Marine Biomaterial-Based Bioinks for Generating 3D Printed Tissue Constructs

Abstract

Biologically active materials from marine sources have been receiving increasing attention as they are free from the transmissible diseases and religious restrictions associated with the use of mammalian resources. Among various other biomaterials from marine sources, alginate and fish gelatin (f-gelatin), with their inherent bioactivity and physicochemical tunability, have been studied extensively and applied in various biomedical fields such as regenerative medicine, tissue engineering, and pharmaceutical products. In this study, by using alginate and f-gelatin's chemical derivatives, we developed a marine-based interpenetrating polymer network (IPN) hydrogel consisting of alginate and f-gelatin methacryloyl (f-GelMA) networks via physical and chemical crosslinking methods, respectively. We then evaluated their physical properties (mechanical strength, swelling degree, and degradation rate) and cell behavior in hydrogels. Our results showed that the alginate/f-GelMA hydrogel displayed unique physical properties compared to when alginate and f-GelMA were used separately. These properties included high mechanical strength, low swelling and degradation rate, and an increase in cell adhesive ability. Moreover, for the first time, we introduced and optimized the application of alginate/f-GelMA hydrogel in a three-dimensional (3D) bioprinting system with high cell viability, which breaks the restriction of their utilization in tissue engineering applications and suggests that alginate/f-GelMA can be utilized as a novel bioink to broaden the uses of marine products in biomedical fields.

Keywords: 3D bioprinting; alginate; bioink; fish gelatin; hydrogel; marine products; tissue engineering.

Figure 1

Schematic illustration of fabrication of alginate/f-GelMA hydrogel and mechanical properties. (A) Schematic diagram showing the process of fabrication for alginate/f-GelMA hydrogel. Step 1: Hydrogel solution containing alginate, f-GelMA, and 0.5% photoinitiator. Step 2: Polymerization of alginate in the presence of calcium ions (Ca²⁺) diffused from agarose-calcium chloride gel. Step 3: Polymerization of f-GelMA in the presence of photoinitiator under UV illumination. Step 4: photograph of alginate/f-GelMA hydrogel with 8 mm diameter and 2 mm thickness (Scale bar = 5 mm). The composition of alginate/f-GelMA hydrogel was 1%, 2%, 3%, and 4% alginate mixed with 4%, 5%, and 6% f-GelMA, respectively. (B) Compressive modulus of different concentrations of pure alginate (n = 3 per group and *** p < 0.001 was considered statistically significant). (C) Compressive modulus of alginate/f-GelMA hydrogels with different compositions.

Figure 2

Physical characteristics (swelling ratio, degradation property, and morphology obtained with SEM) of alginate/f-GelMA hydrogels. (A) Mass swelling ratios of pure alginate hydrogels at 1%, 2%, 3%, and 4%. (B) Mass swelling ratios of alginate/f-GelMA hydrogels consisting of 1%, 2%, 3%, and 4% and 4%, 5%, and 6% f-GelMA. (C) Rehydrated ratios of alginate/f-GelMA hydrogels consisting of 1%, 2%, 3%, and 4% and 4%, 5%, and 6% f-GelMA. (* p < 0.05, ** p < 0.01, *** p < 0.001 was considered statistically significant and n = 5 per group). (D) Degradation property of alginate/f-GelMA hydrogel with 2% alginate and 4%, 5%, and 6% f-GelMA. (E) Degradation property of alginate/f-GelMA hydrogel with 4% alginate and 4%, 5%, and 6% f-GelMA (n = 3 per group). (F) A micrograph obtained using SEM shows a random region of alginate/f-GelMA hydrogel (2% alginate and 6% f-GelMA). (G) A SEM micrograph showing a random region of alginate/f-GelMA hydrogel (4% alginate and 6% f-GelMA) (Scale bar = 1 mm).

Figure 3

Cell adhesion and 3D cell encapsulation in alginate/f-GelMA hydrogel. (A) NIH-3T3 cells in 1%, 2%, 3%, and 4% alginate with 4%, 5%, and 6% f-GelMA (24 h). The live cells are shown in green and the dead cells in red (Scale bar = 100 µm). (B) Cell viability on 2D alginate/f-GelMA surface. (* p < 0.05, *** p < 0.001 was considered statistically significant and n = 3 per group) (C) NIH-3T3 cell encapsulation in 4% alginate with 4%, 5%, and 6% f-GelMA at day one, three, five, and seven. The live cells are shown in green and the dead cells in red (Scale bar = 100 µm).

Figure 4

Deposition of cell-laden hydrogel in 3D bioprinting process and cell viability. (A) Photograph of printed 3D scaffold. (B) Photograph of printed scaffold under microscopy (from left) and Live/Dead cell staining in a two-layer bioprinted scaffold. The live cells are shown in green and the dead cells in red (Scale bar = 500 µm). (C) Live/Dead staining with increasing concentration of f-GelMA (4%, 5%, and 6%) in a fixed 4% alginate (Scale bar = 100 µm).

Figure 4

Deposition of cell-laden hydrogel in 3D bioprinting process and cell viability. (A) Photograph of printed 3D scaffold. (B) Photograph of printed scaffold under microscopy (from left) and Live/Dead cell staining in a two-layer bioprinted scaffold. The live cells are shown in green and the dead cells in red (Scale bar = 500 µm). (C) Live/Dead staining with increasing concentration of f-GelMA (4%, 5%, and 6%) in a fixed 4% alginate (Scale bar = 100 µm).

Figure 5

Schematic illustration of 3D bioprinting process using alginate/f-GelMA bioink. (A) Left: Photograph of 3D bioprinting system. Right: Photograph of extrusion system of bioprinter. (B) Schematic illustration of two-step crosslinking process for hydrogel fibers. Step 1: Ionic crosslinking of alginate in the presence of calcium ion (Ca²⁺). Step 2: Covalent crosslinking of f-GelMA in the presence of photoinitiator under UV light. (C) Schematic illustration of the 3D bioprinting process. Step 1: The coaxial system where the bioink was delivered from the core and the ionic crosslinking solution containing calcium ions (Ca²⁺) was on the outer side. Step 2: Schematic diagram to show the designed pattern of hydrogel scaffold. Step 3: Photograph of a bioprinted (two-layer) scaffold with cells (150 µm fiber diameter) and Live/Dead staining image (inset) to show cell viability.