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In this study, strontium substituted hydroxyapatite (Sr-HAP) was synthesized using collagen type I and citrate as bi-templates and the obtained nanoparticles with high similarity to natural bone minerals were made into composite scaffolds with interconnected porous structure using a three-dimensional (3D) printing technique. A calcium deficient structure of HAP phase was caused by doping Sr which was verified by Fourier transform infrared, x-ray diffractometer, scanning electron microscopy and transmission electron microscopy. The Sr/(Sr + Ca) molar ratio in Sr-HAP nanoparticles was 5.8% estimated by EDX. Furthermore, both 3D printed scaffolds made of Sr-HAP and HAP had uniform porous structure and porosity of about 60%. Cell culturing indicated that MC3T3-E1 cells could adhere on the surface of the scaffolds and the strontium substitution could enhance cell adhesion, proliferation and alkaline phosphatase activity. The printed composite scaffolds were used to repair critical-sized rabbit calvarial defects with a diameter of 15 mm. The results showed that the Sr-HAP scaffolds had better osteogenic capability and stimulated more new bone formation within 12 weeks. It was suggested that these printed Sr-HAP composite scaffolds possessed high potential as candidates in the application of bone augmentation and regeneration.

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In this study, we have successfully fabricated magnesium (Mg) substituted hydroxyapatite nanocomposites (Mg-HA) by utilizing type I collagen (COL I) and citric acid (CA) through a bitemplate-induced biomimetic mineralization approach. The obtained composite nanoparticles were subsequently mixed with chitosan (CHI) and gelatin (Gel) to prepare porous scaffolds with interconnected structures by three-dimensional (3D) printing technique. The Mg-HA powders and composite scaffolds were characterized. The results showed that the substitution of Mg for Ca ions reduced the crystallinity of HA crystals, but did not significantly affect the size and structure of the nanocomposites. The morphology of Mg-HA scaffolds turned smoother compared with the HA scaffolds with Mg substitution. Furthermore, the biocompatibility of Mg-HA composite scaffolds was evaluated by metal ion release, cell attachment, proliferation, and differentiation of MC3T3-E1 cells.According to the results, as the more Ca 2+ was substituted by Mg 2+ , the more Mg 2+ was released from the samples and the pH in cultured medium was more acidic. It was suggested that Mg-HA scaffolds presented higher cell attachment, proliferation rate, increased expression of alkaline phosphatase (ALP) activity and osteogenic related gene, including osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), and COL I. Therefore, it was indicated that the 3D printed Mg-HA composite scaffolds with excellent biocompatibility and bioactivity were a potential candidate in bone tissue engineering.

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