Abstract:

Objective To print the porous titanium alloy scaffolds by 3D printing technology，and to study the effect of micro arc oxidation （MAO）/alkali treatment and arginine-glycine-aspartic acid（RGD） coating on the biological behavior of osteoblasts. Methods The 3D porous titanium alloy scaffolds were designed and printed， and they were divided into MAO group， MAO/alkali treatment （MN） group， MAO/alkali treatment loaded with RGD peptide coating （MNR） group after different surface treatments，another blank control group was set up.The elastic modulus of the scaffolds in various groups was detected.The microstructures of the scaffold surface in various groups were observed by scanning electron microscope （SEM）， the elemental compositions of the scaffold surface were detected by energy dispersive spectroscopy （EDS）， and the contact angles of water droplets on the scaffold surface were measured by contact angle measuring instrument.The mouse embryonic osteoblast precursor cells （MC3T3-E1 cells） were co-cultured with the scaffolds in various groups.CCK-8 assay was used to detect the cell proliferation activities，Live/Dead cell staining was used to detect the biocompatibility of the scaffolds in various groups， cell adhesion test was used to observe the adhesion of cells on the material surface， and alkaline phosphatase （ALP） kit was used to detect the ALP activities.Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of Runt-related transcription factor 2（Runx2）， osteopontin（OPN） and osteocalcin（OCN） mRNA in the cells in various groups. Results The elastic modulus of 3D printed scaffolds was （1.17±0.62） GPa. The SEM observation results showed that the surface of the scaffolds after MAO treatment showed crater-like morphology. After alkali treatment， small cracks and nano scale structure appeared.The scattered RGD particles were found on the surface of the scaffolds loaded with RGD peptide coating.The ERS detection showed that the RGD peptide coating was successfully loaded on the surface of scaffolds. The contact angle measuring instrument detection results showed that the surface contact angles were MAO group>MN group>MNR group. The cell proliferation activities in three groups showed an increasing trend on the 1st， 3rd and 5th days detected by CCK-8 assay， and there were statistically significant differences in the cell proliferation activities between various groups on the 3rd and 5th days of culture（P<0.05 or P<0.01）. The results of Live/Dead cell staining showed that the scaffolds in three groups had good in vitro compatibility. In cell adhesion test， after 48 h of co-culture， the number and morphological extension of cells in MNR group were better than those in MAO group and MN group. On the 7th day of culture， compared with MAO group， the activity of ALP in the cells in MNR group was significantly increased （P<0.01）； on the 14th day of culture， there were significant differences in the ALP activities between three groups （P<0.05 or P<0.01）.The results of RT-qPCR method showed that the expression levels of Runx2 and OPN mRNA in MN group and MNR group were higher than those in blank control group and MAO group on the 7th day of culture （P<0.01）， and the expression level of OCN mRNA in MNR group was higher than those in blank control group and MAO group （P<0.05）； on the 14th day of culture，there were significant differences in the expression levels of Runx2 and OPN mRNA between three groups（P<0.05 or P<0.01）. Compared with blank control group， the expression levels of OCN mRNA in MAO group，MN group，and MNR group were significantly increased （P<0.01）. Conclusion The 3D printed porous titanium alloy scaffolds hve the elastic modulus matching with the human bone tissue. The surface MAO/alkali treatment and loading with RGD peptide coating are non-toxic to the MC3T3-E1 cells and can promote their osteogenic differentiation.

Key words: 3D printing, Titanium alloy, Micro-arc oxidation, Alkali treatment, Arginine-glycine-aspartic acid peptide