Cross-linking manipulation of waterborne biodegradable polyurethane for constructing mechanically adaptable tissue engineering scaffolds

Abstract

Mechanical adaptation of tissue engineering scaffolds is critically important since natural tissue regeneration is highly regulated by mechanical signals. Herein, we report a facile and convenient strategy to tune the modulus of waterborne biodegradable polyurethanes (WBPU) via cross-linking manipulation of phase separation and water infiltration for constructing mechanically adaptable tissue engineering scaffolds. Amorphous aliphatic polycarbonate and trifunctional trimethylolpropane were introduced to polycaprolactone-based WBPUs to interrupt interchain hydrogen bonds in the polymer segments and suppress microphase separation, inhibiting the crystallization process and enhancing covalent cross-linking. Intriguingly, as the crosslinking density of WBPU increases and the extent of microphase separation decreases, the material exhibits a surprisingly soft modulus and enhanced water infiltration. Based on this strategy, we constructed WBPU scaffolds with a tunable modulus to adapt various cells for tissue regeneration and regulate the immune response. As a representative application of brain tissue regeneration model in vivo, it was demonstrated that the mechanically adaptable WBPU scaffolds can guide the migration and differentiation of endogenous neural progenitor cells into mature neurons and neuronal neurites and regulate immunostimulation with low inflammation. Therefore, the proposed strategy of tuning the modulus of WBPU can inspire the development of novel mechanically adaptable biomaterials, which has very broad application value.

Keywords: central nervous repair; mechanical adaptation; modulus; tissue engineering scaffold; waterborne polyurethane.

Graphical abstract

Figure 1.

(A) Schematic representation of tuning the stiffness of waterborne biodegradable polyurethane (WBPU) and the mechanically adaptable mechanism of 3D porous WBPU scaffolds for regulating cell behavior. (B) DSC curves (in heating) of WBPU films; only the P25T0 film without PCDL and TMP exhibits a melting peak at ∼25°C. (C) Water absorption process of WBPU films. (D) ATR-FTIR spectra of the PxTy films in dry and wet states. The W in PxTy-W designates the wet state.

Figure 2.

(A) 3D Porous microstructures of P17Ty scaffolds. (B) Young’s moduli of P17Ty scaffolds in dry/wet states and the corresponding softening ratios. (C) Photographs of P17Ty scaffolds after water absorption. (D) Typical heating curves of P17T10 water-saturated and water-unsaturated scaffolds. (E) Cyclic compressive curves of P17Ty scaffolds after immersion in PBS for 20 days. (F) Porous microstructures of PxTy scaffolds after immersion in PBS for 20 days and 10 times cyclic compression.

Figure 3.

(A) Cell morphology of PC12 cells in the P17Ty scaffolds. (B) Cell morphology of HUVEC cells in the P17Ty scaffolds. (C) Cell morphology of L929 cells in the P17Ty scaffolds. F-actin is stained with rhodamine-phalloidin, red; cell nuclei are stained with DAPI, blue (scale bars were 100 μm) (D) Statistics of length of PC12 nerves in scaffolds with different modulus (n = 25). (E) statistics of HUVEC cells spreading area in scaffolds with different modulus (n = 25). (F) statistics of L929 cells spreading area in scaffolds with different modulus (n = 25). ***P < 0.001.

Figure 4.

(A) Morphology of PC12 on the P17Ty films, scale bars: 20 μm; (B) the cell differentiation rate of PC12 on the P17Ty films; (C) proliferation of PC12 cells on the P17Ty films after 1, 4 and 6 days. (D) Immunofluorescence of PC12 on PU films (p-FAK: green; F-actin: red; nucleus: blue), the green spot in the cartoon picture represents the anchoring spot by adhesion proteins, scale bars: 25 μm; (E) WB results of PC12 on the P17Ty films. (F) relative expression of GAP-43 on P17Ty films. *P < 0.05, # no significant.

Figure 5.

(A) Morphology of BV2 on the P17Ty scaffolds with (+) and without (−) LPS, scale bars: 100 μm; (B) the concentration of TNF-alpha and IL-10 produced by BV2 on the P17Ty scaffolds, and the ratio of IL-10 to TNF-alpha; (C) WB results and the protein expression of BV2 on the P17Ty scaffolds. *P < 0.05, **P < 0.01, ***P < 0.001, # no significant.

Figure 6.

(A) Schematic procedure of the TBI model showing a scaffold (brownish cylinder) implanted into the traumatic brain cavity and typical digital photographs of brain defects and implanting surgery. (B) Representative photographs of damaged brain tissue treated with different scaffolds post-injury on the 14th day. The damaged zones are pointed out by arrows. GEL and CTR represent the PU hydrogel and control groups, respectively. (C) Microglia (IBA-1, red) and CD 86 cells (green) on scaffolds and control groups. Notably, all the implanting materials are below the white line. (D) Microglia (IBA-1, red) and CD 206 cells (green) on scaffolds and control groups. Microglia and immune cells are mainly located in the inner pores of the scaffolds but located on the boundary of traumatic cavity lesions in the hydrogel and control groups. (E) Endogenous neural progenitor cells (NPCs) (DCX, red) and new neurons (Tuj1, green) in the scaffolds and control groups. Especially some new neurons (marked by the arrow) are located inside the P17T10 scaffold (below the white line). (F) Survival of mature neurons (MAP2, red) and neuronal neurites containing synaptophysin (SYP, green) in the scaffolds and control groups. Survival of mature neurons and neurites (marked by arrow) appearing in traumatic cavity lesions and scaffold pores; scale bars: 200 μm. (G) Fluorescence area of CD 86 cells and CD 206 cells on scaffolds and control groups. (H) Fluorescence area of DCX and Tuj1 on scaffolds and control groups. (I) Fluorescence area of MAP2 and SYP on scaffolds and control groups. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.