Retracted Article: Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dickkopf-1 to activate Wnt/β-catenin pathway

Statement of Retraction We, the Publisher of the journal Bioengineered, have retracted the following article: Zhi Peng et al - Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dickkopf-1 to activate Wnt/β-catenin pathway, Bioengineered (2021) (DOI: https://www.10.1080/21655979.2021.1996015) Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors checked their data and confirmed there are fundamental errors present. Therefore, they have agreed to the retraction of this article. The authors apologise for this oversight. We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.


Introduction
Osteoporosis (OP) is a systematic bone disease characterized by bone loss, imbalance of bone metabolism and destruction of trabecular microstructure, and is the most common geriatric disease, especially in postmenopausal women [1].Several studies have confirmed that bone healing in osteoporotic women and osteoporotic animals is remarkably delayed [2,3].In the past few years, a variety of strategies have been developed to treat osteoporotic defects, such as autologous bone transplantation, allogeneic bone transplantation, and the combination of scaffold materials with growth factors or cells, but the efficacy is not satisfactory.Therefore, there is an urgent need to find new treatments to improve the prognosis of OP.
Bone remodeling consists of two processes, osteoblast mediated bone formation and osteoclast mediated bone resorption.Once the homeostasis of bone remodeling is broken, bone diseases such as OP will develop.Bone marrow mesenchymal stem cells (BMSCs) are regarded as promising seed cells in tissue engineering, due to easy accessibility and multipotent ability to differentiate into adipocyte, osteoblast, cardiomyocytes, and neurons.Recently, emerging evidence has demonstrated that the crosstalk between monocyte-macrophage-osteoclasts and osteoblasts plays a vital role in the pathology of OP [4,5].Osteoblasts could be differentiated from BMSCs, and such a process is finely regulated by several transcription factors [6].A series of bonederived regulators responsible for the cross-talk have already been identified, such as transforming growth factor-β, bone morphogenetic protein-9, runtrelated transcription factor −2 (Runx2), Osterix, and alkaline phosphatase (ALP) [7,8].Therefore, further understanding of the mechanism underlying osteogenic differentiation is crucial for the development of therapeutic approaches for OP.
Interestingly, bone remodeling is regulated by the factors packaged in lipid bilayered membrane vesicles called exosomes [9,10].Exosome, as an important part of the microenvironment, is a membrane vesicle with 40-150 nm in diameter secreted by numerous types of cells, such as dendritic cells, reticulocytes, tumor cells, B cells, T cells, mast cells, epithelial cells, and BMSCs [11,12].Prevailing evidences revealed that exosomes play pivotal role in cell communication to regulate the function and differentiation of homogeneous and heterogeneous recipient cells by transferring biologically active molecules, such as proteins, lipids, mRNA, microRNAs (miRNAs) [5,13].Earlier studies based on animal models have suggested that local transplantation of BMSCs promoted bone regeneration [14].BMSCs actively produce exosomes, and BMSCconditioned medium potently stimulates bone regeneration [15].However, the potential role and underlying mechanisms of BMSC-derived exosomes (BMSC-exo) in bone regeneration have not been fully elucidated.Qin et al. [13] compared the miRNAs in BMSCs and its exosomes by RNA sequencing, and found that three osteogenicrelated miRNAs including miR-196a, miR-27a, and miR-206 were highly enriched in BMSC-exo.Notably, miR-196a has been identified to promote pancreatic cancer cell proliferation [16].However, there are few reports about the role of miR-196a in the occurrence and development of OP.
In summary, we speculate that miR-196a derived from BMSCs-exo may be involved in the regulation of osteogenic differentiation.As expected, we presented in vitro evidence to demonstrate that BMSC-exo could enter osteoblasts to promote osteoblastic differentiation.Moreover, we found that miR-196a was a key exosomal component to promote osteoblastic differentiation via targeting Dickkopf-1 (Dkk1), which is a known negative regulator of Wnt/βcatenin pathway.

Statement
All methods were carried out in accordance with relevant guidelines and regulations, and all experimental protocols were approved by Kunming Medical University.

