TGFβ3 recruits endogenous mesenchymal stem cells to initiate bone regeneration
Authors: Keyu Luo
Jianzhong Xu
Tianyong Hou
Hao Pang
Fei Luo
Bo Yu
Moyuan Deng
Aijun Yang
Shiwu Dong
Tieniu Mei
Journal: Stem Cell Research & Therapy (2017)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: MCP1; Mesenchymal stem cell; Recruitment; TGFβ3; Vascular cells.
Affiliations: National & Regional United Engineering Lab of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, the Third Military Medical University, Chongqing, China., Department of Surgery, Fuzhou Mawei Naval Hospital, Fujian, China. ., National & Regional United Engineering Lab of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, the Third Military Medical University, Chongqing, China. ., Department of Biomedical Materials Science, College of Biomedical Engineering, Third Military Medical University, Chongqing, China. .

Abstract:  Background: The recruitment of a sufficient number of endogenous mesenchymal stem cells (MSCs) is the first stage of in-situ tissue regeneration. Transforming growth factor beta-3 (TGFβ3) could recruit stem or progenitor cells and endothelial cells to participate in tissue regeneration. However, the mechanism of TGFβ3 recruiting MSCs toward bone regeneration has remained obscure. Methods: We estimated the promigratory property of TGFβ3 on human bone marrow MSCs (hBMSCs) cocultured with the vascular cells (human umbilical artery smooth muscle cells or human umbilical vein endothelial cells) or not by Transwell assay. After the addition of the inhibitor (SB431542) or Smad3 siRNA, the levels of MCP1 and SDF1 in coculture medium were tested by ELISA kit, and then the migratory signaling pathway of hBMSCs induced by TGFβ3 was investigated by western blot analysis. In vivo, a 2-mm FVB/N mouse femur defect model was used to evaluate chemokine secretion, endogenous cell homing, and bone regeneration induced by scaffolds loading 1 μg TGFβ3 through qPCR, immunofluorescent staining, immunohistochemical analysis, and Micro-CT, compared to the vehicle group. Results: TGFβ3 (25 ng/ml) directly showed a nearly 40% increase in migrated hBMSCs via the TGFβ signaling pathway, compared to the vehicle treatment. Then, in the coculture system of hBMSCs and vascular cells, TGFβ3 further upregulated nearly 3-fold MCP1 secretion from vascular cells in a Smad3-dependent manner, to indirectly enhance nearly more than 50% of migrated hBMSCs. In vivo, TGFβ3 delivery improved MCP1 expression by nearly 7.9-fold, recruited approximately 2.0-fold CD31+ vascular cells and 2.0-fold Sca-1+ PDGFR-α+ MSCs, and achieved 2.5-fold bone volume fraction (BV/TV) and 2.0-fold bone mineral density, relative to TGFβ3-free delivery. Conclusions: TGFβ3, as a MSC homing molecule, recruited MSCs to initiate bone formation in the direct-dependent and indirect-dependent mechanisms. This may shed light on the improvement of MSC homing in bone regeneration. 

