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This work aims at the bioinspired synthesis of hydroxyapatite (HAp) crystals in the presence of both collagen and l-arginine, in an effort to obtain a homogeneous hybrid material, having a bone-like nanostructure. Collagen (Col) is the most commonly utilized protein in most species of life, while L-arginine (Arg) encourages cell attachment, proliferation, and differentiation on HAp surfaces. Transmission electron microscopy, X-ray diffraction and Fourier transform-infrared spectroscopy were used to analyze surface morphology and structure of nanocrystals obtained under different synthesis conditions. It was shown that collagen and arginine content affect HAp crystallization. Collagen has an inhibition effect since HAp crystal size is reduced with the increase of collagen content. The presence of arginine is crucial as a critical content exists (Ca(2+):Arg = 1:1) under which HAp nanocrystals coexist with brushite. Under the optimum synthesis conditions (HAp/Col weight ratio 70/30 and Ca(2+):Arg molar ratio 1:1) HAp nanoplates of a uniform size (around 10 × 10 nm) were obtained. The biocompatibility of this hybrid powder was assessed using human bone marrow derived mesenchymal stem cells (MSCs). Cell response in terms of MSC attachment (scanning electron microscopy) and viability/proliferation (Alamar Blue) demonstrated a noncytotoxic effect of the new material.

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Bioinspired scaffolds mimicking natural bone-tissue properties holds great promise in tissue engineering applications towards bone regeneration. Within this work, a way to reinforce mechanical behavior of bioinspired bone scaffolds was examined by applying a physical crosslinking method. Scaffolds consisted of hydroxyapatite nanocrystals, biomimetically synthesized in the presence of collagen and l-arginine. Scaffolds were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy (SEM), microcomputed tomography, and nanoindentation. Results revealed scaffolds with bone-like nanostructure and composition, thus an inherent enhanced cytocompatibility. Evaluation of porosity proved the development of interconnected porous network with bimodal pore size distribution. Mechanical reinforcement was achieved through physical crosslinking with riboflavin irradiation, and nanoindentation tests indicated that within the experimental conditions of 45% humidity and 37 °C, photo-crosslinking led to an increase in the scaffold’s mechanical properties. Elastic modulus and hardness were augmented, and specifically elastic modulus values were doubled, approaching equivalent values of trabecular bone. Cytocompatibility of the scaffolds was assessed using MG63 human osteosarcoma cells. Cell viability was evaluated by double staining and MTT assay, while attachment and morphology were investigated by SEM. The results suggested that scaffolds provided a cell friendly environment with high levels of viability, thus supporting cell attachment, spreading and proliferation.

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