1.4 SILK FIBROIN IN TISSUE ENGINEERING
1.4.2 Cartilage tissue engineering
of SF/CS supported cell attachment, growth and chondrogenic phenotype of bovine chondrocytes for 2 weeks of in vitro culture (Bhardwaj et al, 2011). Also, the biomechanical studies revealed that the static and dynamic stiffness at high frequencies was higher in cell-seeded constructs than non-seeded controls.
Growth factor releasing scaffolds are an emerging alternative to autologous or allogenous implants, providing a biologically active template for cartilage tissue (re)- generation. The feasibility of controlled IGF-I releasing SF scaffolds in the context of cartilage repair was evaluated and found that the chondrogenic differentiation of hMSCs in TGF-β supplemented medium was observed on IGF-I loaded scaffolds, starting after 2 weeks and more strongly after 3 weeks, whereas no chondrogenic responses were observed on unloaded control scaffolds (Uebersax et al, 2008). The methanol treatment induced water insolubility of SF scaffolds and allowed the control of bioactive IGF-I delivery without affecting IGF-I potency. The cumulative drug release correlated linearly with the IGF-I load. Thus, IGF-I loaded porous SF scaffolds have the great potential to provide chondrogenic stimuli to hMSCs.
The beneficial effects of microwave-induced argon plasma treatment on cellular behaviors of articular chondrocytes seeded on nanofibrous SF mesh were studied and found that the treatment significantly improved its hydrophilicity and cytocompatibility with the human articular chondrocytes (Baek et al, 2008; Jin et al, 2009). Similarly, the chondrogenic responses on microwave-induced argon plasma treated SF/wool keratose blend e-spun scaffold were evaluated by Cheon et al (2010). The argon plasma treatment increased the hydrophilicity of SF/keratose scaffold and induced deeper and more cylindrical pores than nontreated scaffolds. Also, the attachment and proliferation of neonatal human knee articular chondrocytes on treated SF/ keratose scaffolds increased significantly, followed by increased glycosaminoglycan (GAG) synthesis. Thus, the microwave-induced argon plasma treatment would significantly improve chondrogenic cell growth and cartilage-specific extracellular matrix formation.
Mesenchymal condensation is a pre-requisite of chondrogenesis during embryonic development. The current understanding of chondrogenesis is limited in terms of chondrogenic condensation mechanisms. In particular, the role of matrix stiffness on homotypic cell-cell interactions leading to the establishment of distinctly aggregated chondrogenic morphology from mesenchymal cells is unclear. To assess the interactions
of matrix stiffness on chondrogensis an in vitro biomaterials-based model was described, where, by sensing subtle variation in morphology and stiffness of nanofibrous silk protein matrices, hMSCs migrated and assumed aggregated morphologies, mimicking early stage chondrogenesis (Ghosh et al, 2009). This simple in vitro model system has pot ential to play a significant role to gain insight into underlying mechanisms of mesenchymal condensation steps during chondrogenesis, integrating concepts of developmental biology, biomaterials and TE.
The cellular responses of isolated human chondrocytes, embryonic hMSCs derived from bone marrow and adipose tissue, were assessed for chondrogenic potential in 3D culture (Tigli et al, 2009). The cells were differentiated in two different biomaterial matrices, SF and CS scaffolds, in the presence and absence of BMP-6, along with the standard chondrogenic differentiating factors. It was found that the embryonic stem cells- derived MSCs showed unique characteristics, with preserved chondrogenic phenotype in both scaffolds with regard to chondrogenesis. However, after 4 weeks of cultivation, embryonic stem cells-derived MSCs were promising for chondrogenesis, particularly in the silk scaffolds with BMP6. Thus, the cell source differences are important to consider with regard to chondrogenic outcomes.
The effects of 3D porous SF matrix scaffold properties and hydrodynamic environment in cartilage tissue regeneration, and the biomechanical properties of engineered cartilage are important parameters to investigate (Morita et al, 2003, 2006;
Yamamoto et al, 2007; Wang et al, 2010). Changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into SF sponge were investigated and found that the friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the SF sponge surface (Morita et al, 2006). Also, the friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer.
Chondrocytes seeded on the SF-sponge scaffolds were cultured in the stirring chamber (a bioreactor facilitating mechanical stimulation) for up to 3 weeks (Shangkai et al, 2007). It was found that compared to the control group, the seeded scaffolds subjected cultured in stirring chamber demonstrated significant increases in both DNA content
(38.9%) and GAG content (54.3%) at day 21, in addition to facilitating the maturation of cartilage tissue. In the clinical feasibility studies, large defects on rabbit knee joints were repaired with regenerated cartilage, which resembled hyaline cartilage at 12 weeks after operation. Recently, the effects of perfusion bioreactors on the chondrogenic potential of engineered constructs prepared from porous SF scaffolds seeded with human embryonic stem cell derived MSCs was investigated, where, after four weeks of incubation, in comparison to static culture, constructs cultured in perfusion bioreactors showed significantly higher amounts of GAG, DNA, total collagen and collagen II, along with cartilage-related gene expression (Tigli et al, 2011). Also, the mechanical stiffness of constructs increased 3.7-fold under dynamic culture conditions and distinct differences were noted in tissue morphology, including polygonal extracellular matrix structure of engineered constructs in thin superficial zones and an inner zone under static and dynamic conditions, respectively. Thus, the dynamic culture conditions in bioreactors modulate the growth of tissue-engineered cartilage and enhance tissue growth in vitro, and such mechanically stimulated scaffold/cell constructs have great potential to support chondrogenesis in vivo.