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Boron-containing mesoporous bioactive glass (B-MBG) scaffolds could be capable of promoting osteogenesis by activating Wnt/β-catenin signaling pathway during the process of bone defect repair. Despite this, more involving molecular controls are still largely unclear. In the present study, we identified that the downstream of Wnt/β-catenin signaling pathway named transcription factor 7-like 2 (TCF7L2) served as a key effector to promote boron-induced bone regeneration and osteogenesis through lipocalin 2 (LCN2). TCF7L2 was highly expressed in osteoblasts when treated with B-MBG scaffold extraction than MBG. LCN2, as a secreted bone factor, positively affected osteogenic differentiation of MC3T3-E1 and osteogenesis in vivo, which could be induced by TCF7L2. In addition, interference of TCF7L2 decreased the osteogenic differentiation of osteoblasts. Finally, we identified that rLCN2 could rescue the poor ability of osteogenic differentiation of MC3T3-E1 whose Tcf7l2 gene was knocked down by lentiviral transfection of shRNA. Our findings provide some new insights into the molecular controls of boron-associated bone regeneration and potential therapeutic targets for the treatment of bone defects., Competing Interests: Declaration of competing interest No competing interests exist among all authors., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Controlling of pro-inflammation induced by pro-inflammatory cytokines and anti-inflammatory response induced by M2 macrophages is important for osteogenesis in the process of bone tissue repair. Thus, we fabricated biomimetic anti-inflammatory nano-capsule (BANC) that can block cytokines and promote M2 macrophage polarization, presenting a positive role for bone tissue repair. The BANC is a biomimic nanosystem, coated with lipopolysaccharide-treated macrophage cell membranes with cytokine receptors enveloping gold nanocage (AuNC) as "cytokine blocker", and loaded with resolvin D1 inside into AuNC as "M2 polarization inducer" whose controlled-release could be triggered under near-infrared laser irradiation in sequence, and these chronological events were consistent with the healing process of bone tissue repair. Moreover, in vivo application of femoral bone defects revealed that the BANC composite boron-containing mesoporous bioactive glass scaffolds improved the final effects of bone tissue repair through preventing inflammatory response, promoting M2 polarization in sequence in accord with the in vitro investigation. Hence, cytokine neutralization and M2 macrophage polarization enables the BANC to enhance the bone tissue repair as a biomimetic anti-inflammation effector. Therefore, this study provides potential therapeutic strategies for trauma-mediated or inflammation-related bone defects based on a biomimetic nanomaterial with weakened pro-inflammatory and enhanced anti-inflammatory effects. STATEMENT OF SIGNIFICANCE: Cell membrane-mimic nanomaterials have been popular for blocking natural cell responses for some infection diseases, yet their role in biological process of bone repair is unknown. Here, we fabricated Biomimetic Anti-inflammatory Nano-Capsule (BANC), coated with cell membrane with cytokines receptors on the surface which could neutralize the pro-inflammatory cytokine receptor to block activated pro-inflammation, loaded with Resolvin D1 inside which could be controllably released by NIR irradiation to promote M2 macrophage polarization for the following bone formation during the process of bone repair. Administration of BANC as cytokines blocker and M2 polarization inducer to enhance the bone regeneration, thus presenting a promising potential for the treatment of bone repair and regeneration., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Hypoxia in the vicinity of bone defects triggers the osteogenic differentiation of precursor cells and promotes healing. The activation of STAT3 signaling in mesenchymal stem cells (MSCs) has similarly been reported to mediate bone regeneration. However, the interaction between hypoxia and STAT3 signaling in the osteogenic differentiation of precursor cells during bone defect healing is still unknown., Methods: In this study, we assessed the impact of different durations of CoCl 2 -induced cellular hypoxia on the osteogenic differentiation of MSCs. Role of STAT3 signaling on hypoxia induced osteogenic differentiation was analyzed both in vitro and in vivo. The interaction between cellular hypoxia and STAT3 signaling in vivo was investigated in a mouse femoral bone defect model., Results: The peak osteogenic differentiation and expression of vascular endothelial growth factor (VEGF) occurred after 3 days of hypoxia. Inhibiting STAT3 reversed this effect. Hypoxia enhanced the expression of hypoxia-inducible factor 1-alpha (HIF-1α) and STAT3 phosphorylation in MSCs. Histology and μ-CT results showed that CoCl 2 treatment enhanced bone defect healing. Inhibiting STAT3 reduced this effect. Immunohistochemistry results showed that CoCl 2 treatment enhanced Hif-1α, ALP and pSTAT3 expression in cells present in the bone defect area and that inhibiting STAT3 reduced this effect., Conclusions: The in vitro study revealed that the duration of hypoxia is crucial for osteogenic differentiation of precursor cells. The results from both the in vitro and in vivo studies show the role of STAT3 signaling in hypoxia-induced osteogenic differentiation of precursor cells and bone defect healing., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

