Reinforcement of Mono- and Bi-layer Poly(Ethylene Glycol) Hydrogels with a Fibrous Collagen Scaffold

Abstract

Biomaterial-based tissue engineering strategies hold great promise for osteochondral tissue repair. Yet significant challenges remain in joining highly dissimilar materials to achieve a biomimetic, mechanically robust design for repairing interfaces between soft tissue and bone. This study sought to improve interfacial properties and function in a bi-layer hydrogel interpenetrated with a fibrous collagen scaffold. 'Soft' 10% (w/w) and 'stiff' 30% (w/w) PEGDM was formed into mono- or bi-layer hydrogels possessing a sharp diffusional interface. Hydrogels were evaluated as single-(hydrogel only) or multi-phase (hydrogel + fibrous scaffold penetrating throughout the stiff layer and extending >500 μm into the soft layer). Including a fibrous scaffold into both soft and stiff mono-layer hydrogels significantly increased tangent modulus and toughness and decreased lateral expansion under compressive loading. Finite element simulations predicted substantially reduced stress and strain gradients across the soft-stiff hydrogel interface in multi-phase, bilayer hydrogels. When combining two low moduli constituent materials, composites theory poorly predicts the observed, large modulus increases. These results suggest material structure associated with the fibrous scaffold penetrating within the PEG hydrogel as the major contributor to improved properties and function-the hydrogel bore compressive loads and the 3D fibrous scaffold was loaded in tension thus resisting lateral expansion.

Keywords: Hydrogel; Interface; Mechanical properties; Multi-phase; Osteochondral; Scaffold; Tissue engineering.

Figure 1

Mono-layer hydrogels (height = 5 mm, diameter = 5 mm) were fabricated from soft 10% (w/w) or stiff 30% (w/w) PEGDM and bi-layer hydrogels consisted of a soft layer polymerized onto an underlying stiff layer of equivalent height (each layer height = 2.5 mm). Mono-layer hydrogels consisted of either hydrogel only (single-phase) or included a fibrous scaffold (multi-phase) that largely spanned the width and height of the construct. In the bi-layer hydrogels, the fibrous scaffold spanned the stiff layer and penetrated to ∼ 500 – 1000 μm into the soft layer.

Figure 2

30× (A; scale bar = 500 μm) and 90× (B; scale bar = 200 μm) SEM images of the fibrous scaffold, without any infilling hydrogel showing regularity, high porosity and continuity of the fibers throughout the scaffold as well as interconnected pore spaces. (C) A representative confocal microscopy image of the interface for a bi-layer hydrogel formed with a bottom layer from 30% PEGDM (green) and a top layer from 10% PEGDM (red) where the interface extends beyond the yellow band, and is denoted by the total distance across which both fluorophore channels detected fluorescence signal over the background; scale bar = 150 μm. The interface, a diffusional layer formed by mixing of the 10% (w/w) and 30% (w/w) PEGDM hydrogels, was evaluated to be 74 (16) μm as determined by the intensity; representative intensity plot in panel D. (E) 25× SHG image (Field Width = 359 μm) of the multi-phase, bi-layer hydrogel shows the 10% PEG (red) and 30% PEG (green) in pore spaces within the fibrous, collagen sponge (showing as pink due to image processing artifacts).

Figure 3

Representative stress vs. strain plots for mono-layer, stiff (green) and soft (orange) hydrogels (A). Toughness was measured as the total area under each curve indicate to 20% strain for stiff constructs and 35% strain for soft hydrogels (B); inclusion of the fibrous scaffold imparted greater toughness for both stiff and soft multi-phase hydrogels. Table inset shows mean and (SD) for each material formulation. * indicates statistically significant difference (p < 0.05) between single-phase vs. multi-phase for the same hydrogel formulation.

Figure 4

Inclusion of a fibrous scaffold into multi-phase, mono-layer constructs reduced the lateral expansion under unconfined compressive axial loading in stiff (green) and soft (orange) as compared to single-phase, mono-layer constructs. Lateral expansion data are a ratio of the increase in sample width to the decrease in sample height and were measured at 40 % compressive strain.

Figure 5

Tangent modulus plotted vs. strain increment for single- and multi-phase, bi-layer hydrogels. No statistically significant differences were observed when including the fibrous scaffold within the bi-layer hydrogels (Left). The apparent similarities in measured tangent modulus likely resulted from the high expansion in the soft, 10% (w/w) PEG phase relative to the low expansion of the stiff, 30% (w/w) PEG layer (Right).

Figure 6

Comparison of finite element modeling results for single-phase, bi-layer (Left Column) and multi-phase (hydrogel + fibrous scaffold; middle column) PEG hydrogels showing contour plots of (A, B) maximum principal strain (note: stress contour between two lowest strain values are drawn to permit clear visualization), (C, D) maximum shear strain, (E, F) Von Mises stress (kPa), and (G, H) lateral displacement (mm). Finite element modeling shows a 4.2% increase in the compressive modulus of the bi-layered samples with inclusion of the fibrous scaffold throughout the stiff, 30% (w/w) PEG layer and penetrating 500 micrometers into the top, soft 10% (w/w) PEG layer (denoted by **).

Figure 6

Comparison of finite element modeling results for single-phase, bi-layer (Left Column) and multi-phase (hydrogel + fibrous scaffold; middle column) PEG hydrogels showing contour plots of (A, B) maximum principal strain (note: stress contour between two lowest strain values are drawn to permit clear visualization), (C, D) maximum shear strain, (E, F) Von Mises stress (kPa), and (G, H) lateral displacement (mm). Finite element modeling shows a 4.2% increase in the compressive modulus of the bi-layered samples with inclusion of the fibrous scaffold throughout the stiff, 30% (w/w) PEG layer and penetrating 500 micrometers into the top, soft 10% (w/w) PEG layer (denoted by **).