(726b) 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.