3D Bioprinted Artificial Trachea with Epithelial Cells and Chondrogenic-Differentiated Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

Tracheal resection has limited applicability. Although various tracheal replacement strategies were performed using artificial prosthesis, synthetic stents and tissue transplantation, the best method in tracheal reconstruction remains to be identified. Recent advances in tissue engineering enabled 3D bioprinting using various biocompatible materials including living cells, thereby making the product clinically applicable. Moreover, clinical interest in mesenchymal stem cell has dramatically increased. Here, rabbit bone marrow-derived mesenchymal stem cells (bMSC) and rabbit respiratory epithelial cells were cultured. The chondrogenic differentiation level of bMSC cultured in regular media (MSC) and that in chondrogenic media (d-MSC) were compared. Dual cell-containing artificial trachea were manufactured using a 3D bioprinting method with epithelial cells and undifferentiated bMSC (MSC group, n = 6) or with epithelial cells and chondrogenic-differentiated bMSC (d-MSC group, n = 6). d-MSC showed a relatively higher level of glycosaminoglycan (GAG) accumulation and chondrogenic marker gene expression than MSC in vitro. Neo-epithelialization and neo-vascularization were observed in all groups in vivo but neo-cartilage formation was only noted in d-MSC. The epithelial cells in the 3D bioprinted artificial trachea were effective in respiratory epithelium regeneration. Chondrogenic-differentiated bMSC had more neo-cartilage formation potential in a short period. Nevertheless, the cartilage formation was observed only in a localized area.

Keywords: artificial trachea; bone marrow-derived mesenchymal stem cell; chondrogenic differentiation; three-dimensional bioprinting; tissue engineering.

Figure 1

Relative GAG accumulation and chondrogenic marker gene expression level of MSC and d-MSC. (A) Alcian blue-stained dissolvents in a 96-well assay plate; the d-MSC group showed a deeper blue color than the MSC group; (B,C) Alcian blue absorbance on days 14 and 28. The d-MSC group showed 1.67 ± 0.10 (day 14) and 2.62 ± 0.11 (day 28) times higher values than the MSC group. The relative gene expression levels of SOX9 (D); aggrecan (E); collagen type 1 (F) and collagen type 2 (G) were increased in d-MSC. CON = expression level of chondrocyte as positive control. ** p < 0.01, *** p < 0.001.

Figure 2

3D bioprinted artificial trachea. (A) Longitudinal view, 15 mm in length; (B) Vertical view, five-layered structure with a 5-mm inner diameter and 10-mm outer diameter; (C) Magnified structure of the scaffold (SEM image). (a,c,e) layers are composed of PCL. (b) is alginate layer with MSC or d-MSC and (d) is alginate layer with epithelial cells. The innermost (e) and outermost (a) layers showed a microporous feature. The non-porous third layer (c) separates the epithelial cell layer (d) and the MSC or d-MSC layer (b). The scale bar indicates 200 µm.

Figure 3

Cell distribution in alginate hydrogel with CellTracker™. (A) Epithelial cell in the second alginate hydrogel layer (red); (B) MSC in fourth alginate hydrogel layer (green); (C) A and B (merged). Each cell was well-printed layer by layer and separated completely.

Figure 4

Radiographic findings. Plain lateral thoracic view of the MSC (A) and d-MSC (B) groups and 3D reconstructed CT image of the MSC (C) and d-MSC (D) groups at 12 weeks after 3D bioprinted artificial trachea transplantation. Well-sustained tracheal contour was observed with no signs of stenosis or obstruction.

Figure 5

Bronchoscopic findings. Bronchoscopic images of MSC group (A) and d-MSC group (B) were obtained 12 weeks after the surgery. Tracheal lumen fully covered with epithelial mucosa was observed. Remaining suture materials indicate the implant fixation site.

Figure 6

Histopathologic findings. Microscopic images of MSC group (A) and d-MSC group (B) showed newly formed respiratory epithelium in both groups. Regenerated epithelium in A and B had a rough cell arrangement compared with that of the normal trachea (C) but ciliated columnar epithelium was confirmed (Hematoxylin and eosin staining; all scale bars indicate 50 µm).

Figure 6

Histopathologic findings. Microscopic images of MSC group (A) and d-MSC group (B) showed newly formed respiratory epithelium in both groups. Regenerated epithelium in A and B had a rough cell arrangement compared with that of the normal trachea (C) but ciliated columnar epithelium was confirmed (Hematoxylin and eosin staining; all scale bars indicate 50 µm).

Figure 7

Neo-cartilage formation and neo-vascularization. Neo-vascularization and neo-cartilage formation was observed in the d-MSC group (A,B). However, in the MSC group, neo-vascularization was seen but no cartilaginous islet was observed (C). Newly formed immature cartilage islet (yellow arrows) had higher cellular density and lighter proteoglycan staining compared with the cartilage of the normal trachea (D). A, C and D = hematoxylin and eosin staining; B = safranin O staining. Asterisks (*) indicate fifth polycaprolactone (PCL) layer and all scale bars indicates 50 µm).

Figure 8

Application of 3D bioprinted artificial trachea. (A) Approximately 10 × 10-mm half-pipe-shaped tracheal defect on the ventral part of the trachea. (B) The defect was replaced with 3D bioprinted artificial trachea and sutured with 5–0 absorbable suture material.