Meniscus tissue engineering using a novel combination of electrospun scaffolds and human meniscus cells embedded within an extracellular matrix hydrogel

Abstract

Meniscus injury and degeneration have been linked to the development of secondary osteoarthritis (OA). Therapies that successfully repair or replace the meniscus are, therefore, likely to prevent or delay OA progression. We investigated the novel approach of building layers of aligned polylactic acid (PLA) electrospun (ES) scaffolds with human meniscus cells embedded in extracellular matrix (ECM) hydrogel to lead to formation of neotissues that resemble meniscus-like tissue. PLA ES scaffolds with randomly oriented or aligned fibers were seeded with human meniscus cells derived from vascular or avascular regions. Cell viability, cell morphology, and gene expression profiles were monitored via confocal microscopy, scanning electron microscopy (SEM), and real-time polymerase chain reaction (PCR), respectively. Seeded scaffolds were used to produce multilayered constructs and were examined via histology and immunohistochemistry. Morphology and mechanical properties of PLA scaffolds (with and without cells) were influenced by fiber direction of the scaffolds. Both PLA scaffolds supported meniscus tissue formation with increased COL1A1, SOX9, and COMP, yet no difference in gene expression was found between random and aligned PLA scaffolds. Overall, ES materials, which possess mechanical strength of meniscus and can support neotissue formation, show potential for use in cell-based meniscus regeneration strategies.

Keywords: ECM hydrogel; electrospinning; meniscus; tissue engineering.

Figure 1. Overview of the electrospinning equipment and production of multiple layers

(A) A grounded plate collector produced random PLA fibers and (B) a rotating drum collector was used to deposit aligned PLA fibers. (C) Process of production of multiple layers. Human meniscus cells encapsulated in a hydrogel consisting of collagen type II, chondroitin sulfate and hyaluronan were seeded onto a base aligned PLA scaffold, followed by placement of another scaffold above in the same fiber orientation. This was followed by another layer of cells and one more scaffold layer. To hold the layers together, a layer of 2% alginate was deposited over the entire stack and crosslinked. (D) Geometry and size (mm) of the dog-bone shaped tensile test specimens.

Figure 2. Scanning electron micrographs (SEM) of electrospun (ES) PLA scaffolds and cellular response

(A) SEM of random and (B) aligned ES PLA fibers (Mag. 1250×). (C) SEM of avascular human meniscus cells cultivated on random and (D) vascular human meniscus cells seeded on aligned ES PLA fibers (Mag. 1250×), scale bar: 20 μm in SEM images. (E) Confocal microscopy of vascular human meniscus cells cultured on random scaffolds and (F) avascular human meniscus cells cultivated on aligned scaffolds demonstrating viability (live/dead) and alignment cells cultivated on PLA scaffolds (Mag. 10×; scale bar: 200 μm in confocal images).

Figure 3. Relative fold change in gene expression of human vascular and avascular meniscus cells cultivated on either random or aligned PLA electrospun scaffolds

(A) Increased COL1A1 gene expression. (B) Increased SOX9 gene expression. (C) Reduced Aggrecan expression relative to monolayer controls. (D) COMP expression on random and aligned PLA electrospun scaffolds (n = 4–5 donors). Expression levels are relative to monolayer controls (dotted line).

Figure 4. Mechanical testing of random and aligned ES PLA scaffolds

(A) Young’s modulus (MPa) for random, aligned (along fiber orientation), and perpendicular to fiber orientation. (B) Young’s modulus (MPa) of random & aligned electrospun PLA scaffolds over time in culture with or without cells (one week and three weeks). (C) Stress/strain curve for each condition and (D) random PLA scaffold. (E) aligned PLA scaffold (F) perpendicular oriented PLA scaffold. (G) Ultimate stress readings (MPa) for each condition. (H) Ultimate stress (MPa) for random & aligned electrospun PLA scaffolds over time in culture with or without cells (one week and three weeks).

Figure 5. Histology and immunohistochemistry of multi-layer aligned PLA cell seeded scaffolds

(A–B) Safranin O/Fast-green stain. Arrows indicate three aligned PLA scaffold layers and the arrowhead points to the layer of alginate. (C) Collagen type I immunostain. (D) Isotype control stain. (E) Light microscope image of two weeks cultured construct. (F) DAPI stain. (Mag. A = 10×, scale bar: 200 μm ; Mag. B, C, D, E, and F = 40×, scale bar: 100 μm).