Fabrication and characterization of multiscale electrospun scaffolds for cartilage regeneration

Abstract

Recently, scaffolds for tissue regeneration purposes have been observed to utilize nanoscale features in an effort to reap the cellular benefits of scaffold features resembling extracellular matrix (ECM) components. However, one complication surrounding electrospun nanofibers is limited cellular infiltration. One method to ameliorate this negative effect is by incorporating nanofibers into microfibrous scaffolds. This study shows that it is feasible to fabricate electrospun scaffolds containing two differently scaled fibers interspersed evenly throughout the entire construct as well as scaffolds containing fibers composed of two discrete materials, specifically fibrin and poly(ε-caprolactone). In order to accomplish this, multiscale fibrous scaffolds of different compositions were generated using a dual extrusion electrospinning setup with a rotating mandrel. These scaffolds were then characterized for fiber diameter, porosity and pore size and seeded with human mesenchymal stem cells to assess the influence of scaffold architecture and composition on cellular responses as determined by cellularity, histology and glycosaminoglycan (GAG) content. Analysis revealed that nanofibers within a microfiber mesh function to maintain scaffold cellularity under serum-free conditions as well as aid the deposition of GAGs. This supports the hypothesis that scaffolds with constituents more closely resembling native ECM components may be beneficial for cartilage regeneration.

Figure 1

Dual extrusion electros with rotating mandrel diagram.

Figure 2

Scanning electron micrograph images of the different electrospun scaffold architectures and compositions that were generated: (a, d) Pμ, (b, e) PμPn, (c, f) PμFn. Vertical cross-sections of the different scaffolds show the distribution of nanofibers with respect to microfibers throughout the thickness of the scaffold: (g) Pμ, (h) PμPn, (i) PμFn.

Figure 3

Composite z-stack confocal images showing spreading and arrangement of phalloidin labeled cells highlighting F-actin (red) on day 0 (a) Pμ (b) PμPn and (c) PμFn scaffolds (green). Arrows indicate examples of individual cell morphologies and arrangements.

Figure 4

SEM images of constructs showing cells and ECM deposition on the surface of (a, d) Pμ, (b, e) PμPn, and (c, f) PμFn scaffolds on (a, b, c) day 7 and (d, e, f) day 21.

Figure 5

Safranin O staining for GAGs of (a, b, c) day 0 and (d, e, f) day 21 histological sections for (a, d) Pμ, (b, e) PμPn, and (c, f) PμFnscaffolds. Arrows indicate regions of staining within the interior of the construct. Scale bars represent 100 μm.

Figure 6

Fast Green staining for cytoplasm of day 21 histological sections for (a) Pμ day 21, (b) PμPn day 21, and (c) PμFn day 21 scaffolds. Arrows indicate regions of staining within the interior of the construct. Scale bars represent 100 μm.

Figure 7

Scaffold cellularity as determined by total DNA content per scaffold. In this instance, the group indicated as “Cells” denotes the DNA content of the initial cell suspension used to seed each of the scaffold types on day 0. DNA contents are represented as mean ± standard deviation for n = 4 samples.

Figure 8

GAG content of scaffolds at days 0, 7, 14, and 21 as determined by the DMMB assay. GAG contents are represented as mean ± standard deviation for n = 4 samples. Different scaffold types marked with an asterisk, “*”, indicate significant difference in GAG content for a specific time point. Statistical significance of a particular scaffold type at a specific culture period compared to both of the other scaffold types at every other time point is indicated by a “‡”.

Figure 9

GAG content normalized by total DNA content per scaffold at days 0, 7, 14, and 21 as determined by the DMMB assay. Normalized GAG contents are represented as mean ± standard deviation for n = 4 samples. ss