The treatment of massive bone defects is still a significant challenge for orthopedists. Here we have engineered synthetic porous AuPd alloy nanoparticles (pAuPds) as a hyperthermia agent for in situ bone regeneration through photothermal therapy (PTT). After being swallowed by cells, pAuPds produced a mild localized heat (MLH) (40-43 °C) under the irradiation of a near-infrared laser, which can greatly accelerate cell proliferation and bone regeneration. Almost 97% of the cranial defect area (8 mm in diameter) was covered by the newly formed bone after 6 weeks of PTT. RNA sequencing analysis was used to obtain insight into the molecular mechanism of the MLH on cell proliferation and bone formation. These results demonstrated that the Wnt signaling pathway was involved in the MLH. This Letter provides a unique strategy with mild heat stimulation and high efficiency for in situ bone regeneration.

The regeneration of bone tissue is regulated by both osteogenic and angiogenic growth factors which are expressed in a coordinated cascade of events. The aim of this study was to create a dual growth factor-release system that allows for time-controlled release to facilitate bone regeneration. We fabricated core-shell SF/PCL/PVA nanofibrous mats using coaxial electrospinning and layer-by-layer (LBL) techniques, where bone morphogenetic protein 2 (BMP2) was incorporated into the core of the nanofibers and connective tissue growth factor (CTGF) was attached onto the surface. Our study confirmed the sustained release of BMP2 and a rapid release of CTGF. Both in vitro and in vivo experiments demonstrated improvements in bone tissue recovery with the dual-drug release system. In vivo studies showed improvement in bone regeneration by 43% compared with single BMP2 release systems. Time-controlled release enabled by the core-shell nanofiber assembly provides a promising strategy to facilitate bone healing.

