Biocompatibility and bioactivity of an FGF-loaded microsphere-based bilayer delivery system

Abstract

Many drug delivery systems rely on degradation or dissolution of the carrier material to regulate release. In cases where mechanical support is required during regeneration, this necessitates composite systems in which the mechanics of the implant are decoupled from the drug release profile. To address this need, we developed a system in which microspheres (MS) were sequestered in a defined location between two nanofibrous layers. This bilayer delivery system (BiLDS) enables simultaneous structural support and decoupled release profiles. To test this new system, PLGA (poly-lactide-co-glycolic acid) microspheres were prepared using a water-in-oil-in-water (w/o/w) emulsion technique and incorporated Alexa Fluor-tagged bovine serum albumin (BSA) and basic fibroblast growth factor (bFGF). These MS were secured in a defined pocket between two polycaprolactone (PCL) nanofibrous scaffolds, where the layered scaffolds provide a template for new tissue formation while enabling independent and local release from the co-delivered MS. Scanning electron microscopy (SEM) images showed that the assembled BiLDS could localize and retain MS in the central pocket that was surrounded by a continuous seal formed along the margin. Cell viability and proliferation assays showed enhanced cell activity when exposed to BiLDS containing Alexa Fluor-BSA/bFGF-loaded MS, both in vitro and in vivo. MS delivered via the BiLDS system persisted in a localized area after subcutaneous implantation for at least 4 weeks, and bFGF release increased colonization of the implant. These data establish the BiLDS technology as a sustained in vivo drug delivery platform that can localize protein and other growth factor release to a surgical site while providing a structural template for new tissue formation. STATEMENT OF SIGNIFICANCE: Localized and controlled delivery systems for the sustained release of drugs are essential. Many strategies have been developed for this purpose, but most rely on degradation (and loss of material properties) for delivery. Here, we developed a bilayer delivery system (BiLDS) that decouples the physical properties of a scaffold from its delivery kinetics. For this, biodegradable PLGA microspheres were sequestered within a central pocket of a slowly degrading nanofibrous bilayer. Using this device, we show enhanced cell activity with FGF delivery from the BiLDS both in vitro and in vivo. These data support that BiLDS can localize sustained protein and biofactor delivery to a surgical site while also serving as a mechanical scaffold for tissue repair and regeneration.

Keywords: Basic fibroblast growth factor; Bilayer Delivery System; Electrospun nanofiber; Microspheres; Tissue Engineering.

Fig. 1.

SEM images of microspheres (A, D, scale bar = 10 μm). Size distribution for each group of microspheres (B, E). 3D images of each group of microspheres (C, F, scale bar = 100 μm). (A, B, C: Alexa-BSA MS group and D, E, F: Alexa-BSA/bFGF MS group)

Fig. 2.

A schematic of the BiLDS (A). SEM images of Alexa-BSA-loaded microspheres on a single PCL nanofiber scaffold (scale bar = 1 mm) (B). Top view of sealed BiLDS (C). Cross-sectional view of BiLDS immediately after fabrication and sectioning in half (scale bar = 1 mm) (D). Higher magnification image of BiLDS cross-section showing MS within BiLDS (scale bar = 0.5 mm) (E).

Fig. 3.

Image of BiLDS construct after 30 days of incubation in rat serum (A). SEM image of cross-sectioned BiLDS after 30 days in rat serum (scale bar = 1 mm) (B). High magnification SEM image of MS inside the BiLDS pocket after 30 days in rat serum (scale bar = 30 μm) (C). Release profile from Alexa-BSA-loaded MS within the BiLDS compared to free Alexa-BSA-loaded MS over 30 days in rat serum (n = 3) (D).

Fig. 4.

Cell viability and proliferation with direct culture of tenocytes on the BiLDS construct measured using the MTT assay (*indicates p < 0.05 vs. control BiLDS, n = 5) (A). Actin staining with time in direct culture for each group (Scale = 50 μm) (B) SEM images of scaffold surface (Scale = 50 μm) and BiLDS cross-section (Scale = 100 μm) over 18 days (C).

Fig. 5.

Viability and proliferation of tenocytes with indirect culture of media conditioned by BiLDS constructs over one week, measured using the MTT assay (*indicates p < 0.05 vs. control and Alexa-BSA BiLDS) (A). Actin staining over 7 days with indirect culture of tenocytes in media conditioned from BiLDS (Scale = 10 μm) (B).

Fig. 6.

Images taken after 1, 2 and 4 weeks of subcutaneous implantation of each BiLDS groups (A). Fluorescent images showing retention of Alexa Fluor tagged MS BiLDS for up to 4 weeks after implantation (scale = 100 μm) (B).

Fig. 7.

SEM images showing inner and outer surfaces of BiLDS after subcutaneous implantation for up to 4 weeks (scale = 50 μm) (A). High magnification images of MS in the inner pocket of BiLDS at 2 and 4 weeks after implantation (scale = 20 μm) (B).

Fig. 8.

EdU and DAPI staining of the each BiLDS group (control BiLDS, Alexa Fluor-BSA-MS BiLDS, Alexa Fluor-BSA/bFGF-MS BiLDS) after implantation for 2 and 4 weeks (EdU: green, DAPI: blue) (A). H&E staining of each group at 2 and 4 weeks after implantation (Left: scale = 0.5 mm, Right: scale = 100 μm) (B).