Polysaccharide-based multilayers as reservoir for metal ions programming osteogenic differentiation of stem cells

The modification of biomaterial surfaces with polyelectrolyte multilayers (PEM) represents a promising strategy for regulation of cell proliferation, and differentiation. This study examines how uploading cobalt (Co²⁺), copper (Cu²⁺), or iron (Fe³⁺) ions into chitosan-based PEM assembled with either hyaluronic acid (HA) or alginate (Alg) as polyanions affects the adhesion and osteogenic differentiation of a murine stem-cell line. Metal ion content in PEM is quantified by inductively coupled plasma mass spectrometry (ICP-MS) showing the highest concentration of Fe³⁺, followed by Cu²⁺ and Co²⁺. Interestingly, copper- and iron-doped [Chi/Alg] and [Chi/HA] PEM promote cell adhesion, spreading, and proliferation most, whereas cobalt-doped PEM exhibit reduced cellular interactions. In both PEM systems, metal-ion doping enhances osteogenic differentiation relative to undoped PEM, as shown by RT-PCR with up-regulation of alkaline phosphatase (ALP), Noggin, and Osterix particularly with Fe 3+ and Cu 2+ . Microscopy confirms greater calcium-phosphate deposition and stronger collagen I and osteocalcin staining, after doping with Fe³⁺ and Cu 2+ producing the most pronounced effects. Furthermore, [Chi/HA] PEM doped with metal ions demonstrate higher osteogenic marker expression than [Chi/Alg] PEM. These findings suggest that PEM composed of alginate or hyaluronic acid combined with chitosan doped with Cu²⁺, and Fe³⁺ yield promising bioactive coatings on implants and scaffolds, which may promote regeneration of bone tissue. Doping of multilayers fabricated from chitosan with either hyaluronan or alginate with metal ions before seeding with multipotent mouse cells promotes their osteogenic differentiation in absence of other osteogenic inducers particularly with Fe 3+ or Cu 2+ ions demonstrated by RT-PCR, Alizarin red and immunostaining.