Page 285 - IJB-10-5

International Journal of Bioprinting 3D printing of collagen II-scaffolds

(GAG), and chondroitin. In cartilage tissue engineering, correlation of printing optimization and resolution
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ECM production should be sufficient to maintain the enhancement towards cell activities has yet to be studied
overall mechanical strength of the regenerated tissue. 9–11 for collagen II-based scaffolds, especially compared
Likewise, more cartilage-specific compounds are required to scaffolds of similar structures composed of other
to achieve mechanical properties and loading responses biomaterials.
similar to natural cartilage. 12,13 Accordingly, promoting In this study, we investigated the rheological properties
the proliferation of stem cells is crucial to increase the of collagen II-based hydrogels at cryogenic conditions,
overall production of ECM compounds, while enhancing and we obtained a high resolution exceeding 150 μm. On
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chondrogenic differentiation of stem cells is essential to this basis, we investigated how collagen II composition
increase the proportion of cartilage-specific compounds. enhances chondrogenic differentiation of mesenchymal
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Collagen II is a naturally derived protein with excellent stem cells (MSCs) compared to gelatin-based scaffolds,
biocompatibility and surface arginine-glycine-glutamic in terms of level of condensation and hypoxic conditions,
acid (RGE) sequence that favors cell adhesion, rendering to elucidate the underlying mechanisms. The study
it one of the most ideal biopolymers for tissue engineering. further evaluates scaffolds with different pore sizes for
As the key cartilage-specific compound, collagen II has optimization and explores underlying mechanisms.
reportedly promoted chondrogenic differentiation of Finally, an ideal cartilage-specific scaffold was designed
stem cells in several studies. 16,17 Additionally, collagen based on the optimized composition and structural
II imparts cartilage-specific mechanical properties and characteristics. We anticipate that this study will broaden
behaviors that are essential to mimic the load-bearing our understanding of the role of collagen II, corresponding
properties and mechanical responses of native cartilage scaffold designs, and optimized conditions for cartilage
tissues. These functions are distinct from those of collagen tissue engineering.
I, gelatin, and other natural biopolymers. 18,19 According
to recent animal studies, implantation of collagen II- 2. Materials and methods
based scaffolds promoted cartilage repair better than their 2.1. Materials
collagen I counterparts, reflected by the higher amount Gelatin (gel strength: ~250 bloom; biotechnology grade),
of regenerated cartilage tissue and its connectivity, 6,13,20 alginate (mannuronic acid [M]/guluronic acid [G] = 1:1;
further demonstrating its prospects in cartilage tissue biochemical grade; low-viscosity type), anhydrous CaCl ,
engineering. However, collagen II-containing scaffolds collagen I (from cow calcaneal tendon), collagen II (from
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are mainly in a foam form produced by freeze-drying. In bovine cartilage), and genipin were supplied by Macklin
other studies, collagen II was injected as a bulk hydrogel (China). Cellulose nanofiber (CNF) was supplied by
or used as a film, lacking porosity, and diverging from the Jinjiahao Co. Ltd. (China). Male Sprague-Dawley (SD) rats
concept of a conventional scaffold. 16,20–23 In freeze-dried (2–3 weeks old) were purchased from the Animal Research
foam scaffolds, the lack of symmetry and uniformity Center of Anhui Medical University (China).
in the position, shape, and size of pores limits pore
interconnectivity and increases the tortuosity of the pore 2.2. Fabrication of 3D scaffolds by extrusion printing
channels. These structural defects are not favorable for
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cell migration, penetration, and transportation of oxygen, 2.2.1. Ink preparation
water, nutrition, and metabolic waste necessary for cell Ink solutions of CNF/gelatin/alginate, CNF/collagen I/
proliferation. alginate, and CNF/collagen II/alginate were prepared
by dissolving 3% (w/v) gelatin, collagen I, and collagen
To our knowledge, collagen II-based mesh scaffolds II in the readily-purchased 2 wt% CNF solution at 37°C,
have been scarcely reported, despite their potential to respectively; after dissolution, 15% (w/v) alginate was
address the aforementioned issues. The rheological added to the solutions. The collagen II/alginate solution
properties (e.g., viscoelastic, shear-thinning behavior, and was prepared by dissolving 3% (w/v) collagen II and 15%
solidification kinetics) of collagen II-based hydrogel inks (w/v) alginate in deionized water.
remain unclear. In particular, when collagen I and gelatin- All four solutions were continuously stirred with
based hydrogels were excessively used and printed with a magnetic bar at 37°C for 1 h. The mixtures were then
a good shape fidelity under a cryogenic condition, 25–28 stirred by a motor-driven propeller under 25°C and at 150
the temperature effects on the rheological properties of RPM for 15 min.
collagen II-based hydrogels require further evaluation for
printing optimization. Besides that, scaffold resolution has 2.2.2. Preparation of the crosslinking solution
been reported to significantly influence cell proliferation The crosslinking solution was prepared by dissolving 30%
and chondrogenic differentiation. However, the direct (w/v) CaCl in distilled water in a 100 mL flask placed
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Volume 10 Issue 5 (2024) 277 doi: 10.36922/ijb.3371