Deficient bone regeneration causes bone defects or nonunion in a substantial proportion of trauma patients that urges for novel therapies. To develop a reliable therapy, we investigated the effect of negative pressure wound therapy (NPWT) on bone regeneration in vivo in a rat calvarial defect model. Negative pressure (NP) treatment in vitro was mimicked to test its effect on osteoblast differentiation in rat mesenchymal stem cells (MSCs) and MC3T3-E1 cells. Transcriptomic analyses, pharmaceutical interventions, and shRNA knockdowns were conducted to explore the underlying mechanism and their clinical relevance was investigated in samples from patients with nonunion. The potential application of a combined therapy of MSCs in hydrogels with negative pressure was tested in the rat critical-size calvarial defect model. We found that NPWT promoted bone regeneration in vivo and NP treatment induced osteoblast differentiation in vitro. NP induced osteogenesis via activating macroautophagy/autophagy by AMPK-ULK1 signaling that was impaired in clinical samples from patients with nonunion. More importantly, the combined therapy involving MSCs in hydrogels with negative pressure significantly improved bone regeneration in rat critical-size calvarial defect model. Thus, our study identifies a novel AMPK-ULK1-autophagy axis by which negative pressure promotes osteoblast differentiation of MSCs and bone regeneration. NPWT treatment can potentially be adopted for therapy of bone defects. Abbreviations: ADP, adenosine diphosphate; AICAR/Aic, acadesine; ALP, alkaline phosphatase; ALPL, alkaline phosphatase, biomineralization associated; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ARS, alizarin red S staining; ATG7, autophagy related 7; ATP, adenosine triphosphate; BA1, bafilomycin A₁; BGLAP/OCN, bone gamma-carboxyglutamate protein; BL, BL-918; BS, bone surface; BS/TV, bone surface per tissue volume; BV/TV, bone volume per tissue volume; C.C, compound C; CCN1, cellular communication network factor 1; COL1A1, collagen type I alpha 1 chain; COL4A3, collagen type IV alpha 3 chain; COL4A4, collagen type IV alpha 4 chain; COL18A1, collagen type XVIII alpha 1 chain; CQ, chloroquine; GelMA, gelatin methacryloyl hydrogel; GO, Gene Ontology; GSEA, gene set enrichment analysis; HIF1A, hypoxia inducible factor 1 subunit alpha; HPLC, high-performance liquid chromatography; ITGAM/CD11B, integrin subunit alpha M; ITGAX/CD11C, integrin subunit alpha X; ITGB1/CdD9, integrin subunit beta 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; micro-CT, microcomputed tomography; MSCs, mesenchymal stem cells; MTOR, mechanistic target of rapamycin kinase; NP, negative pressure; NPWT, negative pressure wound therapy; PRKAA1/AMPKα1, protein kinase AMP-activated catalytic subunit alpha 1; PRKAA2, protein kinase AMP-activated catalytic subunit alpha 2; PTPRC/CD45, protein tyrosine phosphatase receptor type C; ROS, reactive oxygen species; RUNX2, RUNX family transcription factor 2; SBI, SBI-0206965; SPP1/OPN, secreted phosphoprotein 1; THY1/CD90, Thy-1 cell surface antigen; SQSTM1, sequestosome 1; TGFB3, transforming growth factor beta 3; ULK1/Atg1, unc-51 like autophagy activating kinase 1.

