Evaluation of Self-Assembled Nanofibrous Aggregates (SNAs) for Assessing Osteogenic Potential in the Development of Bioinks for Bone Tissue Regeneration.

A long-standing challenge in regenerative medicine is replicating the extracellular matrix (ECM) of bone. Polyelectrolyte complexes (PECs) have been widely studied in tissue engineering; however, their potential in forming nanofibrous architectures that closely resemble the ECM of bone remains unexplored. This study evaluates the potential of self-assembled nanofibrous aggregates (SNAs) in developing bioinks for facilitating bone repair and growth using bone marrow mesenchymal stem cells (BM-MSCs). Five SNA variants were synthesized via self-assembly: chitosan-chondroitin sulfate (CCS), chitosan-casein (CCas), chitosan-gelatin type B (CG), gelatin type A-carrageenan (GCarr), and chitosan-carrageenan (CCarr). These SNAs were then incorporated separately into a thermosensitive chitosan-based hydrogel. The hydrogels were characterized for their physicochemical, rheological, mechanical, and biological properties. Depending upon the composition of the polyanion and polycation used, the overall properties of the hydrogel varied significantly. FESEM revealed a highly porous architecture of the lyophilized scaffolds. Confocal microscopy confirmed the cell viability and proliferation of encapsulated BM-MSCs over a 14-day culture period. Biological characterization also revealed significant biocompatibility, with cell viability exceeding 90% across all groups, as measured by the MTT assay. Among the SNA incorporated hydrogels, gelatin type A-carrageenan hydrogels (GelA-Carr) exhibited superior osteogenic differentiation, with an increase of 28.9 ± 0.9 units/mL in alkaline phosphatase (ALP) activity, a 0.53 ± 0.01 optical density enhancement in collagen secretion, and a calcium mineralization level of 45.5 ± 6.03 μg/mg. These values were significantly higher than those observed in other SNAs, indicating the superior osteoinductivity of GelA-Carr. This research highlights the influence of different SNAs on stem cell fate, offering the potential for developing advanced bioinks for bone tissue engineering applications.