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Biomacromolecules 2008, 9, 2662–2669
Cartilage Regeneration by Novel Polyethylene Oxide/Chitin/ Chitosan Scaffolds Yung-Chih Kuo* and I-Nan Ku Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan 62102, Republic of China Received June 16, 2008; Revised Manuscript Received July 25, 2008
This study presents the application of novel PEO/chitin/chitosan scaffolds for the cultivation of bovine knee chondrocytes (BKCs). The results reveled that the composition strongly affected physicochemical characteristics of the ternary scaffolds. Based on the contours of porosity, the percentage of void space in these scaffolds was estimated to be higher than 90%. In regard to mechanical strength, the composition of 50% chitin and 50% chitosan in the scaffold led to the maximum of Young’s modulus. Moreover, large extensibility of the scaffolds occurred at the following range of the composition: PEO > 37.5%, chitin < 25%, and chitosan (a) > (c). This suggested the following two points. First, the weight percentage of acetyl amine in the PEO/chitin/chitosan matrices was consistent with the composition. Second, a higher level of acetyl amine caused a larger amount of residual primary amine in the matrices. The reason for the second point was that acetyl amine could hinder the cross-linking of primary amine with genipin. Also, 1153 cm-1 represented the absorption position of the polysaccharide structure in chitin and chitosan.32 Mechanical Property. Young’s modulus and the percentage of elongation at break of the PEO/chitin/chitosan scaffolds are shown in Figure 4. As displayed in this figure, an increase in the weight percentage of PEO reduced Young’s modulus and improved the extensibility of the scaffolds. The rationale behind this behavior was that PEO was a linear elastomeric polymer with high flexibility.33 In regard to block copolymers of polylactide/PEO films, the elongation at break could increase 6-fold when the weight percentage of PEO in the materials was 16.7%.34 On the other hand, when the weight percentage of chitin in the scaffolds was smaller than 50%, an increase in the weight percentage of chitin enhanced Young’s modulus, as indicated in Figure 4. This was because chitin was a stiff ingredient in these biomaterials. When the weight percentage of chitin was larger than 50%, a further increase in the weight percentage of chitin reduced Young’s modulus. This was because a smaller amount of primary amine and a larger amount of acetyl amine led to a lower capacity of cross-linking by genipin. In addition, an increase in the weight percentage of chitin decreased the extensibility of the scaffolds because the chitin matrix was fragile. Large Young’s modulus occurred at the following range of composition: 25% < chitin < 75%, 25% < chitosan < 75%, and PEO ) 0%. The composition of 50% chitin and 50% chitosan yielded the maximum of Young’s modulus. Also, large elongation at break occurred at the following range of composition: PEO > 37.5%, chitin < 25%, and chitosan < 62.5%. The composition of 50% PEO and 50% chitosan led to the largest elongation. Based on these results, it could be concluded that a tough PEO/chitin/chitosan scaffold with a proper composition might be produced. Also, these scaffolds would not be brittle upon the physical influences such as normal and shear stresses. In regard to polyglycolide/
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poly(lactide-co-glycolide) scaffolds, Young’s modulus ranged from 1.65 to 2.35 MPa.35 This indicated that these PEO/chitin/ chitosan scaffolds could be much stronger in the mechanical resistance. Morphology. SEM images of the 3D PEO/chitin/chitosan scaffolds are displayed in Figure 5. As shown in this figure, the composition played a significant role in the pore structure of the scaffolds. For a high weight percentage of PEO, nonuniform pores with diameter of 50-150 µm were commonly observed, as revealed in Figure 5a. Surfaces containing wrinkles, pleats, and closed pores could be also identified in Figure 5a. As exhibited in Figure 5b, the typical cross-section exhibited slim pores with width of 50 µm and length of 200 µm. For a high weight percentage of chitin, cuboid-shaped and rugged pores with a diameter of 100-150 µm were obtained, as presented in Figure 5c. In the case of pure chitin, a flimsy matrix with distorted pores was generally produced, and the