Radiation-induced bone injury (RIBI) is one of the complications after radiotherapy for malignant tumors. However, there are no effective measures for the treatment of RIBI in clinical practice, and the mechanism of RIBI is unclear. We use a single high-dose ionizing radiation (6Gy) to analyze the effect of radiotherapy on osteoblast function. Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) were cocultured with irradiated osteoblasts to examine their therapeutic effects and mechanisms on osteoblast injury. The hUCB-MSC transplantation mouse model is used to confirm the in vivo role of hUCB-MSC treatment in radiation bone injury. Western blot analysis, qRT-PCR, immunohistochemistry, and immunofluorescence staining were used to analyze gene expression and angiogenesis. The apoptosis and migration of osteoblasts were measured by Hoechst staining, scratch test, and transwell. The differentiation of osteoblasts was measured by ALP and Alizarin red staining and transmission electron microscopy. The bone-related parameters of mice were evaluated by micro-CT analysis. We found that radiation can damage the DNA of osteoblasts; induce apoptosis; reduce the differentiation, migration, and adhesion of osteoblasts, leading to lipogenesis of bone marrow mesenchymal stem cells (BMSCs) and reducing the source of osteoblasts; and increase the number of osteoclasts in bone tissue, while MSC treatment prevents these changes. Our results reveal the inhibitory effect of radiation on osteoblast function. hUCB-MSCs can be used as a therapeutic target for the development of new therapeutic strategies for radiotherapy of bone injury diseases.

Bone resorption diseases, including osteoporosis, are usually caused by excessive osteoclastogenesis. Unc‐51‐like autophagy activating kinase 1 (ULK1), a mammalian serine/threonine kinase, may participate in the regulation of bone homeostasis and osteolytic metastasis. In this study, ULK1 expression during osteoclastogenesis was detected with RT‐PCR. We knocked down or overexpressed ULK1 through siRNA or lentiviral transduction in bone marrow macrophage (BMM). TRAP and phalloidin staining were performed to detect the osteoclastogenesis activity. Ovariectomized (OVX) mouse model of osteoporosis and a mouse of model osteoclast‐induced bone resorption were applied to explore the role of ULK1 in bone resorption in vivo. The results showed that ULK1 expression was downregulated during osteoclast differentiation and was clinically associated with osteoporosis. ULK1 inhibited osteoclast differentiation in vitro. Knockdown of ULK1 expression activated phosphorylation of c‐Jun N‐terminal kinase (JNK) and spleen tyrosine kinase (Syk). Docking protein 3 (DOK3) was coexpressed with ULK1 during osteoclastogenesis. Downregulation of DOK3 offsets the effect of ULK1 on osteoclastogenesis and induced phosphorylation of JNK and Syk. Activation of ULK1 impeded bone loss in OVX mice with osteoporosis. Additionally, upregulation of ULK1 inhibited osteoclast‐induced bone resorption in vivo. Therefore, our study reveals a novel ULK1/DOK3/Syk axis that regulates osteoclast differentiation and bone resorption, and targeting ULK1 is a potential therapeutic strategy for osteoporosis.

Objective This study aims to explore the effect of daidzein, which is a natural isoflavone compound mainly extracted from soybeans, on osteosarcoma and the potential molecular mechanism. Material and Methods 143B and U2OS osteosarcoma cells were treated with gradient concentrations of daidzein, and MTT assay was used to determine the cell proliferation capacity and IC₅₀. Hoechst 33342 staining and Annexin V-FITC/PI detection were used to determine apoptosis. Cell cycle was analyzed by flow cytometry, and migration ability were detected by transwell assays and scratch wound assay. An osteosarcoma xenograft mice model was applied to investigate the effect of daidzein on osteosarcoma in vivo. Systematic pharmacology and molecular modeling analysis were applied to predict the target of daidzein to osteosarcoma, and the target Src was verified by western blotting. We also observed the effect of daidzein on cell proliferation and apoptosis of Src-overexpressing osteosarcoma cells. Results In vitro, daidzein significantly inhibited 143B and U2OS osteosarcoma cell proliferation and migration, and induced cell cycle arrest. In vivo, daidzein exerts antitumor effects in osteosarcoma xenograft mice. After systematic screening and analysis, Src-MAPK signaling pathway was predicted as the highest-ranked pathway. Western blot demonstrated that daidzein inhibited phosphorylation of the Src-ERK pathway in osteosarcoma cells. Also, overexpression of Src could partially reverse the inhibitory effects of daidzein on osteosarcoma cell proliferation. Conclusion Daidzein exerts an antitumor effect on osteosarcoma, and the mechanism may be through the Src-ERK pathway.