A Doubly Fmoc-Protected Aspartic Acid Self-Assembles into Hydrogels Suitable for Bone Tissue Engineering

Abstract

Hydrogels have been used as scaffolds for biomineralization in tissue engineering and regenerative medicine for the repair and treatment of many tissue types. In the present work, we studied an amino acid-based material that is attached to protecting groups and self-assembles into biocompatible and stable nanostructures that are suitable for tissue engineering applications. Specifically, the doubly protected aspartic residue (Asp) with fluorenyl methoxycarbonyl (Fmoc) protecting groups have been shown to lead to the formation of well-ordered fibrous structures. Many amino acids and small peptides which are modified with protecting groups display relatively fast self-assembly and exhibit remarkable physicochemical properties leading to three-dimensional (3D) networks, the trapping of solvent molecules, and forming hydrogels. In this study, the self-assembling fibrous structures are targeted toward calcium binding and act as nucleation points for the binding of the available phosphate groups. The cell viability, proliferation, and osteogenic differentiation of pre-osteoblastic cells cultured on the formed hydrogel under various conditions demonstrate that hydrogel formation in CaCl₂ and CaCl₂-Na₂HPO₄ solutions lead to calcium ion binding onto the hydrogels and enrichment with phosphate groups, respectively, rendering these mechanically stable hydrogels osteoinductive scaffolds for bone tissue engineering.

Keywords: amyloid fibrils; biomineralization; building block; calcium ion binding; composite; fluorenyl methoxycarbonyl (Fmoc); injectable hydrogel; osteogenesis; self-assembly; single amino acid.

Scheme 1

Chemical formula of the doubly protected aspartic residue (Asp) with fluorenyl methoxycarbonyl (Fmoc) protecting groups, Fmoc-Asp-OFm.

Figure 1

Macroscopic images of self-assembled Fmoc-Asp-OFm (3 mg/mL) hydrogels (a) and after syringe extrusion (b). Determination of Young modulus for Fmoc-Asp-OFm hydrogels (c) and the compressive stress–strain diagram of three measurements (curves shown in different colors) (d).

Figure 2

Self-assembly of Fmoc-Asp-OFm (3 mg/mL) depicted by means of FESEM after 1 and 7 days of formation (a and b, respectively). Different magnification TEM images of Fmoc-Asp-OFm nanofibers (c,d). Stereoscopic evaluation of amyloid fiber formation via Congo red stain with bright field (e) and cross polarizers (f).

Figure 3

Evaluation of Ca²⁺ binding on Fmoc-Asp-OFm. FESEM images of the hydrogels formed in H₂O (a) compared to the one formed in CaCl₂ solution (b). TEM images of gels formed without calcium (c) and in the presence of calcium (d). Arrows in (d) point to dark nodules of higher electron density that most probably correspond to calcium deposits.

Figure 4

Evaluation of calcium mineralization by means of EDS (a). Arrows in spectrum (a) indicate the Ca²⁺ peaks. Alizarin red staining of hydrogels formed in the absence (b) and in the presence of CaCl₂ solutions (c) confirm the stained calcium deposits in (c).

Figure 5

Evaluation of the calcium phosphate deposition on Fmoc-Asp-OFm formed in CaCl₂ solution after HPO₄²⁻ deposition by means of FESEM (a). Nanoparticle diameter distribution is determined using analysis of FESEM images (b). EDS analysis (c), the arrows in the spectrum indicate the calcium and phosphorus peaks. XRD diffractogram of mineralized Fmoc-Asp-OFm/Ca²⁺/HPO₄²⁻ samples with dots displaying characteristic hydroxyapatite peaks (d).

Figure 6

Representative SEM images visualizing the cell adhesion and morphology on Fmoc-Asp-OFm, Fmoc-Asp-OFm/Ca²⁺ and Fmoc-Asp-OFm/Ca/HPO₄²⁻ hydrogels after 5 days in culture (upper panel at 200× magnification, lower panel at 1000× magnification).

Figure 7

Cell viability and proliferation assessment indicating the cell number of pre-osteoblastic cells seeded on hydrogels formed in CaCl₂, CaCl₂-Na₂HPO₄ solution, and H₂O after 3, 7, and 10 days (a). Normalized by cell viability (OD values) ALP activity of the pre-osteoblastic cells cultured on the peptide hydrogels for 7, 14, and 21 days (b). Calcium production levels from the pre-osteoblastic cells after 7, 14, and 21 days in culture (c). Determination of cumulative collagen production by the pre-osteoblastic cells until days 7, 14, and 21 (d). Statistical analysis was performed for each composite compared to the control sample formed in H₂O, using one-way ANOVA (bars with asterisks *) and the Friedman non-parametric test (bar with hash #) (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).