Regarding cartilage regeneration, the ideal cell carrier substance is one that closely mimics the natural environment in the cartilage-specific ECM [12]. Cartilage ECM mainly consists of type II collagen and glycosaminoglycans (GAGs). Given the importance of GAGs in enhancing chondrogenesis in vitro, the uses of GAGs or GAGs-like biomaterials as components of a scaffold material are likely to be a reasonable approach for enhancing chondrogenesis [11,12,22].

Chitosan is a partially deacetylated derivative of chitin, the primary structural polymer in arthropod exoskeletons. This natural material is a linear polysaccharide consisting of β(1→4) linked D-glucosamine residues with a variable number of randomly located N-acetyl-glucosamine groups. The average molecular weight ranges from 50 to 1,000 kDa. The potential of chitosan as a biomaterial is based on its cationic nature and high charge density in solution. Madihally et al. [9] reported that the cationic nature of chitosan allowed for electrostatic interactions with anionic GAGs, proteoglycans (PGAs), and other negatively charged species. These ionic interactions may serve as a mechanism for retaining and recruiting cells, growth factors, and cytokines within a tissue scaffold. Therefore, chitosan has been used as a good compatible material for tissue repair and wound healing [11,23-25]. In cartilage tissue engineering, previous studies have shown that this material possesses promising potential as a carrier material for the transplant of chondrocytes [12,26-30].

Since chitosan is considered to be a cationic polysaccharide showing excellent cell supporting properties, a hybrid material composed of chitosan combined with GAGs may be a novel class of polyion complex effective for cartilage specific scaffolds [11]. Hyaluronan is a linear GAG composed, on average, of 1 × 10⁴ disaccharide units of glucuronic acid and N-acetylglucosamine, with a molecular weight of 1,000–5,000 kDa. This GAG is a main component of the ECM of articular cartilage and plays a crucial role in regulating the behavior of chondrocytes. Concerning the biological roles of HA, Zimmermann et al. [31] demonstrated that HA is an adhesion modulator molecule, which can mediate the early stage of cell-substrate interaction. CD 44 acts as a cell surface receptor for HA [32,33]. Murdoch et al. [34] showed a dramatic increase in CD44 expression on chondrocytes isolated from the cartilage. These data indicate that scaffold materials introducing HA can provide excellent adhesivity for seeded chondrocytes and enhance the biological behavior of the chondrocytes on the scaffolds.

In articular cartilage tissue engineering, we must address that the articular cartilage is subject to excessive mechanical stress. Consequently, mechanical strength with high cellular adhesivity to maintain the number of seeded chondrocytes is a requirement for scaffold materials. To meet this requirement, we developed a novel chititosan-based HA introduced hybrid polymer fiber as a fundamental material for 3D fabricated scaffolds. Polymer fibers were developed by the wet spinning method as described by Tamura et al. [35] with the following modification. Viscosity average molecular weight of HA was 2,400 kDa. The degree of deacetylation of the chitosan was 81%, and viscosity average molecular weight was 600 kDa. To prepare the polymer fibers, 7 g of chitosan powder was dissolved in 200 mL of 2% aqueous acetic acid solution to give 3.5% of polymer concentration. Dope of chitosan was spun into a calcium coagulant bath (64% CaCl₂ dissolved in 50% aqueous methanol solution) through a stainless steel spinnlet (0.1 mm diameter, 50 holes) at a winding speed of 4.4 m/min at room temperature. Then, 50% aqueous methanol solution was used as a second coagulation bath and hyaluronic acid dissolved in 50% aqueous methanol solution as a third coagulation bath. Using an original roller system (Okada Co., Inc., Sapporo, Japan), the resulting fibers were stretched and treated with 0.8% sodium hydroxide (NaOH) dissolved in 90% aqueous methanol solution to neutralize the acidity of the fibers (Figure 1). The fibers wound in the roller were washed with methanol and dried at room temperature. The diameter of each fiber was 30 μm (Figure 2). The developed hybrid fibers were sterilized in an autoclave at 135 °C for 20 minutes. The tensile strength of the hybrid polymer fiber was determined by the concentration of the introduced HA. The values of the HA hybrid fiber significantly increased compared with that of the chitosan fiber (87 N/mm² in the chitosan fibers, 168 N/mm² in the chitosan introduced with HA 0.04% fibers, and 144 N/mm² in the chitosan introduced with HA 0.07% fibers).

Regarding the diameter of fibers for fabricated scaffolds used in cartilage tissue engineering, Neurnberger et al. [36] suggested that fiber sizes smaller than chondrocytes are beneficial in terms of cellular adhesion and maintainance of the chondrocyte phenotype. The diameter of chondrocytes is approximately 10 to 30 μm. As mentioned above, the diameter of our fibers in dry condition is 30 μm. Due to biodegradability, the diameter in a culture medium or living joints must be smaller than 30 μm. In view of this point, we determined the diameter of the developed fibers.

