Significant type I and type III collagen production from human periodontal ligament fibroblasts in 3D peptide scaffolds without extra growth factors

Abstract

We here report the development of two peptide scaffolds designed for periodontal ligament fibroblasts. The scaffolds consist of one of the pure self-assembling peptide scaffolds RADA16 through direct coupling to short biologically active motifs. The motifs are 2-unit RGD binding sequence PRG (PRGDSGYRGDS) and laminin cell adhesion motif PDS (PDSGR). RGD and laminin have been previously shown to promote specific biological activities including periodontal ligament fibroblasts adhesion, proliferation and protein production. Compared to the pure RADA16 peptide scaffold, we here show that these designer peptide scaffolds significantly promote human periodontal ligament fibroblasts to proliferate and migrate into the scaffolds (for approximately 300 microm/two weeks). Moreover these peptide scaffolds significantly stimulated periodontal ligament fibroblasts to produce extracellular matrix proteins without using extra additional growth factors. Immunofluorescent images clearly demonstrated that the peptide scaffolds were almost completely covered with type I and type III collagens which were main protein components of periodontal ligament. Our results suggest that these designer self-assembling peptide nanofiber scaffolds may be useful for promoting wound healing and especially periodontal ligament tissue regeneration.

Figure 1. Molecular model of designer peptides and nanofiber.

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).

Figure 2. Cell morphology on the different scaffolds after two weeks culture.

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.

Figure 3. Cell densities on the different scaffolds of different mix ratio of designer PRG/PDS and pure RADA16 after two weeks culture.

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.

Figure 4. Constructed images of 3-D confocal microscopy images of periodontal ligament fibroblasts on the different scaffolds.

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.

Figure 5. Type I and type III Collagens fluorescent immunostaining images of periodontal ligament fibroblasts on the different scaffolds after six weeks culture.

Fluorescent immunostaining with Anti-collagen type I and Alexa fluor 488 goat anti-rabbit IgG for collagen type I (green) in A1) RADA16, B1) PRG and C1) PDS, and Anti-collagen type III and Alexa fluor 594 goat anti-mouse IgG for collagen type III (red) in A2) RADA16, B2) PRG and C2) PDS. Mix ratio of designer PRG/PDS and RADA16 scaffold is 1∶9. In case of PRG and PDS, periodontal ligament fibroblasts drastically produced type I and type III collagens which were extra-cellular matrix components of periodontal ligament. The scale bar represents 100 um for all images.

Figure 6. Schematic illustration of the results.

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.