Biological designer self-assembling peptide nanofiber scaffolds significantly enhance osteoblast proliferation, differentiation and 3-D migration

Abstract

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.

Figure 1

Molecular models of pure and designer peptide nanofibers. A) Models represent peptide RADA16, ALK, DGR and PRG from Table 1. B) Model representing a β-sheet double-tape of a self-assembling peptide nanofiber with PRG motif (4∶1). Note the sequences PRG extending out from the nanofiber double-tape.

Figure 2

Tapping Mode AFM images of 1% (w/v) peptides solution of A) RADA16, B) PRG alone, C) PRG+RADA16 (1∶1). The bar represents 100 nm. The nanofiber formation is seen in A) RADA16 and C) PRG+RADA16 (1∶1). An increase in the fiber thickness in C) PRG+RADA16 (1∶1) (D = 29.5±3.1 nm) from A) RADA16 (D = 16.3±1.4 nm) was observed, which correlated to the width of the peptide fiber modeled in Fig. 1.

Figure 3

Cell numbers are calculated from DNA measurement on various scaffolds. MT3T3-E1 cells were cultured for 2 weeks on different scaffolds. RADA16∶RADA16 1% (w/v), ALKmx∶ALK 1% (w/v)+RADA16, DGRmx∶DGR 1% (w/v)+RADA16, PRGmx: PRG 1% (w/v)+RADA16 (all mix ratio is 1∶1). For peptide scaffolds containing active peptides, the cell proliferation rate is higher than that of pure RADA16. *p<0.01; suggesting it is significant against the number of cells grown in pure RADA16 scaffold.

Figure 4

A) ALP activity normalized by DNA amount cultured on the different hydrogels for two weeks. RADA16∶RADA16 1% (w/v), ALKmx: ALK 1% (w/v)+RADA16-I, DGRmx∶DGR 1% (w/v)+RADA16, PRGmx∶PRG 1% (w/v)+RADA16 (all mixture ratio is 1∶1). ALK, DGR and PRG show the higher ALP activity compared to RADA16-I. *p<0.01 suggesting it is significant against the ALP activity in pure RADA16-I. B) Osteocalcin content secreted in culture medium after culturing on the different hydrogels for two weeks. RADA16∶RADA16 1% (w/v), ALKmx∶ALK 1%(w/v)+RADA16, DGRmx∶DGR 1%(w/v)+RADA16, PRGmx∶PRG 1% (w/v)+RADA16 (all mixture ratio is 1∶1). All modified peptides (ALK, DGR and PRG) show the higher osteocalcin contents compared to pure RADA16. PRG has a significantly higher concentration compared to the other scaffolds. *p<0.01 suggesting it is significant against Osteocalcin in RADA16 scaffold.

Figure 5

ALP Staining images after culturing on the different hydrogels for two weeks. The bar represents 100 µm. RAD-I∶RADA16 1% (w/v), ALKmx∶ALK 1% (w/v)+RADA16, DGRmx∶DGR 1% (w/v)+RAD, PRGmx∶PRG 1% (w/v)+RADA16 (all mixture ratio is 1∶1). The bluish color intensity correlates with the high ALP activity. RADA16 shows low cell adhesion to the hydrogel and the cells are aggregated. The cell attachment increases in DGR and PRG scaffolds were considered as a result of RGD cell attachment sequence. ALP, DGR and PRG showed higher ALP activities compared to RADA16-I, especially staining intensity of PRG.

Figure 6

Cell numbers on the different scaffolds of different mix ratio of designer PRG and pure RADA16 scaffolds after 1-week culture. There is an increase in proliferation following the increase PRG % scaffold when increased from 0 to 40%. There was a decrease in cell proliferation at PRG 100% suggesting that there is an optimal ratio of PRG and RADA16 scaffolds.

Figure 7

Cell morphology on the different scaffolds of various mix ratios of RADA16 1% (w/v) and PRG 1% (w/v) using calcein-AM staining. The bar represents 100 µm. A) RADA16 100%∶PRG 0%, B) PRG 1%, C) PRG 10%, D) PRG 40%, E) PRG 70%, F) PRG 100% (RADA16: 0%). B) PRG: 1% shows uniform cell distribution compared to (A) 0%, which shows the increase of cell attachment. There is significant morphological difference in (C) 10% and (D) 40%, as seen by a change in cell shape from elongated form to asteroid form.

Figure 8

Reconstructed image of 3-D confocal microscope image of culturing on the different scaffolds consisting of different mix ratio of RADA16 1% (w/v) and PRG 1% (w/v) using calcein-AM staining. The bar represents 100 µm. A1 and A2: PRG 10% and B1&B2: PRG 70%. A1 and B1 are vertical view and A2 and B2 are horizontal view. In the case of 10% PRG scaffold, the cells were attached on the surface of the scaffold whereas the cells were migrated into the scaffold in the case of 70% PRG scaffold. There is a drastic cell migration into the scaffold with higher concentration of PRG motif.