Engineering meniscus structure and function via multi-layered mesenchymal stem cell-seeded nanofibrous scaffolds

Abstract

Despite advances in tissue engineering for the knee meniscus, it remains a challenge to match the complex macroscopic and microscopic structural features of native tissue, including the circumferentially and radially aligned collagen bundles essential for mechanical function. To mimic this structural hierarchy, this study developed multi-lamellar mesenchymal stem cell (MSC)-seeded nanofibrous constructs. Bovine MSCs were seeded onto nanofibrous scaffolds comprised of poly(ε-caprolactone) with fibers aligned in a single direction (0° or 90° to the scaffold long axis) or circumferentially aligned (C). Multi-layer groups (0°/0°/0°, 90°/90°/90°, 0°/90°/0°, 90°/0°/90°, and C/C/C) were created and cultured for a total of 6 weeks under conditions favoring fibrocartilaginous tissue formation. Tensile testing showed that 0° and C single layer constructs had stiffness values several fold higher than 90° constructs. For multi-layer groups, the stiffness of 0°/0°/0° constructs was higher than all other groups, while 90°/90°/90° constructs had the lowest values. Data for collagen content showed a general positive interactive effect for multi-layers relative to single layer constructs, while a positive interaction for stiffness was found only for the C/C/C group. Collagen content and cell infiltration occurred independent of scaffold alignment, and newly formed collagenous matrix followed the scaffold fiber direction. Structural hierarchies within multi-lamellar constructs dictated biomechanical properties, and only the C/C/C constructs with non-orthogonal alignment within layers featured positive mechanical reinforcement as a consequence of the layered construction. These multi-layer constructs may serve as functional substitutes for the meniscus as well as test beds to understand the complex mechanical principles that enable meniscus function.

Keywords: Electrospinning; Mechanical properties; Meniscus; Nanofibrous scaffold; Tissue engineering.

Figure 1

Schematic of experimental groups. A) Individual scaffolds of varying fiber alignment (fibers parallel to scaffold long axis (0°), fibers perpendicular to scaffold long axis (90°), and comprised of circumferential (C) fibers) were seeded with juvenile bovine mesenchymal stem cells (MSCs) and cultured as single layer or multi-layer constructs. B) Assembly method for the production and culture of multi-layer constructs. C) Illustration of multi-layer groups with different fiber alignment in each layer.

Figure 2

Tensile stiffness of single layer and multi-layer constructs after 3 and 6 weeks of culture (*p<0.05 vs. 3w time point, +p<0.05 between experimental groups at both time points, #p<0.05 between experimental groups at 3 weeks, $p<0.05 between experimental groups at 6 weeks).

Figure 3

Tensile modulus of single layer and multi-layer constructs after 3 and 6 weeks of culture (*p<0.05 vs. 3w time point, +p<0.05 between experimental groups at both time points, #p<0.05 between experimental groups at 3 weeks, $p<0.05 between experimental groups at 6 weeks).

Figure 4

Histological analysis of collagen deposition (picrosirius red staining, top row) and cell infiltration (DAPI staining, bottom row) in single layer constructs after 6 weeks of culture (scale bars = 200 µm).

Figure 5

Histological analysis of collagen deposition (picrosirius red staining, top row) and cell infiltration (DAPI staining, bottom row) in multi-layer constructs after 6 weeks of culture (scale bars = 200 µm).

Figure 6

Collagen alignment within multi-layer constructs assessed by polarized light microscopy. Angled sections show collagen fiber alignment within and between layers (arrows indicate scaffold layer alignment, scale bars = 100 µm).

Figure 7

Collagen content of single-and multi-layer constructs after 3 and 6 weeks of culture (*p<0.05 vs. 3 week time point).

Figure 8

Difference between expected and predicted stiffness and collagen content of multi-layer constructs at 6 weeks of culture based on data from single-layer constructs (#p<0.05 between groups). Positive values indicate a positive interaction effect due to multi-layer culture, while negative values indicate a negative interaction effect.