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The ongoing research on biomaterials that support bone regeneration led to the quest for materials or material modifications that can actively influence the activity or balance of bone tissue cells. The bone biocompatibility of porous chitosan scaffolds was modified in the present study by the addition of calcium phosphates or hemocyanin. The first strategy comprised the incorporation of calcium phosphates into chitosan to create a biomimetic chitosan-mineral phase composite. The second strategy comprised dip-coating of chitosan scaffolds with hemocyanin extracted from crayfish hemolymph. The cytocompatibility was assessed in a mono-culture of human bone marrow stromal cells (hBMSCs) and their differentiation to osteoblasts; in a mono-culture of human monocytes (hMs) and their maturation to osteoclasts; and in a co-culture of hBMSC/osteoblasts-hM/osteoclasts. Mineral incorporation caused an increase in scaffold bioactivity, as shown by reduced calcium concentration in the cell culture medium, delayed differentiation of hBMSCs, and reduced osteoclastic maturation of hMs in mono-culture. Dip-coating with hemocyanin led to increased proliferation of hBMSCs and equivalent osteoclast maturation in mono-culture, while in co-culture, both an inhibitory effect of mineral incorporation on osteoblastogenesis and stimulatory effects of hemocyanin were observed. It was concluded that highly bioactive scaffolds (containing mineral phases) restrain osteoblast and osteoclast development, while hemocyanin coating significantly supports osteoblastogenesis. These influences on the osteoblasts/osteoclasts activity ratio may support scaffold-driven bone healing in the future.

Various methods of chitosan scaffold production are reported in the literature so far. Here, in situ crosslinking with glutaraldehyde is reported for the first time. It combines pore formation and chitosan crosslinking in a single step. This combination allows incorporation of fragile molecules into 3D porous chitosan scaffolds produced by simple and gentle lyophilization. In this study, parameters of in situ crosslinking of porous chitosan scaffold formation as well as their effect on degradation and bioactivity of the scaffolds are examined. The scaffolds are characterized in the context of their prospective application as bone substitute material. The addition of calcium phosphate phases (hydroxyapatite, brushite) to the macroporous chitosan scaffolds allows manipulation of the bioactivity that is investigated by incubation in simulated body fluid (SBF). The bioactivity is significantly influenced by the modus of changing the fluid (static, daily-, and twice-a-week change). Scaffolds are morphologically characterized by means of scanning electron microscopy, and the mechanical stability is tested after incubation in SBF and phosphate-buffered saline. [ABSTRACT FROM AUTHOR]