Microsphere-based scaffolds for cartilage tissue engineering: using subcritical CO(2) as a sintering agent

Abstract

Shape-specific, macroporous tissue engineering scaffolds were fabricated and homogeneously seeded with cells in a single step. This method brings together CO(2) polymer processing and microparticle-based scaffolds in a manner that allows each to solve the key limitation of the other. Specifically, microparticle-based scaffolds have suffered from the limitation that conventional microsphere sintering methods (e.g., heat, solvents) are not cytocompatible, yet we have shown that cell viability was sustained with subcritical (i.e., gaseous) CO(2) sintering of microspheres in the presence of cells at near-ambient temperatures. On the other hand, the fused microspheres provided the pore interconnectivity that has eluded supercritical CO(2) foaming approaches. Here, fused poly(lactide-co-glycolide) microsphere scaffolds were seeded with human umbilical cord mesenchymal stromal cells to demonstrate the feasibility of utilizing these matrices for cartilage regeneration. We also demonstrated that the approach may be modified to produce thin cell-loaded patches as a promising alternative for skin tissue engineering applications.

Figure 1

(A) Coulter multisizer size distribution plot of PLG microspheres of different nominal sizes used in these studies, displaying the monodispersity of the microspheres with discrete peaks (peaks with % volume less than 0.5 have been omitted for the sake of clarity). (B) An image of various shape-specific scaffolds that were produced with PLG microspheres (140 μm) using CO₂ at sub-critical conditions (15 bar for 1 h at 25°C followed by depressurization at ~0.14–0.21 bar/s) utilizing rubber molds of different shapes. From left to right: cylinder, bilayered cylinder, tube, plus-sign, and star. Scale bar: 1 mm.

Figure 2

Characteristic scanning electron micrographs of scaffolds fabricated using different types of PLG microspheres at the typical processing conditions for sintering (CO₂ exposure at 15 bar for 1 h at 25°C followed by depressurization at ~0.14–0.21 bar/s). Sizes of the microspheres used were 240 μm (A, B), 175 μm (C), 140 μm (D, E), and 140 μm together with 5 μm (F). The morphology of a microsphere following the CO₂ sintering (E) is also displayed, where enlarged images of the microsphere connection site (top panel) and sub-micron level surface modifications (bottom panel) are shown. The microspheres were made using PLG (with acid-end group chemistry) of either 0.33 (5, 175, 240 μm) or 0.37 (140 μm) dL/g intrinsic viscosity (i.v.) (see Fig. 1). Scale bar: 100 μm unless labeled otherwise.

Figure 3

Modulus of elasticity of the scaffolds prepared using different microsphere sizes (corresponding to Fig. 1). The differences in the moduli were not statistically significant (p > 0.05).

Figure 4

I. Fluorescence micrographs of Live/Dead dye-stained porcine chondrocytes seeded on scaffolds (175 μm) following a 3 wk cell culture period – A) live (green) and dead (red) cells. Panels B and C show the split of green and red, where all the live cells (B) or dead cells (C) can be seen separately, respectively. There is a mild overlap between the panels B and C, which reveals some cells that are yellow (possibly cells that are dying). Scale bar: 100 μm. II. Immunohistochemistry for collagen types I and II (purple indicates positive stain) and Safranin-O staining for GAGs (orange indicates positive stain) at week 3 (n = 2). HUCMSCs = human umbilical cord mesenchymal stromal cells, CI = collagen type I, CII = collagen type II, and GAG = glycosaminoglycan. Scale bar: 100 μm.

Figure 5

(A) A schematic of producing a microsphere-based cell-loaded scaffold or thin patch is shown. The process of combining the cells and microparticles in a liquid medium results in a melded thin patch (top), whereas mechanically mixing a loose cell pellet in a minimal liquid volume with the microparticles results in a homogeneously seeded scaffold (bottom). Scale bar: 6 mm. (B, C) Fluorescence micrographs of Live/Dead dye-stained HUCMSCs display cell survival during CO₂ sintering of microspheres (120 μm) at sub-critical conditions. Processing conditions (pressure, duration of exposure, depressurization rate, presence/absence of culture medium) for the production of the thin patch (B) and the macroscopic scaffold (C) were (30 bar, 2 min, ~0.21 bar/s, medium present) and (30 bar, 4 min, ~0.21 bar/s, medium absent), respectively. Green indicates live cells and red indicates dead cells. Note the dark circular areas, corresponding to the locations of the microspheres. Scale bar: 100 μm.