Introduction: Synthetic hydrogels are a promising strategy to reproduce the 3D microenvironment of bone tissue. However, most of them lack the required bioactivity to actively influence cell fate. To this end, biochemical cues, such as integrin-binding molecules combined with growth factor derived sequences, may be installed on hydrogels to mimic bone extracellular matrix (ECM) [1-2], thus improving their bioactivity. Herein, we engineered a PEG-based hydrogel modified with a biomimetic peptide containing the cyclic RGD cell adhesive motif (cRGD) and a BMP-2 derived peptide (DWIVA or cDWIVA) in a well-defined chemical manner. The hydrogels also incorporated matrix metalloproteinases (MMPs)-degradable sequences to allow their biodegradation and to ensure cell spreading and differentiation. In vitro studies demonstrated the capacity of such biomimetic hydrogel to support cell survival, growth and spreading and, more interestingly, to trigger human mesenchymal stem cells (hMSCs) osteogenic differentiation.
Methodology: The thiolated biomimetic peptides (cRGD-DWIVA and cRGD-cDWIVA) were synthetized by solid-phase peptide synthesis and anchored to 4-arm-PEG molecules with maleimide groups via Michael addition. Hydrogel crosslinking was also achieved via Michael addition by mixing thiolated-PEG/-MMPs-degradable sequences with functionalized PEG molecules. Physicochemical properties of hydrogels were assessed by means of rheological measurements, scanning electron microscopy as well as degradation and swelling assays, while their biological performance was evaluated with viability, immunostaining, ALP activity and gene expression studies with hMSCs.
Results: Preliminary studies were conducted to find the optimal ratio of biomimetic peptide, biodegradable crosslinking and percentage of PEG in order to engineer advanced hydrogels for bone tissue engineering. After establishing the optimal hydrogel compositions, their physicochemical characterization confirmed the presence of the biomimetic peptides as well as the desired mechanical properties and degradation rates to support cell proliferation and osteogenic differentiation. In this regard, control hydrogels without biomimetic peptides nor biodegradable crosslinking supported cell survival but failed to promote cell spreading or differentiation. On the contrary, the biomimetic-degradable hydrogels promoted high values of hMSCs proliferation and spreading. More interestingly, the functionalized hydrogels triggered hMSCs osteogenic differentiation, as demonstrated by a significant increase in ALP activity and an overexpression of osteospecific genes.
Conclusions: The functionalization of PEG hydrogels with biomimetic peptides combining the cRGD sequence with BMP-2-derived motifs resulted in novel biomaterials with the required mechanical properties, biodegradability rates as well as the biochemical cues to recreate bone ECM and support hMSCs osteogenic differentiation. Thus, this hydrogel may be a promising alternative to the current therapies with stem cells in the field of bone regeneration.
1. Dobre, O. et al., Adv. Funct. Mater. 31, 2010225 (2021).
2. Oliver-Cervelló, Ll. et al., Adv. Healthcare Mater. 10, 2001757 (2021).
Funding: We thank the Agencia Estatal de Investigación for funding (PID2020-114019RB-I00/AEI/10.13039/501100011033)