Functionalized Collagen/Poly(ethylene glycol) Diacrylate Interpenetrating Network Hydrogel Enhances Beta Pancreatic Cell Sustenance

Abstract

Three-dimensional matrices are a new strategy used to tackle type I diabetes, a chronic metabolic disease characterized by the destruction of beta pancreatic cells. Type I collagen is an abundant extracellular matrix (ECM), a component that has been used to support cell growth. However, pure collagen possesses some difficulties, including a low stiffness and strength and a high susceptibility to cell-mediated contraction. Therefore, we developed a collagen hydrogel with a poly (ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN), functionalized with vascular endothelial growth factor (VEGF) to mimic the pancreatic environment for the sustenance of beta pancreatic cells. We analyzed the physicochemical characteristics of the hydrogels and found that they were successfully synthesized. The mechanical behavior of the hydrogels improved with the addition of VEGF, and the swelling degree and the degradation were stable over time. In addition, it was found that 5 ng/mL VEGF-functionalized collagen/PEGDA IPN hydrogels sustained and enhanced the viability, proliferation, respiratory capacity, and functionality of beta pancreatic cells. Hence, this is a potential candidate for future preclinical evaluation, which may be favorable for diabetes treatment.

Keywords: beta pancreatic cells; biomaterial; collagen; hydrogel; interpenetrating network; vascular endothelial factor.

Figure 1

FTIR spectra of acellular collagen hydrogel with PEGDA IPN. Collagen (2 mg/mL) was functionalized with 1, 3, or 5 ng/mL VEGF. The dotted lines with bold numbers correspond to functional groups associated with the VEGF peptide.

Figure 2

Microstructural behavior of collagen/PEGDA IPN hydrogels. (A) Scanning electron microscopy micrographs of functionalized acellular collagen/PEGDA IPN hydrogels (scale bar: 1 µm; 15,000×). (B) Pore size of hydrogels (μm) (n ≥ 3, median with interquartile range, Kruskal–Wallis, p < 0.05, Dunn’s multiple comparisons test * p < 0.05, ** p < 0.01).

Figure 3

Rheological analysis of acellular collagen hydrogel. (A) Amplitude and (B) frequency sweeps of 2 mg/mL of collagen functionalized with 1, 3, and 5 ng/mL of VEGF.

Figure 4

Hydrogel stability over time: (A) swelling (−); (B) water content; and (C) degradation (%) performance of the functionalized collagen/PEGDA IPN hydrogels (n ≥ 3, mean ± SEM; one-way ANOVA, p > 0.05).

Figure 5

Biocompatibility assays of functionalized hydrogels with encapsulated cells: (A) Live and dead assay of encapsulated beta pancreatic cells in VEGF-functionalized collagen (2 mg/mL)/PEGDA IPN hydrogels after 48 h (scale bar in red: 200 µm); (B) Scanning electron microscopy (SEM) images of cells encapsulated in collagen/PEGDA IPN matrix (scale bar: 5 µm); (C) Viability of encapsulated β pancreatic cells in VEGF-functionalized collagen hydrogel crosslinked with PEGDA IPN after 48 h. The combination of 2 mg/mL and 5 ng/mL of VEGF resulted in the highest cell viability; (D) DNA content of encapsulated beta pancreatic cells in VEGF-functionalized collagen/PEGDA IPN hydrogel after 72 h (n ≥ 3, one-way ANOVA, p < 0.05, *** p < 0.001 vs. cell culture plate.

Figure 6

The effect on oxygen flow in all experimental groups was expressed as O₂ flow pmol/(s × 10⁶ cells). One-way ANOVA, n ≥ 3, * p < 0.05, ** p < 0.01, vs. CCP, # p < 0.05 vs. 2 mg/mL.

Figure 7

Functional behavior of beta pancreatic cells in response to low and high glucose concentrations in collagen/PEGDA IPN hydrogels: (A) insulin secretion by encapsulated pancreatic beta cells is expressed as μIU/mL×μg DNA; (B) insulin secretion index. One-way ANOVA, p < 0.05, n ≥ 3, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. CCP. Data points represent the mean ± SEM.