Retracted: No

PubPeer comments:
 Figures 1, 2, and 4 have areas of image overlap. I did not look elsewhere. *** Figure 1 ![file] *** Figure 2 ![file] *** Figure 4 ![file] , 
 Dear Biskrensis, Thank you very much for your kind comment! In the fig.4, the two similar images with blue box you mentioned, illustrated the same migration situation of hBMSCs in the coculture system of hBMSCs and hUASMCs without addition of tgfu03b23(0ng ml). In the fig.4, for the two similar images with yellow box you mentioned, the two images illustrated the same migration situation of hBMSCs in the coculture system of hBMSCs and hUVECs without addition of tgfu03b23(0ng ml). These were explained in figure legend of this article. The query for figure 4 may be a misunderstanding of the content of the article. In fig.1, two similar images marked with red box you mentioned, illustrated the role of tgfu03b23 on migration of hBMSCs after the Tu03b2RI II signaling pathway was blocked by inhibitor and the result of the experiment illustrated that the MSC migration number of two group was no significant difference. The first author made mistake for the image preparation, but result of the experiment would not be affected by this negligence. In fig.2 the two similar images marked with green box you mentioned, illustrated the migration of hBMSCs induced with hUVECs and hUVECs transfected with blank control siRNA. The experiment illustrated that the MSC migration numbers of two groups were significant differences with the Smad3 siRNA group which was not marked with green box. The first author made mistake for the image preparation, but result of the experiment would not be affected by this mistake. We are sorry that we have not notified it, and we have contacted the journal office and request for a correction following normal academic procedures. Again, thank you very much for your kind efforts for the medical science. Sincerely yours moyuan Deng , 
 #2 Thank you for correcting my misunderstanding of Figure 2 (mis-identified above as Figure 4). Your reply in that regard makes sense. However, despite the authors desire to correct the manuscript, the reply above seems as if it blames the u201cfirst authoru201d for the apparent errors, but oddly the response seems to be from the u201cfirst author.u201d Could the person responding in #2 above please identify themselves to avoid further confusion? Regardless of which author is replying, the images above (other than in Figure 2) are described as being treated differently, but appear to be the same. Update: Another reader noticed that I mixed up Figure 2 and 4 in my original illustration. , 
 Dear Biskrensis, thank you very much for your careful correction. Due to our carelessness, we were very sorry for the mistakes in the figure preparation. In order to verify this matter, we checked the original data and found that the errors are due to the fact that all the pictures of each group were placed in the same folder, and so many pictures were opened in the photoshop software at the same time that the pictures were pasted incorrectly when pasting pictures. For proof, we provided the original data of the experiment and made changes to the figures. Express regret for this mistake once again! For the Transwell assay in Fig.1b and fig.4d, we provided the original data as follows: ![file] ![file] For the Transwell assay in Fig.1b and Fig.4d, we provided the original and representative images of each group from three individual experiments. In Fig.1b, the previous figure in 25ng+SB group was mistakenly chosen from the 0ng+SB group. Here, we provided a new picture marked in red box . On the other hand, In Fig.4d, the previous figure in Scrambled hUVEC group was mistakenly chosen from the hUVEC group. Here, we provided a new picture marked in red box. Thank you for your carefulness. We are deeply sorry again for causing the confusion and inconvenience. And we will contact the publisher to correct our mistakes as soon as possible. , 
 #4 ![file] , 
 #4 Thank you for your reply. I await with eagerness and patience the response of the publisher. , 
 Dear Dr. Deng, There are at least two overlapping images in the newly provided originals for Figure 4 that you posted. I have marked these below in cyan and orange. The thin red box is your original marking. It is disappointing that there are still errors in your corrected set of figures. These duplications are between differently labeled cells and treatments. It appears your lab has trouble keeping track of which photo belongs to which experiment, and I have lost faith in all remaining data from this paper. ![file] , 
 To further emphasize my point, the Western blots in Figure 1C and and Figure 3D also appear to show similar lanes. * Green boxes: A TbRII lane in Figure 1C looks similar to a p-Smad3 lane in Figure 3D * Pink boxes: Two GAPDH lanes in Figure 1C look similar to two b-actin lanes in Figure 3D Not only represent these lanes different experimental treatment, they also represent different proteins. ![file] , 
 Dear Biskrensis, thank you for pointing out the errors and we will contact the journal to deal with this article as soon as possible. , 
 Retracted [5 August 2022] . ---------------------------- The Editors-in-Chief have retracted this article. After publication, concerns were raised regarding potential image duplication in the figures. Specifically: - In Fig. 1b, the 0 ngu2009+u2009SB and 25 ngu2009+u2009SB group images appear to originate from the same sample (rotated 90 degrees); - In Fig. 1c, Tu03b2RII lane 3 and GAPDH lanes 2 and 3 appear highly similar to Fig. 3d hUASMC p-Smad3 lane 1 and u03b2-actin (both lanes), respectively; - In Fig. 4d, the images for the hUVEC and Scrambled hUVEC groups appear to contain overlap. The authors have stated that these issues occurred due to errors in data storage. Additionally, they have been unable to provide the raw images of the western blots presented in Fig. 1c. The Editor-in-Chief therefore no longer has confidence in the presented data. Jianzhong Xu has stated on behalf of all authors that none of the authors agree to this retraction. ,