Co-Electrospun Scaffolds with Gradients in Fiber Alignment and Chemistry for Regeneration of the Ligament-Bone Interface

Co-electrospun scaffolds with gradients in fiber alignment and chemistry for regeneration of the ligament-bone interface

Introduction: Grafts currently used for the repair of ligament injuries integrate poorly with bone due to a significant mismatch of mechanical and chemical properties between graft and bone.  This mismatch stems from the grafts' inability to regenerate gradients of mineral content, collagen fiber alignment and cell phenotype that exist at natural ligament-bone transitions.  We envision that an electrospun scaffold, possessing gradients of architecture, mechanical and biochemical properties, will help establish spatial gradients of cell phenotype in bone marrow stromal cells (BMSCs) and thus aid the regeneration and osseointegration of injured ligaments.  As a step towards the larger goal of regenerating complex tissue transitions, we describe herein the fabrication of a scaffold possessing gradients of mineral content and fiber alignment, obtained by co-electrospinning two polymer solutions, with different chemistries, from offset spinnerets onto a dual-drum collector. The dual-drum collector used in this study allows the creation of graded scaffolds with transition regions at physiologically relevant length-scales.

Materials and Methods: Solutions of poly-lactic-co-glycolic acid (PLGA) doped with an amorphous calcium phosphate (ACP) phase and polycaprolactone (PCL) were co-electrospun from offset spinnerets onto a custom-designed slowly rotating dual-drum collector (Figure 1a). Post-electrospinning, the drums were stretched apart to result in strain-induced fiber alignment in the gap region between the drums. This fabrication procedure resulted in the formation of randomly oriented ACP doped-PLGA fibers on one drum, aligned PCL fibers in the gap region between the drums and a transition region consisting of both types of fibers at the interface.  Angular orientation of the fibers from the three regions was characterized from micrographs of the scaffold.  Finally, rat BMSC alignment, cell metabolic activity, deposition of Collagen-I (Col-I) and phenotypic makers of cell differentiation were investigated on all regions of the graded scaffolds.

Results and Discussion: Co-electrospinning onto the custom-designed collector resulted in the formation of a graded scaffold consisting of three different regions: a randomly oriented region possessing fibers that were electrospun from one spinneret (Figure 1b), an aligned region possessing fibers that were electrospun from the other spinneret (Figure 1d) and a transition region possessing a mixture of both types of fibers (Figure 1 c).  After 1 and 3 days of culture, rat BMSCs were found to be aligned in the aligned region of the scaffold (Figure 1e), and randomly oriented on the other regions.  Cells were also found to be metabolically active on all regions of the scaffolds after 1 and 7 days of culture.  Finally, a gradient of cell phenotype and collagen-I (Col-I) orientation was observed on the graded scaffolds.

Figure 1:

(a) Slowly-rotating dual-drum set-up with gap region in between the drums.

(b), (c) and (d): Microscopy images of samples collected from a scaffold fabricated with two PCL solutions from offset spinnerets (proof-of concept).  White arrow in (d) indicates general direction of fiber alignment.

e) Rat bone marrow stromal cells seeded on the aligned PCL section scaffold and imaged with Calein AM after 1 day of seeding. White arrow indicates general direction of cell alignment.

Conclusions: This study demonstrates that scaffolds with complex architectures and gradients in mechano-chemical properties at physiologically relevant length-scales, can be fabricated by co-electrospinning appropriate polymer solutions from offset spinnerets onto a specially designed collector.  The study also indicates that these scaffolds have the potential to be used for the regeneration of graded tissue transitions such as those present at the ligament-bone interface.