Chitosan fibers with improved biological and mechanical properties and intended for tissue engineering applications were prepared by varying the concentrations of acetic acid, ammonia, and the cross-linking agent heparin and by varying the annealing temperature [13Alb]. Optimizing the concentration of acetic acid to 2 % improved fiber strength and stiffness by twofold, and using 25 % ammonia solution during coagulation decreased fiber diameters and increased strength by 200 %. Increase in the strength was attributed to increase in % crystallinity. Annealing also increased strength but resulted in discoloration of the fibers as seen from Fig. 26.3. Cross-linking with heparin decreased fiber strength. Fibers with very low tensile strength of about 0.05 g/den (5 MPa) and breaking strain of 0.2 % were obtained using the optimized fiber production conditions. The physical and chemical treatments did not affect biocompatibility, whereas cross-linking with heparin improved the attachment and proliferation of porcine valvular interstitial cells.

Chitosan fiber scaffolds intended for tissue engineering were coated with colla­gen type I and used to study biocompatibility using human bone marrow stromal cells (hBMSCs) and with murine osteoblast cells [08Hei, 09Hei]. Good adhesion, proliferation, and osteogenic differentiation were observed, and the fibers were considered as excellent scaffolds for hBMSCs. As seen in Fig. 26.4, chitosan fibers coated with collagen (b) showed extensive growth of actin compared to the uncoated fibers (a) after 14 days of culture. The collagen coating is seen in red on the right panel. Similar results were obtained for chitosan fibers cultured with

osteoblasts, but collagen coating did not improve the viability or proliferation [08Hei].

Chitosan fibers (167 den; tenacity 2.1 g/den and elongation of 10.3 %) were N-acetylated, and the influence of acetylation on the in vitro and in vivo biodegra­dation of the fibers was studied [07Yan]. Acetylation was done by immersing the fibers in acetic anhydride at 25 °C. The degree of acetylation was controlled to

7.7 %, 21.6 %, 40.9 %, 61.2 %, 82.5 %, and 93.4 % by varying the reaction time from 10 to 120 min. Degradation of the fibers was studied in pH 7.4 buffer containing 4 mg/ml lysozyme, and it was found that chitosan fibers did not biodegrade, whereas acetylated fibers degraded to different extents depending on the degree of acetylation. Up to 100 % biodegradation was obtained for the fibers acetylated to 93.4 % after immersion in PBS for 9 days. Fibers acetylated to three levels were embedded into mice for 6 months. After 6 months, the mice were

sacrificed, and it was found that the fibers with higher degree of acetylation (93 %) had degraded in the body. As seen in Fig. 26.5, fibers with lower degree of acetylation (7.7 %) were dense and had degraded to a lower extent than the fibers with 93 % acetylation that had completely assimilated in the surrounding tissue.

Obtaining scaffolds with porosity and mechanical properties required for bone tissue engineering has been a challenge [09Lia]. A rapid prototyping and rapid tool technique were used to reinforce calcium phosphate cement composites with chitosan fibers, and the biocompatibility of the scaffold was studied. It was found that attachment and proliferation of mesenchymal stem cells were higher on the scaffolds containing chitosan fibers, particularly at the interface of the chitosan fibers and calcium phosphate cement. As seen in Fig. 26.6, scaffolds containing chitosan fibers showed extensive spreading of the actin filaments indicating better compatibility compared to the fibers without the chitosan fibers.

Porous heart valve scaffolds made from chitosan were reinforced with chitosan fibers (2-10 %) and found to improve the mechanical properties of the scaffolds. Table 26.1 provides a comparison of the properties of the chitosan scaffolds with and without the fibers as reinforcement. To further improve the properties of the scaffolds, cross-linking with heparin was done, but no significant increase in fiber strength was seen but the strength of the scaffold was higher [12Alb]. Properties of

the chitosan and chitosan fiber-reinforced heart valve scaffold are compared with that of human pulmonary and aorta valves in Table 26.2.

