PDGF-BB released from PCL/col/HA scaffolds stimulates MSC chemotaxis.

<p>The lower wells of transwell chambers were filled with either purified PDGF-BB (10 ng/mL), serum-free medium (SFM) or PDGF-BB-containing conditioned media collected from PDGF-BB-coated PCL/col/HA scaffolds after 72 hrs. GFP-MSCs were seeded in the upper chambers and allowed to migrate for 20 hrs. After this interval, MSCs adherent to the underside of the transwells were visualized by fluorescent microscopy (top panel, representative images). In addition, MSC migration to the underside of the filter was quantified by lysing cells and measuring solution fluorescence (lower panel). Three independent experiments were performed for solution fluorescence. Analysis of variance with Tukey’s HSD post-hoc was used to establish significance (* denotes <i>p</i><.01).</p

Standard curve of GFP fluorescent signal from lysed GFP-MSCs.

<p>GFP-MSCs were counted using a hemocytometer and set numbers of cells were spun down in a centrifuge. Cell pellets were lysed and solution fluorescence was measured by a fluorometer. The coefficient of determination for the linear regression was 0.999, showing a very strong linear correlation between GFP-MSC number and solution fluorescence.</p

Chemotactic responses of MSCs.

<p>GFP-expressing MSCs (4×10⁴) were seeded onto the top of transwell chambers, with various cytokines/chemokines placed in the bottom of the chambers; some wells contained serum-free media (SFM) as a negative control. After a 20 hr incubation at 37°C, the GFP-MSCs that had migrated across the transwell membrane were lysed and quantitated by measuring fluorescence intensity of GFP. The following chemoattractants were evaluated: <b><i>A)</i></b> recombinant human PDGF-BB, PDGF-AB, or a mixture of SDF-1α, CXCL16, MIP-1α, MIP-1β, and RANTES, each at the indicated concentrations (ng/mL) (representative of 3 independent runs) <b><i>B)</i></b> PDGF-BB and BMP-2 (representative of 3 independent runs) and <b><i>C),</i></b> varying concentrations of PDGF-BB showing dose response.</p

Released PDGF-BB induces chemotaxis of MSCs in a stringent migration assay. <i>A)</i>

<p>Schematic showing experimental set-up (not drawn to scale). GFP-MSCs were seeded in 8-well rectangular plates. After cell confluence was established, cells were completely removed from the top half of the well by scraping along a pre-drawn central line. Subsequently, a PDGF-BB-adsorbed PCL/col/HA/scaffold, placed on a steel wire mesh, was placed 1.5 cm away from the cell front. As a control, some chambers were set up with PCL/col/HA scaffolds lacking PDGF-BB. <b><i>B)</i></b> After a 72 hr-incubation in the chambers described, MSCs were stained with DAPI and visualized by fluorescence microscopy. The original cell front created is denoted by a white line. <b><i>C)</i></b> GFP-images showing change in cell morphology of MSCs exposed to PDGF-BB. <b><i>D)</i></b> Significantly greater cell number was observed migrating toward PDGF-BB coated scaffolds compared to uncoated scaffolds. <b><i>E)</i></b> DAPI-stained images were further analyzed by counting the number of cells in three defined regions of distance beyond the original cell front. The distribution of cells in the wells with PDGF-BB-coated scaffolds showed that a greater percentage of the total cells that had migrated beyond the cell front had localized to the region beyond 400 µm. In comparison, the greatest percentage of cells in the control wells localized to the region below 150 µm. A total of six samples were analyzed for each condition. An asterisk (*) denotes significant differences observed with <i>p</i><.01, whereas (**) denotes <i>p</i><.0001.</p

Adsorption and release of PDGF-BB from scaffolds. <i>A)</i>

<p>Scaffolds were incubated in PBS containing 1.5 µg PDGF-BB for 24 h at 4°C. ELISA assays were used to measure the unbound PDGF-BB in the supernatants. Adsorption of PDGF-BB to the scaffolds was determined by subtracting the unbound PDGF-BB from the 1.5 µg of PDGF-BB initially added. Data are from three independent experiments (* denotes p<0.01). <b><i>B)</i></b> ELISAs were used to measure the amounts of PDGF-BB in conditioned PBS solution collected from the scaffolds at the indicated time intervals over a period of 8 weeks (for many of the data points, error bars are too small to be visualized).</p

Immunostaining for phosphorylated Focal Adhesion Kinase.

<p>MSCs were seeded onto glass coverslips coated with electrospun nanofibers, or with FBS as a control. After 5 hours, cells were fixed and stained for phosphorylated Focal Adhesion Kinase (red). Cells were counterstained with DAPI to show cell nuclei (blue). Cells seeded onto PCL/col/HA scaffolds were better spread, and exhibited greater amounts of punctuate pFAK staining (site pY397) as compared with cells on PCL or PCL/HA. Cells seeded onto FBS-coated glass coverslips displayed pFAK staining in focal adhesion-type structures (white arrows), as expected for cells grown on 2D surfaces.</p

Live cell imaging of GFP-expressing MSCs seeded onto electrospun scaffolds.

<p>A) Cells were seeded onto scaffolds and imaged over varying time points. Panels a–c: PCL scaffolds; panels d–f: PCL/HA scaffolds; panels g–i: PCL/col/HA scaffolds and panels j–l: col scaffolds. Scale bar = 100 µm. B) Higher magnification images of GFP-expressing MSCs at seven hours on electrospun scaffolds (panels m–p).</p

SEM images of MSCs cultured on nanofibrous scaffolds for 24 hours.

<p>A) Cell spreading was observed on PCL, PCL/HA, and PCL/col/HA scaffolds, but not on 100% collagen I (col). B) Col scaffolds (without cells) were incubated in culture media for 24 hrs to allow the potential release of soluble factors, and then the solution was collected. MSCs were suspended into this conditioned media, seeded onto PCL scaffolds, and allowed adhere in the media for 24 h. Under these conditions cell spreading was extensive, suggesting that lack of cell spreading on col substrates was not due to any soluble factors released from these scaffolds.</p

Adsorption of FN and VN by electrospun scaffolds.

<p>Scaffolds were coated with fetal bovine serum (A), or implanted into rat tibial osteotomies for 30 min (B). Scaffolds were then washed to remove loosely bound proteins, and proteins were subsequently desorbed by incubation in boiling SDS-containing solution. The amounts of FN and VN were evaluated by Western blot.</p

MTS assay quantifying cell proliferation on electrospun scaffolds of PCL, PCL/HA or PCL/col/HA.

<p>At day one, cell number was significantly higher on PCL/HA and PCL/col/HA scaffolds in comparison to PCL. By day four, PCL/HA was still significantly higher than PCL, and PCL/col/HA was significantly higher than PCL/HA and PCL. In addition, cell number on PCL/col/HA was significantly higher on day four than day one. An * denotes p<0.05</p