Bionic Tiger-Bone Powder Improves Bone Microstructure and Bone Biomechanical Strength of Ovariectomized Rats.

OBJECTIVE: To study the curative effect of bionic tiger-bone powder on osteoporosis in ovariectomized rats and investigate its mechanism. METHODS: Overall, a 120 female Wistar rats were randomly divided into Sham (sham-operated group), ovariectomy (OVX, ovariectomized group), TB (bionic tiger-bone powder treatment group after ovariectomy) and TB + VD groups (bionic tiger-bone powder + vitamin D treatment group after ovariectomy). The osteoporotic rat model was established 3 months after ovariectomy, and rats were intragastrically administrated with the corresponding drugs. Serum and bone tissue samples were collected from 10 rats in each group at weeks 4, 12 and 24 after intragastric administration. The bone microstructure of L6 vertebrae was analyzed by MicroCT, the biomechanical strength of left femurs was measured by the three-point bending test, and serum bone metabolism markers (P1NP and CTX) were detected by ELISA. Changes in bone collagen were analyzed by Masson's trichrome staining and hydroxyproline detection, and members of the BMP2/SMAD/RUNX2 and OPG/RANKL/RANK signal pathways were detected by immunoblotting. RESULTS: Compared with the OVX group, the serum level of P1NP in the TB and TB + VD groups was higher (P < 0.05), while the CTX level was lower (P < 0.05). Bone collagen fiber structures in the TB and TB + VD groups were repaired, and the collagen content was significantly higher than that in the OVX group (P < 0.05). In the TB group, BMP-2, P-SMAD1/5, RUNX2 and OPG levels were increased in bone tissue (P < 0.01), RANKL levels were decreased (P < 0.01), and the bone microstructure and biomechanical strength were improved. CONCLUSION: Bionic tiger-bone powder promotes osteogenesis by activating the BMP2/SMAD/RUNX2 signaling pathway, suppresses osteoclasts by downregulating the OPG/RANK/RANKL signaling pathway, increases bone collagen content, and improves bone microstructure and bone biomechanical strength.

Exosomes from bone marrow mesenchymal stem cells enhance fracture healing through the promotion of osteogenesis and angiogenesis in a rat model of nonunion.

BACKGROUND: As important players in cell-to-cell communication, exosomes (exo) are believed to play a similar role in promoting fracture healing. This study investigated whether exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exos) could improve fracture healing of nonunion. METHODS: BMMSC-Exos were isolated and transplanted into the fracture site in a rat model of femoral nonunion (Exo group) every week. Moreover, equal volumes of phosphate-buffered saline (PBS) and exosome-depleted conditioned medium (CM-Exo) were injected into the femoral fracture sites of the rats in the control and CM-Exo groups. Bone healing processes were recorded and evaluated by radiographic methods on weeks 8, 14 and 20 after surgery. Osteogenesis and angiogenesis at the fracture sites were evaluated by radiographic and histological methods on postoperative week 20. The expression levels of osteogenesis- or angiogenesis-related genes were evaluated in vitro by western blotting and immunohistochemistry. The ability to internalize exosomes was assessed using the PKH26 assay. Altered proliferation and migration of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast precursor cells (MC3TE-E1s) treated with BMMSC-Exos were determined by utilizing EdU incorporation, immunofluorescence staining, and scratch wound assay. The angiogenesis ability of HUVECs was evaluated through tube formation assays. Finally, to explore the effect of exosomes in osteogenesis via the BMP-2/Smad1/RUNX2 signalling pathway, the BMP-2 inhibitors noggin and LDN193189 were utilized, and their subsequent effects were observed. RESULTS: BMMSC-Exos were observed to be spherical with a diameter of approximately 122 nm. CD9, CD63 and CD81 were expressed. Transplantation of BMMSC-Exos obviously enhanced osteogenesis, angiogenesis and bone healing processes in a rat model of femoral nonunion. BMMSC-Exos were taken up by HUVECs and MC3T3-E1 in vitro, and their proliferation and migration were also improved. Finally, experiments with BMP2 inhibitors confirmed that the BMP-2/Smad1/RUNX2 signalling pathway played an important role in the pro-osteogenesis induced by BMMSC-Exos and enhanced fracture healing of nonunion. CONCLUSIONS: Our findings suggest that transplantation of BMMSC-Exos exerts a critical effect on the treatment of nonunion by promoting osteogenesis and angiogenesis. This promoting effect might be ascribed to the activation of the BMP-2/Smad1/RUNX2 and the HIF-1α/VEGF signalling pathways.

Exosomes from Bone Marrow Mesenchymal Stem Cells Inhibit Neuronal Apoptosis and Promote Motor Function Recovery via the Wnt/ß-catenin Signaling Pathway.

Severe spinal cord injury (SCI) is caused by external mechanical injury, resulting in unrecoverable neurological injury. Recent studies have shown that exosomes derived from bone marrow mesenchymal stem cells (BMSCs-Exos) might be valuable paracrine molecules in the treatment of SCI. In this study, we designed SCI models in vivo and in vitro and then investigated the possible mechanism of successful repair by BMSCs-Exos. In vivo, we established one Sham group and two SCI model groups. The Basso, Beattie, Bresnahan (BBB) scores showed that BMSCs-Exos could effectively promote the recovery of spinal cord function. The results of the Nissl staining, immunohistochemistry, and TUNEL/NeuN/DAPI double staining showed that BMSCs-Exos inhibited neuronal apoptosis. Western blot analysis showed that the protein expression level of Bcl-2 was significantly increased in the BMSCs-Exos group compared with the PBS group, while the protein expression levels of Bax, cleaved caspase-3, and cleaved caspase-9 were significantly decreased. The results of western bolt and qRT-PCR demonstrated that BMSCs-Exos could activate the Wnt/ß-catenin signaling pathway effectively. In vitro, we found that inhibition of the Wnt/ß-catenin signaling pathway could promote neuronal apoptosis following lipopolysaccharide (LPS) induction. These results demonstrated that BMSCs-Exos may be a promising therapeutic for SCI by activating the Wnt/ß-catenin signaling pathway.

Orthosilicic Acid Accelerates Bone Formation in Human Osteoblast-Like Cells Through the PI3K-Akt-mTOR Pathway.

Silicon is one of the essential trace elements in the human body; the deficiency of which may lead to bone diseases. Numerous animal experiments have shown that an appropriate increase in the intake of silicon is beneficial to enhancing bone density and toughness to prevent osteoporosis. However, the molecular mechanisms of the silicon-mediated osteogenesis process have not been sufficiently clarified. In this study, we determined the possible osteogenesis-related mechanisms of orthosilicic acid at a molecular level. We detected the relevant pathway and osteogenic indicators by immunofluorescence (IF), Western blot, alkaline phosphatase (ALP) staining (using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium [BCIP/NBT]), ALP enzyme labeling method, osteocalcin (OCN), and N-terminal propeptide of type 1 procollagen (P1NP) enzyme-linked immunosorbent assay (ELISA). We found that orthosilicic acid is capable of enhancing the expression of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), phospho-protein kinase B (P-Akt), phospho-mammalian target of rapamycin (P-mTOR), and related osteogenic markers (runt-related transcription factor 2 [RUNX2], type I collagen [COL1], ALP, OCN, and P1NP). However, with the addition of PI3K-Akt-mTOR pathway-specific inhibitor LY294002, the expression of PI3K, P-Akt, P-mTOR, RUNX2, COL1, ALP, OCN, and P1NP decreased. The results indicated that the PI3K-Akt-mTOR pathway played a positive regulatory role in the process of orthosilicic acid-mediated osteogenesis in vitro.