No abstract

The Wnt pathway is a new target in bone therapeutic space. WNT proteins are potent stem cell activators and pro-osteogenic agents. Here, we gained insights into the molecular and cellular mechanisms responsible for liposome-reconstituted recombinant human WNT3A protein (L-WNT3A) efficacy to treat osteonecrotic defects. Skeletal injuries were coupled with cryoablation to create non-healing osteonecrotic defects in the diaphysis of the murine long bones. To replicate clinical therapy, osteonecrotic defects were treated with autologous bone graft, which were simulated by using bone graft material from syngeneic ACTB-eGFP-expressing mice. Control osteonecrotic defects received autografts alone; test sites received autografts treated ex vivo with L-WNT3A. In vivo µCT monitored healing over time and immunohistochemistry were used to track the fate of donor cells and assess their capacity to repair osteonecrotic defects according to age and WNT activation status. Collectively, analyses demonstrated that cells from the autograft directly contributed to repair of an osteonecrotic lesion, but this contribution diminished as the age of the donor increased. Pre-treating autografts from aged animals with L-WNT3A restored osteogenic capacity to autografts back to levels observed in autografts from young animals. A WNT therapeutic approach may therefore have utility in the treatment of osteonecrosis, especially in aged patients.

Background Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) are considered as candidates for osteoporosis (OP) therapy. Estrogen is critical in the maintenance of bone homeostasis. However, the role of estrogen and/or its receptor in BMSC-Exos treatment of OP, as well as its methods of regulation during this process remain unclear. Methods BMSCs were cultured and characterized. Ultracentrifugation was performed to collect BMSC-Exos. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to identify BMSC-Exos. We examined the effects of BMSC-Exos on the proliferation, osteogenic differentiation, mineralization, and cell cycle distribution of MG-63 cells. The protein expression of estrogen receptor α (ERα) and the phosphorylation of ERK were investigated through western blotting. We determined the effects of BMSC-Exos on the prevention of bone loss in female rats. The female Sprague–Dawley rats were divided into three groups: the sham group, ovariectomized (OVX) group, and the OVX + BMSC-Exos group. Bilateral ovariectomy was performed in the OVX and OVX + BMSC-Exos groups, while a similar volume of adipose tissue around the ovary was removed in the sham group. The rats in OVX group and OVX + BMSC-Exos group were given PBS or BMSC-Exos after 2 weeks of surgery. Micro-CT scanning and histological staining were used to evaluate the in vivo effects of BMSC-Exos. Results BMSC-Exos significantly enhanced the proliferation, alkaline phosphatase activity, and the Alizarin red S staining in MG-63 cells. The results of cell cycle distribution demonstrated that BMSC-Exos increased the proportion of cells in the G2 + S phase and decreased the proportion of cells in the G1 phase. Moreover, PD98059, an inhibitor of ERK, inhibited both the activation of ERK and the expression of ERα, which were promoted by administration of BMSC-Exos. Micro-CT scan showed that in the OVX + BMSC-Exos group, bone mineral density, bone volume/tissue volume fraction, trabecular number were significantly upregulated. Additionally, the microstructure of the trabecular bone was preserved in the OVX + BMSC-Exos group compared to that in the OVX group. Conclusion BMSC-Exos showed an osteogenic-promoting effect both in vitro and in vivo, in which ERK-ERα signaling might play an important role.

Exosomes derived from bone marrow mesenchymal stem cells (BMSC-Exos) may modulate the M1/M2 polarization of macrophages during osteoarthritis (OA). However, the underlying mechanisms of BMSC-Exos in this process still need to be elucidated. In this study, the effects of BMSC-Exos on macrophages after induction of lipopolysaccharide (LPS) were determined. The protein expression of Akt, PINK1, and Parkin in macrophages were tested. We prepared an OA model by resecting the anterior cruciate ligament and medial meniscus of SD rats. BMSC-Exos could reduce M1 and promote M2 macrophage polarization, inhibit mitochondrial membrane damage and reactive oxygen species (ROS) production. Akt phosphorylation was downregulated but significantly recovered after treatment with BMSC-Exos (P<0.01). PINK1/Parkin signaling was crucial in mitochondrial damage and subsequent M1 macrophage polarization. The protein expression of PINK1 and Parkin was significantly inhibited by BMSC-Exos (P<0.01). Histological analyses revealed that BMSC-Exos alleviated cartilage damage, inhibited M1 polarization, and promoted M2 polarization in the synovium. The expression of PINK1 and Parkin in the synovium was decreased when BMSC-Exos were used. ELISA results revealed that BMSC-Exos also changed the systematic inflammatory state. These data suggest that BMSC-Exos ameliorate OA development by regulating synovial macrophage polarization, and one of the underlying mechanisms may be through inhibiting PINK1/Parkin signaling. Therefore, BMSC-Exos are a potential useful therapeutic approach for OA.

Background: Exosomes derived from bone marrow stem cells (BMSC-Exos) are considered as candidates for osteoporosis (OP) therapy. Estrogen is critical in the maintenance of bone homeostasis. However, the role of estrogen and/or its receptor in BMSC-Exos treatment of OP, as well as its methods of regulation during this process remain unclear.Methods: BMSCs were cultured and characterized. Ultracentrifugation was performed to collect BMSC-Exos. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting were used to identify BMSC-Exos. We examined the effects of BMSC-Exos on the proliferation, osteogenic differentiation, mineralization, and cell cycle distribution of MG-63 cells. The protein expression of estrogen receptor α (ERα) and the phosphorylation of extracellular signal-regulated kinase (ERK) were investigated through western blotting. We determined the effects of BMSC-Exos on the prevention of bone loss in female rats. The female SD rats were divided into three groups: the sham group, ovariectomized (OVX) group, and the OVX + BMSC-Exos group. Bilateral ovariectomy was performed in the OVX and OVX + BMSC-Exos groups, while a similar volume of adipose tissue around the ovary was removed in the sham group. The rats in OVX+BMSC-Exos group were given BMSC-Exos after 2 weeks of surgery. Micro-CT scanning and histological staining were used to evaluate the in vivo effects of BMSC-Exos.Results: BMSC-Exos significantly upregulated the proliferation, ALP activity, and the ARS staining in MG-63 cells. The results of cell cycle distribution demonstrated that BMSC-Exos significantly increased the proportion of cells in the G2+S phase and decreased the proportion of cells in the G1 phase. Moreover, PD98059, an inhibitor of ERK, downregulated the expression of ERα, which was promoted by administration of BMSC-Exos. Micro-CT scan showed that in the OVX+BMSC-Exos group, BMSC-Exos significantly promoted ERα expression, with ameliorated bone mineral density (BMD), bone volume/tissue volume fraction (BV/TV), trabecular number (Tb. N), and trabecular separation (Tb. Sp). Additionally, the microstructure of the trabecular bone was preserved in the OVX + BMSC-Exos group compared to that in the OVX group.Conclusion: BMSC-Exos showed an anti-osteoporotic role in OVX rats both in vitro and in vivo, which may involve the ERα/ERK signaling pathway.