage constructs have reported mechanical properties at the kPa range, which are not nearly strong enough for physiological loading. Indeed, we have demonstrated that the compressive strength of 3D silica fabrics is 0.2 MPa, which is not stiff enough for use in tissue repair. Since the seeded cells are able to produce ample biological matrix, reasonable scaffold designs should contain more optimized geometry, thereby rendering mechanical properties closer to that of native tissues. The present study shows the extent to which cells seeded on large pore constructs can produce biochemical components while maintaining strong mechanical properties. Even after the scaffolds biodegrade at a later time point, this, theoretically, will allow engineered tissues to withstand high loads immediately after implantation.
We previously demonstrated the high permeability of 3D silica fabrics in culture inserts, which were used for biological investigation of heterotypic cell-cell interaction and co-culture [34]. In fact, it is also suggested that paracrine factors secreted from non-parenchymal cells placed at the top insert were permeated and had enhanced hepatocyte functions in co-culture systems. Therefore, the excellent permeability of 3D silica fabrics seems to be effective for chondrogenesis of stem cells therein. While we only examined chondrogenic differentiation, 3D silica fabrics can be a material that finds advantages in terms of both strength and differentiation, although the performance is inferior to those of spheroids and atelocollagen as highly functional scaffolds.

Conclusions
In this study, chondrogenic differentiation of bone marrow-derived MSCs in 3D silica fabrics was investigated. MSCs under chondrogenic differentiation condition migrated and proliferated inside the 3D silica fabrics. Compared with conventional spheroid and atelocollagen gel culture, the 3D silica fabrics strongly promoted the chondrogenic differentiation of MSCs. These results suggested that 3D silica fabrics are promising scaffolds for the regeneration of cartilage defects using MSCs. This bio-glass based scaffold composed of oxides including SiO 2 , Na 2 O, CaO, and P 2 O 5 has been demonstrated as clinical application use such as bone and cartilage tissue regeneration [59,60] so the application of designed 3D silica fabrics will be increasing in tissue engineering.