Boron (B), a trace element found in the human body, plays an important role for health of bone by promoting the proliferation and differentiation of osteoblasts. Our research group previously fabricated B-mesoporous bioactive glass (MBG) scaffolds, which successfully promoted osteogenic differentiation of osteoblasts when compared to pure MBG scaffolds without boron. However, the mechanisms of the positive effect of B-MBG scaffolds on osteogenesis remain unknown. Therefore, we performed in-vivo experiments in OVX rat models with pure MBG scaffolds and compared them to B-MBG scaffold. As a result, we found that B-MBG scaffold induced more new bone regeneration compared to pure MBG scaffold and examined genes related to bone regeneration induced by B-MBG scaffold through RNA-seq to obtain target genes and epigenetic mechanisms. The results demonstrated an increased expression and affiliation of Setd7 in the B-MBG group when compared to the MBG group. Immunofluorescent staining from our in vivo samples further demonstrated a higher localization of Setd7 and H3K4me3 in Runx2-positive cells in defects treated with B-MBG scaffolds. KEGG results suggested that specifically Wnt/β-catenin signaling pathway was highly activated in new bone area associated with B-MBG scaffolds. Thereafter, in vitro studies with human bone marrow stem cells (hBMSCs) stimulated by extracted liquid of B-MBG scaffolds was associated with significantly elevated levels of Setd7, as well as H3K4me3 when compared to MBG scaffolds alone. To verify the role of Setd7 in new bone formation in the presence of Boron, Setd7 was knocked down in hBMSCs with stimulation of the extracted liquids of B-MBG or MBG scaffolds. The result showed that osteoblast differentiation of hBMSCs was inhibited when Setd7 was knocked down, which could not be rescued by the extracted liquids of B-MBG scaffolds confirming its role in osteoblast differentiation and bone regeneration. As a histone methylase, Setd7 may be expected to be a potential epigenetic target for new treatment schemes of osteoporosis., Statement of Significance: Boron-containing MBG scaffold has already been proved to promote bone regeneration in femoral defects of OVX rats by our research group, however, the epigenetic mechanism of Boron's positive effects on bone generation remains ill-informed. In our present study, we found an increased expression and affiliation of Setd7 and H3K4me3 in Runx2-positive osteoblasts in vivo. And in vitro, the higher expression of Setd7 enhanced osteogenic differentiation of human BMSCs stimulated by extracted liquids of B-MBG scaffold compared to MBG scaffold, which was associated with the activation of Wnt/β-catenin signaling pathway. Above all, it suggests that Setd7 plays an positive role in osteogenic differentiation and it may become a potential epigenetic target for new schemes for osteoporosis., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Bone tissue engineering has become one of the most effective methods for treating bone defects. In this study, an electrospun tissue engineering membrane containing magnesium was successfully fabricated by incorporating magnesium oxide (MgO) nanoparticles into silk fibroin and polycaprolactone (SF/PCL)-blend scaffolds. The release kinetics of Mg2+ and the effects of magnesium on scaffold morphology, and cellular behavior were investigated. The obtained Mg-functionalized nanofibrous scaffolds displayed controlled release of Mg2+, satisfactory biocompatibility and osteogenic capability. The in vivo implantation of magnesium-containing electrospun nanofibrous membrane in a rat calvarial defect resulted in the significant enhancement of bone regeneration twelve weeks post-surgery. This work represents a valuable strategy for fabricating functional magnesium-containing electrospun scaffolds that show potential in craniofacial and orthopedic applications. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) undergo osteogenic differentiation during bone defect healing. However, the role of JAK2/STAT3 in the osteogenic differentiation of MSCs and bone defect healing is still not fully understood. In this study, we aimed to analyze the effect of AG490, a JAK2-specific inhibitor, on MSCs proliferation and osteogenic differentiation as well as in bone defect healing. We used AG490 to inhibit the JAK2/STAT3 signaling in a mice bone marrow stromal cells (BMSCs) culture. AG490 inhibited BMSCs proliferation and osteogenic differentiation markers, i.e. Col1α, Alp and Ocn expression in mRNA and protein levels. Inhibition of JAK2 reduced ALP activity and matrix mineralization in BMSCs culture. Inhibition of JAK2 reduced phosphorylation of STAT3, AKT, P38, and JNK phosphorylation. Immunohistochemistry showed high numbers of pJAK2, pSTAT3 and ALP positive cells and AG490 reduced this effect in vivo. Histology and μ-computed tomography (CT) data showed that AG490 treatment inhibits bone regeneration and bone defect healing. Our results clearly showed the inhibitory effect of AG490 on proliferation and osteogenic differentiation of BMSCs, bone regeneration and bone defect healing. Moreover, AG490 inhibited phosphorylation of STAT3, P38, JNK and AKT. This suggests the possible role of JAK2/STAT3 signaling in hypoxia-induced osteogenic differentiation of MSCs and bone defect healing. [ABSTRACT FROM AUTHOR]

Adenosine (Ade) has been identified to stimulate bone formation. However, the use of Ade is severely limited by the accompanying side effects and its very short half-life in vivo. This study aimed to fabricate an efficient drug-delivery system to reduce the undesirable side effects and enable the clinical application of Ade for treating large bone defects. The fabricated poly(ε-caprolactone) (PCL)/Ade-polyvinyl alcohol (PVA) (0.3/0.4) nanofibrous mats with 0.3:0.4 (w/w) ratio of Ade and PVA showed a sustained and controlled release of Ade and facilitated the osteogenic differentiation of bone mesenchymal progenitor cells (BMSCs). A larger amount of newly formed bone was observed in vivo in the cranial defects of the PCL/Ade-PVA (0.3/0.4) group compared with those of the non-loaded PCL/PVA nanofibrous mats at 4 and 8 weeks after surgery. Moreover, it is the first time to confirm that Ade mediates the osteogenesis of rat BMSCs through the STAT3 signaling pathway and restrains the osteoclastogenesis of rat bone-marrow macrophages (BMMs). These results suggested that this coaxial drug-delivery system loaded with Ade provided a promising and clinically relevant platform to controlled-release Ade and address large bone defects. The adenosine (Ade) that continuously releases from the coaxial PCL/Ade-PVA (0.3/0.4) nanofibers can facilitate the defect healing via interaction with bone mesenchymal progenitor cells (BMSCs) and bone marrow macrophages (BMMs). Unlabelled Image • STAT3 signaling is involved in the process of osteogenesis conducted by adenosine • Adenosine encapsulated into the coaxial nanofibers steadily releases • Coaxial nanofibrous mat loaded with adenosine is a promising drug delivery vehicle for large bone defects therapy • Coaxial nanofibrous mat loaded with adenosine is void of side effects induced by systemic administration of adenosine [ABSTRACT FROM AUTHOR]

