In vitro maturation and in vivo stability of bioprinted human nasal cartilage

Abstract

The removal of skin cancer lesions on the nose often results in the loss of nasal cartilage. The cartilage loss is either surgically replaced with autologous cartilage or synthetic grafts. However, these replacement options come with donor-site morbidity and resorption issues. 3-dimensional (3D) bioprinting technology offers the opportunity to engineer anatomical-shaped autologous nasal cartilage grafts. The 3D bioprinted cartilage grafts need to embody a mechanically competent extracellular matrix (ECM) to allow for surgical suturing and resistance to contraction during scar tissue formation. We investigated the effect of culture period on ECM formation and mechanical properties of 3D bioprinted constructs of human nasal chondrocytes (hNC)-laden type I collagen hydrogel in vitro and in vivo. Tissue-engineered nasal cartilage constructs developed from hNC culture in clinically approved collagen type I and type III semi-permeable membrane scaffold served as control. The resulting 3D bioprinted engineered nasal cartilage constructs were comparable or better than the controls both in vitro and in vivo. This study demonstrates that 3D bioprinted constructs of engineered nasal cartilage are feasible options in nasal cartilage reconstructive surgeries.

Keywords: Skin cancer; bioprinting; hydrogel; nasal cartilage; nude mice; septal chondrocytes; tissue engineering.

Figure 1.

Schematic diagram of experimental design.

Figure 2.

Gross morphology of the FRESH printed structure. (a) 3D model of a right lower lateral nasal cartilage from CT imaging and (b) the preview of the sliced nasal cartilage using Slic3r software. (c) 3D bioprinted lower lateral nasal cartilage in gelatin support bath before and (d) after 30 min incubation in 37°C. Following the 30-min incubation, the support bath was aspirated, and PBS was added.

Figure 3.

(a) Live/dead assay (b) cell viability over culture time. Paired t-tests were done to compare cell viability between day 1 versus 3, 3 versus 6 weeks, and 6 versus 9 weeks. *Represents 0.01 < p < 0.05. Scale bar: 100 µm.

Figure 4.

Histological and biochemical analysis of in vitro constructs across culture time. (a) Safranin-O/Fast green Staining, (b) Masson’s Trichrome staining, (c) GAG/DNA of in vitro constructs and (d) Bern Score of Safranin-O staining. Black arrows indicate tissue areas that have (a) positive Safranin-O staining for aggrecan or (b) positive aniline blue for collagen (b). Data were analyzed by two-way ANOVA and corrected with the Bonferroni post hoc test. GAG: glycosaminoglycan; NS: non-significant; WW: wet weight. Scale bar: 100 µm. Star (*) represent the significant difference with regarding of culture time after Bonferroni post hoc correction: * represents 0.01 < p < 0.05, ** represents 0.001 < p<0.01. Pound (#) represent the significant difference with regarding of scaffold type after Bonferroni post hoc correction: # represents 0.01 < p < 0.05.

Figure 5.

Immunofluorescence of in vitro constructs across culture time. (a) Type I (red) and II (green) collagen and (b) Type × collagen (red). The blue color is from DAPI staining, which indicate cell nuclei. Scale bar: 100 µm.

Figure 6.

(a) Suturability of Chondro-Gide scaffolds and bioprinted constructs across culture time. Images are taken at 0.65x and 1.60x magnification. (b) Bending modulus of in vitro constructs across culture time. Data was analyzed by two-way ANOVA and corrected with the Bonferroni post hoc test. NS: non-significant. Scale bars: 6–3 mm for 0.65x and 1.60x, respectively. Star (*) represent the significant difference with regarding of culture time after Bonferroni post hoc correction: ** represents 0.001 < p<0.01, *** represents 0.0001 < p <0.001, **** represents p < 0.00001. Pound (#) represent the significant difference with regarding of scaffold type after Bonferroni post hoc correction: # represents 0.01 < p < 0.05.

Figure 7.

SEM imaging of in vitro constructs across culture time. Magnification of images is 35x, 100x, 1000x, and 2000x. Scale bars are 100, 10, 2 µm for 35x/100x, 1000x, and 2000x, respectively.

Figure 8.

Gene expression of in vitro constructs. Values shown are 2-ΔCt values from RT-qPCR. Statistics were done using ΔCt values. Data was analyzed by two-way ANOVA and corrected with the Bonferroni post hoc test. Housekeeping genes used were ACTB, B2M, and YWHAZ. n = 6 donors (in duplicate). NS: non-significant. Star (*) represent the significant difference with regarding of culture time after Bonferroni post hoc correction: * represents 0.01 < p < 0.05, ** represents 0.001 < p <0.01, *** represents 0.0001 < p <0.001, **** represents p < 0.00001. Pound (#) represent the significant difference with regarding of scaffold type after Bonferroni post hoc correction: # represents 0.01 < p < 0.05, ### represents 0.0001 < p < 0.001.

Figure 9.

(a) Gross morphology of constructs and scaffolds before and after implantation. (b) Histology and immunofluorescence of in vivo bone formation proteins, including type × collagen (red represents positive type × collagen, which is a marker of chondrocyte hypertrophy), CD31 (green represents positive CD31, CD31 is a marker of angiogenesis), BSP (red represents positive bone sialoprotein formation), and Alizarin Red (orange color represents positive calcification). COL10; type × collagen, CD31; cluster of differentiation 31, BSP; bone sialoprotein. Scale bar: 100 µm.

Figure 10.

Histology and immunofluorescence of chondrogenic related proteins, including Safranin-O/Fast Green straining, Masson’s Trichrome staining, and type I and II collagens immunofluorescence. Scale bar: 100 µm.