Supramolecular Hydrogel Based on an Osteogenic Growth Peptide Promotes Bone Defect Repair

Abstract

Current bone defect treatment strategies are associated with several risks and have major limitations. Therefore, it is necessary to develop an inexpensive growth factor delivery system that can be easily produced in large quantities and can promote long-term bone regeneration. An osteogenic growth peptide (OGP) is a 14 amino acid peptide with a short peptide sequence active fragment. In this study, we developed two OGP-based self-assembling supramolecular hydrogels (F- and G-sequence hydrogels) and investigated the in vitro and in vivo effects on proliferation and osteogenesis, including the mechanism of hydrogel-mediated bone defect repair. The hydrogels presented excellent biocompatibility and cell proliferation-promoting properties (1.5-1.7-fold increase). The hydrogels could effectively upregulate the expression of osteogenic factors, including RUNX2, BMP2, OCN, and OPN, to promote osteogenesis differentiation. Interestingly, 353 differentially expressed genes were identified in hBMSCs treated with hydrogels. The hydrogels were proved to be involved in the inflammatory pathways and folate-related pathways to mediate the osteogenesis differentiation. Furthermore, the therapeutic efficiency (bone volume/total volume, trabecular number, and bone mineral density) of hydrogels on bone regeneration in vivo was evaluated. The results showed that the hydrogels promoted bone formation in the early stage of bone defect healing. Taken together, this study was the first to develop and evaluate the properties of OGP-based self-assembling supramolecular hydrogels. Our study will provide inspiration for the development of delivering OGP for bone regeneration.

Figure 1

Schematic depiction of the mechanisms of OGP-based self-assembly supramolecular hydrogels on promoting bone regeneration.

Figure 2

Characteristics of self-assembled hydrogels. (A) Chemical structure of the F-sequence self-assembling peptide, (B) chemical structure of the G-sequence self-assembling peptide, (C) H-nuclear magnetic resonance (¹H NMR) spectrum of the F-sequence self-assembling peptide, (D) ¹H NMR spectrum of the G-sequence self-assembling peptide, (E) optical and TEM images of the F-sequence hydrogel, (F) optical and TEM images of the G-sequence hydrogel, (G) dynamic frequency sweep of the F hydrogel, and (H) dynamic frequency sweep of the G hydrogel.

Figure 3

F- and G-sequence hydrogel cytotoxicity and proliferation assessment. (A) Cell Counting Kit-8 (CCK-8) data for the F hydrogel and (B) CCK-8 data for the G hydrogel; *p < 0.05 and **p < 0.01.

Figure 4

Osteogenic differentiation of hBMSCs. (A) Western blotting showing the protein levels of RUNX2, BMP2, OCN, and OPN and (B) mRNA levels of RUNX2, BMP2, OCN, and OPN; *p < 0.05 and **p < 0.01.

Figure 5

Effect of F- and G-hydrogels on the expression of different genes in hBMSCs. (A) Volcano plot showing the upregulation and downregulation of differentially expressed genes (DEGs) between the 1 nM F-treated group and the untreated group (UT), (B) volcano plot showing the upregulation and downregulation of DEGs between the 1 nM G-treated group and UT, (C) volcano plot showing upregulation and downregulation of DEGs between the 1 nM G-treated and 1 nM F-treated groups., and (D) heat map showing unsupervised clustering of DEGs within different groups.

Figure 6

GO and KEGG pathway analysis results. (A) GO analyses of DEGs in 1 nM F compared to UT, (B) GO analyses of DEGs in 1 nM G compared to UT, (C) GO analyses of DEGs in 1 nM G compared to 1 nM F, (D) KEGG enrichment analyses of DEGs in 1 nM F compared to UT, (E) KEGG enrichment analyses of DEGs in 1 nM G compared to UT, and (F) KEGG enrichment analyses of DEGs in 1 nM G compared to 1 nM F.

Figure 7

Effect of bone regeneration of F- and G-sequence hydrogels in vivo. (A) Microtomography images of a rat with a right femoral defect and (B) quantitative analysis of bone morphological parameter values in the femoral defect area. Abbreviations: BV/TV: bone volume/total volume, Tb.N: trabecular number, and BMD: bone mineral density.