Investigation of the In Vitro and In Vivo Biocompatibility of a Three-Dimensional Printed Thermoplastic Polyurethane/Polylactic Acid Blend for the Development of Tracheal Scaffolds

Abstract

Tissue-engineered polymeric implants are preferable because they do not cause a significant inflammatory reaction in the surrounding tissue. Three-dimensional (3D) technology can be used to fabricate a customised scaffold, which is critical for implantation. This study aimed to investigate the biocompatibility of a mixture of thermoplastic polyurethane (TPU) and polylactic acid (PLA) and the effects of their extract in cell cultures and in animal models as potential tracheal replacement materials. The morphology of the 3D-printed scaffolds was investigated using scanning electron microscopy (SEM), while the degradability, pH, and effects of the 3D-printed TPU/PLA scaffolds and their extracts were investigated in cell culture studies. In addition, subcutaneous implantation of 3D-printed scaffold was performed to evaluate the biocompatibility of the scaffold in a rat model at different time points. A histopathological examination was performed to investigate the local inflammatory response and angiogenesis. The in vitro results showed that the composite and its extract were not toxic. Similarly, the pH of the extracts did not inhibit cell proliferation and migration. The analysis of biocompatibility of the scaffolds from the in vivo results suggests that porous TPU/PLA scaffolds may facilitate cell adhesion, migration, and proliferation and promote angiogenesis in host cells. The current results suggest that with 3D printing technology, TPU and PLA could be used as materials to construct scaffolds with suitable properties and provide a solution to the challenges of tracheal transplantation.

Keywords: biocompatibility; degradation; inflammatory response; pH; polylactic acid; thermoplastic polyurethane.

Figure 1

(a) Photographic images of the constructed disc-shaped scaffold. Images of TPU, TPU/PLA, and PLA scaffolds from the top view. Scale bar: 1 cm. (b) SEM images of the disc-shaped scaffolds from the surface and cross-sectional views of all scaffolds. Scale bar: 1 mm.

Figure 2

(a) The degradation rate of porous 3D-printed scaffolds. The TPU scaffold showed the highest degradation rate compared to other scaffolds. Moreover, the longer the time period, the higher the degradation rate. (b) pH analysis of the 3D printed scaffold extracts. The PLA extract showed no significant difference compared to PBS. However, in the first two weeks, a significant decrease in pH was observed in both the TPU and TPU/PLA extracts, which gradually reduced to below pH 7 after eight weeks.

Figure 3

(a) Indirect proliferation assay using scaffold extracts. The X-axis represents the incubation time in days, while the Y-axis represents the optical density values of the BEAS-2B cells. In general, the proliferation of BEAS-2B cells was almost consistent from day 1 to day 3 but increased dramatically on day 5 and slowed down on day 7 for all types of scaffolds. Significant proliferation activities were noted between all types of scaffolds over time. (b) The proliferation of BEAS-2B cells in immersion media of all scaffold types. Phase contrast micrographs showing the percentage of confluence of proliferating BEAS-2B cells at days 1, 3, 5, and 7. It was noted that the confluences of the cells in all groups were almost similar. Scale bar: 500 µm.

Figure 4

(a) BEAS-2B cell scratch analysis. (b) The effects of extracts from the 3D printed scaffolds on the migration of BEAS-2B cells. The scratches were made and the cells were treated with different scaffold extracts. Images of the wound area were taken 0, 24, and 48 h after treatment and measured using ImageJ analysis software. Scale bar: 500 µm.

Figure 5

Direct cell attachment assay using Hoechst 33342. The fluorescence image shows that cells had attached to all three scaffold surfaces on day 3. On day 7, more cells were visible, especially between the PLA filaments. The cells appeared slightly out of focus as they were inside the scaffold folds. Scale bar: 200 µm.

Figure 6

SEM images of the cells and tissues following subcutaneous implantation at different time points. (a) TPU at week 1, (b) TPU/PLA at week 1, (c) PLA at week 1, (d) TPU at week 4, (e) TPU/PLA at week 4, (f) PLA at week 4, (g) TPU at week 8, (h) TPU/PLA at week 8, and (i) PLA at week 8. Scale bar 50 µm.

Figure 7

(a) Photomicrograph of tissue adjacent to the implanted scaffold showing inflammatory reaction in rat skin. Histological sections of a representative subcutaneous area were stained with H&E at 4× magnification. (b) Semi-quantitative scoring of the number of inflammatory cells in rats. The number of inflammatory cells at (a) week 1, (b) week 4, and (c) week 8. The X-axis represents the animal groups, while the Y-axis indicates the number of inflammatory cells. At week 1, inflammatory responses were significantly different and highest in all experimental groups compared to the naïve group, significantly reduced at week 4, and gradually decreased at week 8, with mild inflammation present. One-way ANOVA with Tukey’s multiple comparison test was used for comparison between groups. Data are presented as mean ± SD, n = 2 animals per group, **** p < 0.0001.

Figure 8

(a) Vascularisation of the tissue adjacent to the implanted scaffold at week 1. H&E staining shows blood vessels in the tissue adjacent to the implanted scaffold. The green arrow indicates the blood vessels. The highest number of blood vessels was observed in the sham and PLA groups. (b) Semi-quantitative analysis of vascularisation at different time points. Week 1, week 4, and week 8. The X-axis represents the grouping of animals, and the Y-axis represents the number of blood vessels per area. Compared to the naïve group, vascularisation at week 1 showed minimal changes in both the TPU and PLA groups, while it was significantly the highest in the sham group. However, a significant increase was observed in week 4, which was highest in PLA, followed by the TPU/PLA blend, and TPU. Finally, vascularisation was almost back to baseline at week 8. One-way ANOVA with Tukey’s multiple comparison tests was used for comparison between groups. Data are presented as mean ± SD, n = 2 animals per group, * p < 0.05, ** p < 0.01 **** p < 0.0001.