Spiral Layer-by-Layer Micro-Nanostructured Scaffolds for Bone Tissue Engineering

Abstract

This Article reports the fabrication and characterization of composite micro-nanostructured spiral scaffolds functionalized with nanofibers and hydroxyapatite (HA) for bone regeneration. The spiral poly(lactic acid-co-glycolic acid) (PLGA) porous microstructure was coated with sparsely spaced PLGA nanofibers and HA to enhance surface area and bioactivity. Polyelectrolyte-based HA coating in a layer-by-layer (LBL) fashion allowed 10-70 μM Ca²⁺/mm² incorporation. These scaffolds provided a controlled release of Ca²⁺ ions up to 60 days with varied release kinetics accounting up to 10-50 μg. Spiral scaffolds supported superior adhesion, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (MSCs) as compared to controls microstructures. Spiral micro-nanostructures supported homogeneous tissue ingrowth and resulted in bone-island formation in the center of the scaffold as early as 3 weeks in a rabbit ulnar bone defect model. In contrast, control cylindrical scaffolds showed tissue ingrowth only at the surface because of limitations in scaffold transport features.

Keywords: bone tissue engineering; hydroxyapatite; layer-by-layer; nanocomposite; nanofibers; rabbit ulnar defect model; scaffolds.

Figure 1.

Simplified schematic showing the fabrication of the sintered PLGA microsphere sheet, followed by deposition of electrospun nanofibers. The hybrid construct is then rolled into the final spiral shape. HA is deposited in a layer-by-layer polyelectrolyte process, starting with the deposition of cationic chitosan (shown in red) followed by anionic HA (shown in blue). This process is repeated to achieve desired number of HA bilayers.

Figure 2.

(A) Optical micrograph of spiral PLGA microsphere scaffolds and (B) cylindrical PLGA microsphere scaffolds. (C) SEM showing nanofiber coating of PLGA microsphere scaffold and (D) SEM of PLGA sintered microsphere scaffolds, showing microsized pores. Both scaffolds are engineered to have identical pore properties due to similar microsphere size and sintering conditions.

Figure 3.

(A) Quantification of calcium on spiral scaffolds with various numbers of HA bilayers deposited and control adsorbed HA. A direct correlation is shown between a number of bilayers deposited and the total calcium content on scaffolds. (B) Cumulative percent calcium ion release showing the release profiles of spiral scaffolds with 0, 1, 3, and 5 bilayers of HA deposited using the layer-by-layer technique across 60 days. An initial burst release of calcium is seen in all three bilayer scaffolds, followed by a steady, sustained release from day 7–60 with a direct correlation between a number of bilayers and the cumulative amount of calcium ions released.

Figure 4.

MTS assay showing cell attachment and proliferation on cylindrical scaffolds, spiral scaffolds, and spiral/nanofiber scaffolds. (* indicates a significant increase in cell attachment as compared to cylindrical scaffold at day 1; # indicates a significant increase in cell attachment as compared to cylindrical scaffold at day 3; † indicates a significant increase in cell attachment as compared to cylindrical scaffold at day 7; $ indicates a significant increase in cell attachment as compared to the cylindrical scaffold at day 14).

Figure 5.

Cross-sectional view of immunofluorescent staining of differentiated BMSCs seeded on and within layer-by-layer spiral scaffolds showing positive expression for (A) type I collagen at day 14, (B) type I collagen at day 28, (C) osteopontin at day 14, and (D) osteopontin at day 28.

Figure 6.

Alkaline phosphatase (ALP) activity on cylindrical scaffolds, spiral scaffolds, and spiral/nanofiber scaffolds. (* indicates a significant increase in ALP as compared to the cylindrical scaffold at day 1; # indicates a significant increase in ALP as compared to the cylindrical scaffold at day 7; $ indicates a significant increase in ALP as compared to the cylindrical scaffold at day 14).

Figure 7.

Calcium deposition quantified by alizarin red assay. (* indicates a significant increase in calcium as compared to the cylindrical scaffold at day 1; # indicates a significant increase in calcium as compared to the cylindrical scaffold at day 7; $ indicates a significant increase in calcium as compared to the cylindrical scaffold at day 1).

Figure 8.

In vivo implantation of the spiral scaffold in 5 × 7 mm rabbit ulnar defect. Anterior and posterior micro-CT 3-D reconstructions at 10 weeks postimplantation showing regeneration with implanted (A) cylindrical and (B) spiral scaffolds. Spiral scaffolds showed complete regeneration of bone tissue from both anterior and posterior views.

Figure 9.

Cross sections of alcian blue-stained PMMA processed samples of scaffolds 3 weeks postimplantation in rabbit ulna where (A) control cylindrical scaffold (cross section 1×), (B) spiral scaffold (cross section 1×), (C) 10× magnification of A, indicating tissue infiltration, and (D) 10× magnification of B, indicating the formation of new bone in the interior of the tubular structure, (E) 20× magnification of C, indicating fibrous tissue morphology, (F) 20× magnification of D, indicating an osteocyte-like cell morphology in the interior regions of the material. Light pink-stained material indicates cellular cytoplasm and ECM surrounding holes of the dissolved scaffold material. The cells stain deep pink and the GAG stains blue. For cylindrical scaffolds, the cells in the material’s interior are more fibroblastic, with very low GAG and ECM content.

Figure 10.

Longitudinal sections of alcian blue-stained PMMA processed samples of scaffolds, 3 weeks postimplantation in rabbit ulna where (A) control cylindrical scaffold (1×), (B) spiral scaffold (1×), (C) 10× magnification of A, indicating tissue infiltration, and (D) 10× magnification of B, indicating the formation of new bone in the interior of the tubular structure, (D) 20× magnification of C, indicating fibrous tissue morphology, (F) 20× magnification of D, indicating an osteocyte-like cell morphology in the interior regions of the material. Light Pink- stained material indicates cellular cytoplasm and ECM surrounding holes of the dissolved scaffold material. The cells stain deep pink and the GAG stains blue. In the case of cylindrical scaffolds, the cells in the material’s interior are more fibroblastic, with very low GAG and ECM content.