Isolation and identification of BMSCs-exo
Exosomes isolation was performed using an Exoquick reagent (System Biosciences, Pu Mai Technology, Beijing, China) according to the manufacture's protocol with minor modifications.Briefly, BMSCs culture medium was centrifuged at 8,000 g for 30 min.Then, the supernatants containing exosomes were concentrated using a 100 kDa ultrafiltration Vivaflow 200 module to 10-15 ml, and to a final volume of between 0.5 and 1 ml by a 100 kDa ultracentrifuge tube (3,000 g × 30 min).Next, exosomes were precipitated by adding Exoquick reagent (at 1:4 ratio) and incubated overnight at 4°C.Then, the exosome precipitation was obtained by centrifugation at 1,500 g for 30 min, and resuspended in PBS [17].
All procedures are performed at 4°C.Isolated exosomes were immediately used or stored at −80°C for subsequent analysis.The isolated exosomes were observed and identified by transmission electron microscopy (TEM) [18].Briefly, BMSC-exo was fixed with 2% paraformaldehyde at room temperature for 30 min.Then, 8 μL of sample was added drop by drop to an EM grid pretreated with UV light.After drying for 30 min, the exosomes were stained twice with 1% uranylic acid for 6 min each.Finally, TEM was performed at 120 kV using an H-7650 model (Hitachi, Tokyo, Japan).In addition, BMSC-exo was analyzed by Western blotting analysis for characteristic surface marker proteins including CD63, CD81, CD9, ALIX, and TSG101, and the proportion of positive CD9 and CD63 of BMSCexo was detected by flow cytometry [19].

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA samples were extracted according to TRIzol reagent instructions (Lifetech, USA).The extracted mRNA was reversely transcribed to complementary Deoxyribose Nucleic Acid (cDNA) according to the instructions of First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA).For miRNA cDNA synthesis, RNA was reverse transcribed using the TaqMan MicroRNA reverse transcription kit (Applied Biosystems, China).The expression of mRNA and miRNA was analyzed by the 2 −ΔΔCt method [21]

Flow cytometry
Cells were fixed with pre-cooled 70% alcohol for 24 h, centrifuged at 1,000 r/min for 5 min, followed by 2 washes with PBS.Next, cells were incubated with propidium iodide (PI) (Dongren Chemical Technology, Shanghai, China) containing RNase in the dark at 4°C.Next, cells were incubated with Annexin V-FITC (Partec GmbH, CyFlow Space) and PI at room temperature for 15 min and washed twice with phosphate buffer saline (PBS).Cell apoptosis was analyzed using a flow cytometer at an excitation wavelength of 488 nm.

Alizarin red staining
After osteoblast induction for 21 days, cells were fixed in 60% isopropanol for 1 min, followed by washing with PBS for 2 min.Subsequently, cells were stained with 10% alizarin red dyestuff (ScienCell, USA) for 10 min, followed by washing with PBS for 3 times.The cells were observed under optical microscope (Olympus, Tokyo, Japan).

Statistical analysis
Data were expressed as mean ± standard deviation (SD) and analyzed with Graph-Pad Prism 5.0 software (GraphPad Software, San Diego, CA).Statistical analyses were performed using one-way ANOVA, followed by Turkey's posttest.Differences with p < 0.05 were considered statistically significant.

BMSC-exo can enter HFOB1.19 cells
To confirm whether the vesicles extracted from BMSCs were exosomes, we identified the pair shape, size, and exosome markers of vesicles.The extracted vesicles from BMSCs culture supernatants showed small membrane vesicles sized from 40 to 100 nm (Figure 1a).Further, we examined

