Cryogels are advantageous scaffolds for bone regeneration applications due to their high mechanical stability and macroporous structure. Anatomically, bone is composed of collagen and hydroxyapatite and during remodeling, these structural components are replaced. However, early forms of mineralization include calcium salts which take up to months to be converted to the desired hydroxyapatite form. Thus, it is beneficial to provide a primary source of hydroxyapatite within the scaffold, expediting the process of mineralization during bone regeneration. In this study, chitosan-gelatin (CG) cryogels were incorporated with various forms of hydroxyapatite to evaluate effects on the standard characteristics of cryogels, as well as the potential for increased mineralization. Testing included the comparison of porosity, swelling, mechanical integrity, cellular infiltration, and mineralization potential between all types of cryogels. The addition of bone char to CG cryogels produced scaffolds with appropriate porosity and interconnectivity. Additionally, the bone char cryogels exhibited an adequate swelling potential, suitable mechanical properties, excellent cell attachment, and increased mineralization. These properties support this cryogel for such an application in tissue engineering.

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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.

Cell encapsulating scaffolds are necessary for the study of cellular mechanosensing of cultured cells. However, conventional scaffolds used for loading cells in bulk generally fail at low compressive strain, while hydrogels designed for high toughness and strain resistance are generally unsuitable for cell encapsulation. Here we describe an alginate/gelatin methacryloyl interpenetrating network with multiple crosslinking modes that is robust to compressive strains greater than 70%, highly biocompatible, enzymatically degradable and able to effectively transfer strain to encapsulated cells. In future studies, this gel formula may allow researchers to probe cellular mechanosensing in bulk at levels of compressive strain previously difficult to investigate.