pore geometry could be modified by the addition of chitosan.16 As shown in Figure 5d, cylindrical pores with a diameter of 150-200 µm were observed for a high weight percentage of chitosan. In addition, smooth surfaces with interconnected pores were discerned in Figure 5d. As demonstrated in Figure 5e, pore surfaces of the scaffold were covered with proliferated BKCs and secreted ECMs. This induced that these ternary scaffolds could be appropriate for the culture of chondrocytes. As compared with small pores (diameter of 100 µm), a scaffold with average pore diameter greater than 200 µm was concluded to favor the secretion of type II collagen.36 Based on the pore size, these scaffolds with a high weight percentage of PEO or chitin would not be appropriate for cartilage regeneration. Biodegradation. Effects of the composition on the percentage of biodegradation of the scaffolds are presented in Figure 6. As revealed in this figure, an increase in the weight percentage of PEO promoted the percentage of biodegradation. This was because PEO with ether oxygen was highly hydrophilic. It was observed that PEO could absorb a large amount of water.37 Thus, for a high weight percentage of PEO, water erosion led to a fast deterioration of the scaffold. This result also suggested that during cultivation of BKCs, the transport of nutrition and metabolite in the scaffolds could be accelerated by biodegradation. As displayed in Figure 6, the average percentage of biodegradation was 58.84% when the weight percentage of PEO was 80%. This indicated that PEO existed in the scaffold after the culture of BKCs over 4 weeks. The reason for this result was that hydrogen bonds and chain entanglements of the macromolecules could entrap PEO in the scaffold. As shown in Figure 6, an increase in the weight percentage of chitin reduced the percentage of biodegradation. This was because N-acetyl glucosamine in chitin was difficult to be decomposed by enzymes.38 For a constant weight percentage of PEO, an increase in the weight percentage of chitosan enhanced the percentage of biodegradation, as exhibited in Figure 6. This was because deacetylated chitosan was easier to be biodegraded than chitin. On the contrary, an increase in the weight percentage of chitosan reduced the percentage of biodegradation for a fixed weight percentage of chitin, as indicated in Figure 6. This result induced that chitosan was more difficult to be biodegraded than PEO. After the cultivation of BKCs over 4 weeks, the percentage of biodegradation of the PEO/chitin/chitosan scaffolds ranged generally from 30 to 60%. This inferred the aptness of the scaffolds for the application to cartilage tissue engineering. Note that the percentage of biodegradation of the chitin/chitosan scaffolds (the weight percentage of PEO ) 0%) was typically lower than 40%, as displayed in Figure 6. Hence, it could be
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concluded that biodegradability of the PEO/chitin/chitosan scaffolds was improved. Biological Assessment. Effects of the composition on the cultivation of BKC-scaffold constructs are shown in Figure 7. As revealed in this figure, an increase in the weight percentage of PEO enhanced the amounts of BKCs, GAGs, and collagens when the weight percentage of PEO was smaller than 40%. The reverse was true when the weight percentage of PEO was greater than 40%. An increase in the weight percentage of chitin enhanced the amounts of BKCs, GAGs, and collagens at the following range of the composition: chitin < 50%, PEO < 25%, and PEO > 40%. The reverse was true when the weight percentage of chitin was greater than 50%. Also, an increase in the weight percentage of chitin enhanced the amounts of BKCs, GAGs, and collagens at the following range of the composition: chitin < 25% and 25% < PEO < 40%. The reverse was true when the weight percentage of chitin was greater than 25%. In addition, an increase in the weight percentage of chitosan enhanced the amounts of BKCs, GAGs, and collagens when the weight percentage of chitosan was smaller than 50%. On the contrary, an increase in the weight percentage of chitosan reduced the amounts of BKCs, GAGs, and collagens when the weight percentage of chitosan was greater than 50%. Based on these results, it could be concluded that the range of the composition for a better culture of BKCs was 25% < PEO < 40%, 12.5% < chitin < 37.5%, and 30% < chitosan