R E T R A C T E D
the expression of exosome markers in the extracted vesicles and supernatants that did not contain vesicles.The results indicated that extracted vesicles all expressed exosome markers CD63, CD81, CD9, ALIX, and TSG101, however, CD63, CD81, CD9, ALIX, and TSG101 were not expressed in the supernatant (Figure 1b).Flow cytometry also revealed that the positive rate of CD9 and CD63 were 95.06% and 99.82% in extracted vesicles, respectively (Figure 1c).All the above features suggest that the extracted vesicles are exosomes.As expected, compared to HFOB1.19 cells, the exosomes extracted from BMSCs showed higher miR-196a expression, while the expression of miR-196a further increased in the exosomes extracted from BMSCs transfected with miR-196a mimic, but decreased in those transfected with miR-196a inhibitor (P < 0.05, Figure 1d).In addition, we labeled exosomes by PKH-67 to explore whether exosomes can be taken up by HFOB1.19 cells.The results exhibited that exosomes were able to enter HFOB1.19 cells with or without exogenous regulation of miR-196a expression in exosomes, and there was no significant difference in uptake (Figure 1e).These results indicate that exosomes of BMSCs were successfully extracted, and HFOB1.19 was able to take up exosomes.

miR-196a enriched exosomes promoted osteoblast differentiation
To investigate the effect of exosome-derived miR-196a on osteogenic differentiation of HFOB1.19 cells, we exogenously regulated the expression level of miR-196a in exosomes and co-cultured them with HFOB1.19 cells.Alizarin red staining results showed that compared with normal HFOB1.19cells, a large number of calcified nodules appeared in HFOB1.19 cells after exosome treatment (Figure 2a).Compared with the Exosome group, overexpressing miR-196a of exosome could increase the calcified nodules in HFOB1.19 cells, while knockdown of miR-196a had the opposite result (P < 0.05, Figure 2a).As shown in Figure 2b, the expression of OPN, OCN, ALP and RUNX2 was significantly augment in HFOB1.19 cells after exosome treatment (P < 0.05).As expected, compared with the exosome group, the expression of OPN, OCN, ALP and RUNX2 significantly elevated in HFOB1.19 cells treated with the exosomes extracted from miR-196a mimic transfected BMSCs (P < 0.05, Figure 2b), but markedly dwindled in cells treated with the exosomes extracted from miR-196a inhibitor transfected BMSCs (P < 0.05, Figure 2b).When BMSCsexosome was taken up, the percentage of HFOB1.19 cell apoptosis significantly decreased compared to the HFOB1.19group, the take-up of miR-196a enriched exosomes further decreased the percentage of cell apoptosis (P < 0.05, Figure 3a).When the expression of miR-196a in the exosomes was decreased, the percentage of cell apoptosis increased (P < 0.05, Figure 3a).The expression of apoptosis-related proteins caspase-3 and caspase-6 showed the same trend (P < 0.05, Figure 3b).These results suggest that miR-196a derived from exosome facilitates osteogenic differentiation and inhibits apoptosis of HFOB1.19 cells.

Dkk1 is a direct target of miR-196a
Next, we used TargetScan to identify putative targets of miR-196a.Among potential targets we focused on Dkk1 because previous study revealed that Dkk1 was a target of miR-196a [23].The potential binding sequence of Mir-196a and Dkk1 was displayed in Figure 4a.To confirm whether miR-196a targets Dkk1, we examined the effects of miR-196a mimic and inhibitor on luciferase activity in HFOB1.19 cells transfected with luciferase reporters containing wild type (WT) or mutant (Mut) 3′ UTR of Dkk1.The results showed that luciferase activity of WT was significantly decreased by miR-196a mimic but increased by miR-196a inhibitor (P < 0.05, Figure 4b).Meanwhile, miR-196a mimics and inhibitor had no significant effect on luciferase activity of mutant construct.Moreover, we examined the effect of miR-196a mimic and inhibitor on miR-196a expression and Dkk1 protein expression in HFOB1.19 cells.The results demonstrated that miR-196a mimic increased miR-196a expression, while miR-196a inhibitor decreased miR-196a expression (P < 0.05, Figure 4c).In addition, miR-196a mimic decreased Dkk1 expression, while miR-196a inhibitor increased Dkk1 expression

R E T R A C T E D
(P < 0.05, Figure 4d).Dkk1 expression was significantly down-regulated in HFOB1.19 cells after Exosome treatment, and Dkk1 mRNA was significantly down-regulated in the Exo-miR-196a mimic group and up-regulated in the Exo-miR-196a inhibitor group compared to the Exosome group (P < 0.05, Figure 4d).These results confirmed that Dkk1 is a target of miR-196a.

