Effect of the secretome of mesenchymal stem cells overexpressing BMP-9 on osteoblast differentiation and bone repair.

The secretome present in the conditioned medium (CM) of mesenchymal stem cells (MSCs) is a promising tool to be used in therapies to promote bone regeneration. Considering the high osteogenic potential of the bone morphogenetic protein 9 (BMP-9), we hypothesized that the secretome of MSCs overexpressing BMP-9 (MSCsBMP-9 ) enhances the osteoblast differentiation of MSCs and the bone formation in calvarial defects. CM of either MSCsBMP-9 (CM-MSCsBMP-9 ) or MSCs without BMP-9 overexpression (CM-MSCsVPR ) were obtained at different periods. As the CM-MSCsBMP-9 generated after 1 h presented the highest BMP-9 concentration, CM-MSCsBMP-9 and CM-MSCsVPR were collected at this time point and used to culture MSCs and to be injected into mouse calvarial defects. The CM-MSCsBMP-9 enhanced the osteoblast differentiation of MSC by upregulating RUNX2, alkaline phosphatase (ALP) and osteopontin protein expression, and ALP activity, compared with CM-MSCsVPR . The CM-MSCsBMP-9 also enhanced the bone repair of mouse calvarial defects, increasing bone volume, bone volume/total volume, bone surface, and trabecular number compared with untreated defects and defects treated with CM-MSCsVPR or even with MSCsBMP-9 themselves. In conclusion, the potential of the MSCBMP-9 -secretome to induce osteoblast differentiation and bone formation shed lights on novel cell-free-based therapies to promote bone regeneration of challenging defects.

Mesenchymal stem cells overexpressing BMP-9 by CRISPR-Cas9 present high in vitro osteogenic potential and enhance in vivo bone formation.

Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and  protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-ß/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.

Modulation of bone morphogenetic protein-9 expression and processing by insulin, glucose, and glucocorticoids: possible candidate for hepatic insulin-sensitizing substance.

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance (HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting (72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology.