Peptide Hydrogel for Sustained Release of Recombinant Human Bone Morphogenetic Protein-2 In Vitro

Abstract

This study aimed to investigate the impact of varying the formulation of a specific peptide hydrogel (PepGel) on the release kinetics of rhBMP-2 in vitro. Three PepGel formulations were assessed: (1) 50% v/v (peptides volume/total volume) PepGel, where synthetic peptides were mixed with crosslinking reagents and rhBMP-2 solution; (2) 67% v/v PepGel; (3) 80% v/v PepGel. Each sample was loaded with 12 µg of rhBMP-2 and incubated in PBS. Released rhBMP-2 was quantified by ELISA at 1 h, 6 h, and 1, 2, 4, 7, 10, 14, and 21 days. To explore how PepGel formulations influence rhBMP-2 release, the gel porosities, swelling ratios, and mechanical properties of the three PepGel formulations were quantitatively analyzed. The results showed that rhBMP-2 encapsulated with 50% v/v PepGel exhibited a sustained release over the 21-day experiment, while the 67% and 80% v/v PepGels demonstrated significantly lower rhBMP-2 release rates compared to the 50% formulation after day 7. Higher histological porosity of PepGel was significantly correlated with increased rhBMP-2 release rates. Conversely, the swelling ratio and elastic modulus of the 50% v/v PepGel were significantly lower than that of the 67% and 80% v/v formulations. In conclusion, this study indicates that varying the formulation of crosslinked PepGel can control rhBMP-2 release rates in vitro by modulating gel porosity, swelling ratio, and mechanical properties. Encapsulation with 50% v/v PepGel offers a sustained rhBMP-2 release pattern in vitro; if replicated in vivo, this could mitigate the adverse effects associated with burst release of rhBMP-2 in clinical applications.

Keywords: biomaterials; bone morphogenetic protein; controlled release; drug delivery; peptide hydrogel.

Figure 1

A schema of rhBMP-2 encapsulation with PepGel and in vitro release assessment.

Figure 2

(A) In vitro rhBMP-2 cumulative release kinetics from three different formulations of PepGel. (B) Comparison of rhBMP-2 cumulative release rates among the three groups at 1 h, 6 h, 1 d. (C) Comparison of rhBMP-2 cumulative release rates among the three groups at 2 d, 4 d, 7 d. (D) Comparison of rhBMP-2 cumulative release rate among three groups at 10 d, 14 d, 21 d. *, **, and *** indicate significant differences with p < 0.05, p < 0.01, and p < 0.001, respectively. h = hour, d = day.

Figure 3

(A) Microscopic images (×100) of PGmatrix 3D shape nanoweb network. (B) Microscopic images (×400) magnified from the box in (A). (C) Microscopic images (×400) processed by ImageJ. (D) ImageJ analysis of the pore size of the three PepGel groups. Hematoxylin and Eosin Staining; scale bar = 100 µm; *, **, and *** indicate significant differences with p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 4

Correlation analyses of gel porosity and cumulative rhBMP-2 release rate at each timepoint. Note: higher gel porosities were significantly correlated with higher rhBMP-2 release rates from day 1 to day 21.

Figure 5

(A) Comparison of the swelling ratio among three groups. (B) Comparison of the equilibrium water content (EWC) among three groups. h = hour, n = 6 per PepGel formulation/timepoint. # p < 0.05, 80% v/v PepGel compared to 50% v/v PepGel; * p < 0.05, 80% v/v PepGel compared to 67% v/v PepGel; & p < 0.05, 50% v/v PepGel compared to 67% v/v PepGel.

Figure 6

(A) Correlation analysis of PepGel porosity and swelling ratio. (B) Correlation analysis of PepGel porosity and equilibrium water content (EWC). (C) Correlation between PepGel-swelling ratio and rhBMP-2 release rate, indicating that PepGel-swelling ratio was negatively correlated with rhBMP-2 release rate.

Figure 7

In vitro dynamic rheological properties of PepGel formulations. (A) Viscosity and shear rate of PepGel solution (without trigger solution in each formulation). (B) Distribution and ratio of shear elastic modulus (G′) and shear loss modulus (G″) ratio for each PepGel group across the three PepGel formulations. (C) Shear elastic modulus across the three PepGel groups. (D) Self-healing (sol–gel reversible) viscoelastic properties of PGmatrix 3D shape peptide hydrogel. (E) Stress–strain curve at break for PepGel in each group. * Indicates p < 0.05, n = 3.

Figure 8

(A) Correlation analysis of gel strength and equilibrium water content (EWC). (B) Correlation analysis of gel strength and gel porosity.