Study On Biomimetic Preparation Of Calcium Phosphate Based Bone Repair Material And Its 3D Printing

Calcium phosphate cement can be used as a bone repair material due to its excellent biocompatibility,biological activity,bone conduction and osteoinduction.Although calcium phosphate（CaP）powder synthesized by the traditional method has almost the same composition as the natural bone,it lacks the organic components of the natural bone,thus the bionic preparation of CaP powder has attracted widespread attention.Three dimensional（3D）printing is one of the research and commercial hotspots.In the field of bone repair,the main research and application direction is to use 3D printing technology to personalize the fine structure of bone repair implants,but 3D printing“ink”need high injectability and solidification,while the existing CaP printing“ink”has poor printability,which can not meet the requirements of 3D printing CaP.The principle of using mechanical activation method to modify the biomimetic preparation of CaP powder is that the solid particles will change its crystal structure and physical properties under mechanical force,and produce cracks and various defects,thereby increasing its injectability and reactivity.Therefore,bionic CaP powder was prepared mechanical activation and 3D printing technology to develop printable biomimetic CaP“ink”.（In the text,the abbreviations of powders and scaffolds:Gel is added in the solid phase before DCPD and CaP,Gel is added in the solidification solution after CaP,M is mechanical activation）.Using diammonium hydrogen phosphate and calcium nitrate tetrahydrate as raw materials,gelatin（Gelatin,Gel）as the organic template,to prepare biomimetic Gel-DCPD powder via wet synthesis.Gel-CaP powder prepared via calcium phosphate cement Biocement D formula was mechanical activated with ethanol solution and gelatin aqueous solution（solidifying solution）to produce Gel-MCaP as 3D printing“ink”.Different 3D printing“ink”was stirred with different solid-liquid ratio.Biomimetic Gel-DCPD,Gel-CaP powder and CaP scaffold were characterized with X-ray diffractomer（XRD）,fourier transform infrared spectroscope（FTIR）,thermo gravimetric analyzer（TGA）,universal capability testing machine and Gilmore double needle;The scaffolds of MCaP-RO,MCaP-2.4%Gel,60%Gel-MCaP-RO and 60%Gel-MCaP-2%Gel were put into the simulated body fluid in order to conduct in vitro degradation test.The mechanisms of hydration and in vitro degradation of the four group biomimetic CaP scaffolds were investgated.The investigation of the printability of Ca P“ink”were conducted by a series of characterization methods;3D printing biomimetic 60%Gel-CaP scaffolds of different particle sizes were co-cultured with human osteosarcoma cells（MG-63）.The growth of MG-63 cell was evaluated by different approaches.XRD results of biomimetic preparation of CaP powder showed that the addition of gelatin didn’t change the phase composition of DCPD;The infrared spectrum results showed that the adsorption of Ca²⁺in DCPD with gelatin;TGA results showed that the content of gelatin in 60%Gel-DCPD was only 13 wt.%,and the content of gelatin in 60%Gel-CaP powder was calculated to be 3.3 wt.%;The particle size of CaP powder gradually decreased with mechanical activation time,and the particle size stabilized basically after 10h,which was 2.2±0.4μm.The aqueous solution of gelatin improved the injectability of CaP pastes significantly,shortened the setting time of CaP pastes,while reducing the compressive strength of CaP scaffolds.In summary,the formula of the prepared CaP“ink”is able to meet 3D printing satisfies and which is composed of 60%Gel-CaP powder as solid phase after mechanical activation for 10 h,2 wt.%gelatin aqueous solution as solidifying solution,and the solid-liquid ratio was 0.7 g/m L.The injection rate of the CaP paste was 86.0±3.0%and the compressive strength was 2.1±0.1 MPa.Whereafter a nine-grid graphic（30 mm on a side and 5 mm on a small square）was printed by 3D printer.The paste prepared according to above-mentioned formula can be employed as an effective 3D printing.Hydration and degradation studies showed that the 60%Gel-MCaP and 60%Gel-MCaP-2%Gel scaffolds added with gelatin in the solid phase was more soluble,and it was easier to convert to the hydroxyapatite during the hydration process,and intermediate octacalcium phosphate wasn’t produced.The p H values of the degradation of the four group of scaffolds showed a same trend which increased firstly and then decreased.The losing weight and porosity both increased continuously.Therefore,the compressive strength decreased continuously.The 3D printability of 60%Gel-MCaP-2%Gel with different particle sizes showedthat the smaller the particle size is,the higher injectability of paste will achieve,and the injection rate of the paste was 93.0±3.1%after mechanical activation for 10 h.XRD results showed that the diffraction peak intensity of the 3D printing scaffold was stronger than that of the conventional mold scaffold,the diffraction peak of the 3D printing scaffold was sharper than that of the conventional mold scaffold.The 3D printing 60%Gel-MCaP scaffold had a lower compressive strength than that of the conventional mold scaffold,and a higher porosity as well.The microscopic appearance of the 3D printing scaffold showed that the porosity of the scaffold decreased with decreasing particle size.The surface of 3D printing scaffolds with different particle sizes showed that the surface roughness of the scaffolds with mechanical activation for 0,10,and 16 h was relatively rough,whereas the scaffold surface of mechanical activation for 2 h was smoother.The hollow humeral head has an internal structure could be printed into a shape with the length range from the top surface to the bottom is 38 and 90 mm,the width is 46 mm,the height is 88 mm.Cytological researches showed that the proliferation of MG-63 cells was positively correlated with culture time.The scaffolds of mechanical activation for 10 and 16 h were more conducive to cell growth and proliferation,and there was significant difference between the other two groups.In addition,the cell near the 3D printing CaP scaffold during co-culturation grew well and spread evenly.With the prolongation of culture time,the number of cells increased.The cells adhesion,spreading and growth in scaffold surface were observed by fluorescence microscopy and scanning electron microscope after co-cultured for 3 d.In this study,the preparation method of CaP powder was investigated and optimized.It provided a promising bone replacement material with higher injectibility,3D printing technology to develop printable biomimetic CaP“ink”.