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. 2023 Feb 24:26:77-87.
doi: 10.1016/j.bioactmat.2023.02.007. eCollection 2023 Aug.

3D-printed fish gelatin scaffolds for cartilage tissue engineering

Abudureheman Maihemuti  1   2 , Han Zhang  3 , Xiang Lin  3 , Yangyufan Wang  1   2 , Zhihong Xu  1   2 , Dagan Zhang  4 , Qing Jiang  1   2   5
Affiliations

3D-printed fish gelatin scaffolds for cartilage tissue engineering

Abudureheman Maihemuti et al. Bioact Mater. .

Abstract

Knee osteoarthritis is a chronic disease caused by the deterioration of the knee joint due to various factors such as aging, trauma, and obesity, and the nonrenewable nature of the injured cartilage makes the treatment of osteoarthritis challenging. Here, we present a three-dimensional (3D) printed porous multilayer scaffold based on cold-water fish skin gelatin for osteoarticular cartilage regeneration. To make the scaffold, cold-water fish skin gelatin was combined with sodium alginate to increase viscosity, printability, and mechanical strength, and the hybrid hydrogel was printed according to a pre-designed specific structure using 3D printing technology. Then, the printed scaffolds underwent a double-crosslinking process to enhance their mechanical strength even further. These scaffolds mimic the structure of the original cartilage network in a way that allows chondrocytes to adhere, proliferate, and communicate with each other, transport nutrients, and prevent further damage to the joint. More importantly, we found that cold-water fish gelatin scaffolds were nonimmunogenic, nontoxic, and biodegradable. We also implanted the scaffold into defective rat cartilage for 12 weeks and achieved satisfactory repair results in this animal model. Thus, cold-water fish skin gelatin scaffolds may have broad application potential in regenerative medicine.

Keywords: 3D printing; Cartilage defect repair; Fish skin gelatin; Sodium alginate; Tissue engineering.

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Conflict of interest statement

The authors declare no competing financial interests and agreed to author contributions statements below.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Scheme of 3D-printed cold-water fish skin gelatin scaffolds for cartilage tissue regeneration.
Fig. 2
Fig. 2
Rheological and mechanical properties of the fish gelatin/sodium alginate inks: (a) flow behavior; (b) viscosity-shear rate; (c) shear moduli-angular frequency; (d) printability tested by a series of different % W/V ink compositions; (e) stress-strain; (f) Young's modulus of the respective biomaterial inks with different % W/V; G0A6: 6% sodium alginate, G15A0: 15% fish skin gelatin, G15A3: 15/3% fish gelatin/sodium alginate, G15A6: 15/6% fish gelatin/sodium alginate, G15A10: 15/10% fish gelatin/sodium alginat.
Fig. 3
Fig. 3
Macro and micro views of the 3D-printed scaffolds: 3D-printed scaffolds, after crosslinking (a I), freeze-dried (a II), crosslinked and dried at room temperature (a III); stereo microscopic (b I-II) and SEM (b III) image of the scaffolds (scale bars: green: 500 μm, red: 200 μm); (c) front and side views (c I-II) of “ NJU ", the abbreviation of Nanjing University, various kinds of shapes (c III-VI). All scaffolds above were 3D-printed with 15/6% fish gelatin/sodium alginate biomaterial ink.
Fig. 4
Fig. 4
Evaluation of the biocompatibility of the 3D-printed fish gelatin scaffolds: (a) FDA/PI staining of C28 cells cultured on the 3D-printed scaffolds for 5 days, 6% sodium alginate (a I), 5/6% fish gelatin/sodium alginate (a II), and 15/6% fish gelatin/sodium alginate (a III) scaffolds; Phalloidin and DAPI staining of cells co-cultured on the 15/6% fish gelatin/sodium alginate (GA) scaffolds: (b I) fluorescence microscopic images of C28 cells cultured for 7days and (b II, III) confocal microscopic images of the primary chondrocytes cultured for 21 days; (c) the cell growth (OD value) of C28 cells cultured on C, A, and GA scaffolds for 5 days; western blot analysis (d) and relative gray scale (n = 3) (e) of Collagen II, Aggrecan, and SOX9 of the C28 cells cultured on the C, A, and GA scaffolds for 5 days; relative mRNA levels (n = 3) of SOX9 (f), Collagen II (g), and Aggrecan (h) of the C28 cells cultured on the C, A, and GA scaffolds for 5 days. C: control; A: 6% sodium alginate; GA: 15/6% fish gelatin/sodium alginate; scale bars: white (a I) - (a III) 100 μm, black (b I) 200 μm, green (b II) - (b III) 50 μm, p < 0.05.
Fig. 5
Fig. 5
Evaluation of immunogenicity (a)–(b) and degradation (c)–(e) of the biomaterial inks and scaffolds: H&E staining of the skin (a) and routine blood examination (b) of mice implanted with or without crosslinked biomaterial inks for 2 weeks (n = 4); examined parameters including white blood cell (WBC) count, red blood cell (RBC) count, lymphocyte (LYM), monocyte (MON), neutrophils (N) and their percentages; (c) small-animal imaging of the rats implanted with three kinds of Cy7 labeled biomaterials for 37days (n = 5); (d) in vitro degradation of the biomaterials immersed in enzymes for 2 weeks (n = 3); (e) quantification of small-animal imaging (c) (n = 3); (C: control, A: 6% sodium alginate 3D-printed scaffolds crosslinked with Ca2+, G: 15% cold-water fish skin gelatin crosslinked with EDC/NHS, GA: 15/6% fish gelatin/sodium alginate 3D-printed scaffolds crosslinked with Ca2+ and EDC/NHS). The scale bar is 100 μm.
Fig. 6
Fig. 6
Gross view and MRI image of the animal experiment (n = 5): (a) the creation of the rat knee cartilage defect model; (b) 3 months after implantation; (c) MRI of harvested knee joints of the rats at 3 months after implantation; (d) the statistical analysis of the Total ICRS score of the three groups; (e) the quantification and statistical analysis of the defected areas in MRI images. the groups were S (sham), C (control), A (6% sodium alginate scaffold), and GA (15%/6% fish gelatin/sodium alginate scaffold). The red circles in (b) demonstrate the initial range of the holes; the red arrows in (c) point to the defected part of the repaired holes.
Fig. 7
Fig. 7
(a–b), Representative H&E staining, Masson staining, and Safranin O-Fast Green staining (S–F), and their corresponding quantification of relative DNA content, relative collagen content, and relative GAG content (n = 3); (c–d) immunohistochemical staining of Aggrecan (ACAN) and collagen 2 (COL2), and their corresponding relative quantification (n = 3); (e–f) immunofluorescence staining of Aggrecan (ACAN) and collagen 2 (COL2), and their corresponding relative quantification (n = 3) at 3 months after implantation. The groups were S (sham), C (control), A (6% sodium alginate scaffold), and GA (15%/6% fish gelatin/sodium alginate scaffold). The yellow scale bar is 100 μm, and the white and black scale bars are 50 μm (p < 0.05).
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