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Evidence indicates that microRNAs (miRNAs) play vital roles in regulating osteogenic differentiation and bone formation. Methods : Here, we show that a polyethyleneimine (PEI)-functionalized graphene oxide (GO) complex efficiently loaded with the miR-214 inhibitor is assembled into silk fibroin/hydroxyapatite (SF/HAP) scaffolds that spatially control the release of the miR-214 inhibitor. Results : SF/HAP/GO scaffolds with nanosized GO show high mechanical strength, and their hierarchical microporous structures promote cell adhesion and growth. The SF/HAP/GO-PEI scaffolds loaded with mir-214 inhibitor (SF/HAP/GPM) were tested for their ability to enhance osteogenic differentiation by inhibiting the expression of miR-214 while inversely increasing the expression of activating transcription factor 4 (ATF4) and activating the Akt and ERK1/2 signaling pathways in mouse osteoblastic cells (MC3T3-E1) in vitro . Similarly, the scaffolds activated the osteoblastic activity of endogenous osteoblast cells to repair critical-sized bone defects in rats without the need for loading osteoblast cells. Conclusion : This technology is used to increase osteogenic differentiation and mineralized bone formation in bone defects, which helps to achieve cell-free scaffold-based miRNA-inhibitor therapy for bone tissue engineering., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Osteoporosis is a disease characterized by the degeneration of bone structure and decreased bone mass. Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) have multiple advantages that make them ideal seed cells for bone regeneration, including high-level proliferation, multi-differentiation potential and favorable immune compatibility. Distal-less homeobox (DLX)3, an important member of the DLX family, serves a crucial role in osteogenic differentiation and bone formation. The present study aimed to evaluate the effects of DLX3 on the proliferation and osteogenic differentiation of human iPSC-MSCs. iPSC-MSCs were induced from iPSCs, and identified via flow cytometry. Alkaline phosphatase (ALP), Von Kossa, Oil Red O and Alcian blue staining methods were used to evaluate the osteogenic, adipogenic and chondrogenic differentiation of iPSC-MSCs. DLX3 overexpression plasmids were constructed and transfected into iPSC-MSCs to generate iPSC-MSC-DLX3. iPSC-MSC-GFP was used as the control. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to measure the expression of DLX3 2 days after transfection. Subsequently, cell proliferation was assessed using a Cell Counting Kit-8 assay on days 1, 3, 5 and 7. RT-qPCR and western blotting were used to analyze osteogenic-related gene and protein expression levels on day 7. ALP activity and mineralized nodules were assessed via ALP staining on day 14. Statistical analysis was performed using an unpaired Student's t-test. Flow cytometry results demonstrated that iPSC-MSCs were positive for CD73, CD90 and CD105, but negative for CD34 and CD45. iPSC-MSC-DLX3 had significantly lower proliferation compared with iPSC-MSC-GFP on days 5 and 7 (P<0.05). mRNA expression levels of osteogenic markers, such as ALP, osteopenia (OPN), osteocalcin (OCN) and Collagen Type I (COL-1), were significantly increased in iPSC-MSC-DLX3 compared with iPSC-MSC-GFP on day 7 (P<0.05). Similarly, the protein expression levels of ALP, OCN, OPN and COL-1 were significantly increased in iPSC-MSC-DLX3 compared with iPSC-MSC-GFP on day 7 (P<0.05). The number of mineralized nodules in iPSC-MSC-DLX3 was increased compared with that in iPSC-MSC-GFP on day 14 (P<0.05). Thus, the present study demonstrated that DLX3 serves a negative role in proliferation, but a positive role in the osteogenic differentiation of iPSC-MSCs. This may provide novel insight for treating osteoporosis. [ABSTRACT FROM AUTHOR]