Abstract: Background: As a model animal, the mouse has already been widely used in bone-related research. However, there is a lack of ideal long bone segmental defect mouse model. Since external fixation has disadvantages of heavy weight, penetrating the skin, and hampering mobility, an internal fixation device is probably more preferable to maintain the segmental bone defect. The aim of this study was to establish a simple, reproducible, and standardized murine critical-size defect model through designing an internal fixation system, verifying its adaptability, and investigating the critical size of femoral segmental defect. Methods: By utilizing computer-aided measuring and processing system, anatomical data of adult C57BL/6 mouse femur was obtained, and a plate-bolts system was designed for rigid fixation. The plate and screws were fixed in 67 mice and 1.5 or 2.0 mm defect gaps were created in the femoral midshaft. Compression and three-point bending of bone-implant construct were tested in mice at 0, 2, 5, and 12 wk postoperative to test the biomechanical stability. X-ray, micro-computed tomography, and histology were used to investigate the defect healing process. Results: The plate- and screws-fitted mouse femur and unilateral or bilateral operation had seemingly no adverse impact on the mouse in general. Mechanical tests indicated that there were no significant differences between the bone-implant construct and intact femur in compression and three-point bending loading. Micro-computed tomography scanning showed the bone mineral density had not been affected by the implantation of fixation device. There was no union of the 2.0 mm segmental defect in 12-wk period. Conclusion: Using the specifically designed rigid internal fixation device, a segmental defect size of 2.0 mm in C57BL/6 mouse femur will show nonunion and can serve as a critical defect size for bone tissue engineering and bone regeneration research. [Copyright &y& Elsevier]

[Display omitted] Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEV) have shown promising prospects as a cell-free strategy for bone tissue regeneration. Here, a bioactive MSCs-sEV-loaded electrospun silk fibroin/poly(ε-caprolactone) (SF/PCL) scaffold was synthesized via a mussel-inspired immobilization strategy assisted by polydopamine (pDA). This pDA modification endowed the as-prepared scaffold with high loading efficiency and sustained release profile of sEV. In addition, the fabricated composite scaffold exhibited good physiochemical, mechanical, and biocompatible properties. In vitro cellular experiments indicated that the MSCs-sEV-loaded composite scaffold promoted the adhesion and spreading of preosteoblast and endothelial cells, as well as enhanced osteogenic differentiation and angiogenic activity. In vivo experiments showed that the functionalized electrospun scaffolds promoted bone regeneration in a rat calvarial bone defect model. Results suggest that the developed MSCs-sEV-anchored pDA-modified SF/PCL electrospun scaffolds possess high application potential in bone tissue engineering owing to their powerful pro-angiogenic and -osteogenic capacities, cell-free bioactivity, and cost effectiveness. [ABSTRACT FROM AUTHOR]

• Exosomes mediate cell-free regenerative therapy. • ATDC5-derived exosomes induce osteoblastic differentiation of MSCs in vitro. • Engineered exosomes encapsulating VEGF165 promote vascular bone regeneration. • Extending exosome applications from simple carriers to enhanced therapy. Exosome as an advanced carrier has been extensively used in gene and drug delivery due to its excellent biocompatibility, efficient cell uptake and convenient targeted modification. Apart from the conventionally controlled release of bioactive molecules, some other exosome roles should be further explored. Herein, we report to construct a specifically gene-activated engineered exosome that not only can effectively modulate the gene release of the vascular endothelial growth factor 165 (VEGF165), but also can play a pivotal role in enhancing therapy in vascular bone regeneration. Our findings revealed that the transfection efficiency of the VEGF165 plasmid gene was extremely elevated via the exosome-based vector. Both alone exosomes and engineered exosomes exhibited a certain osteoblastic differentiation of precursor cells (MC3T3-E1) in vitro. More importantly, the engineered exosomes that internalized the VEGF165 gene combined with electrospun nanofiber films played a dual role in osteogenesis and angiogenesis based on the evaluation of a rat critical-sized calvarial defect model in vivo. Consequently, our current work creates a bifunctional engineered exosome-based biomaterial that can direct progenitor cell differentiation in vitro and promote the vascularized bone regeneration in vivo, extending exosome applications from simple biomolecular carriers to exosome-enhanced therapy. [ABSTRACT FROM AUTHOR]