Dkk1 knockdown promoted osteoblast differentiation
To investigate the effect of Dkk1 on osteogenic differentiation, we knockdown the expression level of HFOB1.19 cells by si-Dkk1 and co-cultured the treated HFOB1.19 cells with exosomes.The expression of Dkk1 was significantly reduced after transfection with si-Dkk1 in HFOB1.19 cells (P < 0.05, Figure 5a).Notably, the expression of Dkk1 was significantly lower in the Exosome+si-Dkk1 group compared to the si-Dkk1 group (P < 0.05, Figure 5a).Alizarin red staining indicated that knockdown of Dkk1 significantly reduced calcified nodules in HFOB1.19 cells, and further reduced calcified nodules in HFOB1.19 cells after co-culture with exosomes (Figure 5b).In addition, the expression levels of OPN, OCN, ALP and RUNX2 were significantly reduced after transfection with si-Dkk1 in HFOB1.19 cells (P < 0.05, Figure 5c).As expected, the expression levels of OPN, OCN, ALP and RUNX2 were lower in the Exosome+si-Dkk1 group compared

R E T R A C T E D
to the si-Dkk1 group (P < 0.05, Figure 5c).The above results suggest that the promotion of osteogenic differentiation of HFOB1.19 cells by exosomes is achieved, at least in part, through inhibition of Dkk1 expression in HFOB1.19 cells.

BMSCs-exo targeted Dkk1 to activate Wnt/βcatenin pathway in HFOB1.19 cells
Wnt/β-catenin pathway play a key role in osteoblast differentiation, Dkk1 as a negative regulator of this pathway has been confirmed to be a direct target of miR-196a.Therefore, we detected the levels of Dkk1 and other components of Wnt/βcatenin pathways in HFOB1.19 cells by Western blotting analysis (Figure 6a).Densitometry analysis showed that Dkk1 levels decreased significantly in HFOB1.19 cells treated with exosomes compared to control cells, and further decreased in HFOB1.19 cells treated with miR-196a mimic (Figure 6b).Consistently, Wnt, Dvl, β-catenin levels increased significantly while p-GSK3β levels decreased significantly in cells treated with exosomes and miR-196a mimic compared to control cells (Figure 6c).Collectively, these results indicated that BMSCs-exo targeted Dkk1 to activate Wnt/β-catenin pathway.

Discussion
OP is a metabolic bone disease characterized by decreased bone mass and microstructural destruction of bone tissue, resulting in increased bone fragility and fracture [24].The proliferation and differentiation of BMSCs are closely related to bone metabolism.One aspect of OP is low potential of osteoblast differentiation [25].
The growing evidence has revealed that miRNAs play an important role in osteoblast differentiation.Kim et al. [26] found that miR-196a positively regulated osteoblast differentiation but the mechanism remained unclear.Exosomes can directly transfer various bioactive molecules including mRNAs, miRNAs and proteins from donate cells to recipient

R E T R A C T E D
cells.In this study, we found that BMSC-exo can be taken by recipient HFOB1.19 cells, and osteoblast differentiation of HFOB1.19 was promoted by BMSC-exo.Moreover, osteoblast differentiation was enhanced when miR-196a enriched BSMCsexo was taken up, but was antagonized when miR-196a was deprived.Therefore, miR-196a as one of the osteoblast-related miRNAs found enriched in BMSC-exo [13], may play an indispensable role in osteoblast differentiation.Moreover, we found that miR-196a directly inhibited the expression of its target Dkk1, a known negative regulator of Wnt/β-catenin pathway [27,28].
Osteoblast differentiation involves multiplesignaling pathways.Wnt signaling participates in cell proliferation, differentiation, migration, apoptosis to maintain the dynamic balance of cells [29,30].We found that Dkk1 was downregulated while Wnt/β-catenin signaling was activated when BMSCs-exo was delivered to HFOB1.19 osteoblast cells.Taken together, these results suggest that BMSCs-exo promotes osteoblast differentiation by delivering miR-196a which activates Wnt/βcatenin signaling via targeting Dkk1.BMSCs-exo could be considered as a new therapeutic approach to alleviate OP.