Cartilage tissue engineering with silk scaffolds and human articular chondrocytes
Introduction
Adult articular cartilage is an avascular tissue with limited self-repair capacity. Autologous cell-based cartilage tissue engineering provides a promising option for the repair of severe cartilage damages caused by trauma or aging-related degeneration such as osteoarthritis. Successful cartilage tissue engineering requires cells capable of undergoing chondrogenic differentiation upon treatment with appropriate biochemical factors and a three-dimensional (3-D) porous scaffold capable of providing a favorable environment for chondrogenic cell growth and new cartilage-specific ECM formation. Developmentally, skeletal cells [1], [2], [3], [4], [5] like osteoblasts and chondrocytes are derived from a common cell source, mesenchymal stem cells (MSCs) [6]. MSCs provide an attractive cell source for cartilage tissue engineering in vitro and in vivo [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21].
Another major cell source used to generate engineered cartilage tissue is chondrocytes, commonly isolated from articular cartilage tissues [22], [23]. Freshly isolated MSCs and chondrocytes are normally present in limited numbers. The cells have to be expanded in vitro in order to obtain enough cells for cartilage tissue engineering applications or for related cell therapeutic strategies. The expansion of MSCs can be achieved by culturing the cells in the presence of basic fibroblast growth factor (bFGF) without significantly losing their multi-lineage differentiation potential [24], [25]. In contrast, the in vitro expansion of freshly isolated chondrocytes in monolayer culture tends to induce their dedifferentiation, even in the presence of chondrogenic factors like transforming growth factor β (TGF-β1) [26], [27], [28]. These dedifferentiated chondrocytes lose the spherical morphology that chondrocytes possess in the physiological environment and acquire a fibroblast-like morphology [26], [29]. The dedifferentiation of chondrocytes during expansion in monolayer culture is also characterized by a dramatic decrease in the synthesis of cartilage-specific ECM molecules like collagen type II (Col-II) and aggrecan (AGC) [26], [29]. These dedifferentiated chondrocytes can be induced to redifferentiate by culturing the cells in a 3-D environment favoring a spherical morphology and the synthesis of cartilage-specific ECM [26], [30], [31]. Culture-expanded autologous articular chondrocytes have been used to treat full-thickness cartilage defects [22], [23]. Culture-expanded chondrocytes are not as multipotent as MSCs; therefore, they are less likely to generate unwanted cell types when used for the repair of severely damaged cartilage tissue.
Synthetic and natural polymers are the primary scaffold materials used to create the 3-D environment for tissue engineering applications [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. Silks as naturally occurring degradable fibrous proteins with unique mechanical properties, excellent biocompatibility and processability have been identified as a suitable scaffold material for skeletal tissue engineering [1], [2], [3], [4], [5], [32], [33], [34]. Silk fibroin hydrogel-derived sponge has been combined with freshly isolated rabbit chondrocytes for in vitro cartilage tissue engineering [32], [33], [34]. The remaining questions with these reports are (a) whether these sponges will be able to support the differentiation of culture-expanded chondrocytes, as freshly isolated chondrocytes are often in limited numbers and quickly dedifferentiate during in vitro expansion; (b) whether sufficient cell condensation and cell–cell interaction needed for chondrogenic differentiation can be achieved in the rather irregular, small pore spaces (∼80 μm); and (c) whether cartilage-like tissues with more uniform extracellular matrix deposition can be regenerated by overcoming the mass transfer constraints in the rather small pores in the spongy scaffolds. Engineered 3-D porous silk fibroin scaffolds with larger pore size (500–700 μm) have been derived from a process involving the use of hexafluoroisopropanol (HFIP) as an organic solvent and used for osteogenesis and chondrogenesis of bone marrow-derived MSCs [35], [36], [37]. Recently, we developed an all-aqueous process to prepare a new type of highly porous scaffolds with controlled pore structure, morphology and mechanical properties [38]. These new ductile, sponge-like scaffolds contain pores with more homogeneous pore size distribution, a rougher surface, improved mechanical properties, and more controllable degradation than the HFIP-derived scaffolds [38]. The aqueous-derived scaffolds have been successfully used for MSC-based cartilage and bone tissue engineering in vitro [7], [39]. However, it is unknown whether these scaffolds can also be used for skeletal tissue engineering in combination with specific primary cell types such as chondrocytes and osteoblasts. Thus, these types of studies will help diversify cell sources for silk-based tissue engineering applications, and also improve our understanding of the fundamental differences between stem cell-based and primary cell-based tissue engineering related to scaffold structure and morphology. As a first step for such studies, in the present work the feasibility of combining aqueous-derived silk fibroin scaffolds with adult human chondrocytes (hCHs) for in vitro cartilage tissue engineering was examined, following methodologies described in our previous report with MSCs [7]. The hCHs used in the present study were isolated from adult normal articular tissues, expanded in monolayer culture, and seeded in the aqueous-derived silk fibroin scaffolds. The hCHs were subsequently cultured for 3 weeks in a serum-free, chemically defined medium containing TGF-β1 [40]. The attachment, proliferation and differentiation of hCHs in the 3-D aqueous silk fibroin scaffolds were evaluated by confocal microscopy, real-time RT-PCR for cartilage-specific gene markers, histology and immunohistochemistry.
Section snippets
Preparation of aqueous-derived silk fibroin scaffolds
3-D aqueous-derived silk fibroin scaffolds were prepared according to the procedure described in our previous studies [7], [38]. Briefly, a 6 w/v% silk fibroin solution was prepared from Bombyx mori silkworm cocoons. The cocoons were extracted in a 0.02 m Na2CO3 solution, dissolved in a 9.3 m LiBr solution and subsequently dialyzed against distilled water. To form the scaffolds, four grams of granular NaCl particles (600–700 μm) was added to 2 ml of a 6 w/v% silk fibroin solution in Teflon cylinder
Cell attachment to aqueous-derived silk fibroin films
The attachment of hCHs to aqueous-derived silk fibroin films was assessed by microscopy at varying time points after cell plating and compared to cell attachment to TCP (Fig. 1). On aqueous-derived silk fibroin films, no significant cell attachment was observed until 1 h after cell plating (Figs. 1E and F). The attachment of hCHs to TCP was significantly faster than to silk fibroin films. On TCP, hCHs started to attach in 30 min upon cell plating (Fig. 1A). At the 2-h point post plating, most
Discussion
Successful cartilage tissue engineering requires a thorough understanding of the cellular response of autologous cells on appropriate biocompatible polymers in terms of cell attachment, proliferation and differentiation. The nature of the cellular response depends on the type of cell and the type of materials used. The engineered silk materials used in the present study are derived from an all-aqueous process [38]. To evaluate the cellular response to these new engineered materials, cell
Conclusions
hCHs have been successfully combined with 3-D aqueous-derived silk fibroin scaffolds for in vitro cartilage tissue engineering. Culture-expanded hCHs attached to, proliferated and differentiated in the scaffolds in a serum-free, chemically defined medium containing TGF-β1. The hCHs cultured in the 3-D scaffolds regained the spherical morphology, which is similar to the cell morphology of chondrocytes in the physiological environment of articular cartilage tissue and freshly isolated
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