Spirulina protein concentrate hydrogel reinforced by hydroxyapatite nanoparticles and hydroxychloroquin: A novel platform for bone regeneration

Among the fabrication methods, hydrogels have received significant attention due to their excellent ability to mimic the extracellular matrix structure. Spirulina is a type of unicellular blue-green algae. It is an excellent source of protein and a wide variety of other nutrients. Hydroxychloroquin (HCQ) reduces bone resorption and osteoclasts growth. This study hypothesized that the hydrogel, which is composed of Spirulina protein cross- linked by transglutaminase and reinforced by hydroxyapatite (HAP) nanoparticles loaded with HCQ, could potentially aid in bone regeneration. The protein from Spirulina was extracted using the probe sonication method. Afterwards, hydrogels were prepared by incorporating the Spirulina protein with gelatin and cross-linked with varying percentages of transglutaminase. HCQ-loaded HAP nanoparticles were used to enhance not only the mechanical properties of the hydrogels but also to stimulate bone mineralization. Hydrogels were studied for their mechanical and rheological properties in vitro, as well as their ability to swell, break down, injectability, and release HCQ. Cell adhesion, proliferation, and bone mineralization on MG-63 osteoblast cells were also studied using Alizarine red and alkaline phosphatase markers. The loading efficiency of HCQ in HAP nano- particles was about 97 %. The HCQ release rate from hydrogel containing HAP was 85 % in 9 hours. The tensile strength and injectability force of the optimized hydrogel were in the range of 530.75 KPa and 21 N, respectively. The results showed that hydrogels made with 10 % protein, 150 units of transglutaminase, 6 % gelatin, and HAP loaded with HCQ had the best cell behavior and improved markers of bone regeneration. The promising results obtained suggest that the prepared hydrogel may act as a suitable construct for accelerating bone regeneration.