Surface structure and calcium release of the composite scaffold BG40.

<p>Disc shaped polymer/bioglass scaffold BG40, with a diameter of 5 mm and a thickness of 1 mm (A). A representative SEM image of BG40 is shown in (B). The composite demonstrates a relatively smooth surface with a thin fibrous texture and small pores (arrows). Scale bar indicates 6 µm. The release of ionic calcium to medium by BG40 (B4), BG20 (B2), PLA (P) and β-TCP (T) is shown in C. The most ionic calcium was released within the first two days. * = p<0.05 BG20, BG40 vs b-TCP, PLA; $ = p<0.05 BG40 vs BG20, n = 5.</p

Calcium released by BG40 induces EPC elongation.

<p>EPCs were incubated in the presence of either 30/mL, 10 mM Ca²⁺ or BG40 conditioned medium+EGTA [3 mM]. Cell length was determined on day five (A) by means of phase contrast microscopy and subsequent histomorphometric evaluation. In (B) corresponding micrographs of EPCs are shown. The elongation of the EPCs was significantly lower in the presence of the calcium chelator EGTA [3 mM]. The uptake of DiL-ac-LDL demonstrated the endothelial phenotype and EPC vitality. The mean DiL-ac-LDL amount per cells was not significantly altered among the groups (control, 10 mM calcium, BG40, BG40+EGTA) (C). * = p<0.05 vs control; $ = p<0.05 vs BG40+EGTA, n = 5. Scale bars indicate 200 µm.</p

BG40+EPC increase vascularization one week after implantation.

<p>Representative vWF-stained histological sections of the defect site of male rats treated with BG40 (A, n = 6) and BG40+EPC (B, n = 6). The blood vessel density increased significantly in animals treated with BG40+EPC, compared to BG40 alone (B). vWF-positive structures appear brown (arrows). The isotype control is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079058#pone-0079058-g006" target="_blank">figure 6C</a>. The absence of staining in blood vessel structure (arrows) indicates the specificity of the vWF-staining (C). An overview of the defect area is provided in d (original magnification 50x). The BG40 implant could not be preserved during sample sectioning and left a gap corresponding to the BG area (defect site). The skull bone is located in the right corner (skull). The encircled area marks the region shown at higher magnification (original magnification 200x) in (A) and (B). The quantitative evaluation of vWF-positive blood vessel structures is depicted in (e). All vWF-positive, blood vessel like structures in the neighborhood of the whole defect site were counted. Red scale bars: 100 µm (A, B, C), 500 µm (D), *: p = 0.015.</p

Seeding efficacy and EPC phenotype on the composite scaffold.

<p>Seeding efficacy of EPCs on BG40, BG20, PLA and β-TCP (A). The percentage of initially adhering EPCs on β-TCP was significantly increased compared to PLA. Appearance of EPCs seeded on BG40 on day 1 and day 5 (B). Representative SEM images are shown. EPCs demonstrated a spherical structure on the first day after seeding on BG40 whereas a flattened appearance was seen when EPCs were seeded on PLA. On day 5 EPCs presented a necrotic phenotype when seeded on PLA, whereas they appear vital on BG40. Scale bars indicate 60 µm (day 1: PLA, BG40) respectively 20 µm (day 5: PLA, BG40). *: p = 0.045 β-TCP vs. PLA, n = 5. Confirmation of increased necrosis of EPC on PLA compared to BG20 and BG40, representative fluorescence microscopy images were shown in (C) at an original magnification of 100 fold. DiL-ac-LDL prestained EPCs were seeded onto the scaffolds. DAPI was added over a period of 3 min to discriminate between live (red cytoplasm, no blue nucleus) and necrotic EPCs (red cytoplasm, blue nucleus) on day 3 or 5 after seeding. The quantitative evaluation of this experiment is depicted in (D). The number of EPC was significantly lowered on PLA on day 3 (p = 0.02 vs BG20, p = 0.01 vs BG40) and the percentage of necrotic EPCs differed significantly between PLA and BG40 on day 3 (p = 0.02) and day 5 (p = 0.02). *: p<0.05 vs. PLA, $: p = 0.045 BG40 vs. BG20; P: PLA; B2: BG20; B4: BG40, n = 5. Scale bar indicates 200 µm.</p

Coincubation of EPCs with BG40 and BG20 leads to EPC elongation.

<p>Incubation with BG20 (B2) and BG40 (B4) lead to a significantly sustained increase in EPC length compared to EPCs incubated with β-TCP (T), PLA (P) or the medium control (C) during the whole observation period (A). The length of EPCs incubated with BG20 and BG40 increased significantly from day 2 to day 10 (A). Representative micrographs of EPCs incubated with either medium, PLA or BG40 demonstrated EPC elongation (B). * = p<0.05 BG20, BG40 vs control, β-TCP, PLA; $ = p<0.05 d6, d10 vs d2; # = β-TCP vs control, n = 5.</p

Gene expression of VEGF and vWF in EPC cultured on PLA, BG20 and BG40 over a period of 5 days.

<p>The values are given as fold change to the expression of the GAPDH gene which served as housekeeping gene (median value (25% quartile/75% quartile). Day 0 demonstrates the gene expression of the EPC before seeding on the biomaterials. Generally, gene epression of VEGF declined over the time whereas gene expression of vWF increased during the observation period. The experiment was performed with 4 independent EPC preparations. ↑ = increased gene expression; ↓ = decreased gene expression.</p