In another study, heart valves with desired properties could be obtained by controlling the amount, length, and tensile properties of the fibers. Core-shell fibers with chitosan as core and calcium phosphate as shell and intended for bone tissue engineering with different properties were developed by varying the coagulation bath conditions. Analysis of the fibers showed that the Ca and P atoms were distributed mostly on the surface of the fibers. Tensile properties of the fibers were found to increase with increasing concentration of chitosan. No porosity or cell culture studies were done [04Mat].

3D fibrous chitosan scaffolds containing poly(lactic acid-co-glycolic acid) nanocapsules loaded with the bone morphogenetic growth factor BMP-2 and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles loaded with BMP-7 were used to culture rat bone marrow mesenchymal stem cells (MSCs) for bone tissue engineering [09Yil]. Fibers were prepared using two concentrations (4 and 6 %) of chitosan, and chitosan was also blended with polyethylene oxide (PEO). The addition of PEO resulted in a rough fiber surface but improved stability of the fibers. MSCs showed higher initial cell proliferation and increased ALP activity for the chitosan fibers containing PEO fibers. Figure 26.7 shows SEM images of the MSCs growing on the chitosan scaffolds (top) and chitosan-PEO scaffolds (bottom) after 21 days of incubation clearly demonstrating that the inclusion of PEO promoted cell growth. In addition to adding PEO, growth factor containing nanocapsules made from PLGA and PHBV were inserted onto the fibers via mixing with the spinning solution or seeded onto the fibers after fiber produc­tion. Higher release rate was obtained by loading the nanocapsules on the surface after fiber formation. PLGA nanocapsules were found to have better release com­pared to PHBV nanocapsules after 21 days of testing.

3D fibrous mesh scaffolds were also prepared from chitosan for tissue engineer­ing applications, and the biocompatibility and cytotoxicity were evaluated using mouse fibroblasts and human osteoblasts [04Tuz]. Fibers obtained had strength of about 205 MPa and elongation of 8.5 % and swelled rapidly to about 110 % within 30 min. Immersion of the fibers in simulated body fluid for 30 days led to the formation of films composed of calcium phosphate as shown in Fig. 26.8 indicating the bioactivity of the fibers. 3D fiber meshes had swelling of up to 160 % within 50 min of immersion in NaCl solution but preserved their integrity. Extensive attachment and growth of cells and formation of bridges between cells were observed suggesting that the fibers were not cytotoxic.

Table 26.2 Comparison of the tensile properties of chitosan-based scaffolds with the human pulmonary and aorta valves (reproduced from Albanna et al. [12Alb]) Pulmonary valve Aorta valve Property Chitosan Reinforced Radial Circumferential Radial Circumferential Tensile strength (kPa) 58 ±28 220 ±17 290 ±60 2,780 ±1,050 320 ±40 1,740 ±290 Strain {%) 90 ±30 55 ±10 30 ±4 19.4 ±3.91 24 ±4 18.4 ±7.6 Modulus of elasticity (kPa) 70 ±10 400 ±140 1,320 ±930 16,050 ± 2,020 1,950 ±150 15,340 ±3,480

Chitosan fibers were proposed to serve as bioartificial nerve grafts to treat peripheral nerve injuries and to evaluate this possibility, Schwann cells were cultured on chitosan fibers and compared to chitosan films for cell attachment, growth, and proliferation. Schwann cells were found to have a spherical and long shape, grew contact extensions, and migrated faster on the fibers compared to films [04Yua]. After 14 days of culture, cells formed chains on the chitosan fibers as seen in Fig. 26.9.

Collagen fibers were coated with hyaluronic acid (HA) and used for ligament tissue engineering. It was reported that incorporating HA increased mechanical properties of the fibers and also promoted the attachment and proliferation of fibroblasts [05Fun].

Alcogels were developed by coagulating chitosan fibers in hydroalcoholic solutions than the conventional approach of using alkali baths or ammonia vapors. Chitosan was dissolved using acetic acid into which an alcohol such as 1,2, propanediol was added, and fibers shown in Fig. 26.10 were extruded by hot-air drying. It was proposed that the alcohol system increased the entanglement density and chitosan chain interactions that led to improved mechanical properties [13Des]. Fibers produced using the hydroalcohol approach had a large proportion of anhydrous crystals. However, information on the properties of the hydroalcohol fibers is not available.