Effect of the secretome present in the conditioned medium of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone rapair of mouse calvarial defects

Cell-free therapy using the cellular secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) has been reported in the scientific literature as a promising therapeutic strategy in the field of regenerative medicine with a view to repairing bone tissue. This secretome consists of a set of factors and molecules secreted into the extracellular space, which allows cell communication, biological regulation and modulation of various tissue physiological conditions. Among one of the secreted molecules is the bone morphogenetic protein 9 (BMP-9), important components of endochondral and intramembranous bone tissues, reported as one of the most osteoprogenitor of the BMPs family. Based on this, this study aimed to investigate the potential of the cellular secretome present in the CM by genetically edited MSCs cells for overexpression of the BMP-9 protein in inducing osteoblastic differentiation in vitro of primary MSCs and in vivo bone repair in critical defects created in the mouse calvarial. MSCs were obtained from the bone marrow of 8-week-old male C57BL/6 mice and underwent the gene editing process using the CRISPR-Cas9 technique, with one group infected only with the empty vector (MSCsVPR) while the other group received the combination of SP-dCas9-VPR endonuclease plus the customized guide RNA for the overexpression of BMP-9 with positive GFP reporter (MSCsBMP-9). After confirming the effectiveness of this process through the analysis of the gene expression of Bmp-9 by the PCR technique in real time and the positive labeling of GFP by flow cytometry, these cells were used to obtain the CM in different times. The secreted presence of BMP-9 in the collected CMs was confirmed by means of an ELISA immunoenzymatic test, and its highest expression peak occurred after 1 hour of culture without the presence of fetal bovine serum (FBS), the period of election of the CM to be used in the following experiments. To evaluate the effect of CMs on the osteoblastic differentiation of MSCs, the cells were grown in contact with the CM by MSCsVPR (CM-MSCsVPR) and by MSCsBMP-9 (CM-MSCsBMP-9) in the proportion 1:1 with fresh non-inducing growth medium (MEM 20%) always in the last 48 hours before the experiment to be carried out. Cell migration capacity was evaluated by the scratch method, cell viability by MTT colorimetric assay, gene expression of bone markers (Runx2, Alp and Opn) by real-time PCR, protein expression of bone markers (RUNX2, ALP and OPN) by western blot and the phenotypic activity of ALP in situ by the Fast Red method. The positive effect of CM-MSCsBMP-9 on osteoblastic differentiation of MSCs was noticeable, which was able to induce a phenotypic osteoblastic profile in vitro. As for the evaluation of the effect of CMs on bone repair, one week after the creation of critical defects of 4 mm created in the mouse calvarial, the defects were treated by injecting a volume of 25 µL of CM-MSCsVPR and CM-MSCsBMP-9. Four weeks after the treatment, it was noticed through the three-dimensional reconstructions of the morphometric analysis of µCT, that the treatment with CM-MSCsBMP-9 favored the repair of the bone tissue compared to the defects treated with CM-MSCsVPR and the animals that were not submitted to no treatment, generating a greater bone volume, percentage of bone volume, bone surface and a greater trabecular number in bone tissue in vivo. These data were corroborated by histological analysis, showing that MSCs genetically edited for BMP-9 overexpression were able to form bone tissue with similar quality to native bone, without inflammatory indicators. Furthermore, we also observed that treatment with CM-MSCsBMP-9 favored bone tissue repair in comparison of defects treated with MSCsBMP-9. Finally, this study showed that CM-MSCsBMP-9 has a high osteogenic and osteoinductive potential, being an interesting methodology for clinical application in the field of Dentistry and Medicine, in which, in addition to its advantages at the molecular level for tissue repair challenging sites, has low production cost and the possibility of carrying out a guided therapy for specific therapeutic effects. (AU)