RESUMO
Objective:]To investigate the role of Arpin protein in bone repair by mediating migration of host bone marrow mesenchymal stem cells (BMSCs) to the bone defect area after transplantation of tissue engineering bone (TEB).Methods:Immunofluorescence was used to observe the expression and relative localization of Arpin and Arp2/3 proteins in BMSCs. Lentiviruses that ware designed to interfere with Arpin expression were constructed to transfect BMSCs for knockdown Arpin expression. Knockdown efficiency was verified by real-time quantitative reverse transcription PCR ( qRT-PCR) and Western blot. According to different levels of Arpin protein expression, experiments were divided into empty vector control group and an Arpin expression inhibition group in vitro and in vivo. In vitro experiments: the cell migration model was established with a migration chamber, then the cells from both groups were seeded on the up chamber, and the number of migrated cells were detected by fluorescence microscopy. Cells from both groups were seeded on six-well plates. Model of wound healing experiment was established and wound healing ratio was examined by microscopy. In vivo experiments: 8-week-old C57BL/6 mice were selected and assigned to empty vector control group and Arpin expression inhibition group according to the random number table, with 6 rats per group. Diaphysis of 2 mm and periosteum in the middle femur were excised to make a large segment of bone defects. Then, TEB was transplanted into the defect area and fixed.Green fluorescein-labeled BMSCs (1 million cells per mouse) from empty vector control group and Arpin expression inhibition group were injected through the tail vein. Number of BMSCs homing to the bone defect area was detected by immunofluorescence staining at day 2 and 7 after operation. At 4 weeks after operation, the femur was taken for a Micro-CT scan to analyze bone mass density(BMD), bone volume density (BV/TV), trabecular spacing (Tb.Sp) and trabecular thickness (Tb.Th). Then, the specimens were stained with pathological HE and MASSON staining to observe the quality of bone formation. Results:Mouse BMSCs expressed Arpin protein, which was located at the cell edge relative to Arp2/3. After transfection of lentivirus, BMSCs expressed green fluorescent protein, and the expression of Arpin gene and protein in Arpin expression inhibition group were decreased compared to empty vector control group ( P<0. 01). BMSCs migration was enhanced in Arpin expression inhibition group compared to empty vector control group [(76.6±6.6) vs. (105.7±6.5)] ( P<0. 01). Wound healing was accelerated in Arpin expression inhibition group compared to empty vector control group [(43.8±0.19)% vs. (62.6±3.2)%]( P<0.01). At day 2 after operation, immunofluorescence results showed no significant difference in cell migration between the two groups and almost no labeled cells migrated. At day 7 after operation, more cells migrated to the transplanted area in Arpin expression inhibition group compared to empty vector control group [(5.7±1.5) vs. (11.3±1.5)] ( P<0.01). At 4 weeks after operation, Micro-CT results showed that Arpin expression inhibition group had better bone formation quality than empty vector control group [BMD: (172.7±6.0)mg/cm 3vs. (140.0±6.0)mg/cm 3, BV/TV: (28.8±1.3)% vs. (23.4±0.9)%, Tb.Sp: (0.33±0.01)μm vs. (0.28±0.01)μm, Tb.Th: (0.11±0.01)μm vs.(0.15±0. 01)μm]( P<0.05). Pathological staining showed there were more new bone tissue in Arpin expression inhibition group ( P<0.01). Conclusion:Silencing Arpin protein expression promotes BMSCs to migrate to the bone defect area and improves bone repair effect.