Integrin and syndecan binding peptide-conjugated alginate hydrogel for modulation of nucleus pulposus cell phenotype.

Biomaterial based strategies have been widely explored to preserve and restore the juvenile phenotype of cells of the nucleus pulposus (NP) in degenerated intervertebral discs (IVD). With aging and maturation, NP cells lose their ability to produce necessary extracellular matrix and proteoglycans, accelerating disc degeneration. Previous studies have shown that integrin or syndecan binding peptide motifs from laminin can induce NP cells from degenerative human discs to re-express juvenile NP-specific cell phenotype and biosynthetic activity. Here, we engineered alginate hydrogels to present integrin- and syndecan-binding peptides alone or in combination (cyclic RGD and AG73, respectively) to introduce bioactive features into the alginate gels. We demonstrated human NP cells cultured upon and within alginate hydrogels presented with cRGD and AG73 peptides exhibited higher cell viability, biosynthetic activity, and NP-specific protein expression over alginate alone. Moreover, the combination of the two peptide motifs elicited markers of the NP-specific cell phenotype, including N-Cadherin, despite differences in cell morphology and multicellular cluster formation between 2D and 3D cultures. These results represent a promising step toward understanding how distinct adhesive peptides can be combined to guide NP cell fate. In the future, these insights may be useful to rationally design hydrogels for NP cell-transplantation based therapies for IVD degeneration.

Copper-Free Azide-Alkyne Cycloaddition for Peptide Modification of Alginate Hydrogels.

Alginate, a biocompatible polymer naturally derived from algae, is widely used as a synthetic analogue of the extracellular matrix in tissue engineering. Integrin-binding peptide motifs, including RGD, a derivative of fibronectin, are typically grafted to the alginate polymer through carbodiimide reactions between peptide amines and alginate uronic acids. However, lack of chemo-selectivity of carbodiimide reactions can lead to side reactions that lower peptide bioactivity. To overcome these limitations, we developed an approach for copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated conjugation of azide-modified adhesive peptides (azido-cyclo-RGD, Az-cRGD) onto alginate. Successful conjugation of azide-reactive cyclooctynes onto alginates using a heterobifunctional crosslinker was confirmed by azido-coumarin fluorescent assay, NMR, and through click reactions with azide-modified fluorescent probes. Compared to cyclo-RGD peptides directly conjugated to alginate polymers with standard carbodiimide chemistry, Az-cyclo-RGD peptides exhibited higher bioactivity, as demonstrated by cell adhesion and proliferation assays. Finally, Az-cRGD peptides enhanced the effects of recombinant bone morphogenetic proteins on inducing osteogenesis of osteoblasts and bone marrow stromal stem cells in 3D alginate gels. SPAAC-mediated click approaches for peptide-alginate bioconjugation overcome the limitations of previous alginate bioconjugation approaches and potentially expand the range of ligands that can be grafted to alginate polymers for tissue engineering applications.