Tables 2 and 3 summarize the data regarding the biological effects of the developed hybrid polymer fibers on cellular behaviors [16]. The DNA content of the chondrocytes at seven days after cultivation indicates the degree of chondrocyte proliferation on the scaffold materials. The obtained data suggested that adhesion, proliferation, and ECM products of the chondrocytes significantly increased on the hybrid polymer fibers as compared to the non-hybrid chitosan fiber. Light microscopy and scanning electron microscopy (SEM) showed the maintenance of the characteristic round morphology of the cultured chondrocytes on the hybrid fibers (Figure 3). The SEM images also revealed dense fibrous tissue indicating type II collagen on the hybrid fibers (Figure 3). The obtained data suggest the superior biological effects of our novel hybrid fibers on chondrocyte activities.

Fiber materials can provide adequate mechanical strength for a 3D scaffold of cartilage tissue engineering. Additionally, they easily control the macroscopic and microscopic scaffold structures. One of the most important factors for developing a 3D cartilage tissue engineering scaffold is the pore size of the scaffold material. To determine an adequate pore size, we performed an experiment to clarify the effects of pore size of the developed scaffolds on chondrocyte behavior. From the chitosan-based HA hybrid polymer fibers, which are introduced with 0.07% hyaluronic acid, the 3D scaffolds with three different pore sizes (100, 200, and 400 μm diameter) were woven by using the original apparatus (Figure 4). The effects on the ECM products of each scaffold material are summarized in Table 4 [17]. The obtained data suggested that the current scaffold with 400 μm pore size significantly increased ECM synthesis by cultured chondrocytes.

Previous studies have shown that the scaffold pore size is highly interconnected with cell proliferation and ECM synthesis in chondrocyte culture [10,37,38]. Nehrer et al. [10] clarified the effects of scaffold pore sizes, from 20 to 86 μm, on chondrocyte behavior using collagen sponges. They suggested that the cultured cells on the material with small pore diameter lost the chondrocytic morphology over time. Using poly (ethylene-glycol)-terephthalate/poly (butylenes terephthalate) scaffolds with different pore sizes (182 μm and 525 μm), Malda et al. [38] demonstrated that large pore size scaffolds significantly increased the GAG production of cultured chondrocytes. These previous and our results suggest great promise for the future of a fabricated scaffold with a relatively large pore size, such as 400 μm, for cartilage regeneration.

In clinical usage of the developed scaffold material, we must inhibit inflammatory reactions and an acute decrease in mechanical strength of the implanted tissue during the biodegradation process of the material. One of the ideal strategies for addressing this issue is to reduce the volume of scaffold material in engineered constructs with mechanically mature cartilage. For this strategy, we developed two types of 3D scaffolds fabricated with the chitosan-based HA hybrid polymer fibers [18]. One was a cushion-type scaffold, consisting of two sheets, which were 8 × 8 mm wide and 1 mm thick. The other was a 10 mm high cylinder-type scaffold, which had a 6 mm diameter (Figure 5). Based on the obtained data, the pore size of both scaffolds was 400 μm. Although the volume of each scaffold material was minimized, both scaffolds retained the initial shape under a dynamic culture condition.

To prepare tissue-engineered constructs, chondrocytes were isolated from eight week-old Japanese white rabbits according to sequential enzyme digestion under sterile conditions as described previously [39]. A chondrocyte suspension containing 3 × 10⁵ and 7 × 10⁵ cells was seeded onto the cushion-type (cushion group) and the cylinder-type (cylinder group) scaffold. The samples were placed on a 48-well plate in a 37 °C, humidified 5% CO₂ incubator for 1 hour and then overlaid with 1 mL of the culture medium. After one-week static culture, these samples were transferred into a disposable high aspect ratio vessel bioreactor (HARV, 50 mL; Synthecon, Inc., Houston, TX, U.S.) and dynamically cultured for a further four or seven weeks. Table 5 summarizes biochemical and biomechanical evaluations of engineered cartilage in both material groups at each time point [18]. At eight weeks after cultivation, macroscopic (Figure 6) and histological (Figure 7) findings suggested successful hyaline-like cartilage regeneration with rich GAG and type II collagen products. Regarding the biomechanical properties of regenerated tissues, the cushion-shape scaffold significantly increased the Young's modulus of regenerated tissue from five to eight weeks after cultivation. On the other hand, the cylinder-shape scaffold did not alter the value during the culture period. Although the Young's modulus of the cylinder group was significantly inferior to that of the cushion group, the value was comparable to that of normal rabbit cartilage. Regarding the reason for the high stiffness of the engineered cartilage in the cushion-type scaffold, the stiffness of the scaffold material may affect the value of the engineered tissue. The obtained results indicate that our novel 3D scaffolds regenerate histologically and mechanically mature tissue.