Abstract
OBJECTIVE: To investigate the effects of titanium modified by ultrasonic acid etching/anodic oxidation (UAT) loaded with endothelial progenitor cells-exosome (EPCs-exo) on proliferation and osteogenic and angiogenic differentiations of adipose-derived stem cells (ADSCs). METHODS: The adipose tissue and bone marrow of 10 Sprague Dawley rats were harvested. Then the ADSCs and EPCs were isolated and cultured by collagenase digestion method and density gradient centrifugation method, respectively, and identified by flow cytometry. Exo was extracted from the 3rd to 5th generation EPCs using extraction kit, and CD9 and CD81 were detected by Western blot for identification. The three-dimensional printed titanium was modified by ultrasonic acid etching and anodic oxidation to prepare the UAT. The surface characteristics of UAT before and after modification was observed by scanning electron microscopy; UAT was placed in EPCs-exo solutions of different concentrations (100, 200 ng/mL), and the in vitro absorption and release capacity of EPCs-exo was detected by BCA method. Then, UAT was placed in DMEM medium containing different concentrations of EPCs-exo (0, 100, 200 ng/mL), and co-cultured with the 3rd generation ADSCs to construct UAT-ADSCs-exo. Cell morphology by laser confocal microscopy, live/dead cell staining, and cell proliferation were observed to evaluate biocompatibility; alkaline phosphatase (ALP) staining and alizarin red staining, RT-PCR detection of osteogenesis-related genes [osteocalcin (OCN), RUNT-related transcription factor 2 (Runx2), ALP, collagen type 1 (COL-1)] and angiogenesis-related gene [vascular endothelial growth factor (VEGF)], immunofluorescence staining for osteogenesis (OCN)- and angiogenesis (VEGF)-related protein expression were detected to evaluate the effect on the osteogenic and angiogenic differentiation ability of ADSCs. RESULTS: Scanning electron microscopy showed that micro-nano multilevel composite structures were formed on the surface of UAT. About 77% EPCs-exo was absorbed by UAT within 48 hours, while EPCs-exo absorbed on the surface of UAT showed continuous and stable release within 8 days. The absorption and release amount of 200 ng/mL group were significantly higher than those of 100 ng/mL group ( P<0.05). Biocompatibility test showed that the cells in all concentration groups grew well after culture, and the 200 ng/mL group was better than the other groups, with fully spread cells and abundant pseudopodia, and the cell count and cell activity were significantly higher than those in the other groups ( P<0.05). Compared with the other groups, 200 ng/mL group showed enhanced ALP activity and mineralization ability, increased expressions of osteogenic and angiogenic genes (OCN, Runx2, COL-1, ALP, and VEGF), as well as increased expressions of OCN and VEGF proteins, with significant differences ( P<0.05). CONCLUSION: EPCs-exo can effectively promote the adhesion, proliferation, and osteogenic and angiogenic differentiation of ADSCs on UAT surface, the effect is the most significant when the concentration is 200 ng/mL.