Molecular model of designer peptides and nanofiber.

<p>A) Molecular models of designer peptides RADA16, PRG and PDS. B) Molecular model of self-assembling peptide nanofibers formation with PRG peptide, representing a beta-sheet structure. Note the sequences PRG extending out from the nanofiber. C) Typical AFM morphology of a self-assembling peptide nanofiber scaffold PRG mixed with RADA16. (Photograph by Akihiro Horii).</p

Designer self-assembling peptides used in this study.

<p>The sequences are from N→C. Ac  =  acetylated N-termini, -CONH₂  =  amidated C-termini. The peptide motif souces from various protein origins.</p

Cell morphology on the different scaffolds after two weeks culture.

<p>Fluorescence microscopy image of periodontal ligament fibroblasts A) on RADA16, B) on PRG, C) on PDS and D) on rat type I Collagen as a positive control. Fluorescenct staining with Rhodamin phalloidin for F-actin (red) and SYTOX Green for nuclei (green) showed the cell attachments and distributions. The scale bar represents 100um for all images.</p

Schematic illustration of the results.

<p>A) Periodontal ligament fibroblasts on the peptide scaffold RADA16, B) on the functionalized peptide scaffold PRG and C) on the functionalized peptide scaffold PDS. In case of the functionalized peptide scaffold PRG and PDS, periodontal ligament fibroblasts showed cell proliferation, migration into the scaffolds and type I and type III collagen productions required to regenerate periodontal ligament.</p

Cell densities on the different scaffolds of different mix ratio of designer PRG/PDS and pure RADA16 after two weeks culture.

<p>Initial seeding density (255 cells/mm²) was used to calculate fold changes in cell densities after two weeks in culture for each of the scaffolds. There is a tendency of periodontal ligament fibroblasts to proliferate on functionalized peptide scaffolds PRG and PDS. The fibroblasts proliferated significantly on PRG 10% and 30% compared to RADA16 (#ρ<0.01 vs RADA16). PRG/PDS concentration in the mix as low as 10–30% seems to be effective for growing the fibroblasts.</p

Constructed images of 3-D confocal microscopy images of periodontal ligament fibroblasts on the different scaffolds.

<p>Fluorescent staining with Rhodamin phalloidin and SYTOX Green. A) RADA16, B) PRG and C) PDS. A1, B1,C1) Vertical and A2, B2,C2) horizontal images after five hours culture. A3, B3, C3) Vertical and A4, B4, C4) horizontal images after two weeks culture. There were significant cell migrations into the functionalized peptide scaffolds PRG and PDS after two weeks. The scale bar represents 200 um for all images.</p

The thermal behavior of d-EAK16.

<p>A d-EAK16 peptide sample was measured at 218 nm and 208 nm from 4°C to 110°C with incremental 2°C change. The temperature equilibration time is 30 seconds and averaging time is 1 second. After reaching 110°C and waiting for 1-minute, the cooling from 110°C to 4°C was initiated. The changes of ellipticity from 4°C to 110°C and from the 110 to 4°C were shown. After the temperature cycles, [θ]_218nm of d-EAK16 decreased ∼2%, but increased ∼13% at [θ]_208nm. A and B profiles refer to d-EAK16 ellipticity [θ]_218nm from 4°C to 110°C and back from 110°C to 4°C. On the other hand, C and D profiles refer to ellipticity [θ]_208nm from 4°C to 110°C and back from 110°C to 4°C.</p

pH effect on the d-EAK16 and l-EAK16 structural transition.

<p>The d-EAK16 was incubated in solutions of various pH range from 0.76–12.0. A) The structural behavior from the d-EAK16 spectra in different pH showed the variations. The structures were different at pH0.76, pH12.0 and neutral pH. The d-EAK16 beta-sheet content is fairly similar at pH 5.2, pH6.1, pH7.2, pH8.1 and pH10.5. The d-EAK16 at pH2.8 and pH10.5 exhibits somewhat different spectra. There seemed to be either an intermediate structure or mixture with both alpha-helix and beta-sheet. B) The d-EAK16 spectra suggest four variations of structures at pH0.76, pH2.8, and pH10.5 and pH12.0. C) The structural behavior from l-EAK16 spectra in different pH showed variations. D) The l-EAK16 spectra suggest four variations of structures at pH0.76, pH2.8, pH10.2 and pH12.0.</p

Circular dichroism spectra of peptides d-EAK16 and l-EAK16 were in water at 25°C.

<p>X-axis is wavelength in nm and Y-axis is expressed as mole residue ellipticity [θ]. The mirror images of d-EAK16 and l-EAK16 reflect the molecular chirality.</p

The temperature effect on the d-EAK16 and l-EAK16 (100 µM). Each spectrum was collected at indicated temperature from 10°C to 110°C, using the same samples covered with mineral oils to prevent water evaporation. Every temperature point was averaged from 3 measurements.

<p>The sample was incubated at indicated temperatures for 15 minutes because 3 at 5-minute each measurements took 15 minutes. After measurement at 10°C, then the temperature was increased to 20°C, 30°C, 40°C, etc in the CD instrument to reach the indicated temperature and eventually elevated to 110°C. A) The CD spectra of d-EAK16 from 10°C to 110°C. At 20°C, it formed a stable beta-sheet. This beta-sheet structure was stable until 80°C. It then underwent two distinctive transitions. During the first transition, the beta-sheet content was not only reduced, but also changed its twist. The second structural transition occurs between 80°C to 90°C and drastically between 90°C to 110°C. Here, the beta-sheet structure is converted to an alpha-helical structure. B) d-EAK16 exhibits 3 distinctive structures at different temperatures, 60°C, 80°C, and 90°C. The isosbestic point was at 208nm as indicated by an arrow. C) The CD spectra of l-EAK16 from 10°C to 110°C. It was a very stable beta-sheet. The beta-sheet content was reduced, but no obvious structural transition was observed. D) l-EAK16 was very stable at different temperatures, 80°C, 90°C, 100°C and 110°C. No drastic structural transition was observed.</p