Hydrogel modification of 3D printing hybrid tracheal scaffold to construct an orthotopic transplantation

Abstract

Objective: To evaluate the biological properties of modified 3D printing scaffold (PTS) and applied the hybrid graft for in situ transplantation.

Methods: PTS was prepared via 3D printing and modified by Pluronic F-127. Biocompatibility of the scaffold was examined in vitro to ascertain its benefit in attachment and proliferation of bone marrow mesenchymal stem cells (BMSCs). Moreover, a hybrid trachea was constructed by combining the modified PTS with decellularized matrix. Finally, two animal models of in situ transplantation were established, one for repairing tracheal local window-shape defects and the other for tracheal segmental replacement.

Results: The rough surface and chemical elements of the scaffold were improved after modification by Pluronic F-127. Results of BMSCs inoculation showed that the modified scaffold was beneficial to attachment and proliferation. The epithelial cells were seen crawling on and attaching to the patch, 30 days following prothetic surgery of the local tracheal defects. Furthermore, the advantages of the modified PTS and decellularized matrix were combined to generate a hybrid graft, which was subsequently applied to a tracheal segmental replacement model.

Conclusion: Pluronic F-127-based modification generated a PTS with excellent biocompatibility. The modified scaffold has great potential in development of future therapies for tracheal replacement and reconstruction.

Keywords: 3D printing scaffold; hydrogel carrier; surface modification; tissue engineering tracheal transplantation.

Figure 1

The process for creating porous tracheal scaffolds by 3D-printing technology (A: Tubular porous structure). The different rotation angles produced different printing structures, which were displayed from (B-E). The stitching (F) and forming (G) of hybrid scaffold.

Figure 8

The specimens of group D3 harvested at day 30 (A, B). Profiles of the epithelium’s states following fiberoptic bronchoscopy (C: group D3 at 30 days. D: D3 group at 60 days). Postoperative IHC staining of CK-18 (E, F: substitution site in group D3, black arrows indicate the same part inside the lumen. Magnification: C: ×40, D: ×400).

Figure 2

The EDS analysis of the composition ratio of each element on the surface of the scaffold (A: Unmodified scaffold, D: Modified scaffold). The results of SEM observation (B, C: Unmodified PCL scaffolds. E, F: Modified PCL scaffolds. G, H: Appearance of the gel at high magnification. Magnification: B, E: ×80, F: ×600, G: ×5000, H: ×30,000).

Figure 3

The cell appearance on the scaffold of each group for 48 h after inoculation. From top to bottom: A1, B1, C1 (Magnification: A, E and I: ×1,000; B, F and J: ×3,000). The appearance of stained cells following their inoculation around the scaffold. (Magnification: C, G and K: ×40; D, H and L: ×100).

Figure 4

Intraoperative conditions across in group C2 (A: The suturing was done), and the representative photographs of harvested specimen (B: At POD 30 in group C2). The view of fiberoptic bronchoscopy (C: Fresh trachea, D: Trachea of individuals in group C2 at POD 30, “M” indicates the tracheal membrane). H&E staining for identifying repair site in segments of the cartilage ring. The consecutive photographs of the repaired area in group C2 (E-G: Black arrow indicated the native tracheal epithelium, the yellow arrow is the new epithelial cell layer on the inner surface of the matrix. Magnification: ×40). The appearance of cell strips outside the matrix of group C2 (H: The red arrow indicates, the “C” shape: cartilage rings. Magnification: ×200).

Figure 5

IHC staining of CK-18 consecutive photos of repair sites (yellow arrow) in group C2 (A), partial enlargement of three different areas of (A) (B, C and D, black arrow: the native tracheal epithelium. Magnification: A: ×40, B-D: ×100). The postoperative IHC staining of type-II collagen. (Red arrows: positive cells; “C” shape: cartilage rings. Magnification E: ×100, F: ×400).

Figure 6

Intraoperative conditions across in group C2 (A: Addition of the gel containing growth factors and stem cells onto the surface). The photograph of harvested specimen (B: At POD 90 in group C2). The view of fiberoptic bronchoscopy (C: Trachea of individuals in group C2 at POD 90, “M” indicates position of the tracheal membrane). HE staining revealed newer blood vessels and glandular tissues between the two cartilage rings (the repair site) compared to those at 30 days (D, E, red mark). CK-18 staining showed that the continuous epithelial cell covered in the lumen (F: yellow arrow, “C” shape: cartilage rings; black arrows indicate the native tracheal epithelium. Magnification: D, F: ×40, E: ×200).

Figure 7

The appearance of tissues during the operation following anastomosis (A: group A3, B: group B3, C: group C3, D: group D3). Post-operative tissue harvesting (E: Bioengineered matrices harvested at the observation time point across the four groups). Postoperative H&E staining (F: group A3, G: group B3, H: group C3, I: group D3. Black arrow indicates the inside lumen, “C” shape: cartilage rings. Magnification: ×100).