(H-307) Enhancing Biological Properties of 3D Printed Chitosan/Hydroxyapatite Hybrid Scaffolds through Alginate Impregnation for Cartilage Regeneration

Three-dimensional (3D) printing shows great promise as a technique for cartilage tissue engineering, providing exceptional control over important structural aspects such as porosity, pore size, and pore interconnectivity [1]. Chitosan, a natural polymer widely used in tissue engineering applications, either alone or in combination with other polymers, offers benefits such as high biodegradability, non-toxicity, and antimicrobial properties. Its glycosaminoglycan content makes it a promising candidate for cartilage tissue engineering as it promotes chondrogenesis. However, challenges arise from its low bioactivity when dissolved in high-concentration acetic acid and difficulties in 3D printing at low concentrations, as indicated in a recent study[2].

The study addressed the issue of poor cell attachment in chitosan-based scaffolds for cartilage regeneration by modifying chitosan with alginate. Alginate, a hydrophilic polysaccharide, is widely used in tissue engineering due to its biocompatibility, biodegradability, and cost-effectiveness, providing a suitable matrix for chondrocyte delivery[3]. Nano hydroxyapatite (nHA), a bioactive bioceramic and the main component of bone mineral, into the hydrogel matrix. This approach successfully enhanced the mechanical properties and introduced bioactive characteristics to the hydrogel network[4].

The main goal of this study is to enhance the biological properties of chitosan-based cartilage scaffolds by incorporating alginate as an impregnating solution to create a hybrid structure. Additionally, nano hydroxyapatite (nHA) was strategically used to further improve the essential properties of the chitosan/alginate hybrid scaffolds for cartilage tissue engineering applications.

In this study, the materials used were chitosan with low molecular weight (75–85% deacetylation), nano hydroxyapatite (nHA) powder (< 200 nm particle size (BET), ≥97%, synthetic, and Molecular Weight: 502.31g/mol), and sodium alginate (medium viscosity) obtained from Sigma Aldrich Company. The crosslinkers used were calcium chloride and sodium hydroxide (molecular weight: 40.00), and the chemicals acetic acid 100% (molecular weight: 60.05 g/mol) were also purchased from Sigma Aldrich Company. A 10% (w/v) chitosan solution containing 10% w/v nHA was prepared in acetic acid. Cubic scaffolds (20 × 20 × 5 mm³) with 10 layers were 3D-printed using the chitosan/nHA mixture, employing a conical needle with a 250 μm exit diameter, and air-dried at room temperature for 24 hours. The chitosan/nHA scaffolds were crosslinked by immersing them in a 5% w/v sodium hydroxide solution at 25°C for 2 hours, followed by neutralization and drying at 37°C with 65% humidity for 24 hours. For the impregnation process, sodium alginate solution was used to create hybrid chitosan-sodium alginate scaffolds, and chemical crosslinking with CaCl2 was applied.

Cell viability:

Figure 1 illustrates that chitosan scaffolds had few attached ATDC5 cells, but live cells were observed on the strands. Incorporating nHA improved cell attachment, resulting in higher cell numbers on the chitosan/nHA scaffolds, where the presence of nHA particles attracted more cells to the strands. In impregnated scaffolds, cells were more prominent within the pores than on the strands, as the alginate solution effectively filled the pores, providing a favorable cell attachment environment within the alginate hydrogel where both alginate and cell-friendly nHA were present. The last tested sample, a chitosan/nHA scaffold impregnated into an alginate hydrogel, exhibited a high number of live cells attached to the strands.

Morphology of scaffolds:

In Figure 2, the architectural images of chitosan/nHA impregnated into alginate solution with 2-mm and 3-mm pore sizes reveal interconnected pores in all samples. Figure 3 displays SEM images of the chitosan scaffold, chitosan impregnated into alginate with a 2-mm pore size, and the presence of nHA on the surface of the chitosan/nHA scaffold.

Contact angle:

According to the results in Figure 4, the scaffold with the highest contact angle (chitosan/nHA with 2-mm pore size) exhibited the lowest wettability (109.8°), while the scaffolds with chitosan (45.2°) and impregnated chitosan/nHA into alginate solution (39.6°) showed higher wettability. The presence of nHA decreased the wettability, and scaffolds with sodium alginate-based impregnation displayed increased hydrophilicity, indicating that impregnating chitosan scaffolds with alginate resulted in hybrid scaffolds with improved wettability. The decrease in wettability in scaffolds containing nHA may be attributed to changes in the crystallinity behavior of hydrogels.

Conclusion:

The results show promising biocompatibility, with few dead cells found after chondrocyte seeding on the scaffolds. The inclusion of nHA and alginate improved cell viability and attachment, and impregnated chitosan/nHA scaffolds with alginate hydrogel exhibited the best properties with a higher density of live cells attached to the strands. This research suggests that chitosan/nHA/alginate hybrid scaffolds hold promise for cartilage regeneration and the developed 3D printing and impregnation techniques can be extended to fabricate scaffolds for other tissue engineering applications.

References:

[1]      D. X. B. Chen and D. X. B. Chen, Extrusion bioprinting of scaffolds. Springer, 2019.

[2]      A. Sadeghianmaryan et al., “Extrusion-based printing of chitosan scaffolds and their in vitro characterization for cartilage tissue engineering,” Int. J. Biol. Macromol., vol. 164, pp. 3179–3192, 2020.

[3]      F. You, X. Chen, D. M. L. Cooper, T. Chang, and B. F. Eames, “Homogeneous hydroxyapatite/alginate composite hydrogel promotes calcified cartilage matrix deposition with potential for three-dimensional bioprinting,” Biofabrication, vol. 11, no. 1, p. 15015, 2018.

[4]      B. Zhang et al., “3D printing of calcium phosphate bioceramic with tailored biodegradation rate for skull bone tissue reconstruction,” Bio-Design Manuf., vol. 2, pp. 161–171, 2019.